IN PARTIAL FULFILLMENTS FOR THE DEGREE OF ...prr.hec.gov.pk/jspui/bitstream/123456789/14886/1/Misbah...iii EXAMINATION COMMITTEE The thesis viva MISBAH SULTANA (REG.# DBC -0212300

ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND

EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN

IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY




INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY







UPDATE FROM PAKISTAN

SUBMITTED BY:

MISBAH SULTANA

(REG. # DBC


(Ph.D) IN BIOCHEMISTRY


THE UNIVERSITY OF LAHORE




SUBMITTED BY:

MISBAH SULTANA

(REG. # DBC-021230





2012-2017




SUBMITTED BY:

MISBAH SULTANA

02123009)





2017
















In the name of Allah the most gracious the most mercifulIn the name of Allah the most gracious the most merciful

"Bismillah al rahman al rahim"

In the name of Allah the most gracious the most merciful

i








In the name of Allah the most gracious the most mercifulIn the name of Allah the most gracious the most merciful

ii

SUPERVISORY COMMITTEE

We, the Supervisory Committee, certify that the contents and form of thesis submitted by MISBAH SULTANA (DBC-02123009) have been found satisfactory after receiving all the evaluation reports (External/Internal) and recommended it for the award of the degree of Doctor of Philosophy (PhD) in Biochemistry under title “ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN UPDATE FROM PAKISTAN”.

SUPERVISOR: PROF. DR. ARIF MALIK ___________________

Institute of Molecular Biology and Biotechnology (IMBB/

Center for Research in Molecular Medicine (CRiMM)

The University of Lahore.

CO-SUPERVISOR: PROF. DR. M. H. QAZI____________________

Institute of Molecular Biology and Biotechnology (IMBB/

Center for Research in Molecular Medicine (CRiMM)

The University of Lahore.

iii

EXAMINATION COMMITTEE

The thesis viva MISBAH SULTANA (REG.# DBC-02123009) was held on 00-00-2015 at the Institute

of Molecular Biology and Biotechnology/ Center for Research in Molecular Medicine (CRiMM), The

University of Lahore. The Supervisor/Co-Supervisor and the examiner gave satisfactory remarks on thesis

and viva and were approved for the award of the degree of Doctor of Philosophy (PhD) in Biochemistry

under title “ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS

AND EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN

PUNJAB. AN UPDATE FROM PAKISTAN”.

______________________________________ ______________________________________

EXTERNAL EXAMINER INTERNAL EXAMINER

PROF. DR. ARIF MALIK

INSTITUTE OF MOLECULAR BIOLOGY AND

BIOTECHNOLOGY (IMBB/CENTER FOR

RESEARCH IN MOLECULAR MEDICINE (CRIMM).

____________________________________________

PROF. DR. M. H. QAZI

DIRECTOR,

Institute of Molecular Biology and Biotechnology (IMBB/Center for research in molecular medicine (CRiMM)

iv

ACKNOWLEDGEMENTS

ALL PRAISES FOR ALLAH, WHO GUIDES US FROM DARKNESS TO LIGHT AND HELP

US IN ALL HARDSHIPS, AND ALL MY REVERENCE IS FOR HIS HOLY PROPHET

MOHAMMAD (P.B.U.H) WHO ENABLES US TO RECOGNIZE OUR CREATOR. I AM GRATEFUL

TO THAT SPIRITUAL POWER WHO HELPS ME AT EVERY STAGE OF MY LIFE. GREAT

PERSONALITIES HAVE BROUGHT IMPECCABLE TRANSFORMATION AND AMAZING

CHANGES IN THE USUAL ROUTINES. WHETHER IT IS POLITICS OR ARTS OR SCIENCE,

WHETHER IT IS RELIGIOUS OR PHILOSOPHICAL, WHETHER IT IS BIG OR SMALL, THE

IMPACT MADE BY PEOPLE MADE THEM FAMOUS PERSONALITIES. PROF. DR. ARIF MALIK

“INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY/CENTER FOR RESEARCH

IN MOLECULAR MEDICINE” MY RESPECTED SUPERVISOR; HIS IDEAS HAVE MANAGED TO

COME THROUGH AND HAVE REVOLUTIONIZED THE FACE OF MODERN SCIENCE. IT IS MY

FOREMOST DUTY TO EXPRESS MY HEARTIEST AND SINCEREST GRATITUDE TO MY

RESPECTED CO-SUPERVISOR PROF. DR. M.H. QAZI, DIRECTOR, INSTITUTE OF

MOLECULAR BIOLOGY AND BIOTECHNOLOGY FOR THEIR KIND GUIDANCE DURING MY

STUDIES. I AM MUCH OBLIGED TO HIM FOR PRODUCTIVE GUIDANCE.

MISBAH SULTANA

REG.# DBC-02123009

v

DECLARATION OF ORIGINALITY

NAME OF STUDENT: MISBAH SULTANA

REG.#: (REG.# DBC-02123009)

DECLARATION

1-understand what plagiarism is and I am aware of the University’s policy in this regard.

2-I declare the thesis is my own original work. Where other people’s work has been used (Either from a

printed source, Internet or any sources), this has properly acknowledged and referenced in accordance

with departmental requirements.

3-I have not used work previously produced by another student or any other person to hand in as my own.

4-I have not allowed, and will not allow, anyone to copy my work with the intention of passing it off as

his or her own work.

SIMILARITY INDEX ( %) ON DAY (0-0-2017 At 0:00 PM)

SIGNATURE OF STUDENT: -----------------------------------------------------------------------------

SIGNATURE OF SUPERVISOR: ------------------------------------------------------------------------

SIGNATURE OF CO-SUPERVISOR: ------------------------------------------------------------------

SIGNATURE OF LIBRARIAN: --------------------------------------------------------------------------

vi

DEDICATION

Dedicated

To

MY LOVELY FATHER, Whose Prayers and Constant Encouragement Enabled Me to

Complete this Task of Learning

vii

LIST OF ABBREVIATIONS

TRH Thyrotropin releasing hormone

TSH Thyroid stimulating hormone

VIP Vasoactive inhibitory peptide

PAF Prolactin activating factor

PIF Prolactin inhibitory factor

MATI/II Methionine adenosyltransferase

SAM S-adenosyl methionine

GNMT Glycine N-methyltransferase

SAH S- adenosylhomocysteine

SAHH S- adenosylhomocysteine hydrolase

CSβ Cystathionine beta

MS Methionine synthase

MSR Methionine synthase reductase

THF Tetrahydrofolate

MTHFR Methlene tetrahydrofolate reductase

CH2-THF 5, 10-Methlenetetrahydrofolate

CH3-THF 5-methltetrahydrofolate

PLP Pyridoxal Phosphate

SHMT Serine methylhydroxytransferase

VitB6 Vitamin B6 (pyridoxine)

VitB9 Vitamin B9 (Folic acid)

VitB12 Vitamin B12 (Cobalamine)

NAC N-acetylcysteine

Glu Glutamate

r-Glu-Cys Gamma-Glutamate-Cysteine

Glu-Cys ligase Glutamate-Cysteine ligase

GSH synthase Glutathione synthase

viii

NADPH Nicotinamide adenine dinucleotide Phosphate

FADPH Flavin adenine dinucleotide Phosphate

8-oxodG 8-oxo- 7, 8 dihydro-2’-deoxyguanosine

8-oxoGuo 8-oxo- 7, 8 dihydroguanosine

I- Iodide ion

I2 Iodine molecule

MIT Mono-iodotyrosine

DIT Di-iodotyrosine

RT3 Reverse tri-iodothyronine

T3 Tri-iodothyronine

T4 Tetra-iodothyronine

ALT Alanine aminotransferase

AST Aspartate aminotransferase

BUN Blood urea nitrogen

CAT Catalase

GSH Glutathione

GSH Glutathione

GSSH Glutathione disulphide

GSHr Glutathione reductase

GSHPx Glutathione peroxidase

CAT Catalase

O2- Superoxide free radical

H2O2- Hydrogen peroxide free radical

SOD Superoxide dismutase

MDA Malondialdehyde

ONOO- Peroxynitrate

TPP Total Plasma Peroxidases

NO Nitric oxide

NOS Nitric oxide synthase

ix

LPO Lipid peroxidation

ROS Reactive oxygen specie

SOD Superoxide dismutase

TBARS Thiobarbituric acid reactive substances

TNF Tumor necrosis factor

x

LIST OF SYMBOLS

SYMBOLS NAME

°C Degree centigrade

> Greater than

< Less than

rmp Rotation per minute

µl Micro liter

% Percentage

nm Nano meter

mg/dl Mille gram per deci liter

mg/kg Mille gram per killo gram

g/dl Gram per deci liter

IU/L International units per liter




ACKNOWLEDGEMENT

DEDICATION


LIST OF SYMBOLS

CHAPTERS

CHAPTER ONE

CHAPTER TWO

CHAPTER THREE

CHAPTER FOUR

CHAPTER FIVE

CHAPTER SIX

CHAPTER SEVEN




ACKNOWLEDGEMENT

DEDICATION


LIST OF SYMBOLS

CHAPTERS

CHAPTER ONE

CHAPTER TWO

CHAPTER THREE

CHAPTER FOUR

CHAPTER FIVE

CHAPTER SIX

CHAPTER SEVEN




ACKNOWLEDGEMENT


LIST OF SYMBOLS

INTRODUCTION

REVIEW OF LITERATURE

MATERIALS AND METHODS

RESULTS

DISCUSSION

SUMMARY

CONCLUSION

LITERATURE CITED

APPENDIX

TABLE OF CONTENTS

INTRODUCTION



RESULTS

DISCUSSION

SUMMARY

CONCLUSION

LITERATURE CITED

APPENDIX

xi

TABLE OF CONTENTS

DESCRIPTION

INTRODUCTION



DISCUSSION

CONCLUSION

LITERATURE CITED

TABLE OF CONTENTS

DESCRIPTION



LITERATURE CITED

MATERIALS AND METHODS MATERIALS AND METHODS

i

ii

iii

iv

v

vii

ix

PAGE #

1

11

23

59

131

146

90

92

120

xii

TABLE OF CONTENTS

S.# DESCRIPTION PAGE. #

1.0 INTRODUCTION 01

2.0 REVIEW OF LITERATURE 11

2.1 OXIDATIVE STRESS MARKERS 11

2.2 INFLAMMATION IN UMBILICAL CORD IN

BLOOD

11

2.3 NON-PROTEIN BOUND IRON AS

PREDICTIVE MARKER OF NEONATAL

BRAIN DAMAGE

12

2.4 HUMAN MILK ENHANCES ANTIOXIDANTS 12

2.5 LEUKOCYTOSIS IN PRETERM INFANTS

WITH INTRAVENTRICULAR

HEMORRHAGE

13

2.6 INFLAMMATION AT BIRTH 13

2.7 NITRIC OXIDE THERAPY IN PREMATURE

INFANTS

14

2.8 NPBI AND F2-ISOPROSTANES IN NEWBORN 14

2.9 MELATONIN PROTECTS AGAINST

OXIDATIVE DAMAGE

14

2.10 MATERNAL ANEMIA 15

2.11 IRON STATUS AT BIRTH 15

2.12 MORTALITY OF PRETERM BIRTHS 15

2.13 IRON DEFICIENCY ANEMIA AND SEVERE

PREGNANCY OUTCOME

15

2.14 PREGNANCY OUTCOME ASSOCIATED

WITH ANEMIA AND IRON

16

2.15 IRON IN PREGNANCY 16

2.16 NEGATIVE EFFECT OF OXIDATIVE STRESS

IN FETUS AND NEWBORN

18

2.17 HYPOXIA AND NITRIC OXIDE 20

xiii

2.18 PARTURITION AND OXIDATIVE STRESS 20

2.19 OXIDATIVE STRESS AND DIABETIC

PREGNANCY

21

2.20 ROLE OF OXIDATIVE STRESS IN

MENOPAUSE AND AGE RELATED

FERTILITY DECLINE

21

2.21 STRATEGIES TO OVERCOME OXIDATIVE

STRESS IN ASSISTED REPRODUCTION

21

2.22 PHYSIOLOGICAL STATUS OF MOTHERS 22

2.23 ROLE OF OXIDATIVE STRESS AND LIPID

PEROXIDATION IN PRETERM DELIVERY

23

3.0 MATERIALS AND

METHODS

31

3.0 MATERIALS AND METHODS 32

3.1 INCLUSION CRITERIA 32

3.2 EXCLUSION CRITERIA 32

3.3 CHEMICALS 33

3.4 BIOCHEMICAL ASSAYS 33

3.4.1 ESTIMATION OF GLUTATHIONE (GSH) 33

3.4.2 ESTIMATION OF CATALASE (CAT) 33

3.4.3 ESTIMATION OF THIOBARBITURIC ACID

REACTIVE SUBSTANCES (TBARS)

33

3.4.4 ESTIMATION OF SUPER OXIDE

DISMUTASE (SOD)

34

3.4.5 ESTIMATION OF GLUTATHIONE

REDUCTASE (GR)

34

3.4.6 ESTIMATION OF VITAMIN C 34

3.4.7 DETERMINATION OF ADVANCED

OXIDATIVE PROTEIN PRODUCTS (AOPPs)

35

3.4.8 PREPARATION AND DETERMINATION OF

(AGEs)

35

xiv

3.4.9 DETERMINATION OF GAMMA

GLUTAMYL TRANSFERASE (GGT)

35

3.4.10 DETERMINATION OF NITRIC OXIDE 36

3.4.11 DETERMINATION OF TUMOR NECROSIS

FACTOR BY ABCAM’s TNF ALPHA

HUMAN ELISA KIT

36

3.4.12 ESTIMATION OF VITAMIN-E 36

3.4.13 DETERMINATION OF VITAMIN A 37

3.4.14 DETERMINATION OF INTERLEUKIN-6 “IL-

6” LEVELS

37

3.4.15 ESTIMATION OF C-REACTIVE PROTEIN 38

3.4.16 DETERMINATION OF GPx 38

3.4.17 DETERMINATION OF TOTAL

CHOLESTEROL (TCh)

38

3.4.18 DETERMINATION OF TRIGLYCERIDES

(TG)

38

3.4.19 ESTIMATION OF LOW DENSITY

LIPOPROTEIN (LDL)

39

3.4.20 DETERMINATION OF HIGH DENSITY

LIPOPROTEINS (HDL)

39

3.4.21 ESTIMATION OF TOTAL BILIRUBIN 39

3.4.22 DETERMINATION OF

MYELOPEROXIDASE (MPO)

39

3.4.23 ESTIMATION OF SODIUM AND POTASSIUM 40

3.4.24 DETERMINATION OF NEUTROPHILS 40

3.4.25 DETERMINATION OF MAGNESIUM AND

CALCIUM

40

3.4.26 DETERMINATION OF COMPLETE BLOOD

COUNT (CBC) IN THE SERUM

40

3.4.27 ESTIMATION OF SERUM FOLATE

(VITAMIN B9) AND VITAMIN B12

57

xv

3.4.28 DETERMINATION OF TOTAL IRON

BINDING CAPACITY

41

3.4.28 DETERMINATION OF RENAL FUNCTION

TESTS (RFTs)

41

3.4.29 ESTIMATION OF VITAMIN D (Vit D) IN THE

SERUM OF PRETERM INFANTS

41

4.0 RESULTS 42

4.1 LEVEL OF HEMOLYTIC CELLS IN PRETERM INFANTS (PTI)

43

4.2 LEVEL OF RFT and LFT IN PRETERM INFANTS (PTI)

44

4.3 LEVEL OF LIPID PROFILE IN PRETERM INFANTS (PTI)

44

4.4 LEVEL OF STRESS BIOCHEMICAL MARKERS IN PRETERM INFANTS (PTI)

45

4.5 LEVEL OF DIFFERERENT TYPES OF VITAMINS (B12, A, C, E AND D) IN PRETERM INFANTS (PTI)

45

4.6 EFFECT OF PHYSICAL MARKERS ON PRETERM INFANTS (PTI)

46

4.7 LEVEL OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) IN PRETERM INFANTS (PTI)

46

4.8 LEVEL OF ELECTROLYTES (HCO3, Ca, Na, K, Mg, CL-, Zn, Cu AND P) IN PRETERM INFANTS (PTI)

46

4.9 LEVEL OF INFLAMMATORY BIOMARKERS IN PRETERM INFANTS (PTI)

47

5.0 DISCUSSION 49

6.0 SUMMARY 65

7.0 CONCLUSION 69

8.0 PART II 71

9.0 INTRODUCTION 72

10.0 REVIEW OF LITERATURE 79

10.1 SELENIUM ROLE IN PRETERM LABOR 81

10.2 VITAMIN D AND SPONTANEOUS PRETERM

BIRTH

82

10.3 ROLE OF THYROID HORMONE IN 84

xvi

PRETERM DELIVERY: ANATOMY AND

PHYSIOLOGY OF THYROID GLAND

10.4 THYROID FUNCTION DURING PREGNANCY 86

10.5 MATERNAL THYROID FUNCTION AND

FETAL BRAIN DEVELOPMENT

87

10.6 IMPACT OF THYROID AUTOANTIBODIES 87

10.7 PRETERM PREMATURE RUPTURE OF

MEMBRANES (PPROM)

88

10.8 COMMON BIOLOGICAL PATHWAYS TO

PRETERM BIRTH

89

10.9 INTRAUTERINE INFLAMMATION AND

INFECTION PATHWAY

89

10.10 EXTRACELLULAR MATRIX DEGRADATION PATHWAY

90

10.11 FETAL STRESS PATHWAY 91

10.12 FETAL ANOMALIES PATHWAY 92

10.13 ESTROGEN METABOLISM PATHWAY 92

11.0 MATERIALS AND

METHODS

94

11.0 MATERIALS AND METHODS 95

11.1 SOURCE OF DATA 95

11.2 INCLUSION CRITERIA 95

11.3 EXCLUSION CRITERIA 95

11.4 CHEMICALS 95

11.5 FOLLOWING PARAMETERS WILL BE

ESTIMATED

95

11.5.1 ESTIMATION OF THYROID PROFILE IN

SERUM SAMPLES

96

11.5.2 ESTIMATION OF T3 (Tri-iodothyronine)

ELISA

96

11.5.3 ESTIMATION OF T4 (Thyroxine) ELISA 96

xvii

11.5.4 ESTIMATION OF TSH (Thyroid stimulating

hormone) ELISA

96

11.5.5 ESTIMATION OF TPO (Thyroid peroxidase)

IgG ELISA

96

11.5.6 ESTIMATION OF Thyroglobulin IgG ELISA 96

11.5.7 ESTIMATION OF STRESS BIOMARKERS IN

SERUM SAMPLES

96

11.5.8 ESTIMATION OF THIOBARBITURIC ACID

REACTIVE SUBSTANCES (TBARS)

96

11.5.9 ESTIMATION OF SUPER OXIDE

DISMUTASE (SOD)

96

11.5.10 ESTIMATION OF GLUTATHIONE (GSH) 97

11.5.11 ESTIMATION OF CATALASE (CAT) 97

11.5.12 DETERMINATION OF NITRIC OXIDE 97

11.5.12.1 PRINCIPLE 97

11.5.12.2 CHEMICALS REQUIRED 98

11.5.12.3 PROTOCOL 98

11.5.13 ESTIMATION OF INTERLEUKIN-6 (IL-6) (BY

ELISA KIT)

98

11.5.13.1 PROCEDURE 98

11.5.14 DETERMINATION OF TUMOR NECROSIS

FACTOR BY ABCAM’s TNF ALPHA HUMAN

ELISA KIT

99

11.5.15 DETERMINATION OF GLUTATHION

PEROXIDASE (GPx)

100

11.5.15.1 PROCEDURE 101

11.5.16 ESTIMATION OF GLUTATHIONE

REDUCTASE (GR)

101

11.5.17 DETERMINATION OF AOPPs 101

11.5.18 DETERMINATION OF VITAMIN 102

xviii

11.5.19 ESTIMATION OF VITAMIN-E IN SAMPLE 102

11.5.19.1 PRINCIPLE 102

11.5.19.2 REAGENTS 102

11.5.19.3 PROCEDURE 103

11.5.20 ESTIMATION OF VITAMIN-C IN LIVER

SAMPLE

103

11.5.20.1 PRINCIPLE 103

11.5.20.2 REAGENTS 103

11.5.21 EXTRACTION OF VITAMIN-C 103

11.5.21.1 PROCEDURE 104

12.0 RESULTS 105

12.1 THYROID BIOMARKERS PROFILE OF

PRETERM MOTHER PATIENTS Vs

CONTROL

106

12.2 CIRCULATING STRESS BIOMARKERS

PROFILE OF PRETERM MOTHERS VS

CONTROL

106

12.3 ANTIOXIDANT BIOMARKERS PROFILE OF

PRETERM MOTHER PATIENTS Vs

CONTROL

107

13.0 DISCUSSION 111

14.0 CONCLUSION 118

15.0 LITERATURE CITED 120

xix

LIST OF TABLES


TABLE-01 DEMOGRAPHIC DISTRIBUTION OF DATA 32

TABLE-02 VARIABLES OF MEDICAL IMPORTANCE IN THE PRETERM INFANTS (PTI) SUFFERING FROM ANEMIA

47

TABLE-03 THYROID BIOMARKERS PROFILE OF PRETERM DELIVERY

FEMALE PATIENTS Vs CONTROL

108

TABLE-04 STRESS BIOMARKERS PROFILE OF PRETERM DELIVERY

FEMALE PATIENTS Vs CONTROL

108

TABLE-05 ANTIOXIDANT BIOMARKERS PROFILE OF PRETERM

MOTHERS Vs CONTROL

109

xx

LIST OF FIGURES


FIGURE 01 PATHOGENESIS OF PRETERM INFANTS 62

FIGURE 02 MECHANISM OF DIFFERENT PARAMETERS INDUCING

PRETERM BIRTH

117

CHAPTER ONE INTRODUCTION MISBAH, 2017

1

INTRODUCTION


2

1.0 INTRODUCTION

Infants born before completion of 37 weeks of pregnancy are termed as premature.

Almost 12% of deliveries in normal population are thought to be premature. ‘Preterm’ and

‘preemie’ are the terms used for premature babies. Short gestational period/premature birth is

responsible for underdeveloped organs in such babies, therefore they are at higher risk to develop

multiple problems, such as problems associated with respiratory and digestive system etc.

Additionally the development and growth of brain, lungs also remain improper. In premature

birth fetus will also be improperly developed. Immature development of lungs such as lungs will

not be able to make ‘surfactant’ also termed as respiratory distress syndrome (RDS). Normally

alveoli plays essential role in lungs surfactant as the level decreases it effects breathing whereas,

it is very difficult to reopen the alveolar sac. Such babies are aided with artificial surfactant.

Likewise, bronchopulmonary dysplasia is also one of the lungs disease can be seen in babies

born prematurely it is characterized by injured lungs hence, also known as chronic lung disease.

In 22-25 weeks of pregnancy the lungs are not properly formed. If baby is born in this condition

would not be able to survive outside the womb whereas, the respiratory problems can be

managed in the following ways

a) Proper inhalation of oxygen by the help of nasal cannulas, regulating air ways, b) Aided by

alveoli function as only oxygen cannot deliver continuous positive airway pressure (CPAP). c)

Addiitional support is delivered by intubation; in that case tube is being connected with the

aiding machine termed as ventilator. If breathing centre is not properly developed it may result in

breathing pauses (apnea). Apnea is a reason for reduction of heart rate and that apnea is

maintained by the help of nasal canula, ventilator, CPAP and other breathing aids. Essentially

after 38 weeks of pregnancy changes can be observed in the babies include change in their heart

and lungs whereas, these changes may not occur in the case of preterm babies. Hence, they may

suffer severe infectious state. Such babies may have low blood pressure, improperly developed

intestine and several other medical conditions that may be effectively treated with the

administration of proper medication only. So, minor quantities of feed may be provided to them

which may be even less than one teaspoon. In such condition breast feed (milk) is considered

best for the babies (especially premature babies) it may best nourish and lest exposed to

infections, fewer initial complications and improved growth. Mothers are hence boosted to breast

feed her baby.


3

The prevalence of necrotizing enterocolitis (NEC) is higher in newborns and is believed to be

the reason for consequential problems. Therefore, such babies are fed with the help of tiny tubes

by the nose and mouth, blood vessels of immature babies are more fragile and can be ruptured

easily bleeding problems may be from minor to severe which may be the reason for lifetime

developmental issues. Retina is usually developed at 34th week of pregnancy hence in premature

babies they may have undeveloped retina, breathing centre and due to such issues they are kept

in oxygen tanks. Ophthalmologists regularly observe these preterm babies for the overgrowth of

the blood vessels of retina. Newborns are highly prone to infections; therefore, they are given

with antibiotics to accelerate immune system to overcome infections. There are numerous factors

involved in premature birth of which some directly affect mother and babies ultimately leads to

early labor. Maternally inherited causes remain preeclampisa (because of toxemia during

pregnancy), chronic diseases of heart, kidney or liver etc. Certain kind of infections (infections

caused by B streptococcus, urinary tract and vaginal infections, fetal/placental tissue infections),

Abuse of drugs (such as cocaine), abnormality in structure of the uterus, cervical inability

(failure of the cervix to remain closed during pregnancy), preterm birth history, factors

concerning the pregnancy, abnormally placental functioning, polyhydramnios, placental

abruption, placenta Previa as well as premature rupture of uterus membrane. These factors

influence the fetus, its behavior and contribute as unfavorable environment for uterus, and serve

as reason for several gestation (twins, triplets, etc.) and hereditary abnormalities.

Globally, in 2010 15 million preterm births were estimated of which 1 million died

(Blencowe et al., 2012). Premature birth remains second largest risk factor for death in babies

under the age of 5 years. Another set of studies show premature babies died in their early four

months of life (Liu et al., 2012). Premature babies are prone to several health issues throughout

their life span. In developed countries rate of premature birth is one major health issue whereas,

underdeveloped countries lack evidence about the preterm deliveries. Born Too Soon (report)

tells us about the preterm births country wide and the records shows that it occurrence is

increasing. The effects of premature birth usually spread from birth to whole life. Preterm babies

are not actually with properly developed organs hence, they should be provided with an extra

care and attention as they are more prone to undergo health issues such as, intellectual

deficiency, chronic diseases of kidneys, lungs and liver, impaired vision and hearing

(Anonymous, 2007).


4

Special attention is required in this sector there should be proper gynecological diagnosis

and monitoring of NCDs, premature babies are at higher risk of hypertension and diabetes (Hovi

et al., 2007). There is a great relationship among preterm births and NCDs throughout the world.

About 9 million people below 60 years of age usually die because of NCDs, and the rate is

increasing day by day in Africa and other low-income counties (Anonymous, 2011). According

to WHO approximatly 135 million infants were delivered in 2010 of which 15 million almost

11.1% of deliveries were premature of which significant higher rates of premature births were

observed in Africa and Asia (Blencowe et al., 2012). According to the Millennium Development

Goal Regions in South Asia estimated highest rates of preterm deliveries to about 13.4% of all

live births. The rate is predominantly increased in Asia and states of Africa. Figures about

premature birth-related death are indefinite in relevant countries. In 88 countries the estimated

preterm delivery rate is less than 10%, at the same time in 11 countries the preterm birth rate is

15% or more. There are at least 10 countries including India, China, Nigeria, Pakistan, Indonesia,

United States, Bangladesh, the Philippines, Democratic Republic of the Congo and Brazil, which

have highest preterm birth rates. About 60% of all premature births belong to the upmentioned

countries. Death rates are higher in low gestational age (Qiu et al., 2011). 16% of total preterm

babies born were of less than 32 weeks of gestational age. Rate of death and disease are highest

in the premature babies. In high-income countries the developments of medical care enhanced

the rate of survival and coped with the outcomes of newborn infants efficiently (Saigal and

Doyle, 2008). In 1990, only two-third of premature born babies survived without any deficiency

(Mohangoo et al., 2011). Consesus in underdeveloped countries suggested that only 30% babies

were born in between 28-32 weeks of gestation but rate of mortatlity was increased in under-

developed countries as compared to developed ones.

On the chart of 65 countries about 10,000 living births are reported annually in Europe,

America and Australia. From the year 1999-2010 rates of preterm deliveries have increased

drastically, it is almost increased from 2 million to 2.2 million in year 2010. Rate of mortality in

newborns were reduced only in three countries that include Ecuador, Croatia and Estonia.

Preterm birth rates are constant in fourteen countries like less then 0.5%. In several under-

developed countries rate of preterm birth is either similar or in some cases it is bit higher. Late

and moderate preterm [LAMP] at 32 to 35 weeks, increase the percentage from previous reports

in low-income conteries (Davidoff et al., 2006). According to the previous reports in these


5

countries no change was observed in preterm births from 1990 to 2010. The data obtained from

many little- and medium-income countries rate of preterm should be carefully understood as

there was substantial data gap and the obtained information was not enough to show the

characteristics of relevant inhabitants. Rate of preterm birth in low and medium income states

such as China and South Asia have increased like other countries.

The NICU is an intensive care unit, arranged for the special care of preterm babies or

who have complications soon after birth. The NICU staff includes the following, Neonatologists,

Pediatricians, Neonatal nurse practitioners (NNPs), Nurses and respiratory therapists, Dietitians,

Physical therapists, and includes many other health care professionals for the care of a premature

baby. NICU babies heart rate, breathing rate, and oxygen levels monitored continuously. Most

premature babies are placed in incubator after birth, which provides babies with warm moist air.

Anemia has been defined as lower number of RBC’s than normal. Main function of RBC’s is to

deliver oxygen to tissues in the body. At the time of preterm birth, major cause of anemia is

declination in the level of erythropoietin (EPO) because of the low pressure of oxygen

(Stockman et al., 1984; Brown et al., 1984). In pregnant women iron deficiency anemia is

common public health problem in developing countries (Pasricha et al., 2008). Two third of

expecting women are affected and have low birth weight, high maternal morbidity and mortality

rate (Baig-Ansari et al., 2008). Mutually low socio economical and educational statuses are the

main reasons for high occurrence of anemia (Mahe et al., 2004). Twenty to fifty (20-50%)

percent of people in the world suffer from iron deficiency anemia. Iron deficiency is the most

significant risk factor of anemia in pregnant females (Breymann, 2006). Deficiency not only

affects mother but it also affects infant particularly his/her intellectual and psychomotor

functions and leads to anemia (Kilbride et al., 1992). According to WHO, anemia is a basic

health problem in countries with occurrence of anemia higher than 40% (Anonymous, 2001).

Anemia is defined by WHO as Hemoglobin less than 11gm in pregnancy, and it is further

divided into three categories:

I. Mild (9.0 - 10.9gm %),

II. Moderate (7.0-8.9gm) and

III. Severe degree (<7.0 gm%) (Marahatta R, 2007).

Anemia is a complication common in young, poor, pregnant women of racial minority

(Scholl et al., 1992). Early diagnosis and management of anemia is significant in direction to


6

prevent the maternal and prenatal morbidity (Aimaku and Olayemi, 2003). The incidence,

etiology and degree of severity of anemia differ in diverse populations worldwide. Almost 35%

women without pregnancy and 51% of women on whole suffer anemia. The rate might vary in

different regions as the occurence of anemia in females is 3 to 4 time higher in under developing

or low-income regions (Adiba et al., 2007). The frequency of anemia in pregnant women is as

much as 65% in South Asia (Schultink et al., 1993). In order to decrease anemia in pregnant

women an effective care should be taken at primary level. Sufficient supply of drugs to remove

the causes of anemia should be provided to improve the levels of Hb in pregnant women it may

also contribute in the plan to reduce the chances of anemia in infants (Massawe et al., 1999).

Vast number of pregnant women visited and got admitted in gynecological units with multiple

complications as the current study aims to rule out the causes and estimate the regularity of

anemic cases. Normally there are three main causes of anemia, First is the insufficient production

of RBC’s, Physiologic anemia is the example of less production of RBC’s because of growth of

the babies and Anemia is common in premature babies than full-term babies. Second, Break

down of red blood cells are so fast i.e., it is problematic when the mother and baby have different

blood types (Rh/ABO incompatibility). These babies usually suffer from jaundice

(hyperbilirubinemia) or Infections or genetic disorders can also cause this problem. And finally,

internal bleeding in premature babies can transfer blood from the baby to the mother in the

womb. Symptoms of anemia includes palness of skin, lethargy because of low energy, feeding is

not proper as baby gets tired, rapid heart rate and quick breathing when resting etc.

Tests for the diagnosis of anemia include estimation of following factors in the blood od

the patients;

• Hemoglobin - oxygen carrying protein in red blood cells

• Hematocrit - % of blood comprises red blood cells

• Reticulocyte count - % of immature red blood cells present in the blood. It measures new cells

in the blood which is produced in the body.

In the first few weeks of life, preterm infants may suffer from anemia due to the

following reasons, (a) Because of rapid growth in first months of life needs sufficient amount of

blood to maintain or stablize the levels of hemoglobin (Collard, 2009), (b) In new born babies

the formation of epo primarily produced from liver have no ability to generate the red blood cells

due to hypoxic condition (Ziajani et al., 1981 and Eckardt et al., 1992) subsequently the liver


7

have less ability regarding to anemia because it is the major factor for the development of

preterm infants (c) Erythropoiesis is the rate limiting enzyme which is expressed instantaneously

at the time of birth because it inhibit the transcription factor of hypoxia like hypoxia-inducible

factors (HIF) (Kling et al., 1996), (d) there are number of of iatrogenic phlebotomies in preterm

infants to cause the anemia as in such conditions the levels of red blood cells decreases due to

proginator cells (Strauss, 1995), (e) anemia can occur by certain pathological circumstances that

can cause prematurity (Carroll and Widness, 2012).

The severe type of anemia may be caused by lower gestational period. According to the

recent reports gestational period have positive correlation with the concentration of hemoglobin

in first 4 weeks of life because desired levels of hemoglobin have significant role in biological

system (Jopling et al., 2009). In first 28 days of life infants with gestational period of 4 to 5

months have significantly decreased levels of Hb as compared to infants with gestational period

of 35-42 weeks (Jopling et al., 2009). In premature infants deterioration of hemoglobins and

decreased potential to carry oxygen contributes in the occurrence of anemia. During the first 3

month of gestation the levels of hemoglobin were increased in fetus. During the gestational

period the levels of hemoglobin F type progressively moves to the type of adult hemoglobin (Hb

A). At 40 weeks gestational age, the Hb F and Hb A present in the same quantity in flow

(Stockman and Pochedly, 1988), but in premature infant the circulation of Hb F type is higher.

The potential of oxygen affinity is higher in Hb F type as compare to Hb A type and this

potential is differ due to the action of 2,3-diphosphoglycerate (Nathan and Oski, 1993).

Subsequently the binding affinity of oxygen were high in the F type hemoglobin as compare to

Hb A in this results the delivery of oxygen is low and cause the tissue damage and hypoxia.

According to the result of previous work the newborn babies get inferior due to the higher

concentration of 2,3-diphosphoglycerate (Delivori et al., 1971). The desire levels of hemoglobin

have the significantly role to carry the oxygen from lungs to tissue for proper function.

Experimental trials in future can deliver complete info about prematurity.

Multiple iatrogenic phlebotomies contribute to the anemia in first few weeks of life in

premature babies (Widness, 2008). According to one study during the first month of preterm

infant life conducted that the phleobotomies average were held in reserve. The physicians

collected the blood from the veins of newborns to determine the parameters that includes; type of

blood, concentration of hemoglobin, arterial blood gases, level of lactic acid, level of antibioties


8

in blood, calcium ionization, blood culture, level of glucose, complete blood count, level of

phosphorus, level of theophylline, platelet count, thyroid perameters, concentration of bilirubin

and electrolytes. Levels of phlebotomies were high in newborn babies analyzed from drawn

blood and were compared with normal infants (Freise et al., 2010).

Generally for the therapy of premature anemic infants five main approaches are

employed, treatment involves iron supplementation, delayed cord clamping, low blood count,

transfusion of red blood cells. Moreover, it is nutritionally insensitive in poor infants (DeAlarcon

and Werner, 2005). Iron intake incareses the activity in blood (Ohle, 1999, Maier et al., 1996).

Therefore sufficient iron is supplemented to preterm infants in common practice by clinicians.

For clamping of umbilical cord (a part through which baby and mother are attached) two clamps

are joined and cord will be cut down. An observation shows postponement (i.e., 30-180 seconds)

in clamping of umbilical cord significantly increased the Hb levels (McDonald and Middleton,

2008). Delaying in the clamping of umbilical cord also resulted in iron over load and reduction

in the RBC production (Rabe et al., 2008). In contrast there are various studies that indicate

delay in the clamping of umbilical cord have no effect on the reduction of RBCs (Strauss et al.,

2008). There are some risks associated with the clamping of cord still unclear. Another side

effect associated with the delayed cord clamping is polycythemia, such effects can be deffered

efficiently by clamping in time. Nicholl et al. determined delaying in cord clamping has more

beneficial than risky effects in a moderate condition (Nicholl et al., 2010). Mechansim of

delaying cord clamping reduces iatogenic blood loss which is valuable approach in removing the

anemic prematurity (Strauss, 1995; Widness et al., 1996; Kling et al., 1997 and Ohls, 2002).

A link has been discussed in the studies where blood transfusion estimation of

phlebotomy infants during initial weeks ranging from 11 to 22 Ml/KG resembles 15 to 30 % of

total blood count in the infants (Hume, 1999; Widness, 2008). Estimation of blood volume with

the help of blood analyzer signifies decreased volume of blood in premature infants (Alves-

Dunkerson et al., 2002). Therefore monitors are used to corrext the performace of many trsts by

blood nalayzer so the complete volume reduces and has been significantly reduced.In a clinical

trials of blood monitors decvice performed by 93 infants (birth weight <1000g) to regulate the

possible phlebotomy diminiation (Widness et al., 2005). Clinical trials show 33% reduction in

the count of RBCs through transfusion. Furthermore, blood clinical monitoring is used to analyze

the clinical trials in preterm infants for first two weeks (birth weight>1000g) proved 46%


9

reduction with 30% production in phlebotomics (Madan et al., 2005). If the blood monitors are

able to investigate glucose, bilirubin, blood urea nitrogen and creatinine will reduce the

iatrogenic phlebotomies. Almost about 80% theoretical phlebotomy reduction is possible with

blood monitors which would significantly result in reduced blood transfusions in preterm infants

(Alves-Dunkerson etal, 2002). Transfusions in infant increases toxic possible factors related to

circulatory overload (TACO) and Transfusion-related acute lung injury (TRALI). Reserchers

indicate transfusions in infants can be accomplished by overload activity in fluid, balanced

electrolyte and red blood cell transfusion (Ohlson and Aher, 2006).

CHAPTER TWO REVIEW OF LITERATURE MISBAH, 2017

10



11

2.0 REVIEW OF LITERATURE

2.1 OXIDATIVE STRESS MARKERS

Reactive oxygen species are responsible for the production of free radicals having

unpaired electron in their outermost shell. Basically they are the oxidizing agents that are

produced as consequences of oxygen metabolism. Free radicals due to the unpaired electron are

highly reactive molecules. These reactive oxygen species consist of wide range of radicals such

as superoxide ion (O2-), hydroxyl ion (OH-), nitric oxide (NO), lipid peroxyl (LOO) and peroxyl

(RO2) and from non-radicals molecules it contains hydrogen peroxide (H2O2), singlet oxygen

(O2), hypochloric acid (HOCL), ozone (O3) and lipid peroxide (LOOH). The free radicals of

oxygen and its derivatives have significant potential for the intiation of damaging process by

binding of free radicals with macromolecules that includes proteins, carbohydrates, DNA and

lipid molecules. Free radicals from reactive oxygen specis readily binds with polyunsaturated

fatty acids (PUFA) and results in lipid peroxidation and its products such as malondialdehyde

(MDA), MDA is also known as universal oxidative stress biomarker. Concentration of MDA

was measured in terms of TBARS. Pro-oxidants can also bind with protein molecules and lead to

the modification in secondary and tertiary structure. Oxidants can also directly damage proteins

like albumins and results in products such as advanced oxidative protein products (AOPPs) by

the activation of enzyme known as myeloperoxidase (MPO). As per some prior studies the levels

of antioxidants effects vaginal delivery (VD) and elective cesarean section (CS) on antioxidant

status (the level of glutathione (GSH) and ferric lowering the capacity of (FRAP)), the

antioxidant activities includes superoxide dismutase (SOD), glutathione peroxidase (GPx),

catalase (CAT), the concentration of lipid peroxidation (LP), protein and DNA damage in the

umbilical cord blood. Lipid peroxides and carbonyl proteins are crucial product of oxidative stess

mechanism. A need to protect DNA from oxidants is necessary as it will contribute in the

formation of DNA adducts and ultimately lead to DNA damage.

2.2 INFLAMMATION IN UMBILICAL CORD IN BLOOD

An enzyme system is present in RBCs, which prevents the cell from accumulation of

non-functional proteins and its fragments (Fagan, 1986). Malondialdehyde (MDA) was measured

in erythrocytes of maternal and cord blood to check the lipid peroxidation. Oxidative damage to

proteins was estimated by proteolytic degradation of and measuring lipid peroxidation end-

products. Levels of stress markers, reduced levels of glutathione reductase (GR), interleukin-6


12

(IL-6) in preterm infants is responsible for the inflammation of umbilical cord and damage to

neighbouring tissues and such infants are provided with the appropriate dosages of antenatal

corticosteroids for the treatment and for accelerating maturation of fetal lungs (Miracle et al.,

2008). Increased oxidative stress, formation of oxygen radicals in pretermie’s is reason for the

tissue and organ damage as gestation is a physiological state in which tissue oxygen requirement

and metabolic demands are increased (Buonocore et al., 2002 and Saugstad, 2005). Increased

oxidative stress is defined as imbalance in the levels of oxidants and antioxidants in the body

increased stress and free radicals can cause damage to fetus and can suppress the antioxidant

defense system (Gitto et al., 2002). In relation to this aspect, it is necessary to rule out production

of intracellular antioxidant components (Davis and Auten, 2010). Two different comparative

analyses of antenatal corticosteroid (betamethasone) were conducted: the first compared the

results in between those who were administered and those not administered whereas, second

compared mothers who received the complete cycle with those given only a partial antenatal

corticosteroid cycle.

2.3 NON-PROTEIN BOUND IRON AS PREDICTIVE MARKER OF NEONATAL

BRAIN DAMAGE

Out of 130 million births worldwide each year four million are reported as asphyxia and

million will die. It is necessary to develop effective interventions for the proper understanding of

pathophysiological mechanisms and ultimately leads to brain injury. According to the set of

studies traditional and new markers for oxidative stress were studied to develop relationships in

neurodevelopmental stages and stress markers.

2.4 HUMAN MILK ENHANCES ANTIOXIDANTS

Preterm infants show immature activity of antioxidant defense mechasim. Hydroxyl

radiclas are very aggressive and they directly reduce levels of antioxidants. Practically by

products of DNA oxidation or phenylalanine in urine serve as the major marker for reliable

activity. Human milk contain is enriched with the large amounts of antioxidants (Ledo et al.,

2009). Oxidative stress may be related with the prematurity as discussed human milk is enriched

with antioxidants, therefore feeding with mother milk could diminish related risks and can serve

as best prognostic measure in such infants. Number of studies signifies antioxidative property of

human milk and its use in the case of premature infantsm (Friel et al., 2002) An observation in

the study confirms the human milk provided better antioxidant protection and less ascorbate as


13

increased ascorbate is responsible for increased oxidants in the extracellular matrix (ECM). Not

only antioxidants, but human milk is also enriched with vitamin C that is essential antioxidant for

defense mechanism in neonates. Premature infants have lower elimination of radicals that

increases lipid peroxidation and oxidative stress which can only be reduced by consumption of

enough antioxidants (Silvestre et al., 2008).

2.5 LEUKOCYTOSIS IN PRETERM INFANTS WITH INTRAVENTRICULAR

HEMORRHAGE

The most common type of intracranial hemorrhage is intraventricular hemorrhage which

exists in premature newborn babies. IVH is common factor for increased morbidity and mortility

in infants with low birth weight. Most intracerebral hemorrhage may occur in first 3 days of birth

(Fanaroff et al., 2007; Fanaroff et al., 2003; Rezvanian, 2011). A relationship can be observed in

between intravenous hemmorage and leukocytes. For the development of relationship CBC was

done within 1 hour of such infants, it signified increased rate of leukocytes which increases the

risks of blood injury along with the increased oxidative stress. Moreover, leukocytosis may be

responsible for subclinical maternal or neonatal infection (Fanaroff et al., 2003 and Wilson-

Costello, 2007)

2.6 INFLAMMATION AT BIRTH

In the response of increased oxidative stress, systemic inflammation is increased. Levels

of pro-inflammatory cytokines were recorded and they were in higher concentration in the cord

blood or in the blood of neonatal which is responsible for neuronal disorders such as white

matter brain damage (WMD) (Duggan et al., 2001 and Minagawa et al., 2002). As per the

number of studies increased secretion of pro-inflammatory cytokines have direct relationship

with cerebral/brain damage. Hansen-Pupp et al., (2008) postulated that as the concentration of

pro-inflammatory cytokine increase, it increases adverse developmental outcomes after birth.

TNF-α is a type of cytokine responsible for the increased adverse effects after birth such as it

causes brain injury and is involved in both types of mechanisms including tolerance and

sensitization (Rosenzweig et al., 2007). TNF- α inhibits proliferation and migration of neuronal

precursor cells, triggers of apoptosis which in turn influence white matter and causes injury

(Downen et al., 1999; Ben-Hur et al., 2003 and Feldhaus et al., 2004). As these infants are

particularly deficient in antioxidants and in anti-inflammatory defenses, they are more prone to

have potentially harmful long-term effects on their developing lungs and brain (Yeung, 2006).


14

2.7 NITRIC OXIDE THERAPY IN PREMATURE INFANTS

The rate of survival in premature infants can be efficiently improved with the

administration of appropriate ratio of nitric oxide (NO) and by inhalation therapies which are

responsible for the protection of bronchopulmonry dysplasia in premature infants (Philipp et al.,

2007). Administration of NO and inhalation therapy not only improved the antioxidant status but

NO was responsible for immature lungs vasoconstriction and influx of plasma air (Saugstad,

2003). Nitric oxide (NO) significantly improved secondary mechanisms such as airways

structure and alveolarization as well (McCurnin et al., 2005; Bland et al., 2005 and

Balasubramaniam et al., 2006).

2.8 NON-PROTEIN BOUND IRON (NPBI) AND F2-ISOPROSTANES IN NEWBORN

Non protein bound iron (NBI) and F2 Isoprostanes in premature infants are also

responsible for the increased oxidative stress and stimulates prooxidants in plasma (Gutteridge

and Halliwell, 2000; Thibeault, 2000). In perinatal time especially immature infants, low evel of

stored form of iron, decrased iron binding sites and low level of ceruplasmain may be associated

with the appearance of NBPI in aminotic fluid. F2 isoprostanes contain a series of prostaglandins

like ponds in vivo and in vitro by catalyzed peroxidation of phospholids bound archiodionic acid

(Morrow and Roberts, 1996 and Delanty et al., 1996). Signorini et al., (2008) confirms the

association of lipid peroxidation NPBI concentration.

2.9 MELATONIN PROTECTS AGAINST OXIDATIVE DAMAGE

Oxidative stress remains responsible for the pathogenesis of bronchopulmonary dysplasia

(BPD). Bronchopulmonary dysplasia (BPD) is a chronic pneumopthay observed in newborn

infants with prolonged respiratory tract. Melatonin detoxifies free radicals under the influence of

mechanisms directly and indirectly. Melatonin (MT) or N-acetyl-5-metoxy-tryptamine is the

indole synthesized from the pineal gland during night. The fetus receives MT from the mother,

followed by premature delivery and leads to prolonged (MT) deficiency. Defined deficiency may

be harmful because neurohormone has important functions. Blood MT influences the sleep-wake

cycle and changes the physiology from day time to night time in a well-coordinated manner and

aids in the synchronization of circadian rhythmicity in all tissues. Pan et al., (2009) investigated

the factors of affecting the MT or imbalancement of antioxidants and oxidants in the lung.


15

2.10 MATERNAL ANEMIA

Pregnancy is often refered to number of diseases/complications such as anemia. Maternal

anemia is the most common disorder. Incidence of maternal anemia in developing countries such

as Pakistan is significantly increasing (Ahmad et al., 2011). Under the influence of oxidative

stress (OS) and scavenging activity of free radicals, physiological and metabolic functions are

altered. Pregnancy itself is a very stressful condition, levels of antioxidants decreases (Khanna et

al., 2010).

2.11 IRON STATUS AT BIRTH

Defined relationship in between low birth weight and the level of hemoglobin helps to

explain complications such as iron deficiency in infants (Salsbury, 2001). Almost ninty five

percent (95%) of premature infants (26-34 weeks of gestational age) show low birth weight, iron

deficiency and decreased growth. Maternal anemia is the reason for premature delivery of infants

lead to iron deficiency at the time of birth (Sichieri et al., 2006).

2.12 MORTALITY OF PRETERM BIRTHS

There are different factors involved in the very low birth weight (VLBW) in pretermies

including perinatal, neonatal, postnatal morbidity and mortality and is responsible for premature

complications and infections. Neonates with very low birth weight (VLBW) are 30 times at

greater risk of mortality (Kabir et al., 1995). Due to the advancement in neonatal and perinatal

cares rate of VLBW are significantly decreased (Chowdareddy et al., 2014). Anemia is the major

risk factor to cause preterm delivery. The rate of mortality has the negative relationship with

gestational age. However, obstetric and perinatal complications in this population of pregnant

women could be significantly reduced by providing better antenatal care and timely interventions

where needed.

2.13 IRON DEFICIENCY ANEMIA AND SEVERE PREGNANCY OUTCOME

Iron deficiency and anemia results in impaired transport of hemoglobin. Supply of

oxygen to uterus, placenta and fetus is also impaired that causes cellular dysfunction. Mechanism

can be explained by impaired myometrial contractility leads to atonic uterus and placental

dysfunction contributes to preterm birth and growth restriction in newborns. Impaired delivery of

oxygen contributes in impaired wound healing (Jaleel and Khan, 2008).

Erythropoietin (Epo) is a 30.4 kD glycoprotein hormone that contains 165 amino acids

and is produced by peritubular fibroblast cells that are located in the kidney. Erythropoetin (Epo)


16

is produced by the kidney in response of decreased circulating oxygen in the blood which is

sensed through hypoxia-inducible factor. The primary function of Epo is to produce erythrocytes

through erythropoiesis. Premature infants may develop anemia in their first few weeks of life and

results blood loss, shortened red blood cell lifespan, low plasma erythropoietin levels and

inadequate erythropoiesis.

2.14 PREGNANCY OUTCOME ASSOCIATED WITH ANEMIA AND IRON

Maternal anemia may be diagnosed before mid-pregnancy and it contributes as one of the

primary risk factor for the preterm delivery. Moreover, at the later stages particularly third

trimester high concentration of hemoglobin increases the process of hematocrit and serum

ferritin levels in pregnant females. These factors collectively are responsible for the preterm

delivery. Several studies established positive correlation in between maternal anemia and

preterm delivery (gestation age < 36 weeks). Anemic pregnant females required extra medical

care with special emphasis on the levels of hemoglobin. Physical paramters such as education,

ethnicity, gestational age, and smoking all collectively increase incidence of preterm delivery.

Studies explain concentration of Hematocrits ≥ 40% before 20 weeks of pregnancy, it

was also documented that 18% of women released vaginal bleeding at the time of the first

antenatal. Risks of preterm delivery increases upto five folds due to vaginal bleeding because of

iron deficiency and increases risks of preterm delivery. The prevlance of preterm delivery was

increased 1.6 times in such women those were having levels of hemoglobin in between 100 and

109 g/L. Improved levels of hemoglobin reduced the risks of preterm deliveries.

2.15 IRON IN PREGNANCY

As discussed maternal anemia is one of the common problems during pregnancy

therefore, iron supplementation is made necessary during the course of pregnancy. Iron

deficiency increases the risks of preterm delivery, fetal growth restriction leading to the cause of

preeclampsia to many folds (Scholl, 2005). Apart from that iron supplementation and increased

reserves of iron is found to increase some gestational complications i.e., gestational diabetes and

increased oxidative stress hence, iron supplementation to females who are not iron deficient may

increase their risk factors.

Among low-income minority women in their reproductive years are believed to be iron

deficient such as low level of hemoglobin or hematocrit. Women belonging to poor and ethnic

minorities are reported with that the adverse outcomes of pregnancy in respect to low levels of


17

hemoglobin and increased storage of iron. However, desired levels of iron must be attained and

were significantly helpful in pregnant women. Most frequent relationship was in between

nutrients and ethnicicty whether females belong to developed or undeveloped countries.

Menstruation cycle is the major factor in woman for loss of iron in form of menstrual blood.

Prevalence of iron deficiency and IDA was increased in women belonging to group of

minorities, below poverty line and with less education. Iron deficiency was even higher in

women with more than 2 children (Looker et al., 1997). Another estimate shows fifty percent of

women were not having adequate iron stores during pregnancy (Yip et al., 2001; Perry et al.,

1995). During pregnancy (3-4mg/d) is substantial amount of iron required and risks of iron

deficiency and IDA increases with gestation. Women belonging to lower income class were

more prone to develop anemia as compared to those belonging to stable income (Perry et al.,

1995).

Anemia is also known as “sickness index” for the body (Hoffman et al., 2000). Anemia

includes pathologically abnormal levels of hemoglobin that may cause the thalassemia some

times, or may be because of abnormal beta chain of hemoglobin therefore, hemoglobin cannot

perform its pathological function and develops thalassemia or folate/B12 deficinency.

Additionally it may also contribute as the lead cause for other chronic diseases such as

cardiovascular diseases, cancer and some infectitious and chronic inflammatory disorders

(Hoffman et al., 2000). The levels of Hemoglobin and hematocrit were reduced throughout the1st

and 2nd trimesters and it reaches to the lowest point by second to early 3rd trimester and

ultimately rises up to the normal level (Anonymous, 1990) but in later pregnancy, it becomes

very hard to distinguish in between physiologic anemia by iron deficiency internally and

externally (Anonymous, 2002).

According to the previous studies it is suggested that during 1st trimester the meternal

anemia affected preterm delivery (Zhou et al., 1998). According to the study of Allen (2001), it

suggested that there are three main factors in regulation of potential mechanisms of maternal

IDA and may be the reason for preterm delivery. In the presence of oxidative stress it leads to

chronic hypoxia and is responsible for the release of CRH from placenta and stress hormone

from fetus. Excess production of free radicals and decreased levels of antioxidants lead to the

damage to maternal fetal unit and is the reason for preterm delivery. These types of free radicals

attain ability to damage organs, tissues and to even cells present in biological system (Halliwell


18

and Gutteridge, 1999). Oxidative stress over time is now thought to be major factor of aging,

reason for the development of diseases such CVD, cancer etc. Iron overload remains associated

with the stress status that further contributes in the pathogenesis of hepatitis and diabetes

(Fernandez-Real et al., 2002; Jiang et al., 2004). Preliminary findings suggest that the

overloading of iron increases excretion of 8OHdG and MDA in the maternal-fetal unit.

2.16 NEGETIVE EFFECT OF OXIDATIVE STRESS IN FETUS AND NEWBORN

As the oxidative stress is increasesd in maternal fetus production of free radicals and

decreased levels of antioxidants contribute in the disruption of cell cycle, fibrinolysis and

signaling transduction (Wagenaar et al., 2004). Well-established fact reveals that reactive oxygen

species (ROS) are imperative for fertilization (Dennery, 2004). In moderate concentration of free

radicals and sufficient supply of antioxidant defense system they continuously maintain the

process of fetal growth and cell aerobic metabolisms whereas, during abnormalities endogenous

or exogenous production of free radicals increases attack to biological macromolecules such as

carbohydrates, lipids, proteins and DNA (Halliwell, 1994 and Buonocore and Perrone, 2006).

Fenton chemistry, Hypoxia, endothelial damage, inflammation, arachidonic acid cascades

are some other mechanisms that results in the synthesis of highly reactive products. Fenton

reaction is responsible for the DNA damage (such as fragmentation, apoptosis, base

modifications and strand breaks), protein, lipid (arachidonic acid), and polysaccharides oxidation

(Saugstad, 1996). Premature born infants have weak defense system therefore, are very sensitive

to oxidative stress under the influence of fenton reaction (FR). Initially at the time of birth

newborn has to compete with the environmental changes and with the maintainence of oxygen

pressure (PO2 100torr) as compare to intrauterine environment (20-25 torr) (Maulik et al., 1999).

Then with the antioxidant defense which helps to copeup with hyperoxic challenges. Finally

pretermies have increased sensitivity against inflammatory infections and increased levels of free

iron in their plasma and tissue (Ciccoli et al., 2003).

Oxidative stress in hypoxic fetuses and neonates initiates damaging processes such as

lipid peroxidation that results in the formation of fragmants of malondialdehyde (MDA) that in

term combines with the DNA and leads to formation of DNA adducts and DNA damage. MDA

further converts stress markers including serum total hydroperoxides, isoprostanes, non protein

bound iron (NBPI) and advanced oxidative protein products (AOPPs). Whereas, serum levels of


19

antioxidants decreases in addition with red blood cells (Ciccoli et al., 2003; Buonocore et al.,

1998).

Isoprostanes belongs to the family of prostaglandins secreted by the oxidation of

arachidonic acid and its derivates by binding to free radicals generated in the condition of

oxidative stress (Morrow and Roberts, 1996). Concentration of isoprostanes can be measured by

the circulating plasma and can be later detected in the urinary excretions. Particularly, 8-iso-

PGF2, is a major isoform of isoprostane which is relatively more stable and can be estimated in

the biofluids, it serves as most reliable oxidative bio-marker (Pratico, 1999). Levels of F2-

isoprostanes were measured with the help of mass spectrophotometer and gas spectrophotometer

in newly born infants. Levels of F2-isoprostanes were significantly higher in preterm babies as

compared to healthy ones (Comporti et al., 2004). Significant inverse relation was observed in

between gestational age and isoprostanes in plasma. Whereas, significance in plasma levels of

isoprostanes in mothers before and after pregnancies were not observed. ROS was also found to

be involved in the damaging of amniotic epithelium and chorioamniotic collagen. Concentrations

of F2-isoprostanes were significantly increased in patients due to the breakdown of membrane as

compared to normal infants (Longini et al., 2006).

ROS induced rupture of membrane in pregnant state; it also abnormally binds free amino

acids and causes abnormal synthesis of proteins. Moreover, it also disturbs membrane integrityby

the oxidation of polyunsaturated fatty acids (PUFA) and leads to modification of chrioamnitic

biology and cell dysfunction (Masumoto et al., 1992 and Ogino et al., 2005). The most abundant

oxygen free radical is superoxide anion that was initially discovered during bacteriocidal activity

and was further involved in production of other oxide derivatives that includes hydrogen

peroxide (H2O2) and hydroxyl ions (OH-). Radicals were neutralized by the action of

antioxidants such as superoxide dismutase (SOD) and catalase (CAT) (Klebanoff, 1980).

Oxidative stress and its permeability of capillaries increases and promotes transportation of

certain cytokines whereas, mechanisms in between oxidative stress and inflammation are not

well-known. The production of cytokines during infection amplifies the induction of hypoxic-

ischemic effects. It was suggested that the production of cytokines were increased in state of

infections and inflammation (Dietrich et al., 2004).


20

2.17 HYPOXIA AND NITRIC OXIDE

During the hypoxic condition the normal physiology of cells were altered by the over

production of Nitric oxide (NO) in the presences of eNO synthase (eNOS) in endothelial cells

(Meini et al., 2003). NO produced by the activation of eNOS in the presenaces of arginine and

oxygen. Once NO is produced it combines with superoxide radicals that converts other toxic free

radicals such as peroxynitrile, hydroxyl radicals, nitrogen dioxide. Cascade reactions of NO2+

activates series of enzymes (Beckman and Koppenol, 1996) as well as some other damaging

metabolites including nitryl chloride (NO2Cl), nitrogen dioxide radical (NO2), sysnthesized

through nitrite reaction. The reaction ends up to final metabolite which is termed as

hypochlorous acid (HOCl) with the help of neutophilic myeloperoxidase (MPO) activation

(Eiserich et al., 1996). There are three types of NOS yet established as follow: endothelia NOS

(eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) (Rothman et al., 1992; Cozzi et al.,

1990 and Kaur et al., 1999). As the hypoxic condition prevails it increased oxidative stress by

the up-regulation of NO and some other varities of cells such as (endothelial cells, macrophages,

neurons and astrocytes) (Dalkara and Moskowitz, 1997). According to the experimental studies

at initial stages NO has shown protective effect against vasoconstriction and it promotes

perfusion by the activation of NOS 3 (Iadecola et al., 1997). However, expression of NOS 1 and

NOS 2 were also increased after ischemic condition (Dalkara et al., 1994). Excess in the amount

of NO was responsible for the increased oxidative injury by change in Ca+ homeostasis that has

major role in cellular signal transduction and in regulation of several cellular processes like

proliferation (Meini et al., 2003).

Another well-known fact states that gene expression may be regulated by the cascade

pathway termed as MAPK pathway. Cascade is as follow Ras/Raf/ERK. All of the cascade

reactions are regulated under the normal concentrations of Ca2 and NO however, any major

fluctuations in these levels may result in cell cycle arrest (Mannick et al., 1999). Hence in the

light of knowledge negative relationship may be observed in between NO, and Ca+ for

regulation of cell cycle.

2.18 PARTURITION AND OXIDATIVE STRESS

Term labor stimulates compensatory defense mechanism that includes enzymatic and

non-enzymatic antioxidants in the compartment of fetal red blood cells (Buhimschi et al., 2003).

The given mechanism shows protection to neonate at the time of birth against hypoxic condition.


21

Reduced levels of antioxidant in preterm neonates, and increased status of free radicals may

cause damage at cell and tissue level including bronchopulmonary dysplasia and retinopathy

(Saugstad, 2003 and Dani et al., 2004).

2.19 OXIDATIVE STRESS AND DIABETIC PREGNANCY

Complications of pregnancy contribute in overproduction of free radicals, lipid

peroxidation and reduced activity of antioxidants which can be measured in terms of levels of

MDA and other antioxidants such as glutathione (GSH), catalase (CAT) and superoxide

dismutase (SOD). Elevated concentraitons of MDA signified increased hemolysis of

erythrocytes in premature infants as compared to heathy ones (Djordjevic et al., 2004).

2.20 ROLE OF OXIDATIVE STRESS IN MENOPAUSE AND AGE RELATED

FERTILITY DECLINE

Reactive oxygen species (ROS) is found to affect the levels of sex hormones. ROS is

found to reduce the levels of estrogen with the passage of time by the over production of free

radicals. During premenopausal to menopausal period the scavenger activity of both glutathione

peroxides and superoxide dismutase were reduced in the ovary (Okatani et al., 1993).

2.21 STRATEGIES TO OVERCOME OXIDATIVE STRESS IN ASSISTED

REPRODUCTION

Under the influence of low oxygen development of embryo and fertilization is not proper

(Burton et al., 2003). Initially, such conditions were avoided that resulted in increase of free

radicals with supplementation of proper antioxidants the oxidative insult may be reduced

effectively (Catt and Henman, 2000). During cryopreservation hydrogen peroxide levels were

reduced with effective therapy of ascorbate (Lane et al., 2002). ROS has harmful effects at the

quality of embryo and oocyte in patients suffering with endometriosis. Levels of inflammatory

cytokines such as; TNF-α, ILs, MMPs significantly increases peritoneal fluids in patients with

endometeriosis and unexplained infertility (Saleh and Agarwal, 2002). Strategies involve sperm

preparation methods that affect ART outcomes. Preparation by centrifugation has been directly

linked with the formation of reactive oxygen species. It has been reported that seminal plasma

has high levels of antioxidants and protects the spermatozoa from DNA damage and lipid

peroxidation (Potts et al., 2000).

Whereas minimum amounts of reactive oxygen species (ROS) are necessary for the

physiological process of sperm’s functions. On the other hand, unbalanced levels of reactive


22

oxygen species have negative impact on spermatozoa and fertility rate. Procreating is a natural

process in the life of couples that relies on special planning but not in intercession. The couples

are tried to be conceive but 15-25% is unable to cause pregnancy and they seek out medical

suggestion to improve the chance of successful fertilization and pregnancy. The guidelines of

World Health Organization suggested that male factors are responsible for infertility which

included variation in the concentration and motility rate of sperm. According to recent work,

oxidative stress is supposed to be a major and credible cause of male infertility. Generally

oxidative stress is a condition in which there is imbalancing of systemic radicals such as reactive

oxygen species and in biological system in which antioxidants acts as defense mechanisms.

According to the some perior studies production of ROS were increased by prolong (16-20

hours) co-incubation of sperm-oocyte as compare to some other prospective randomized

controlled studies to using a shorter time incubation of sperm-oocyte (Kattera and Chen, 2003;

Gianaroli et al., 1996) because the rate of fertilization and implantation will be improved at the

incubation of 1-2 hours which lead to significantly increased the quality of embryos (Dirnfeld et

al., 2003).

2.22 PHYSIOLOGIC STATUS OF MOTHERS

Those factors which are maternal physiological factors and are interested in the study are

as follow: gestational diabetes, Eclampsia, chronic hypertension, pre-pregnancy diabetes,

cervical cerclage, pregnancy weight gain and history of preterm birth. Nevertheless there are

other illness and complications that influence pregnancy results which are not recorded in the US

data base records, like; kidney or bladder (urinary tract) infection; severe nausea, vomiting,

dehydration and vaginal bleeding; cervix had to be sewn shut; hypertension also Pregnancy-

Induced Hypertension [PIH]), pre-Eclampsia; placental complications (such as placental

abruption, placenta previa), car accident and blood transfusion. Pre-pregnancy conditions that

may include diabetes mellitus, hypertension, pre-Eclampsia and asthma are also related to an

increased chance of developing preterm birth (Aly et al., 2011; Madan et al., 2010). About 30%,

the reasons for preterm births are either fetus or mother related; such as maternal diseases or

uterine fetal retardation (Gravett, Rubens, & Nunes, 2010). From 1989 to 2000, there has been an

increase of 55% in the incidence of preterm births in US (Ananth, Joseph, Oyelese, Demissie, &

Vintzileos, 2005). With the increase in the intensity of pregnancy induced hypertension there is

also in an increase in the risk of developing preterm births (Ye, Li, Ma, Ren, & Liu, 2010).There


23

are many studies which support the concept that pregnant women with vaginal bleeding have

double the risk of developing preterm birth. In early pregnancy, vaginal bleeding is always

related to the risk factor of preterm birth (Hackney & Glantz, 2011; Ramaeker & Simhan, 2012).

Yet, Yang et al supported that in African women vaginal bleeding is not associated with preterm

birth (Yang et al., 2004).

2.23 ROLE OF OXIDATIVE STRESS AND LIPID PEROXIDATION IN PRETERM

DELIVERY

When the rate of the production of the free radicals becomes more than the removal rate

by the cellular defense mechanism, this state is known as Oxidative Stress (Burton & Jauniaux,

2010). There has been a strong relation between the oxidative stress and the preterm labor and

other diseases of pregnancy such as PROM, Early Deliveries, IUGR, Pre-Eclampsia and many

other diseases associated with preterm infants (Buhimschi et al., 2003; O’Donovan & Fernandes,

2004). Not only in placenta, fetus and newborns the numbers of cells divide rapidly, but there is

a rapid growth of division in the maternal cells compartments as well. One of many pregnancies

is a state where there is a need of an antioxidant which has to be present in the body for

combating this stress. (Kaiser and Allen, 2002) Free radicals are produce during pregnancy, also

the other oxidant molecules which exceeds the value of the antioxidant which are available as a

buffer in pregnant mothers and developing fetus. Cellular damage is the end result of the above

mentioned process, in which not only the preterm labor and early deliveries are associated, but

also it includes IUGR, PROM and Pre-Eclampsia and many other serious issues for mature

infants (Joshi et al., 2008). The excess of these free radicals attacks the endothelial lining of

vessels if these are not are not buffered and many more system also by acquiring the nucleic

acids of many lipids, carbohydrates and proteins and hence they denature the DNA of these cells

(Burton and Jauniaux, 2010). Normal placental growths are greatly affected by these free

radicals and may cause stillbirths OR miscarriages, also causing number of chromosomal

disorders at the same time (Stein et al., 2008).

Pre-Eclampsia can also occur by the disturbance in the vessels vasodilator mechanism

caused by the oxidative stress, resulting in the elevation of the maternal blood pressure, hence

placental blood flow is impaired (Buhimschi et al., 2003) Alteration in the genetic metabolic

detoxification is also associated with the oxidative stress by the cytochrome P450 1A1 gene and

many others (Agarwal et al., 2005). Cytotoxic damage to the proteins may be caused by the


24

oxidative oxygen and nitrogen, DNA OR Lipids, until unless the non-enzymatic and enzymatic

oxidants equalize there harmful effects. There are many for free radicals to act, e.g. ROS formed

by the mediation of the iron which causes the Lipids and DNA damage is due to the fluctuation

of the iron molecules from their normal values; that is the transport of oxygen to the tissues,

results in the cellular damage due to the iron induced free radicals. Macrophages and Neutrophils

release free radicals because of the presence of the inflammation and infection in the body. Free

radicals of specific area are referred as Reactive Oxygen Species (ROS) because many free

radicals which biologically important are derived from oxygen (Saugstad, 1996).

Reactive Oxygen Species (ROS) is defined as mutual consideration about including not

only the superoxide anion and hydroxyl radicals but also including the other radicals such as

hydrogen peroxide, is a derivation of molecular oxygen. Reactive Oxygen Species (ROS) have

many different mechanism for its generation, like the normal movement of electron in the fatty

acid and the mitochondria, metabolism of the prostaglandin, ischemic re-perfusion, low levels of

oxygen at cellular level (HYPOXIA), state in which there is an excess of oxygen at cellular

levels (HYPEROXIA), activation of macrophages and neutrophils (INFLAMMATION),

hypoxanthine-xanthine oxidase system in the endothelial cells (ADINOSINE TRIPHOSPHATE

DEGRADATION), high levels of free circulating metals and Fenton reaction (FERROUS to

FERRIC ACID) (Shoji and Koletzko, 2007). Due to development and growing of placenta the

rate of the flow of the blood in the vessels increases and as gestational period advances in its

later life, if there will be a damage to the endothelial cells of the blood vessels then it would

cause a very harmful and particularly a very serious damage to the vessels. During vasodilatation

blood vessels usually uses nitric oxide (NO) signaling to initiate the process. There are near to

circumference of muscle cells that surrounds the wall of the blood vessels which dilate the blood

vessels itself in order to control the additional flow of the blood. The increased blood flow for

the growing fetus and placenta is provided by the vasodilation effect in the blood vessels

(Edemann and Schiffrin, 2004).

The process of vasodilatation effect in the blood vessels is disrupting by the amount lf

free radicals present in the blood. Hence, there is a low supply of blood to the fetus and placenta

due to vasoconstriction in the blood vessels, which result in IUGR, Pre-Eclampsia and Preterm

Labor (PTL) (Stein et al., 2008) Chromosomal disorders, may arise due to the presence of

oxidative stress that may lead to fetal death. There is also an effect of oxidative stress in a


25

reversal manner that it may cause the disability of the body to detoxify the oxygen radicals

present in blood (Blondi et al., 2005). The Cytochrome P450 A1A gene (CYP1A1), Glutathione

S-transferases µ1 (GST m1), and 01 (GSTT1), they all can interfere with the detoxification

process. There is a cascade of fatty acid that is triggered by Reactive Oxidative Species (ROS)

such as arachidonic acid that lead to premature contractions, dilatation of the cervix and delivery

of baby before the 37th week of gestation (Joshi et al., 2008).The only management of this

process is to balance the free radicals of oxygen and nitrogen in the blood is by balancing the

values by giving external oxygen and nitrogen via supplementation. The enzyme Superoxide

Dismutase (SOB) is included in the defense system of anti-oxidants, glutathione and catalase.

Those infants who are premature in their births are at high risk for oxidative stress, it is due to

the both endogenous as well as acquired exogenous antioxidant defenses that do not activate in

maturation in the third trimester (Finer & Leone, 2009; Baba & McGrath, 2008).

Through the production of free radicals oxidative stress is a contributing factor in tissue

injury and by leading inflammatory cytokines by the reactive oxygen/nitrogen and end up in

preterm labor (PTL) (Pressman, 2011). There is a lot of evidence that is increasing about the

oxidative stress that it is the end point of a very complex process that either they are determined

genetically or they are activated by an in-utero stressor. Those new born infants which are born

premature or they are born preterm labor are highly at risk for induced oxidative stress tissue

damage. Oxygen therapy is the first choice of management in the premature infants and

HYPEROXIA which is known as high levels of oxygen and in intrauterine environment where

the oxygen is in much less in amount as compared to hyperoxic state results in exposing the

infants to a very high free radicals zone. (Maulik et al., 1999) Later, the mechanism of

antioxidant defense is induced by hyperoxia and it is comparatively damaged in infants who are

premature (Speer and Silverman, 1998). Thirdly, there is an increased risk for preterm infants to

be induced for inflammation and infection which causes the effect of oxidative stress (Saugstad,

1988). There is a presence of free iron in the tissues and plasma of premature infants as

compared to full term babies. There is a variety of disease which is caused by the oxidative stress

to an individual organ or sometime to a group of organs frequently in the neonates which include

retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC), bronchus-pulmonary

dysplasia (BPD), periventricular leukomalacia (PVL) and intra-ventricular hemorrhage (IVH)


26

(Clyman, 1989; Archer et al., 1989 and Sanderud, 1993).In spite of their timing preterm and full

term were considered to be similar process which follows a same and common pathway.

However, the basic reason and the initial process of preterm births is still unclear,

Romero et al (2006) used the term syndrome for describing every possible pathological

reasoning which cause preterm labor (PTL). Although, the activation of these pathways in

physiological manners results in full term labor while the preterm labor occurs when there is an

alteration in these common pathways due to the presence of many pathological reasons which

induces pathological conditions from multiple causes and risk factor (Romero et al., 2006).

Preterm labor is sub-divided into two types;

1. Indicated (Usually associated with maternal and fetal conditions)

2. Spontaneous

One of the four primary pathogenic pathways is the prime responsible factor for the preterm

birth. They may include over-distension of the uterine, cervical disease, infection, endocrine

disorders (Romero et al., 2006) and ischemia, decidual hemorrhage and activation of maternal

and fetal hypothalamic-pituitary axis (Behrman and Bulter, 2007). Intra-Uterine infection and

inflammation is to be considered as the main contributor to preterm birth (Norman et al., 2007).

These are the mechanisms that meet on a mutual pathway which involve in an increase in the

uterotonin and protease expression. In a given woman more than one process can take place at a

time. In some women the combination of inflammation and genetics response are the area of

research that could explain the process of preterm labor with some common risk factors (Challis

and smith, 2001 and ACOG, 2010). Labor is a process which encourages change in chorio-

amniotic membranes that are reliable to acute inflammatory response, regardless the absence of

the inflammatory response (Haddad et al., 2006). NF-Kappa B is activated by the reactive

oxygen species that is responsible for the stimulation of COX-2 expression and it also promotes

inflammation with succeeding parturition.

There is a decrease in the Gx protein in both the preterm and full term labor women and

this was reported by (Khan et a l.., 2010), as compare to non-labor groups. If these data taken

together, they suggest that state of labor whether preterm or full term requires the actions of GPx

for limiting lipid oxidation and is also associated with the reduction in the ROS of antioxidant

defenses (Khan et al., 2010). Mustafa et al., (2010) discovered remarkably increased stages of 8-

OHdG and MDA and suggestively decreased stages of glutathione (GSH) in the blood of


27

mothers having preterm labor as compared to those mothers having full term labor. This

conclusion proposed that those women who were in preterm labor have reduced antioxidant

capabilities to secure against the damage cause by the induced Oxygen specie (OS).

Furthermore, abridged events of FRAP, it is an assay which is present in the body to measure the

body ability to defend against the oxidative damage and, also GST, has also been seen in the

women with preterm labor (Hong et al., 2002; Frosali et al., 2004; Mustafa et al., 2010 and

Pathak et al., 2010). Furthermore, the results support that the increase amount of OS in the

maternal environment and low levels of antioxidants extracts both the mother and fetus more

vulnerable to ROS-induced damage. Inflammation persuades the uptake of ROS and can be

reason of apparent OS, which results in tissue injury and ensuing preterm labor (Chadha et al.,

2007).

As a protective response the concentration of Mn-SOD increases to inflammation and OS,

and decrease down the levels of MAPK pathways, NF-kappa B and activator protein-1 (Oberley,

2005). In women having preterm labor were observed with high levels of Mn-SOD in the

membranes of their fetus as compared to those women who were in spontaneous labor at term,

that may recommend a larger degree of OS and inflammatory process in the former. Chorio-

amnionitis is suggested to be linked with preterm labor and histological infection was found to

be related with raised expression of Mn-SOD mRNA in the membranes of fetus of those women

having preterm labor (Than et al., 2009). The increased amount of the Mn-SOD mRNA

expressions may be a compensatory response to the existence of high levels of OS and

inflammation in preterm labor. Suggestively increased amounts of the pro-inflammatory

cytokines IL-1 beta, IL-6, and IL-8, have been detected in the amnion and chorio-decidua of

those women having preterm labor as compared to those women who were having spontaneous

labor. There is an activation of membrane inflammatory response in women having preterm

labor (Keelan et al., 1999). Levels of TAS are slightly lower in women as compared to those

women with simple pregnancies having similar gestational period, and this may indicate the

presence of OS in women with preterm labor (Cinkaya et al., 2010). In comparison to those

women having simple pregnancies those women having preterm labor were observed to have

low levels of PON 1 activity in them (Bakar et al., 2010). This discovery proposed that there are

increase levels of lipid peroxidation and a decrease in the levels of antioxidant activity of PON-

1that may collectively generate a pro-oxidant setting and enhance the hazards for developing


28

preterm birth. Moreover, there is a remarkable decrease level of GSH in patients having preterm

labor (Lee and Davis, 2011).

Preterm birth in early gestational age has been associated with the low levels of the

maternal serum selenium (Rayman et al., 2011). GST polymorphism was found to be

expressively greater in those patients who were in preterm labor, demonstrating that these

patients are more susceptible to oxidative damage (Mustafa et al., 2010). Maternal infection

caused by inflammatory setting is concomitant with preterm birth and a state of OS is produced

and the resulting in the decrease of antioxidant defenses and likely to elevate the chances for

preterm birth. The offered suggestion involves the inflammation and repressed the levels of

antioxidant defenses in the pathophysiology of preterm labor. It seems reasonable that in

preventing preterm labor antioxidants supplementation may be associated and also inflammation

associated with birth. Temma-Asano et al 2011 conducted a study and established that NAC was

operative in minimizing OS induced by chorio-amnionitis, and hence protecting preterm labor

(Temma-Asano et al., 2011). Nevertheless, those women with nulliparous complication having

vitamin C and E supplementation in early gestational ages did not reduce the chances of

developing preterm labor. As the results of these studies were contradictive, it is still unclear that

supplementation of antioxidants by the mother is helpful in reducing the event of preterm labor

(Rumbold et al., 2006 and Hauth et al., 2010). Those substances which decrease the variety of

OS by producing free radicals or by reducing the damage which is responsible for creating free

radical chain reactions.

They work in a sense by slowing down or by preventing the damage to the cells from

infection by enhancing the immune system. There are three major categories in which

antioxidants are classified:

(1) Low molecular antioxidants [glutathione (GSH), vitamins C and E, bilirubin, and urate]

(2) Enzymes that neutralize free radicals [superoxide-dismutase, catalase, glutathione peroxidase

(GPx), DTdiaphorase, and peroxiredoxin], and

(3) Non-enzymatic proteins (thioredoxin, glutaredoxin, and metllothionines).

The classification of antioxidants can be in primary and secondary manner. Endogenous

enzymes are classified as primary antioxidants [e.g. Catalase, GPx and superoxide dismutase

(SOD)] and the site of their action is the site where free radicals are produced. Those

antioxidants which are obtained through foods are known as exogenous antioxidants and are


29

classified as secondary antioxidants (e.g. Carotenoids and Vitamins A, C, E).Antioxidant

enzymes contribute in a complex communication in oxidizing and reducing those molecules

which defines the cell setting that is essential for keeping cellular, placental, fetal, and postnatal

growth (Dennery, 2004). Such enzymes have low activity in preterm infants and cannot balance

excessive ROS production. The most important antioxidant enzymes are copper-zinc superoxide

dismutases (SOD), which are found in cytoplasm as well as peroxisomes, and manganese SOD

from mitochondria. SOD catalyzes the dismutation of superoxide anion to H2O2. Glutathione

peroxidase (GPx) in mitochondria and catalase (CAT) in peroxisomes catalyze the reaction of

H2O2 to molecular oxygen and water. Together with Vitamin E these enzymes plays an

important part in per-oxidation of those fatty acids which are still poly-unsaturated in the cell

membranes (Thibeault, 2000).

Non- Enzymatic pathways are the other pathways which produces resistance in the

process of oxidative stress. Some of the nutrients like zinc, copper and selenium may have

antioxidants functions as a vital component of antioxidant enzymes. Ceruloplasmin, glutathione

(GSH), transferrin, bilirubin and Vitamins A, E, and C are to be known to have properties of

antioxidants enzymes. ROS induced injury to the full term infants may took place due to the

premature infants because of the deficiency of utero-placental movement of nutrients which are

significantly vital for antioxidant defenses (Shah MD & Shah SR, 2009).Many seleno-enzymes

may include selenium in them having in them GPx as most important part. There is an

association between the serum selenium and Glutathione per-oxidase (GPx) activity and the

weight of the newborns (Trindade, 2007). Preterm diseases may have the involvement of

selenium deficiency in them but this data is not more than enough to hypothesize this theory

(Darlow & Austin, 2003). Vitamin A part is likely intermediated by the action of this vitamin on

retinol-binding protein and also the receptors of retinoic acid, apart from showing its direct

effects as anti-oxidants. In preterm infants the levels of serum vitamin A are consistently to be

shown as low (Darlow& Graham, 2007). With an increase in the gestational age there is an

increase in the concentrations of vitamin C also known as ascorbic acid. Prevention of tera-

togenic effects of maternal diabetes with the supplementation of vitamin C and vitamin E has

been suggested by number of studies but these studies do not show more evidence about other

measurable benefits (Dheen et al., 2009).


30

Transferrin, ferroxidase and Ceruloplasmin contribute in the process of iron metabolism

which is a potent oxidizing agent, when bio-availability OP the functions are diminished then an

increase in the susceptibility of oxidative stress. Free iron existence is the sole indicator of the

production of ROS (Brion et al., 2003). There is a decrease in the concentrations of these non-

enzymatic antioxidants that influences those infants which are born in preterm conditions to

difficulties with increased concentrations of ROS. The reduced levels of ceruloplasmin and

transferrin have been detected in those infants which are preterm births with conditions like

asphyxia prior to brain hemorrhage (Lindeman et al., 2000). Glutathione reduction or reduced

synthesis has been anticipated to elaborate the risk of oxidative stress to the newborn infants. A

low level in the bronchi-alveolar fluid foresees that in intubated premature infants have later

developments of BPD in them (Grigg et al., 1993). In vitro, bilirubin has been demonstrated as a

powerful antioxidant vulture of per-oxy radicals. Bilirubin degenerates to its predecessor

biliverdin in the oxidative process as a non-toxic product. In full term as well as preterm births

there is a direct relationship between the plasma concentrations of serum bilirubin and total anti-

oxidants status (Wiedemann et al., 2003).

CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017

31



32

3.0 MATERIALS AND METHODS

For the current study one hundred and seventy one (n=171) preterm infants and about one

hundred (n=100) age and sex matched controls were enrolled. All of the patients were reviewed

after a signed consent form and experimental work was carried out and approved by the

Research Ethics committee of institute of molecular biology and biotechnology (IMBB), The

University of Lahore-Pakistan. Five ml (5ml) blood was drawn from the cuboidal vein and stored

in the vial at -75 degree centigrade for the future analysis.

TABLE 01: DEMOGRAPHIC DISTRIBUTION OF DATA

SEX (n=) MALE 84

FEMALE 87 INFANTS RESUSCITATED (n=)

Yes 57 No 114

SMOKERS (n=) YES 49 NO 122

SEPSIS (n=) YES 56 NO 115

DELIVERY (n=) NORMAL 41

C-SECTION 130 BLOOD TRANSFUSION (n=)

YES 39 NO 132

IRON THERAPY (n=) YES 28 NO 143

DIABETIC (n=) YES 35 NO 136

3.1 INCLUSION CRITERIA

Preterm infants with anemic profile were added in the following study

3.2 EXCLUSION CRITERIA

Infants to mothers with any congenital diseases or diabetes were excluded out of the current study


33

3.3 CHEMICALS

Reagents and chemicals of high analytical grade were acquired from Sigma Chemical

Co.(St. Louis, Mo, USA).

3.4 BIOCHEMICAL ASSAYS

3.4.1 ESTIMATION OF GLUTATHIONE (GSH)

Glutathione (GSH) was evaluated in the blood of patients according to the method of

Moron et al., 1979. Glutathione is first reacted with the reagent in term to produce a

chromophore TNB taking its absorbance at 412 nm. Amount of Glutathione (GSH) is

calculated according to the formula ([GSH]t = [GSH] + 2 x [GSSG]). The rate of absorbance

change (ΔA 412nm/min) is repeated after fixed interval. Amount of sample is determined by

generating a standardized equation from several standards of glutathione (Tietze, 1969).

3.4.2 ESTIMATION OF CATALASE (CAT)

Catalase was determined according to the generalized procedure of Aebi, 1974.

Evaluation was made with the help of spectrophotometer by reducing the absorbance to 230nm.

Sample was then homogenized by the help of phosphate buffer (pH 7.0) at 1-40C and sample

was then centrifuged at 5000 rpm. The mixture contained phosphate buffer, H2O2 and the

enzyme extract. The specific activity of Catalase will be expressed in terms of units/gram.

Procedure was repeated with the standardized dilutions of Catalase and absorbance was

measured to generate the standard curve.

3.4.3 ESTIMATION OF THIOBARBITURIC ACID REACTIVE SUBSTANCES

(TBARS)

Malondialdehyde (MDA) is the end producduct of oxidative stress that is secreat in the

activation of lipid peroxidation by the binding of free radicals with unsaturated fatty acid s and

the levels of MDA were estimated in subject blood through Thiobarbituric acid reactive

substances (TBARS) method. This method was introduce by Ohkawa et al., 1979. For this

method the take 200µl to 0.2ml of sample in test tube, then add the 0.2ml of 8.1% Sodium

dodecyl sulfate (SDS), then further add the 20% acetic acid by 1.5 ml, finally add the 1.5 ml of

0.8% TBA was purpose about 200µl to 0.2ml of sample 0.2ml of 8.1% Sodium dodecyl sulfate

(SDS) and it stand at centrifugation for 10 min at 3000rpm. After the centrifugation the two

layers were formed in test tube. 2 ml of upper solution ware taken from each wells to take

absorbance at 532nm.


34

3.4.4 ESTIMATION OF SUPER OXIDE DISMUTASE (SOD)

Activity of Superoxide dismutase (SOD) was estimated by the method of Kakkar, 1972.

It explained the how appropriate amount of samples and buffer were mixed to maintain the

appropriate levels of pH and 0.052M concentration. About 100µl of phenazine methosulfate

was then added followed by the 0.3ml of nitro blue tetrazolium (300μ m) and 0.2 ml of NADH

(750 μmol). As the NADH was added it initiated the reaction instantly afterwards it was

incubated at 300oC for 90 seconds. Finally 0.1ml of glacial was allowed to stand for few

minutes to stop the reaction and it was then centrifuged and n-butanol layer was separated. The

color intensity of the chromogen in butanol layer was then measured at 560 nm. Absorbance

values were then compared with a standard curve generated from known SOD.

3.4.5 ESTIMATION OF GLUTATHIONE REDUCTASE (GR)

Glutathione reductase was determined by the method of David and Richard (1983). It is

employed in the conversion of oxidized glutathione into reduced form Glutathione reductase

catalysis the conversion of oxidized glutathione to reduced glutathione employing NADPH, so

the amount of NADPH is a direct measure for the following enzyme activity. Reagents such as

PBS, EDTA and sodium azide are used in following ratios (0.12 M, 15 milli mole, and 0.1ml),

0.1 ml oxidized glutathione and 0.1 ml sample were added into the cuvette and 600 micro liter

distilled water was added to make the volume up to 2ml, it was then incubated for 3 minutes

and 0.1 ml NADPH. Absorbance was measured at 340nm with help of spectrophotometer after

every 15 seconds interval for 2-3 minutes. Controls contained water instead of oxidized

glutathione. The activity of GR represented as micro moles of NADPH oxidized/minute/g of

sample.

3.4.6 ESTIMATION OF VITAMIN C

The estimation of vitamin C was follow the protocol of Roe and Keuther (1943). It

involvs the conversation that ascorbate is converted into dehydroasorbate whn it is treated with

activated charcoal and later reacted with 2,4-dinitrophenylhydrazine and incubating it for three

hours at 37°C that results in the formation of osazone crystal. The crystals were dissolved in

2.5ml of 85% sulphuric acid and then tubes were cooled in ice and again absorbance was

measured at 540nm with the help of spectrophotometer. A standard graph was made and the

concentration of ascorbate in the samples were observed and expressed in terms of mg/g o

f sample.


35

3.4.7 DETERMINATION OF ADVANCED OXIDATIVE PROTEIN PRODUCTS

(AOPPs)

AOPPs were estimated in the plasma of patients using semi-automated method

discussed briefly, AOPPs were measured by the help of spectrophotometer (model MR 5000,

Dynatech, Paris, France) and were then calibrated with help of chloramine-T (Sigma, St. Louis,

MO). For the purpose about 200 ml of plasma which was diluted with 1/5 PBS was placed on

microtiter plate (Becton Dickinson Lab ware, Lincoln Park, NJ), and it was then added with

20ml of acetic acid. The absorbance of the reaction mixture is immediately read at 340 nm on

the microplate reader against a blank containing 200ml of PBS, 10 ml of potassium iodide, and

20ml of acetic acid.

3.4.8 PREPARATION AND DETERMINATION OF (AGEs)

The AGE-HSA were determined in vitro by incubating HSA (type V; Sigma; 50 mg/ml)

with 500 mM glucose in PBS for about 65 days at 37°C under sterile conditions. The AGE-

pentosidine which is considered as a marker of non-enzymatic glycation of proteins employing

a modified method by Odetti et al. Plasma proteins or AGE-HAS was allowed to precipitate on

TCA. The pellets were then hydrolyzed in 2 ml of 6 N HCl and were then dissolved in 250ml

0.01 M heptafluorobutyric acid (Sigma). 4 mg of plasma protein was then injected into an

HPLC system (Waters Division of Millipore, Marlborough, MA). A 25-30.46-cm C18 Vydac

type 218TP (10mm) column was used (Separations Group, Hesperia, CA). HPLC was

programmed with a linear gradient of 10 to 17% acetonitrile from 0 to 35 min. Pentosidine was

eluted out almost after thirty minutes by fluorescence excitation at 335 nm and emission at 385

nm.

3.4.9 DETERMINATION OF GAMMA GLUTAMYL TRANSFERASE (GGT)

In the determination of GGT a simple principle is involved method identified by the

Persijin and Van der Silk (1978) that measures the activity of GGT in the given sample for the

reason about 1.0ml of the reagent was incubated at room temperature for about one minute

followed by the addition of the sample in cuvette. Initially the contents were mixed and

absorbance was measured at 405nm by the help of spectrophotometer. The applied step was

repeated after every one, two and three minutes and enzyme activity was calculated and

expressed in IU/L.


36

3.4.10 DETERMINATION OF NITRIC OXIDE

Nitric Oxide was determined in the group of arthritic patients. NO which is produced in the

response of cells such as picomolar and nanomolar range and they are believed to have a very

shorter half-life (t1/2 <5s) in biological fluids; therefore, their direct assessment is difficult

while they are estimated in the mean of NO2¯ and NO3¯ they are performed to estimate NO

level in biological fluids and cell culture medium (Moncada et al., 1991). Nitrite concentration

is typically measured by a well-known method known as colorimetric Griess assay (Moshage et

al., 1995).

Sulfanilic acid is quantitatively converted to a diazonium salt under the reaction carried out by

reacting nitrite in acid solution. The diazonium salt is then coupled to N-(1-naphthyl)

ethylenediamine, which form a dye which is then measured at 548nm with help of

spectrophotometer and then quantified.

3.4.11 DETERMINATION OF TUMOR NECROSIS FACTOR BY ABCAM’s TNF

ALPHA HUMAN ELISA KIT

TNF alpha is estimated with the help of commercially available ELISA kit by Abcam. The

following kit is designed for the quantitative measurement of TNF alpha in supernatants,

serum, plasma samples and buffered solutions. For the purpose monoclonal antibody specific

TNF alpha has been coated onto the wells of the microtiter strips provided. Samples are added

and the reaction was initiated by adding the appropriate amounts of following reagents in these

wells. When they were incubated for the first time monoclonal antibodies were incubated along

with them. Afterwards they were washed and enzyme Streptavidin-HRP was added which

binds and illuminates it was again incubated followed by washing. A substrate solution was

added which acts on the bound enzyme to induce a colored reaction product. The intensity of

this colored product is directly proportional to the concentration of TNF alpha present in the

samples. And finally absorbance was measured with the help of ELISA reader.

3.4.12 ESTIMATION OF VITAMIN-E

The estimation of vitamin E assessed in blood sample through following method that was

well-established like Emmerie-Engel reaction as reported by Rosenberg in 1992. The method is

followed the reduction method and cause the red colour after the convertion of ferrous from

ferric. The absorbtion of vitamin E was measured at 460nm. About 1.5ml of test sample, abour

standard 1.5ml and 1.5ml of water were pipetted out separately. Abter this step 1.5 ml of ethano


37

and xylene were added in all the tubes and centrifuged after the well mixing. Than take the 1.5

ml solution from each well one by one to estimate the levels of vitamin E at 640nm. Although

Ferric chloride solution was added to each well in same quantity, after the addition of ferric

chloride the color of solution is change into red color due to reduction of ferric into ferrous than

after 15 min the take absorbance of each well solution at 520nm with the help of

spectrophotometry method.

3.4.13 DETERMINATION OF VITAMIN A

Rutkowski et al., (2006) explained a unique method for the determination of vitamin A.

1ml of liquid was taken in test tube I with tight stopper and 1ml of KOH solution and it was

then shaken vigorously for 1 minute and heated in tube in a water bath (60oC, 20 minutes),

then it was cooled under cold water and 1 ml of xylene was added, tube was plugged and

shaken vigorously again and absorbance was measured at 335nm against xylene-irradiate the

extract in the test tube II to the UV light for 30 minutes.

3.4.14 DETERMINATION OF INTERLEUKIN-6 “IL-6” LEVELS

Levels of interleukins i.e., IL-6 in serum was evaluated by the help of commercially

available ELISA kits. 0.03 pg/ml is the minimum measureable levels respective method to

callcolate IL6 that is universal cytokine and released due to the inflamtion by any type of

endogenous and exogenous infactions. For the calculation of ILs levels we will face the very

attention for the preparation of specific ranging of standard concention like 0 to 200 pg/mL.

With the help of pipeting each dilution of Aliquots (100 μl) were added in prepared assay

plate. Afterword these prepared solutions were stand in incubation for 2 hours at room

temperature. After passing the incubation time it will be washed with eight times with the

help of washing buffer by specific type of washer called BioTek's ELx50™ Strip. After each

step of washing it will be inverted at specific absorbing paper to eliminate the extra fluids

from kit for taking significant reading through ELISA reader. All the wells are consisting100

μl of solution. After washing process HRP conjugate and antibody solution were added and

incubated at roome temperature for 60 minutes. After the incubation process the ELISA kits

were washed by previous method for eight type. After incubation 150 μL of Sword

substrate/peroxidase mixture were added in the wells of ELISA palate and than it stand at

room temperature at dark light for 15 mintues. 150 μL of development media was finally

added in the wells of ELISA palate and than it stand at room temperature at dark light for half


38

hour. 100 μL of TMB substrate were added in wells and were incubated for 30 minutes. After

the incubation the 50 μL of stop solution were added to stop the reaction for the taking of

accurate result through ELISA kit reader with specific principle of ELISA reader.

3.4.15 ESTIMATION OF C-REACTIVE PROTEIN

For the determination of CRP, reagent is used determination employs turbid metric

method by using Synchron® ELIZA Kit. In the reaction, C-reactive protein are allowed to

combine with the specific antibody to form insoluble antigen-antibody complexes. The

employed immune complexes causes increased scattering of light which is determined

proportional to the concentration of CRP in serum. The scattering of light is measured by

taking its absorbance at 570nm. The CRP concentration is determined from a calibration

curve developed from CRP standards of known concentration.

3.4.16 DETERMINATION OF GPx

GPx can be estimated by the help of spectrophotometer. Sodium azide solution was

taken. GPx solution 0.1 along with glutathione was added into the NADPH vial. It was then

mixed by inversion and its pH was maintained upto 7.0 at 25oC with 1M HCl. Finally vial

was added with the 48mM sodium phosphate, 0.38mM ethylene diamine-tetra acetic acid,

0.95mM sodium azide, 0.12mM NADPH, 3.2 units of GPx and 0.075 to 0.15 units of GSH.

After 5 minutes absorbance was measured at 340nm (Aebi and Bergmeyer, 1983).

3.4.17 DETERMINATION OF TOTAL CHOLESTEROL (TCh)

For the estimation of total cholesterol in the serum sample following protocol was

followed reagent and samples were taken in respective amounts reagent at 1 ml and sample

10µl similarly standards were added separately. The mentioned contents were then mixed

together and incubated for ten minutes at the temperature of 37 degree centigrade and finally

absorbance was measured at 505nm and results were expressed in units (mg/dl).

3.4.18 DETERMINATION OF TRIGLYCERIDES (TG)

For the evaluation of triglycerides in the serum sample in the very first step TG gets

hydrolyzed by an enzyme named lipase which converts TG into glycerol and fatty acids. For

the purpose three tubes were taken and were named as blank, calibrator and assay tube. They

were then added with buffer and sample at the rate of 300 and 3µl contents within these tube

were allowed to mix and then stand for ten minutes at 37 degree centigrade and then their


39

absorbance was measured at 546nm by the help of spectrophotometer against the blank and

results were expressed in units (mg/dl).

3.4.19 ESTIMATION OF LOW DENSITY LIPOPROTEIN (LDL)

Low density lipoproteins (LDL) were estimated in the serum samples of preterm infants

according to the principle LDL gets hydrolyzed with to fatty acids and free cholesterol and

then oxidized to (CHOD) cholesterol oxidase. Hydrogen peroxide gets reacted with the PAP

and ultimately gives rise to the purple colored compound. Intensity of the color directly

signifies the concentration of LDL in the sample. And finally with the formula of Friedwald’s

levels of LDL were determined.

3.4.20 DETERMINATION OF HIGH DENSITY LIPOPROTEINS (HDL)

High density lipoproteins were determined with the help of special assay known as

HDL-cholesterol assay. First mixture of polyanions and polymers were formed and they were

allowed to attach with the surface of vLDL, chylomicrons and lipodystrophy (LD). These

complexes are may be considered as lipoprotein in nature. pH of the reaction was maintained

with the help of detergent (Reagent II) but HDL remains unstable and separated.

3.4.21 ESTIMATION OF TOTAL BILIRUBIN

Total bilirubin was determined in the serum sample by the commercially available kit.

For the estimation diazotized sulfanilic acid was reacted with bilirubin ti produce a compound

named azobilirubin and then its absorbance was measured at 550nm. The measured

absorption remained directly related with the concentration of total bilirubin in the sample

which was measured in the units (mg/dl). Bilirubin reagent and nitrite reagent were added and

mixed with samples in the well. Titration was done with the help of pipetting up and down for

four to five times. It was then incubated at room temperature for five minutes and at last

absorbance was measured at 550nm.

3.4.22 DETERMINATION OF MYELOPEROXIDASE (MPO)

Standard and samples were prepared by diluting in standard diluents. 100 µl of assay

diluent RD1-27 was added to each well. 50 µl of sample and standard were added in each well

and covered with adhesive strips provided incubated for 2 hours at room temperature. Each

well was aspirated and washed for 4 times with wash buffer. 200 µl of human MPO

Conjugate was added to each well and covered with new adhesive strip and incubated for 2

hours at room temperature. 50 µl of stop solution was added to each well (color change from


40

blue to yellow). Wavelength was recorded at 450nm (Klebanoff, 2005). 50 µl of dilutions of

samples and standard were added into appropriate wells. 50 µl of detection reagent A was

added immediately in each well. Plate was shaken using microplate shaker and covered with

plate sealer and incubated at 37Oc for 1 hour. Wash with 350 µl of 1X wash solution and let it

sit for 1-2 minutes. 100 µl of detection reagent B working solution was added to each well.

Incubate it for 30 minutes at 37°c after covering then washed it for 5 times. Then 90 µl of

substrate solution was added to each well and covered with a plate sealer. Incubated for 10-20

minutes at 37Oc. 50 µl of stop solution was added and solution turned yellow. Absorbance

was recorded at 450nm.

3.4.23 ESTIMATION OF SODIUM AND POTASSIUM

For the estimation of sodium and potassium mixture of MgCl2, NaCl, KCl and Tris-HCl

was assayed together in respective amounts 5, 80, 20 and 40mM respectively whereas, pH was

maintained upto 7.4 total volume was made upto 200µl. The reaction was later initiated by

addition of ATP and concentration remained 3.0mM. Similarly controls were processed. At last

activity was estimated by the difference in both assays.

3.4.24 DETERMINATION OF NEUTROPHILS

For the determination of neutrophils whole blood from the patients were obtained and

stored in EDTA vial and were analyzed on the automatic hematological analyzer (Sysmex 3000)

3.4.25 DETERMINATION OF MAGNESIUM AND CALCIUM

The estimation of Mg and Ca was done by their respective protocols explained by Lin

and Way, (1984). Medium of assay was set to about 1ml which contain 50mM of imidazole-HCl

buffer (pH 7.5) after all the additions mixture was incubated for fifteen minutes at the

temperature of 37 degrees n then stop solution was added into it which is ice cold ten percent

TCA. In following the activity of enzymes Mg+2 ATPase and Ca+2 ATPase were determined to

estimate levels of calcium and magnesium.

3.4.26 DETERMINATION OF COMPLETE BLOOD COUNT (CBC) IN THE SERUM

Complete Blood Count includes the determination/estimation of red blood cells,

hemoglobin, values of hematocrit, determination of white blood cells in term to define the

infectious status of patients and platelet count. Estimation of (CBC) was done with the help of

hematology analyzer by the help of automated Coulter (LH780) by Beckman Coulter, Inc.


41

3.4.27 ESTIMATION OF SERUM FOLATE (VITAMIN B9) AND VITAMIN B12

Serum folate (folic acid) and vitamin B12 was determined in the serum preterm infants

by their respective protocols that employs the use of HPLC (high performance liquid

chromatography) for the purpose standard and working solutions were prepared and the required

pH and volume was maintained and after the HPLC they were measured for their respective

absorptions for vitamin B9 wavelength was set to (200nm-290nm) while for vitamin B12 was

(210nm-260nm).

3.4.28 DETERMINATION OF TOTAL IRON BINDING CAPACITY

Total iron binding capacity was measured with the help of calorimeter method that

involves two reagents sequentially. First one is acidic reagent that contains iron-binding dye and

excess iron. While the second involved reagent is responsible for increasing pH and restoring the

transferrin affinity for iron. Transferrin is responsible for the extraction of iron from dye-iron

complex and it becomes 100% saturated while the remainder iron stays intact with the dye.

Hence in the particular way decrease in the absorbance of that color dye is then measured with

the help of spectrophotometer by measuring its absorbance at 660nm and this is proportional to

the total iron binding capacity of the serum sample.

3.4.29 DETERMINATION OF RENAL FUNCTION TESTS (RFTs)

Renal function test includes the determination of creatinine, Uric acid, and levels of

electrolytes with the serum sample of preterm infants for the sake of analysis of kidney and its

function. Urinalysis was done which comprise on the investigation of color, blood and turbidity

or any other visual appearance of the urine. Estimation of glomerular filtration rate (GFR), level

of urea and creatinine from serum, for the purpose three to four samples of each subject were

selected and considered for the analysis. Creatinine is the waste material that is given out under

the normal action of muscles in the body these samples are then thoroughly examined under the

microscope for the physical appearances as mentioned up and the levels are then revealed in the

following results.

3.4.30 ESTIMATION OF VITAMIN D (Vit D) IN THE SERUM OF PRETERM

INFANTS

Vitamin D was determined in the serum of preterm infants with the help of commercially

available ELISA kits by (Cayman chemicals USA).

CHAPTER FOUR RESULTS MISBAH, 2017

42

RESULTS


43

4.0 RESULTS

4.1 LEVEL OF HEMOLYTIC CELLS IN PRETERM INFANTS (PTI)

The infants were categorized in different niches such as resuscitation, smoking, gender,

sepsis, mode of delivery, blood transfusion etc Table 02, shows that preterm infants with

significantly decreased levels of RBCs when were compared with control group. Mean of red

blood cells (RBCs) in preterm infants was recorded as (4.35±0.27 s/L) where as in the healthy

group the value remained (4.65±0.15 s/L, p-value=0.013). The levels of white blood cells in

preterm infants ware significantly increased as compare to controle group such as (10.04±1.78)

and (7.57±0.41, p-value=0.042) respectivity shown in table 02. The mean value of platelets in

preterm infants was recorded as (16.34±1.72) but in control group the value remained (307.

±5.27) and it show very signifent results where the p value was (0.035). Table 02 shows that

preterm infants executed significantly decreased levels of Hct when were compared with control

group. Mean of hematocrit (Hct) in preterm infants remained as (35.34±1.67) significantly

decreased as compared to controls (41.29±1.59, p-value=0.012). Neutrophils were significantly

decreased in the preterm infants recorded as (3.35±0.43) as compared to (4.65±0.58, p-

value=0.025) shown in table 02.

Data in Table 02 and regarding hematological biochemical markers reticulocytes, MCH,

MCHC, rHuEPO, serum folate, hemoglobin, sferritin, serum MPO and SFe show highly

prominent results as compare to negative control group because the P<0.05. The consistent

increasing trend in reticulocytes levels (8.35±1.55) were recorded in preterm infants as compared

to control (4.64±0.93) where the p value (0.042). The consistent decreasing trends in case of

MCH, MCHC and increased the value of s-ferritin from groups of preterm infants were recoded

(3.24±0.06), (18.41±3.48) and (6.95±1.79) as compared to healthy group such as (114.87±4.20),

(31.99±2.93) and (1.95±1.05) respectively. The level of rHuEPO and serum folate ware also

shows the consistent decreasing trend (5.45±0.79) and (23.34±2.57) in preterm infants as

campare to normal group such as (7.62±0.83) and (34.63±2.17) respectively. Where the p value

also p<0.05. Table 02 shows that preterm infants executed significantly increased levels of serum

MPO when were compared with control group. Mean of serum MPO in preterm infants was

recorded as (30.84±2.35) where as in the healthy group the value remained (4.81±2.03) where p

value was (0.042). The levels of hemoglobin and sFe in preterm infants ware significantly

decreased (30.84±2.35), (20.56±1.35) as compare to healthy group likewise (14.11±0.87) and


44

(85.28±3.26) respectivity. Where the p value was (0.039 and 0.040) that is show in table 02.

Levels of TIBC is significantly (p=0.012) decreased in subjects (32.82±4.03) that is the symptom

of hemolysis/anemia as compared to the control (42.23±5.63). There was a significant decrease

(p=0.018) in the level of STFR has been observed in infected (8.77±1.26) as compared to control

(13.21±1.66). ZPP is the significant biomarker of hematolgic profile, our study resulted in the

significant increased (p=0.032) in the level of ZPP in subjects (2.37±1.24) as compared to

normal controls (0.00024±0.000094).

4.2 LEVEL OF RFT and LFT IN PRETERM INFANTS (PTI)

Table 02 shows that preterm infants executed significantly increased levels of uric acid

when were compared with control group. Mean of uric acid in preterm infants was recorded as

(5.37±1.18) where as in the healthy group the value remained (3.85±0.85) where p value was

(0.037). The value of creatinine in preterm infants was recorded as (1.60±0.27) but in control

group the value remained (0.72±0.04) and it show very significant decreased results in pateint

group as compare to healthy group, where the p value was (0.026). The levels of blood urea

nitrogen (BUN) in preterm infants ware significantly increased as compare to controle group

such as (25.59±5.32) and (14.67±2.17) respectivity. Where the p value was (0.035) that is show

in table 02. Furthermore, the levels of other RFT and LFT were increased in the preterm infants

in which including TBILI (3.31±2.64), total protein (6.45±0.81), GGT (53.74±7.98) and hsCRP

(1.40±0.32) as compare to normal group like (0.89±0.36), (6.43±0.29), (43.12±6.82) and

(1.04±0.02) respectively. Where the p value were <0.05.

4.3 LEVEL OF LIPID PROFILE IN PRETERM INFANTS (PTI)

The results that were depicted in Table 02, the levels of lipid profile including

triglycerides (Tg), cholesterol (TCh), high density lipoprotein (HDL) and low density lipoprotein

(LDL) were significantly differ with negative control groups (P=0.038, 0.012, 0.046, .017

respectively). the level of HDL and LDL have the negative correlation with to each other

because if the levels of LDL were increased in this response the levels of HDL automatically

decreased so LDL and triglycerides are also called the bad cholesterol but the higher levels of

HDL were most beneficial effects for human health so it also called good cholesterol. An

increasing trend of TCh (5.32±0.72), Tg (2.53±0.40), LDL (2.96±0.76) was observed in preterm

infants as compared to health group like (4.39±0.32), (1.30±0.13) and (2.29±1.18) respectivitly.

Show in table 02 the mean value of HDL in preterm infants was recorded as (1.37±0.16) but in


45

control group the value remained (1.69±0.16) and it show very significant increased results in

pateint group as compare to healthy group, where the p value was (0.042).

4.4 LEVEL OF STRESS BIOCHEMICAL MARKERS IN PRETERM INFANTS (PTI)

According to the result of present study (table 02) the levels of oxidative biomarkers were

altered due to the oxidative stress. The major prominent biomarkers including SOD (Superoxide

dismutase), GSHpx (glutathione peroxidase), MDA (Malondialdehyde), GSH (Glutathione),

CAT (Catalase), GSHpr (glutathione reductase) and NO (nitrogen oxide) shows highly

significant outcomes after comparing with negative control group. The consistent increasing

trend in MDA levels (4.07±1.08) were recorded in preterm infants as compared to control

(1.43±0.36) where the p value (0.025). The consistent decreasing trends in case of Glutathione,

GSHpx and increased the value of GSHpr from groups of preterm infants were recoded

(3.35±1.37), (6.64±0.59) and (3.49±1.58) as compared to healthy group such as ( 9.79±1.22),

(8.22±0.69) and (1.47±0.22) respectively. The level of Catalase and SOD ware also shows the

consistent decreasing trend (1.75±1.14) and (0.101±0.004) in preterm infants as campare to

normal group such as (3.9±0.81) and (0.49±0.14) respectively. Where the p value also p<0.05.

The lowest value (0.06 ng/ml) of SOD was recorded in group B (diabetic patients). Table 02

shows that preterm infants executed significantly increased levels of NO when were compared

with control group. Mean of NO in preterm infants was recorded as (58.05±10.385) where as in

the healthy group the value remained (19.82±1.42) where p value was (0.020).

4.5 LEVEL OF DIFFERERENT TYPES OF VITAMINS (B12, A, C, E AND D) IN

PRETERM INFANTS (PTI)

Data in Table 02 and regarding different types of vitamins such as vit B12, vit A,

vit C, vit E and vit D has significant altered their normal pattern after combine with negative

control group. The consistent trend in vit A levels (82.42±4.58) as compared to health group

such as (44.52±6.15). The levels of vitamin A were shows the decreased trends, where it p value

was (0.031). Table 02 shows that preterm infants executed significantly decreased levels of vit

B12 and vitamin C when were compared with control group. Mean of vit B12 and vitamin C in

preterm infants was recorded as (48.49±2.56) and (0.36±0.11) where as in the healthy group the

value remained (58.31±6.65) and (0.56±0.08) respectively. Where the p value ware (0.034 and

0.041 respectivily). Show in table 02 the mean value of vitamin E and D in preterm infants was

recorded as (0.23±0.07) and (9.72±1.25) but in control group the value remained (0.29±0.05) and


46

(13.22±0.81) respectively. It show very significant decreased results in pateint group as compare

to healthy group, where the p value was (0.011 and 0.029 respectivily).

4.6 EFFECT OF PHYSICAL MARKERS ON PRETERM INFANTS (PTI)

The data presented in table 02 shows that variables of physical marker such as body

weight and age differed significantly (≤0.05). The higher levels of body weight (1.47±0.27) and

age (28.07±3.36) were recorded in preterm infants as compared to normal healthy controls group

such as (2.92±0.33) and (37.65±1.70) respectively.

4.7 LEVEL OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) IN PRETERM

INFANTS (PTI)

Table 02 shows that preterm infants executed significantly decreased the levels of G6PD

when were compared with control group. Mean of phosphate in preterm infants was recorded as

(3.05±2.89) where as in the healthy group the value remained (4.13±4.06) where p value was

(0.0k33).

4.8 LEVEL OF ELECTROLYTES (HCO3, Ca, Na, K, Mg, CL-, Zn, Cu AND P) IN

PRETERM INFANTS (PTI)

Data in Table 02 and regarding stress biochemical markers bicarbonate (HCO3-), sodium

(Na), zinc (Zn), magnesium (Mg), calcium (Ca), chaloride (Cl), cupper (Cu), phosphate (P) and

potassium (K) shows highly significant results as compare to standard group. The levels of

biocarbonate (21.94±3.80) were consistent reduced in preterm infants as compared to control

(28.55±1.63) where the p value (0.015). Table 02 shows that preterm infants executed

significantly decreased levels of Ca nd Cu when were compared with control group. Mean of Ca

and Cu in preterm infants was recorded as (9.03±0.71) and (0.92±0.37) where as in the healthy

group the value remained (9.69±0.32) and (1.60±0.08) respectively. Where p value ware (0.021

and 0.027 respectivily). The levels of Mg in preterm infants ware significantly decreased

(1.53±0.32) as compare to controle group such as (1.71±0.17). Where the p value was (0.029)

that is show in table 02. The consistent increasing trends in case of Na, K and Cl from groups of

preterm infants were recoded (5.28±0.36), (5.92±1.03) and (90.59±6.18) as compared to healthy

group such as (4.37±0.34), (4.04±0.48) and (1.03±1.31) respectively. Where the p value were

(p<0.05). The level of Zn was also shows the consistent increasing trend (0.28±0.08) in preterm


47

infants as campare to normal group such as (0.016±0.06). The p value also recorded (0.012).

Table 02 shows that preterm infants executed significantly increased the levels of P when were

compared with control group. Mean of phosphate in preterm infants was recorded as (5.75±0.72)

where as in the healthy group the value remained (3.92±0.58) where p value was (0.047).

4.9 LEVEL OF INFLAMMATORY BIOMARKERS IN PRETERM INFANTS (PTI)

The levels of inflammatory biomarkers are also disturbed in the preterm infants due to the

oxidative stress and some other complication. The levels of inflammatory biomarkers ware

increased in preterm infants including IL6 (6.80±0.78) and TNFα (31.63±4.42) versus the

normal healthy group like (5.64±0.51) and (29.90±1.14) respectively. Where the p value were

(0.043 and 0.038 respectively). Table 02 depicts that there was an increased level of AOPP in

subjects (1.36±0.40, p=0.040) that is significantly higher than controls (0.84±0.04). Present study

also resulted in the higher level AGE in infected subjects (2.73±0.20) as compared to normal

controls (2.56±0.10). Where the p value was (0.036).

TABLE 02: VARIABLES OF MEDICAL IMPORTANCE IN THE PRETERM INFANTS (PTI) SUFFERING FROM ANEMIA VARIABLES CONTROL (n=100) PATIENTS (n=171) P˂(.05) RBCs/L 4.65±0.15 4.35±0.27 0.013 WBCs/L 7.57±0.41 10.04±1.78 0.042 PLTs/L 307.83±5.27 16.34±1.72 0.035 Hct% 41.29±1.59 35.34±1.67 0.012 Neutrophil Counts/L 4.65±0.58 3.35±0.43 0.025 ReticulocytesK/mm3 4.64±0.93 8.35±1.55 0.042 MCHpg 114.87±4.2 3.24±0.06 0.022 MCHCg/L 31.99±2.93 18.41±3.48 0.021 rHuEPO IU/L 7.62±0.83 5.45±0.79 0.041 Serum Folate 34.63±2.17 23.34±2.57 0.029 Hbg/L 14.11±0.87 9.90±1.82 0.039 SFerritin ng/ml 1.95±1.05 66.95±1.79 0.028 Serum MPO 4.81±2.03 30.84±2.35 0.042 SFe ng/ml 85.28±3.26 20.56±1.35 0.040 UricA mg/dL 3.85±0.85 5.37±1.18 0.037 Creatinine mg/dL 0.72±0.04 1.60±0.27 0.026 BUN mg/dL 14.67±2.17 25.59±5.35 0.035 TBILI mg/dL 0.89±0.36 3.31±2.64 0.021 TotalP g/L 6.43±0.29 6.45±0.81 0.019 GGT U/L 43.12±6.82 53.74±7.98 0.016 hs-CRP mg/L 1.04±0.02 1.40±0.32 0.042 TCH mg/dl 4.39±0.32 5.32±0.72 0.038 Tg mg/dl 1.30±0.13 2.53±0.40 0.012 LDL mg/dl 2.29±0.18 2.96±0.76 0.046 HDL mg/dl 1.69±0.14 1.37±0.16 0.017 MDA nmol/ml 1.43±0.36 4.07±1.08 0.025


48

SOD µg/ml 0.49±0.14 0.101±0.004 0.001 GSH µg/ml 9.79±1.22 3.35±1.37 0.008 CAT nmol/mol 3.95±0.81 1.75±1.14 0.017 IL-6 pg/ml 5.64±0.51 6.80±0.78 0.043 TNF-α pg/ml 29.9±1.14 31.63±4.42 0.038 AOPPs 0.84±0.04 1.36±0.30 0.040 AGEs 2.56±0.10 2.73±0.20 0.036 TIBC mcg/dl 42.23±5.63 32.82±4.03 0.012 STfR 13.21±1.66 8.77±1.26 0.018 ZPP µmol/mol 0.00024±0.000094 2.37±1.24 0.032 GSHPx µg/ml 8.22±0.69 6.64±0.59 0.027 GSHPr µg/ml 1.47±0.22 3.49±1.58 0.033 NO µmol/L 19.42±1.42 58.05±10.38 0.020 BWeight kg 2.92±0.33 1.47±0.27 0.028 Age year 37.65±1.70 28.07±3.36 0.042 VitB12 mg/g 58.31±6.65 48.49±2.56 0.034 VitA mg/g 44.52±6.15 82.42±4.58 0.031 VitC mg/g 0.56±0.08 0.36±0.11 0.041 VitE mg/g 0.29±0.05 0.23±0.07 0.011 VitD mg/g 13.22±0.81 9.72±1.25 0.029 HCO3 mmol/L 28.55±1.63 21.94±3.80 0.015 Ca mmol/L 9.67±0.32 9.03±0.71 0.021 Na mmol/L 4.37±0.34 5.28±0.36 0.037 K mmol/L 4.04±0.48 5.92±1.03 0.039 Mg mmol/L 1.71±0.17 1.53±0.32 0.044 Cl mmol/L 1.03±1.31 90.59±6.18 0.041 Zn mmol/L 0.16±0.06 0.28±0.08 0.012 Cu mmol/L 1.60±0.08 0.92±0.37 0.027 G6PD IU/L 4.13±4.06 3.05±2.89 0.033 P mmol/L 3.97±0.58 5.75±0.72 0.047

CHAPTER FIVE DISCUSSION MISBAH, 2017

49

DISCUSSION


50

5.0 DISCUSSION

Status of oxidative stress was reviewed in preterm infants suffering from anemia.

Different antioxidative enzymes namely, glutathione peroxidase (GSH-Px), superoxide

dismutase (SOD), reduced glutathione (GSH), malondialdehyde-hemoglobin (MDA-Hb),

catalase (CAT), and biomarkers viz., IL-8, IL-6 and TNF-alpha including many other blood

factors are responsible for the preterm labor and anemia. Pregnancy is normally a time frame of

felicity and anticipation, but due to many reasons it can contribute in the preterm delivery. About

12% of total pregnancies are thought to have increased risk for the premature birth but with

proper knowledge risks of preterm can be decreased. In normal cases pregnancy usually last for

about 40 weeks but in the cases of pretermies labor may be commenced before the completion of

37 weeks due to physical and biochemical reasons. Uterine contraction opens the cervix that

increases the chanes for preterm delivery. Other risk factors for the development of preterm birth

includes high blood pressure, excessive use of alcohol, are reason for certain cervical or uterine

abnormalities, high blood pressure, smoking, use of alcohol, drugs etc. Anemia is characterized

as reduced numbers of red blood cells (RBCs), hematocrit and/or hemoglobin concentration.

Reasons of anemia may include increase loss of erythrocyte or inadequate production of red

blood cells. At gestational period of 2 weeks, erythropoiesis may occur in the yolk sac that

generates cell expresses embryonic hemoglobin.

At gestational period of 6 weeks, red blood cells production initiates in liver that later

expresses fetal hemoglobin. At 6 months of gestation, bone marrow becomes the site of

hematopoiesis. During fetal life, size of erythrocytes decreases and number increases, 30-40% of

hematocrit increases on onset of second trimester. In later gestation RBCs shift from fetal to

adult hemoglobin production under the influence of increased oxygen concentration. Red blood

cell counts continue to decrease unless delivery of oxygen is inadequate and production of

erythropoietin is induced again. Preterm babies, after birth also go through reduction in

concentration of hemoglobin. After birth, level of hemoglobin in preterm infants declines

gradually and progressively in the two to three months of age. Anemia of prematurity is due to

lower level of hemoglobin at birth, reduced lifespan of red blood cells and a suboptimal response

of erythropoietin. This anemia of prematurity possibly exaggerated through factors that include

blood sampling frequently for laboratory tests, or through substantial clinical signs and

symptoms. Oxidative stress production is generally in two ways. First way is when reactive


51

molecules are created by endogenous metabolites and second way is when environmental

exogenous reactive contents get into biological system. Cell physiology changes because of these

two ways. The instability between generation and excretion of reactive oxygen species is known

as “oxidative stress”. Reactive oxygen species are unstable type of molecules having oxygen that

can react easily. Reactive oxygen species can damage RNA, DNA and lipid proteins, and even

cell death can occur. The reactive oxygen species (ROS) synthesized during metabolic pathways

commonly are superoxide anion, hydroxyl radicals, hydrogen peroxide and singlet oxygen Many

studies revealed that reactive oxygen species are generated from mitochondrial oxidative

phosphorylation (Trachootham et al., 2009).

Throughout oxidative phosphorylation, O2 is utilized and reduced into water and

approximately 1% oxygen changes to superoxide anion (O2 −). Cellular organs are damaged

through reactive oxygen species (ROS) molecules. Reactive oxygen species affects lipids,

proteins and DNA molecules. Reactive oxygen species effects mainly mitochondrial DNA as it

dont have the ability to repair like nuclear DNA. Thus, function of mitochondrial DNA is lost

due to repeated hurts. Defense system of antioxidant is generated by living cell which also

protects cellular membranes, nucleic acids and proteins. These may include compounds viz

lipoic acid, coenzyme, glutathione and enzymes etc. Enzymes viz catalase, glutathione

peroxidase, glutathione reductase and superoxide dismutase protects against oxidative stress.

Lipid peroxides are released through unsaturated lipid membrane components that undergo

oxidative damage. Studies revealed that isoprostanes are produced when arachidonic acid is

injured by oxidative stress. DNA guanosine is oxidized to 8-hydroxydeoxyguanosine (8OHdG),

that is closely related to neuronal oxidative stress. (Pandey and Rizvi, 2010 b).

Oxidative stress damages the erythrocyte membrane and also causes hemoglobin

oxidation. During aerobic respiration different oxidants come through endogenous and

exogenous means, which altogether known as called Reactive Oxygen Species (ROS). The

endogenous source of ROS includes mitochondrial respiratory chain, resistance reactions,

enzymes like nitric oxide synthase, transition metal interceded oxidation and xanthine oxidase.

(Lykkesfeldt, 2007). Erythrocytes have such enzymes which limits ROS effect. The main cause

of oxidative stress is the superoxide of ROS present in the cells. Red blood cells are mainly

responsible for transport of oxygen and carbon dioxide between tissues and lungs. Antioxidant

protection is provided to erythrocytes through anti-oxidative system and red blood cells also


52

move freely which facilitate other organs and cells of the body. (Siems et al., 2000). As red

blood cells have high O2 and hemoglobin concentration, hence oxidative stress chances increases

(Bryszewska et al., 1995). As RBCs lack mitochondria, so in mature red blood cells formation of

adenosine triphosphate takes place through glycolysis. Various studies proved that normally 90%

glucose produces ATP through glycolysis it was proved by different studies that in normal

conditions about 90% of glucose produced ATP by glycolysis as remaining 10% get into hexose

monophosphate shunt. In tissues glutathione is reduced via HMP shunt by using NADPH

pathway (Sivilotti, 2004). In vivo various studies explained that high level of oxidative stress

negatively affects red blood cells parameters which includes deactivation of enzymes and

membrane bound receptors (Halliwell and Gutteridge, 2007), high glutathione (GSH), lipids and

proteins oxidation (Pandey and Rizvi, 2009; Pandey and Rizvi, 2010a).

Red blood cells membrane has increase concentration of polyunsaturated fatty acids that

can oxidize easily. The cell membrane of red blood cells has two fields viz: lipid bilayer and

cytoskeleton. In the bilayer, phospholipids are spread asymmetrically while in entire lipid

domain cholesterol is dispersed (Bryszewska et al., 1995). Lipids are considered vital for shape

protection of red blood cells. For normal and regular functioning, fluidity is very essential for

plasma membrane of erthrocytes. Reactive oxygen species assail membrane and via lipid

peroxidation various unnecessary products are formed. (Pandey and Rizvi, 2010b).

Malondialdehyde (MDA) is the universal oxidative stress marker and it secreated in the blood by

the process of lipid peroxidation. Pryor and Stanley (1975) introduced that MDA is produced as

product of decomposition through lipids oxidation and the mechanism is believed to involve

prostaglandins formation, just like from PUFA, endo-peroxides are formed with two or more

double bonds. Alkaline phosphatase (ALP) enzyme is present in each cell of the body. The major

concentration of ALP is present in bone, liver and bile ducts. Alkaline phosphatase test is done

for the diagnosis of liver or bone problem. As liver damages, cells of liver releases ALP in

concentration more than normal in the blood. Alkaline phosphatase test is done to diagnose any

bile duct blockage. With the increase in bone tissues activities, ALP level also increases.

Aspartate aminotransferase (AST) enzyme is present in heart, muscle cells and liver. Aspartate

aminotransferase was formally known as glutamic oxaloacetic transaminase. Aspartate

aminotransferase level indicates stage of liver or other organ damage. Through the level of AST,

it can be diagnosed that jaundice is due to the disorder of liver or blood (Nyblom et al., 2006).


53

Bilirubin test is done to measure the bilirubin level in the blood. Bilirubin, a yellow

pigment, is present in bile which is formed through liver. Premature babies have to face number

of complications. Hyperbilirubinemia is one of the common problems of infants.

Hyperbilirubinemia condition is such in which babies have elevated bilirubin level, which is

produced by normal red blood cells breakdown. Elevated level of bilirubin contributes to

jaundice. Extremely raised bilirubin level causes damage to brain, thus preterm infants are

rapidly treated for jaundice before bilirubin attains life-threatening level (Liu et al., 2008).

Albumin formation is through liver cells and it’s continues circulation in the blood gives balance

to the fluid of the body. Albumin is one of the blood plasma protein and also play role as a

carrier in the body. Albumin level can be altered due to any liver abnormality. Serum albumin

test is helpful in diagnosis of many problems like hepatic disorders, deficiency of nutrition,

chronic pancreatitis and renal disease (Suzuki et al., 2006). Catalase is an enzyme, which act as

catalyst. Catalase enzyme through catalyzes reaction decomposes hydrogen peroxide into oxygen

and water. It is present in those organisms which can survive in presence of oxygen. Catalase

plays its role to prevent from accumulation and cellular tissues and organelles are protected from

peroxide damage that is produced continuously through various metabolic reactions. Glutathione

reductase is an enzyme which acts as catalyst. Glutathione reductase also regulates the reduction

of glutathione (GSSG) that exists in the oxidized form and reduced to glutathione (GSH).

Glutathione reductase is substantive for redox cycle of glutathione through which reduced

cellular glutathione (GSH) level is maintained.

Glutathione (GSH) have the ability to reduce the levels of oxidative stress by reacting

with organic peroxides and free radicals, it can also act as a substrate for glutathione S-

transferases and glutathione peroxidases in organic peroxides detoxification and xenobiotics

metabolism (Deponte, 2013). Glutathione transferase (GST) or glutathione S-transferases

enzymes are mainly present in the cytosol. Glutathione transferase has different activities and is

involve in various reactions. Glutathione transferase plays an important role in transformation of

compounds like cancerous products, therapeutic drugs and also produces compounds against

oxidative stress (Tew et al., 2011). Adenosine triphosphatase (ATP-ase) enzyme acts as catalyst

and decomposes ATP into ADP and releases free phosphate ion. Energy releases during

dephosphorylation reaction. Calcium use to control metabolic activities and enzymes of various


54

cells. Calcium ions regulate soluble enzymes activities in the liver. Calcium ATPase enzyme is

P-ATPase form which after contraction of muscles transfer the calcium (Jensen et al., 2004). In

the fetus, the main origin of Epo is liver macrophage (Falke et al., 1987). There comes a point in

between the gestational age of third trimester and first month, where Epo produces from

peritubular cells inside the kidney, instead of liver macrophage and reason of this alteration in

still unknown. Either Epo reduces through kidney and/or liver, there is high demand of

hypoxemia for Epo to be produce. Epo production is regulated by physiological or non-

physiological stimulants through increase gene expression of Epo (Bondurant and Koury, 1986

and Schuster and badiavas, 1989). Rich (Rich, 1986), found out that Epo could be produced

through macrophages of bone marrow. Researchers influenced that macrophages were reactive to

alterations inside oxygen tension. They also contended that macrophages beneath firm condition

can play its role to regulate erythropoiesis.

Preterm infants fail to produce Epo significantly on anemia contempt an evident demand

towards oxygenation of tissue (Bifano et al., 1992). Still, it is not known whether premature

babies rely on production of Epo through liver / kidney and/or both. This study resulted that

production of Epo mRNA and proteins seem to be integral in macrophages isolated of premature

infants cord blood. Production of Epo from macrophages of preterm infants is as like term

infants, it brings doubt that defective production of Epo through macrophages is responsible for

anemia of prematurity. To understand the molecular level of premature anemia, studies proposed

to determine regulation from gene expression of Epo in the development of peritubular renal

cells. Erythropoietin (Epo) is the cytokine that play its role in stimulation of erythropoiesis, and

is available since 1985 for clinical usage and studies. Strauss et al, (1995) established the criteria

that describe the hematocrits in infants suffering with respiratory problem, it indicated the need

of blood transfusion is necessary in such infants also receiving Epo. They concluded, it would be

wrong to say that tried medicines reduces blood transfusions rather critical health condition of

the patients make it more frequent to be blood transfuse. In 1996 Bechensteen et al,(1996)

reported that patients whether receive Epo or not, it does not affect the length, weight and head

perimeter of the infants, while the reason of this observation in unknown. It is clear that through

increase erythropoiesis stimulation, need of protein and vitamin also increases, as protein and

vitamin are not supplied through diet by preterm infants. In 1990 Shannon et al, described that

treatment with Epo caused weight gain more faster in patients while most researches did not


55

shown any significant difference in weight gain in between the treated and non-treated groups.

Vera et al drew in 2001 that treatment with EPO increased the level of hematocrits significantly.

They also proposed that count of reticulocytes increases with the treatment, which proves that

preterm babies have the ability to react with exogenic EPO through increase erythropoiesis,

Although interruption may come from endogenic EPO production, which may results from either

raise hemoglobin or absence of hemoglobin reduction which is require for stimulation of Epo.

Similar report was given by Bechesteen et al, (1993) that treatment affects the reticulocyte count.

At any stage of life, erythropoiesis depends mostly on enough nutrition, particularly

intake of iron and protein, deficiencies of iron and protein are significant determining factors

towards erythropoiesis (Brown and Howard, 1996). For newborns nutritional adequacy is

important due to their fast growth (Doyle, 1997). It is necessary to adjust intake of protein in

accordance with neonatal growth, however enough quantity of protein for erythropoiesis

stimulation during treatment of Epo is not yet defined (Brown and Howard, 1996). Authors

described that erythropoiesis enhances as intake of protein increases, as Epo dosage decreases.

Preterm babies induce lower iron stores because of reduce gestational age and decrease intake of

iron. Epo usage increases deficiency of iron in this means so supplementation of iron becomes

important during treatment of Epo. Presently, it has been confirmed that necessary amount of

iron supplemented for effective treatment of Epo should be no more less than 6mg/kg/day

(Donato et al., 1996 and Soubasi et al., 1995). Studies have been conducted on administration of

oxygen at resuscitation through variation in titration schemes of oxygen within the first ten

minutes after delivery. (Escrig et al., 2008). Kumar et al, 2014 modified this to fix the oxygen

concentration at resuscitation. The study was designed to fine the unbalance of oxidative stress

markers and how exposure to oxygen affects it at the time of birth. In 2005 according to NRP

(Neonatal Resuscitation Programme) preterm infants were resuscitated in between 21% to 100%

of O2 and objective saturation was not defined for the initial ten minutes. All preterm infants

with 100% of O2 received positive-pressure ventilation (PPV) which shows that ventilation or

hypoxia was responsible for intubation instead of sustained hyperoxia or apnea. Results depicted

that saturated oxygen had higher values as compared with the neonatal resuscitation program

upper limit, thus 100% of O2 concentration is not right for resuscitation of preterm infants

(guidelines of 2010) at the time of birth. Saturated oxygen does not bring any significant change

in between 21% and /or 40% of O2.


56

Preterm infants are suggested to wean at 21% of O2 through six minutes of lifetime.

Gestational age could be the significant factor which influenced resuscitation of O2 at the time of

birth, as extremely preterm infants less than 26 weeks of gestation need approximately oxygen at

the initial stage. Gestation of 28 weeks might get away oxygen for SpO2 (oxygen saturations)

maintenance within the defined range. The issue; exposure of oxygen following through titration

frequently to maintain range of SpO2 at resuscitation have been addressed by many studies. In

case of 100% O2 initial titration concentration was observed more effective than still

concentration in premature infants in maintaining targeted SpO2. (85-92). However, treatment

fails with 21% of O2 at resuscitation. (Rabi et al., 2011). (Kumar et al., 2014) In a study it was

indicated that 100% of O2 at resuscitation had higher oxidative glutathione (GSSG) than reduced

glutathione (GSH), at one month of age. In preterm infants having asphyxia from modest to

control had high total hydroperoxide while redox potential was lowered in resuscitation of 100%

O2 which indicated that is inspired concentration of oxygen is lowered it can help to reduce

oxidative stress (Ezaki et al., 2009). Preterm infants at 30% O2 resuscitation can have reduction

in oxidative stress (Vento et al., 2009). Plasma nitrotyrosine (NT) significantly increases in 40%

and 100% O2 resuscitation. In the plasma, nitrated proteins indicate inflammation and/or form

peroxynitrite from superoxide and nitric oxide (Ischiropoulos, 1998). In an analysis, preterm

infants had high plasma nitrotyrosine (NT) in case of bronchopulmonary dysplasia (BPD) while

NT was found lower infants without BPD (Banks et al., 1998).

At birth, polycythemia of preterm infants is well distinguished along the rapidly

decreasing values of hemogram in the first 8 months of age. After birth, total count of heme

declines (Sisson et al., 1959) and iron gained is maintained in different tissues by baby (Langley,

1951) As the formation of blood rate exceeds the destruction rate, mass of hemoglobin raises and

iron tissue storage re-utilizes (Seip and Halvorsen, 1956). Schulman et al, (1954) show that

preterm infants from different weights retrieve the mass of hemoglobin present at 3 to 4 months

of initial life and at this state preterm infants had relatively excess iron. Preterm infants reveal

minimum quantity iron of non-hemoglobin in the bone marrow at the time of birth, number

increases from 1st to 2nd month and amount disappear by the 3rd month. At birth, if tissue iron

stores reutilized for the formation of novel hemoglobin, it would not anticipate that preterm

infants response to administrated iron until and unless mass of hemoglobin in the circulation gets

equivalent. However, different researches proposed that early anemia ameliorated in preterm


57

infants give oral iron preparations from 1st week of age and few studies show even more speedy

response. (Tuttle and Etteldorf, 1952; Sisson and Whalen, 1958 ). Gaisford and Jennison (1955)

intramuscularly fed iron to ten preterm infants on the third week of life. This group, near 16

weeks of age showed high hemoglobin concentration as compared with the control group.

Preterm infants when parenterally fed iron on the first four weeks of age, concentration of

hemoglobin, counts of hematocrits and erythrocytes rapidly decreases. Same results were find

out from the group without iron supplementation, rather the group supplemented with iron

rapidly recovered from early anemia and also hemoglobin concentration increased on the 3rd

month of age. This proves that at the time preterm infants had raised tissue iron stores, even then

iron administered exogenically accelerate the range of formation of hemoglobin.

Schulman (1958) depicted that erythropoietic resumption time is not linked with

maturity, preterm infant’s birth weight but is associated with initial hemoglobin concentration. In

this study, preterm infants with corresponding birth weight along decreases concentration of

hemoglobin took up erythropoiesis before. The alteration in concentration of hemoglobin was

about 7 or 8mg/100ml at the time of birth and remained for more than four weeks of life.

Gardner et al, (1955) established relationship among activity of erythroid in the bone marrow

and hemoglobin concentration of preterm infants. This study described that occurrence of

erythropoiesis takes place after the level reduces beneath 11gm/100 ml without depending on the

concentration of hemoglobin or birth weight. Seip and Halvorsen (1956), observed that preterm

infants with below 1500gm birth weight had occurred earlier reticulocytosis comparable with the

preterm infants of birth weight 1500gm to 2000gm. This research aimed to associate the

erythropoietic intensity and time response towards the overall preterm infants rate of

development. Denman and Arlene (1960), studied the hemoglobin concentration and found no

significant difference in the first 8 weeks of life. Only the initial deviation in the mass of

hemoglobin was 6gm in preterm infants who had 2000gm birth weight. The range and mean of

birth weight and growth rate was quite same. Both groups resumed erythropoeisis at the same

time of age, the time when concentration of hemoglobin and mass of hemoglobin were nearly

identical. Thus, preterm infant’s hematological behavior differences are not explicated through

any evident underlying deviations among the studied group.


58

The evident consequence of iron supplemented during early anemia of preterm infants is

not explained well. Just a suggestion not establishment that erythropoietic stimulation might be

due to exogenic irons (Josephs, 1953). It could be possible that raised iron readily accelerate

and/or stimulate formation of hemoglobin through action of mass. Shoden and Sturgeon (1959)

point out that formulations of iron-dextran parenterally fed incline to deposit at sites of storage as

ferritin instead of hemosiderin. Heilmeyer and Granick (1958, 1954) proposed that physiological

form of iron is ferritin and is available promptly for body rather than iron of hemosiderin. In this

study the observed effects could be due to these deviations. In preterm infants, iron deficiency

anemia might be the result of various influences. Iron storage of preterm infants might be

influenced by frank parental anemia and /or impaired parental iron. (Sisson and Lund, 1958).

Sturgeon (1959) suggested that no detectable change produce during the period of non-anemic

pregnant mothers by parental supplementation of iron in hemotoligical profile of preterm infants

during the initial 1½ year of life. In the study by Denman and Arlene 1960, no possible change

was seen in between the groups because of parental iron deficiency anemia. Results showed that

one mother had concentration of hemoglobin beneath 9.7gm/100ml while four had under

11gm/100ml. None of the mother effected of erythrocytes microcytosis or hypochromic anemia.

Parental iron storage differences potentially evaluated by measuring iron binding capacity, iron

binding protein saturation, serum iron and free protoporphyrin of erythrocyte. Such findings

indicated an improved total nutritional iron in termed women (Holy, 1953) as compared with the

women with oral supplementation of iron during the time of pregnancy (Sturgein, 1959). Both

groups had good nutritional iron and so no variation was observed that may influence the iron

storage of preterm infants. Preterm infants in this research were properly nourished. To observe

the mothers nutrition, they were also registered. Questionary emphasized on the diet and weight

of preterm infants. Though uner observation preterm infants had no medicative iron after 2-3

months of age they started taking iron riched cereals. The recorded diet indicated that preterm

infants of both groups were identical while feeding. At birth, preterm infants had minimum iron

storage and iron in the mass of circulatory hemoglobin estimates the total available iron for the

formation of hemoglobin. (Langley, 1951). At birth, the most significant determinant is the size

of the mass of hemoglobin for the need of exogenic iron inorder to compete the development

requirement in late infancy. (Sisson et al., 1959).


59

Differences actually in gestational age, weight at birth, weight gain and hemoglobin

concentration were little and so are not likely substantial. if these factors had influence then that

would in the form of nutritional iron towards the control group. Contemporary, experimental

subjects had increased hemoglobin concentration below 12 weeks of age and high level

continues to maintain onwards from the 3rd month of life. In the experimental preterm infants

circulating mass of hemoglobin exceeds than that from control group by 10 weeks of age, and all

through the whole study duration. The reason could be the administration of iron to the

experimental preterm infants. Parenterally iron was administered to assure the consistent and

measurable amount. Research was not dependent on the iron form utilized and/ or administration

route of iron. This study showed how preterm infants absorb compounds of iron easily when

administered orally. (Oettinger et al., 1954). As hemoglobin concentration and absorption of iron

are interlinked instead of iron storage (Hahn et al., 1943), it is expected that preterm infants

would easily absorb oral administration of iron during the period of early anemia. Generally,

course of hematology in the control group which was fed with diet rich of iron underlined that

not necessarily preterm infants become anemic if super nutrition are given. This data reveals that

preterm infants by the age of six weeks take up hematopoiesis and as soon as possible

supplementation of exogenic iron provides benefit to them. On should not wait for iron storage to

be utilized.

Henny et al, 2016 conducted a study on iron profile based on the level of hemoglobin,

transferrin saturation and serum ferritin. Two markers of iron in combination i.e. transferrin

saturation and zinc protoporphyrin ware used for iron deficiency diagnosis, as these markers

have the ability to detect dysregulation of supplemental iron in erythropoiesis before the

occurrence of anemia (Rao and Georgieff, 2009). Serum ferritin is the protein of acute phase

which during any inflammation or infection increases. This study excluded infected patients and

diagnosis of infection was examined clinically and through laboratory tests. C-reactive protein,

the marker of acute infection was also not assesst. Indonesians have recommended

supplementation of iron as standard for preterm infants. Negative iron balance is found in

preterm infants as their need is increases with the rapid development. This usually occurs at 8

weeks of life. At this time period, erythrocytes production along mass of muscles and also

enzymes need more iron for consumption. (Hammond and Murphy, 1960 and Ziegler et al.,

2011). A Cochrane analysis described that preterm infants who were supplemented with iron had


60

higher level of hemoglobin than the preterm infants without supplementation of iron at 6-9

months of age. (Mills and Davies, 2012).

Iron level is lower in breast milk as compared with the formula milk which had high

amount of iron. However, the gut of preterm infants can easily assimilate the iron of mother feed.

A case study reported that preterm infants who were breastfed solely and partially showed a

significant difference of 4.1g/dL in the level of hemoglobin among subjects at 6-8 weeks of age.

(Mills and Davies, 2012). In preterm infants of gestational age less than 36 weeks iron deficiency

is linked with low weight at birth, abrupt duration of fort1ified formula fed and weight gain

initially at 1½ month of life. (Rao et al., 2002). However, it is difficult to figure out how much

iron is needed to prevent preterm infants from iron deficiency. Few studies report that high

amount of iron is needed from formula milk as compared with the mother feed to reduce iron

deficiency but still it’s doubtful that this will prevent preterm infants from iron deficiency.

Preterm infants fed with human milk might have protein mal nutrition.

Preterm infants had deficiency of vitamin E due to hemolytic anemia (Oski and Barness,

1967). This study reports that preterm infants would not have this deficiency if vitamin E is

supplemented with soluble water and the whole study showed normal vitamin E concentration of

plasma. Likewise, in preterm infants no deficiency of folic acid, vitamin C and/or vitamin B12

was found. Hemolytic anemia might be due to low superoxide dismutase along glutathione

peroxidase activity within the erythrocytes. It is indicated that selenium and copper could be

responsible for activity decline in such enzymes. Rudolf et al (1981) reported that hemoglobin

concentration had no correlation with values of selenium RBCs or activity of glutathione

peroxidase in the preterm infants with very low birth weight. Ronnholm and Simes, (1985)

design the study which resulted that selenium intake and RBC selenium concentration

subsequently depend on intake of protein. Therefore, correlation found among the intakes of both

with concentration of hemoglobin is not storming. The level of ceruloplasmin and albumin of

plasma concentration decreases in preterm infants with very low birthweight rather than the full

term normal infants (Raiha et al., 1976). In copper transport chain both ceruloplasmin and

albumin are seem to be active. Albumin transports ingested copper towards liver while

ceruloplasmin is involved in donating copper too extrahepatic tissues (Evans, 1973). Haga et al,


61

(1981) suggested neither activity of superoxide dismutase nor ceruloplasmin play any role in

anemia of prematurity.

CHAPTER FIVE

FIGURE 01consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the mother and fetoplacental unit. of 20-37 weeinclude reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic predisposition, environmental and emoof events are involved such as activation of fetal HPA axis (

CHAPTER FIVE

FIGURE 01: Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the mother and fetoplacental unit.

37 weeks which cause the complication approximately 7include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic predisposition, environmental and emoof events are involved such as activation of fetal HPA axis (

Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the mother and fetoplacental unit. Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration

ks which cause the complication approximately 7include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic predisposition, environmental and emoof events are involved such as activation of fetal HPA axis (

Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the

Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration ks which cause the complication approximately 7

include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic predisposition, environmental and emotional factors. For the initiation of labor, there are various endocrine cascade of events are involved such as activation of fetal HPA axis (

62


Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration ks which cause the complication approximately 7

include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic tional factors. For the initiation of labor, there are various endocrine cascade

of events are involved such as activation of fetal HPA axis (


Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration ks which cause the complication approximately 7-10% of all delieveries.


of events are involved such as activation of fetal HPA axis (hypothalamic


Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration 10% of all delieveries. Major factors of preterm birth


hypothalamic-pituitary-adrenal

DISCUSSION MISBAH, 2017

Labour is the normal mechanism in which the fetus is ejected form the uterus. This process requires the consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the

Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration Major factors of preterm birth


adrenal axis). This cascade of


This process requires the consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother and fetus at the end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the



). This cascade of


This process requires the consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the

nd fetus at the end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the



). This cascade of


63

event is responsible to enhance the levels of estrogen and diminish the concentrations of progesterone. The steroid hormones have important role in the stimulation and activation of myometrial cell by increasing the synthesis of prostaglandin and Oxytocin hormone. Stimulatory event of prostaglandin raise the expression of gap junction protein as well as prostaglandin F receptor and Oxytocin receptor. The expression of contraction associated protein is tightly regulated by estrogen positively, whereas negatively regulated by progesterone hormone. The contraction associated proteins (CAPs) are also expressed on the uterus to regulate the concentrations of Cyclooxygenase, Lipoxygenase and Oxytocin endopeptidase. CRH (Corticotropin releasing hormone) which is the peptide hormone secreted from the hypothalamus that is present on the chorionic and placental membrane as well as decidual and amnionic cells. Corticotropin releasing hormone has the capability to stimulate the secretion of ACTH and cortisol synthesis. In the negative feedback loop, the mother pituitary secrete cortisol hormone which hampers the activity of hypothalamic Corticotropin releasing hormone (CRH). While in positive feedback loop, CRH further involved in the activation of maternal and fetal HPA axis. After the activation of fetal HPA axis, it further triggers to increase the secretion of ACTH (Adrenocorticotropin hormone) which leads toward the release of DHEAS (Dehydroepiandrostenedionsulfate) which is actually the precursor of steroid hormone. DHEAS is basically expressed on the zone of fetal adrenal gland of fetus and mother as well. Dehydroepiandrostenedionsulfate is than further metabolize into Estrone, estradiol and estriol. CRH has also the capability to enhance the production of prostaglandin in decidual, chorionic and amnionic cells. This increase level of prostaglandin has crucial role in parturition. Corticotropin releasing hormone (CRH) has some other actions such as in the contraction of smooth muscle cells and uterine vessels dilation. Moreover the deficiency of 15-OH-PGDH enzyme triggers to enhance the degradation of prostaglandin which is responsible to enhance the levels of PGE2 and cause myometrial contractions. Moreover, genital infections have also important role in the synthesis of inflammatory cytokines. During infections, macrophages and granulocytes are synthesized in the myometrial cell which further release proteases such as matrix metalloproteinases, Lipoxygenase and Cyclooxygenase as inflammatory cytokines including IL-6, IL-8 and TNF-α. Cytokines have the capability to increase the production of prostaglandin that causes contraction of myometrial membrane, preterm prelabour rupture of membrane and cervical ripening. On the other hand, genital infections trigger to reduce the enzyme levels of 15-OH-PDGH, results in preterm labour. Due to oxidative stress and poor nutritional diet in pregnant mother, elevated levels of free radicals and inflammatory cytokines are produced. Furthermore, the disruption in mitochondrial electron transport chain of fatty acid and prostaglandin synthesis as well as ischemic and hypoxic condition have major role in the formation of reactive oxygen species (ROS). Stimulation of macrophages, monocytes and neutrophils are also contributed in the synthesis of free radicals. On the other hand, reactive nitrogen species (RNS) are implicated in the damaging of cellular DNA by disrupting nitric signaling (NO) that cause vasoconstriction. The decrease blood flow in the uterus and placental wall triggers to cause pre-clampsia, preterm labour and PPROM (Preterm prelabour rupture of membrane). Poor nutritional diet of mother such as lack of vitamins, iron and folic acid is also one of the major factors for the progression of reactive oxygen species and reactive nitrogen species. Due to infection or inflammation, macrophages, neutrophils and granulocytes secrete various inflammatory cytokines that are responsible to stimulate NADPH oxidase enzyme which is basically ROS generating enzyme. This enzyme is the main contributor to decline the formation of anti-oxidants and increase the synthesis of free radicals that cause lipid peroxidation by MDA and Isoprostane. MDA forms an adduct in the nucleus and trigger the DNA damaging, stands breakage and chromosomal abnormalities. Oxytocin is a non-peptide hormone which has peripheral and central actions during pathological and physiological conditions including lactation, parturition, ejaculation, erectile dysfunction and material behavior. It has secreted from paraventricular nuclei of hypothalamus and also synthesized via peripheral tissues such as lining of uterus (uterine epithelium), amnion, placenta and corpus luteum. The Oxytocin hormone which is formed by maternal and fetal stress has major effect on myometrium. The Oxytocin binds with its specific receptor OTR (Oxytocin receptor), it belongs to the family of G-protein coupled receptor family. In the uterus, the alpha subunit of Oxytocin receptor Gα/1i couple with PLC-β (Phospholipase C-β) and triggers the hydrolysis of PIP2 (Phosphoinositide-bis-phosphate) into DAG (Diacylglycerol) and IP3 (Inositol-tris-phosphate). IP3 is implicated in the mobilization of Ca2+ in SR (Sarcoplasmic reticulum) whereas DAG has involved in the regulation of PKC (Protein kinase C). The Ca2+ is released from Sarcoplasmic reticulum and form Ca2+ dependent Calmodulin complex that further stimulate MLCK (Myosin light chain kinase). The phosphorylation of myosin chain by myosin light chain kinase enzyme triggers the contraction of myometrium wall. VOCCs (Voltage-operated Ca2+ channels) which are also known as L-type Ca2+ channels that trigger the entry of Ca2+ ions in the myometrial cell and important for impulsive activity. These channels are open during the mechanism of action potentials and consequently open the channels for the Ca2+ entry. The contraction in smooth muscles of uterus builds upon the balance between Myosin light chain phosphates (MLCP) and myosin light chain kinase (MLCK). The phosphorylation of myosin chain has the capability to cause contraction in the smooth muscle cells in myometrium. Whenever Oxytocin binds to its specific receptor, it activates Rho proteins and triggers


64

the interaction between actin and myosin and cause the Ca2+ sensitization. In myometrial cell, the Rho protein is stimulated by the activation of alpha subunit of Oxytocin receptor that further activates ROCK. It is basically Rho kinases which contribute in the inhibition of myosin light chain phosphatase (MLCP) and consequently cause myometrium contraction and lead to preterm labour. Moreover, Rho kinase and protein kinase C enzymes are stimulated by DAG (Diacylglycerol) that activate CP1-17 (C-kinase-activated protein phosphatase-1 inhibitor) and influence MLCP-P. Since, the Ca2+ sensitization by the Oxytocin receptor metabolism to cause smooth muscle cells contraction of myometrium is still debatable. In the body system, iron level is regulated by hepcidin which is bascially peptide hormone and formed in the liver than released into the blood circulation. The levels of hepcidin enhanced due to the response of inflammation/infection and iron concentrations. While its levels declined due to the result of erythropoiesis. The basic purpose of Hepcidin is to regulate iron homeostasis via interacting with exporter protein (Ferroportin). This transmembrane exporter protein is located on the membrane of duodenal Enterocytes as well as on the surface of RE (Reticuloendothelial) macrophages of liver, bone marrow and spleen. In mother, low iron intake in diet and excessive bleeding during delivery are responsible to cause infection and inflammation which trigger macrophages activation and secretes interleukins and tumor necrosis factor. These inflammatory cytokines are the causative factor to enhance the levels of Hepcidin in the liver. The Hepcidin released into the blood circulation and hampers iron absorption in the small intestine of fetus. It has also the capability to bind with Ferroportin receptor and cause its degradation. The increase level of Hepcidin is involved to decrease iron absorption in liver, whereas inflammation trigger to reduce red blood cells survival and cause bone marrow suppression. On the other hand, the preterm babies have immature red blood cells due to preterm delivery. These babies have increased breakdown of red blood cells and decrease formation of blood cells which further lead towards low erythropoietin response and resultantly anemia is occur.

CHAPTER SIX SUMMARY MISBAH, 2017

65

SUMMARY


66

6.0 SUMMARY

Almost 12% of babies born are known as premature babies as they were born before the

thirty seventh week of pregnancy such babies are known to have underdeveloped organs and are

subjected to multiple problems that comprise of issues related to their digestion or respiration,

additionally, they are found to have problems in their brain development and growth. Such

babies do not have fully developed lungs which is not able to produce sufficient ‘surfactant’ this

complication is termed as respiratory distress syndrome. Especially preterm babies who may

suffer any infection at the time of birth usually have low blood pressure and hence they may be

supplemented by the appropriate medication. In digestive system they may not have fully

developed intestines so they are provided with very low quantity of feed which might be as low

as one teaspoon. Breast milk is best for the new born babies especially the premature babies,

such babied fed on breast milk are on less chance to develop any infection or serious intestinal

complications and such babies have encountered to have improved growth. Coming on to the

vessels blood vessels of premature babies are more fragile and are subjected to rupture easily.

Bleeding problem in such babies may be from minor to severe. Premature babies are kept under

the oxygen tanks because of their uncontrolled oxygen pressure and they are also aided with IV

nutrition for the deficiencies. Causes beyond the preterm birth are common out of which one

common maternal cause remains preeclampsia, certain kind of infections, abuse of drug,

abnormality in structure of uterus, preterm birth history, placental abruption and premature

rupture of membrane of uterus. Such babies even if they survive they remain prone to infections

and may suffer through medical conditions throughout their life span.

Moreover, preterm infants are at higher risk of diabetes and hypertension. Rate of

premature births are increasing especially in the developed countries under the reason which

includes modern lifestyle. Hence if such preterm baby is born in high socioeconomic status attain

higher rate to lead healthy life as compared to those who born in relative lower socioeconomic

status. During pregnancy iron deficiency counts for a common cause in developing anemia and

increasing chance to have a preterm birth. Therefore sufficient iron is supplemented in common

clinical practice to preterm infants. Umbilical cord is the importents part of placenta that connect

the mother with baby for transpotation of specific nutrients. Typically, umbilical cord are

attached with two clamps and the cord is cut at this clamp. It has been reported that the levels of


67

Hb secnificantly increased at the time of birth by the two clamps assignment within the 35 to 175

seconds. It has been observed that cord clamping is not associated with the specifically infants or

preterm infants and they have significantly high levels of Hb and due to delayed cord clamping

there is reduce in RBC transfusion. It has been defined by the author that reduce levels of RBC

transfusion leads to induce delayed cord clamping in preterm infants, so the levels of RBC

reduced by the removal of blood that leads to use for various blood tests performed by the blood

analyzer. Clinical trial is obligatory to perform to monitor the device by using 93 preterm infants

having weight less than 1Kg.

Preterm infants fail to produce Epo significantly on anemia contempt an evident demand

towards oxygenation of tissue. Still, it is not known whether premature babies rely on production

of Epo through liver / kidney and/or both. This study resulted that production of Epo mRNA and

proteins seem to be integral in macrophages isolated of premature infants cord blood. Production

of Epo from macrophages of preterm infants is as like term infants, it brings doubt that defective

production of Epo through macrophages is responsible for anemia of prematurity. To understand

the molecular level of premature anemia, studies proposed to determine regulation from gene

expression of Epo in the development of peritubular renal cells. Erythropoietin (Epo) is the

cytokine that play its role in stimulation of erythropoiesis, and is available since 1985 for clinical

usage and studies. Established the criteria that describes the hematocrits in infants suffering with

respiratory problem, it indicated the need of blood transfusion is necessary in such infants also

receiving Epo.

Studies concluded, it would be wrong to say that tried medicines reduces blood

transfusions rather critical health condition of the patients make it more frequent to be blood

transfuse. In 1996 it was reported that patients whether receive Epo or not, it does not affect the

length, weight and head perimeter of the infants, while the reason of this observation in

unknown. It is clear that through increase erythropoiesis stimulation, need of protein and vitamin

also increases, as protein and vitamin are not supplied through diet by preterm infants. In 1990

scientists described that treatment with Epo caused weight gain more faster in patients while

most researches did not shown any significant difference in weight gain in between the treated

and non-treated groups. Treatment with EPO increased the level of hematocrits significantly.

They also proposed that count of reticulocytes increases with the treatment, which proves that


68

preterm babies have the ability to react with exogenic EPO through increase erythropoiesis.

Although interruption may come from endogenic EPO production, which may results from either

raise haemoglobin or absence of haemoglobin reduction which is require for stimulation of EPO.

Similar report support that treatment affects the reticulocyte count. Preterm infants are suggested

to wean at 21% of O2 through six minutes of lifetime. Gestational age could be the significant

factor which influenced resuscitation of O2 at the time of birth, as extremely preterm infants less

than 26 weeks of gestation need approximately oxygen at the initial stage. Gestation of 28 weeks

might get away oxygen for SpO2 (oxygen saturations) maintenance within the defined range.

At birth, polycythemia of preterm infants is well distinguished along the rapidly

decreasing values of hemogram in the first 8 months of age. After birth, total count of heme

declines and iron gained is maintained in different tissues by baby. As the formation of blood

rate exceeds the destruction rate, mass of hemoglobin raises and iron tissue storage re-utilizes.

Studies reveal that preterm infants from different weights retrieve the mass of hemoglobin

present at 3 to 4 months of initial life and at this state preterm infants had relatively excess iron.

Preterm infants reveal minimum quantity iron of non-hemoglobin in the bone marrow at the time

of birth. Therefore, correlation found among the intakes of both with concentration of

haemoglobin is not storming. The level of ceruloplasmin and albumin of plasma concentration

decreases in preterm infants with very low birth weight rather than the full term normal infants.

In copper transport chain both ceruloplasmin and albumin are seem to be active. Albumin

transports ingested copper towards liver while ceruloplasmin is involved in donating copper too

extra-hepatic tissues.

CHAPTER SEVEN CONCLUSION MISBAH, 2017

69

CONCLUSION

CHAPTER SEVEN CONCLUSION MISBAH, 2017

70

7.0 CONCLUSION

The current study concludes that imbalance between oxidant and antioxidants in the body

leads to the overproduction of reactive oxygen species and inflammatory cytokines that are

involved in the preterm delivery of babies. These inflammatory cytokines in the number of ways

are involved in the uptake of several proteins and hampers the iron absorption in liver. Moreover,

preterm babies have immature red blood cells due to preterm delivery. So they may execute

increased breakdown of red blood cells and decrease formation of blood cells that further leads

to low erythropoietin response and resultantly anemia may occur.

PART II MISBAH, 2017

71

PART-II

EXPRESSION OF UPSTREAM MATERNAL VARIABLES REGULATING PRETERM

BIRTH


72

INTRODUCTION


73

9.0 INTRODUCTION

Preterm birth is defined as birth of a baby earlier than 37th week of gestational period and

is one of the leading and major causes of mortality and long-term morbidity in perinatal

medicine (Lawn et al., 2006 and Goldberg et al., 2008). Premature babies are classified as per

their gestational age (GA) and they are; Extremely preterm with Gestational Age < 28 weeks,

second is the Severely preterm with Gestational Age between 28-31 weeks, and the third one is

the Moderately preterm births with Gestational Age between 32-33 weeks and the last one is the

Near term with Gestational Age between 34-36 weeks. The incidence of preterm birth is 5-9 %

of all the deliveries in Europe out of which 20% are those infants who are born before the 28th

week of gestational period (Goldberg et al., 2008 and Muglia and Katz, 2010). Over the last few

decades the number of incidence of preterm births has been rising, and the reason of this is the

increase numbers of preterm deliveries for example the earlier intervention in preeclampsia and

increased number of assisted pregnancies. A very major issue is that premature infancy accounts

for 75% of perinatal mortality and about more than half of the long-term morbidity (Lawnet et

al., 2006 and Muglia and Katz, 2010). In 1970s before the widespread use of assisted ventilation,

there were only a few numbers of survivors among the infants which were born extremely

premature and even more infants who were full mature and they died due to respiratory distress

which is cause by absence of pulmonary surfactant therapy (Saigal and Doyle, 2008).

Usage of antenatal corticosteroids may have improved many methods of assisted

ventilation and also it’s introduces the surfactant administration, as survival rates for preterm

born have improved remarkably. Improvement in the survival rate has not been yet escorted by a

low levels in major neonatal diseases occurs. The National Institute of Child Health & Human

Development (NICHD) Neonatal Research Network reported a decreased numbers in the

mortality of the babies who are born at preterm but also reported an increased numbers in some

major morbidity among those infants who are born with a birth weight of 501-750 grams (Stoll et

al., 2010). The deliveries at preterm commonly known to us as Preterm Births (PTD) are defined

as birth occurring at or before 37 weeks of gestational age and are the major reason of perinatal

diseases and often deaths in the United States (Demissie et al., 2001). Despite a jointly arranged

effort to decrease down the incidence level of Preterm Births, there has been a non-stoppable

increased incidence level of PTB in last two decades. In 2005, specifically the incidences of

preterm births were 12.7% as compared to 9.1% which was in 1981 (Goldberg et al., 2008). The


74

increasing level in the incidence of Preterm Birth and the effect of Preterm deliveries on the

infant survival and health, General Surgeon has made the level of preterm births to a low level of

7.6% and it is a very major goal of the initiative Healthy People 2010. Majority of the women’s

who had a preterm delivery doesn’t have any known risk factor of the specific disease.

The best determinants before pregnancy are history prior to a Preterm delivery. Risk

factors other than these during Pre-Pregnancy may include American-African race, more than

one gestation, short sized cervix, and a BMI (Body Mass Index). When a woman gets pregnant,

the presence of fetal fibronectin and intra uterine infection are also the determinants of preterm

delivery. The success rates of intervention in preventing the rates of preterm births are at limited

rates. In a single study, administration of 17α-hydroxyl progesterone caproate in women with a

history of Preterm Birth results in a low level of repeated Preterm delivery ranging from 55% in

the placebo group up to 36% in women which were receiving α-hydroxly progesterone caproate

(Meis et al., 2003). A recent editorial reported that “our limited ability to stop preterm labor once

it has started is even more disheartening than our frustrated efforts to predict it.”(Lockwood,

2002) As soon as the preterm contraction starts, to decrease down the preterm delivery no

effective intervention exists. Even, if the preterm labor has started no specific anti biotic and

tocolytic therapy has been introduced to reduce the effect of preterm birth (Alex Stagnaro-Green,

2009). At the level of hypothalamus and pituitary the concentration of the circulating thyroid

hormones is regulated by a negative feedback system. Thyroid stimulating hormone (TSH) is

responsible for the production of thyroid hormone by the thyroid gland produced by the anterior

pituitary, and it is itself control by the thyroid-releasing hormone (TRH) in the hypothalamus.

For the production of thyroid hormone Iodide is the main element which is required. Thyroid

peroxidase (TPO) is an enzyme which is expressed by the thyroid stimulating hormone (TSH) in

the thyroid gland, and iodine is oxidized by iodide by this enzyme. Subsequently iodine is

arranged in a glycoprotein known as thyroglobulin (Tg) which in turn is responsible for the

formation of thyroid hormones, formation is in a sense that major biologically inactive form

thyroxine (T4) and lower amounts of the biologically active tri-iodothyronine (T3).

In the circulation T3 and T4 are bind to the circulation due to a protein called Thyroxine-

Binding Protein (TBP). The entrances of free T3 and T4 into the target cells can only be happen

by the mean of the thyroid hormone transporters (MCT8, Oatp1c1and MCT10). Thyroid

hormones can be activated (T4 to T3) and can be inactivated (T3 to T4 or T2) in the target cells,


75

called as de-iodinates (D1, D2 and D3).Transcription can be modulated by the binding of T3

with the nuclear thyroid receptors (TR-alpha and TR-beta) (Miot et al., 2012). For the

development of neonatal and fetal brain thyroid hormone is very essential, it also helps in other

stages of pregnancy such as development of placental and fetal growth. Various physiological

functions occur to ensure the release of optimal amount of thyroid hormones to maintain a

normal pregnancy and fetal development and this all happens as soon as the pregnancy is

initiated. Human chorionic gonadotropin (hCG) stimulates the thyroid glands in the first

trimester due to its structural resemblance to thyroid stimulating hormone (TSH). This may

results in a temporarily higher concentration of free T4 levels and lower concentrations of TSH

in circulation in the first trimester of pregnancy as compared to outside the pregnancy

(Guillaume et al., 1985 and Glinoer et al., 1993). After this period there comes a phase where

free T4 levels began to drop to a lower concentration levels and serum TSH levels rise to a

normal level in circulation. In early gestational age, estrogen levels also increases, which in turn

stimulates the release of thyroxine-binding globulin (TBG) from the liver. During gestation,

there is an increase in the levels of TBG concentrations; there is a peak in the mid gestational age

and maintained afterwards (Glinoer, 1991).

This causes a rise in the concentrations of T3 and T4 levels in the circulation. Renal

blood flow and glomerular filtration rate also increases and it leads to more iodide removal from

plasma and more loss of iodine (Dworkin et al., 1966). This causes an increase in demand of

release of thyroid hormones from the thyroid glands during pregnancy. In the first trimester, all

the fetuses are totally dependent on mother’s thyroid hormone production and their release. After

the 18th to 20th week of gestational age most of the hormones synthesis starts to happen but in the

case of T4 is detected in serum approximately as soon in 10th week to 12th week of gestation.

Maternal thyroid hormone can move across the placental barrier. The conversion of T4 to rT3 is

carried out by de-iodinate which is present in placenta (Burrow et al., 1994). In reproductive age

disorders of thyroid hormones are very common. The estimation of incidence of thyroid hormone

disorder is to be equal to 2% to 3% (Krassas et al., 2010). Hyperthyroidism and Hypothyroidism

are the two most basic thyroid hormone disorder clinically. Hyperthyroidism is to be detected in

about 0.1% to 0.4% of total pregnancy and it has been connected with the risk factors in

pregnancy which may include pre-eclampsia, miscarriages, intrauterine growth restriction,

placental abruption and preterm birth (Casey et al., 2007; Earl et al., 2010 and Reid et al., 2010).


76

The incidence of hypothyroidism is estimated to be 0.3% to 0.5% of total pregnancy (Krassas et

al., 2010). The most common disease which is cause by the hypothyroidism is the Hashimoto’s

disease and it is closely related to the presence of TPO-Ab. Miscarriages, preeclampsia, impaired

psychomotor development and preterm births are some of the major diseases which are closely

associated with hypothyroidism (Haddow et al., 1999 and Reid et al., 2010). The presence of

active thyroid hormones both in fetus and mothers is required for a normal fetal development.

Any process which affects the bio-availability of the free thyroid hormones and intra cellular

interactions of thyroid hormones may cause an abnormal fetal development and its growth

(Kester et al. 2001). In 2006, (Ghafoor et al 2006) assessed the outcomes of pregnancy and

status of TPO antibody in approximately 1500 having normal working thyroid hormone

Pakistani women. For the entire cohort, the prevalence of positive Thyroid Per-Oxidase was

11.2%. Thyroid peroxidase (TPO) positive-antibody women had a remarkable amount of

increase in the levels of Preterm deliveries as compared with the negative-antibody women

(26.8 vs. 8.0%, P < 0.01). In the study, thyroglobulin (TGB) assessment was not performed.

The prevalence of low birth weight in Pakistan is defined as a birth weighing less than

2.5 kg, is 25%. The resemblance in the prevalence of low birth weight and preterm birth was

26.8% and 25%, respectively which encourages the authors to conjure a theory without firm

evidence that Auto-Immune Thyroid Disorder AITD brings about a high incidence of low birth

weight present in Pakistan (Ghafoor et al 2006). There have been remarkable understandings in

the deleterious impacts of thyroid disorders in the last two decades. Specifically, clinical

hypothyroidism has been related with miscarriages in both the first and second trimester (Allan

et al., 2000 and Abalovich et al., 2002). The presence of thyroid antibodies in normal working

thyroid gland women has been linked with increase in numbers of miscarriage in both unselected

women and also in women with a previous history of recurrent (Stagnaro-Green et al., 1990 and

Stagnaro-Green et al., 2004). Although, a low IQ levels of the new born are closely related to the

minor thyroid diseases in mothers (Pop et al., 1999 and Haddow et al., 1999). Finally, in general

population approximately 5% to 10% postpartum thyroidal dysfunction has been reported and

about 25% is reported in those women who were suffering from type I DM (Diabetes Mellitus)

(Alvarez-Marfany et al., 1994 and Stagnaro-Green, 2004). Given the effect of clinical

hypothyroidism and autoimmune thyroid disease (AITD) on multiple features regarding maternal

and fetal health, and the large amounts of Preterm Births across United States, an evaluation of


77

relationship between the thyroid hormones and Preterm Birth is indicated. In 1969 an article

made by Jones and Manin, about 13 different studies have shown analysis between the

relationship of different thyroidal disorders and either preterm Delivery or low birth weight.

Together taken, these different studies have reported fascinated results. Now, to express the

effect of hypothyroidism and Auto-Immune Thyroidal Disease on Preterm birth, the current

documents just show a summary of the present literature. A large-scale prospective study of

approximately 25,765 pregnant women describes that clinical hyperthyroidism does not show to

have any type of effect on the occurrence of Preterm Births (Casey et al., 2006).


78



79

10.0 LITERATURE REVIEW

Before the 24th week of gestation, the loss of pregnancy is known as Miscarriage which

has an effect in every one woman out of five who receive pregnancy due to which it has become

the common complication of pregnancy (NHS, 2010). The delivery of babies between the 24th

week to 37th week of gestation is known as Preterm Birth and it has a incidence ratio of about 6-

10% of all the pregnancies (Creasy, 1991).More than 10% off all the pregnancies are the preterm

babies. The most important cause of neonatal mortality is the preterm birth and it is also the

second most major reason of mortality before the age of 5 (after pneumonia) globally. The main

reason for neonatal short term and long morbidity worldwide is the preterm birth. The incidence

range of preterm birth is approximately 5% to 18% with lowest in Northern European countries

and highest in sub-Saharan African countries (Blencowe et al., 2012).Over the last decades, the

ratio of preterm births have raised in the developed as well as the developing countries

worldwide. The possible debate to this phenomena may include broader use of ultrasound-based

estimation of gestational age, improved registration, infertility treatment, increased maternal

age, multiple pregnancies, maternal health conditions (particularly with increasing age), and

changes in obstetric practice with preterm deliveries induced because of fetal or maternal

indication. The highest rate of preterm births has been recorded in moderate (between 32-33

weeks’ gestation) and also in late preterm (between 34-36 weeks’ gestation) pregnancies

(Shapiro-Mendoza et al., 2012).

The preterm birth rates in Norway decreased down to 7.2% in year from 2000-2002 and

there was another decrease in the rates of preterm to 6.5% in year 2009-2011. There have been

large impacts on preterm infants over the last few decades due to the advance care of neonatal

care. Before labor for maternal and fetal indication about only one third of total preterm birth are

medically induced (Zeitlin et al., 2013). Maternal indications include Eclampsia, Pre-Eclampsia,

HELLP syndrome, uterine rupture, maternal disease and a history of previous caesarean

(Gyamfi-Bannerman et al., 2011). Some of the common fetal indications may include IUGR

(Intra-Uterine Growth Retardation), intrauterine infection, oligohydramnion, a poor flow of

blood to umbilical cord, placenta previa, abnormal fetal heart rate and placental abruption

(Gyamfi-Bannerman et al., 2012). Off all the preterm births about two third are the spontaneous

births, and the causes are largely unknown. It is said that the biological pathways are dependent

upon multiple factors including genetic and environmental which includes the fetal and maternal


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genetic as well as environmental factors such as IUF (intrauterine infection), inflammation and

other immunological processes, placental ischemia or hemorrhage and also uterine distension.

Those mothers who are herself preterm deliver their child too early (Wilcox et al., 2008).

There is a high risk for preterm birth for the fetuses who are male (Zeitlin et al.., 2002)

there is a high risk of preterm birth in those child who had a birth defect during their birth

(Rasmussen et al., 2001).Those mothers who are in the Asian, African and Caribbean region

gives birth to child early as compared to those mothers who are in European region (Patel et al.,

2004) and it suggests a wide range of variation in pregnancy length with social status. There are

several social and other characters which are associated with preterm birth. Those mothers who

have a low social status and are less educated and those with young and advanced age are at risk

of delivering early births (Thompson et al., 2006 and Sukhov et al., 2011) Increased risk of

preterm birth may also include psychological and physical factors for example low BMI before

pregnancy and stressful environment, and also may include depression (Hendler et al., 2005;

Grigoriadis et al., 2013; Witt et al., 2014). From the conception to the weeks around the

expected date of delivery the pregnancy continuum is known as FETAL MATURATION. The

disruption of this process is known as preterm birth, and before the organs of the baby are fully

developed they are exposed to the external environment. Those children who are born in early

stages before its normal duration undergoes neonatal complication due to anatomical and

functional immaturity and above all organs such as brains, digestive organs, lungs and heart are

more vulnerable.

As compare to full term birth the babies with preterm birth are more affected to apnea,

ARDS (Acute Respiratory Distress Syndrome), necrotizing enterocolitis, uncontrolled body

temperature, hypoglycemia, intra-cerebral hemorrhage, difficulty in feeding and bacterial

infections (McIntire et al., 2008; Hibbard et al., 2010). With the decrease in gestational age there

is an increase in the neonatal complication such as morbidity and fetal death (Markestad et al.,

2005). Those children who are born preterm are at risk of developing various numbers of

complications in later life, and with the decrease in gestational age there is an increase in long-

term disabilities (Moster et al., 2008; Saigal et al., 2008) There is a high risk in case of preterm

child for cerebral palsy (CP), asthma, vision and hearing deficit, epilepsy, impaired mental

function, behavioral and psychological disorders, and learning disabilities. (Moster et al., 2008;

Crump et al., 2011; Mackay et al., 2013) There is an extremely increased rate of risk of


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developing early chronic obstructive pulmonary disease in those adults who are born in preterm

births (Vollsaeter et al., 2013). Those preterm babies who are born preterm are different to those

babies which are born at full term in functional manner. Those adults who are born preterm

undergo a lower educational status, lower economic status and have fewer children, and are less

often married. However, it is important to note that despite the increased risk of untoward long-

term outcomes, most individuals born preterm are healthy and have a normal level of function

(Moster et al., 2008).

10.1 SELENIUM ROLE IN PRETERM LABOR

Preterm labor which is less than 37 weeks of gestation is one of the primary cause of

mortality and morbidity of mother and as well as to fetal life which occur in approximately 6%

to 7% of all the total pregnancies in developing world and about 25% in the under-developed

countries (Steer,2005). Number of studies has detected the correlation between the selenium and

the preterm births. Few Cross-sectional researches in Holland, India, Iran and Germany reported

that those women delivering preterm births have tend to have lower levels of selenium in their

serum as compared to those women who are delivering full term births (Sievers et al., 2001;

Gathwala et al., 2003; Iranpour et al., 2009 and Rayman et al., 2011). A study done in Poland

also reported about low levels of selenium concentrations in maternal serum and also low levels

of GPx activity in those women who deliver preterm births as compared to those mothers who

delivered at full term. The low GPx activity and reduced concentrations of selenium in the blood

of preterm infants may subsidize them to a higher risk of developing sepsis and other premature

diseases (Dobrzynski et al., 1998). There was a study conducted in United States which include

13 infants which were born preterm and 15 those infants who were born at full term and

postulated that there was a low concentration of selenium in the blood of those infants who were

born preterm as compared to those who were born at full term, however the concentration of

selenium was not significant in the mothers blood (Mask and Lane, 1993).

As predicted, intake of selenium in the diet was about 2 to 3 times higher (96-134 g) in

those target subjects which were reported in the Polish population. The intake of selenium

supplementation shows some variations in different studies. Preterm is the premature rupture of

the membranes before the onset of labor (PPROM) is a primary factor in the initial stages of

preterm births and affects approximately 10% to 12% of all the total pregnancies. At a

worldwide scale, PPROM is strongly related to the maternal and fetal morbidity and mortality


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(Parry and Strauss, 1998 and ACOG, 2007). In the pathophysiology of PPROM there is a vital

role of antioxidants activity and different generations of ROS that has been correlated with

augmentation of collagen degradation and succeeding damage to fetal membrane veracity. With

selenium there has been an underlined association between the active potential through a

prospective, placebo-controlled, double blind, a small RCT in Iran, which randomized 166 primi-

gravid pregnant women in their first trimester to receive a100_g/d selenium until their labor

(Wood et al., 2001 and Wall et al., 2002). There was a significant increase in the concentration of

the serum selenium concentration in the women with dietary supplementations of selenium and a

reduce levels in the occurrence of PPROM (Tara et al., 2010).

10.2 VITAMIN D AND SPONTANEOUS PRETERM BIRTH

Before the completion of 37 week of gestation spontaneous preterm birth (SPB) occurs.

There are number of reasons according to pathophysiological point for SPB for developing the

intra-uterine infection and inflammation. Bacterial vaginosis is one of the major factors in

developing the infection in SPB and vaginosis is the disturbance between the normal vaginal

floral balance with a rapid increased growth of anaerobic bacteria that is the main factor that

secretes the inflammatory cytokines, phospholipase A2 and prostaglandins (Hillier, 1993;

Lamont, 2000 and Allsworth and Peipert, 2007). A study was conducted by Bodnar et al., (2009)

and his study reported that in early pregnancy there is an inverse proportion between the

maternal vitamin D status and the prevalence of bacterial vaginosis. Hensel et al., (2011) uses an

intensive data set of about 523 women and proved that there is a strong correlation between the

25 (OH) D3 insufficiencies (< 75 nmol/l) with bacterial vaginosis between the pregnant women.

A cross-sectional study of American-African adolescents shows a strong association between the

vitamin D deficiency (25(OH) D3< 50 nmol/l) with bacterial vaginosis (Davis et al., 2010). As

vitamin D shows anti-inflammatory and immune-dulatory effects like the production and

regulating the functions of cytokines and de-granulation of neutrophils products which are vital

and appropriate in the prevention of invasion of micro-organisms that may induce the risk of

developing the risk of SP birth (Nizet et al., 2001; Liu et al., 2006 and Chesney, 2010). Many

cells in the immune systems are responsible for the expression of the VDRs and they are

controlled by the vitamin D (Muller et al., 1991). In response to auto-immunity vitamin D tends

to diminish the activation of acquired immune system, and innate immune system is enhanced by

this key hormone.


83

It reduces the production of inflammatory cytokines such as IL 1, 6 and TNF that are

involve in the SPB by involving in the cell mediated immunity (Helming et al., 2005; Bouillon et

al., 2005; Liu et al., 2006 and Diaz et al., 2009). Active 1, 25(OH) 2D3 is readily synthesized by

the human decidual cells. Hence, number of studies shows that vitamin D is associated with

innate and as well as acquired immunity responses at maternal-fetal edge crossways gestation

(Diaz et al., 2009 and Liu et al., 2009). By helping the myometrium dormancy vitamin D helps

to reduce the risk of SPB. Myometrium contractility is reliable on calcium secretion in the

muscle and this is controlled by vitamin D (Delorme et al., 1983 and Tribe, 2001). Regarding the

experimental data there is a limited observation available that shows a relationship between

vitamin D and spontaneous preterm birth (SPB). In Canada, a prospective cohort study was

conducted including 221 women and this study does not show any relationship of 25(OH) D3

deficit (< 50 nmol/l) or inadequacy (< 75 nmol/l) with preterm births (Shand et al., 2010) while

there was another observational study which reported that maternal 25(OH) D3 levels of(< 28

nmol/l) at 28th week to 32nd week of gestational period were correlated to 0.7 weeks lesser

gestation in white skin women sample (Morley et al., 2006). Another cohort study with 4,225

women with SPB at ≤34 gestational period shows that the incidence of vitamin D insufficiency

(25 (OH) D3< 50 nmol/l) among those women who deliver preterm births were comparable to

those women who deliver at full term (Baker et al., 2010).

Tanzanian pregnant women with vitamin deficiency (25(OH) D3 levels < 80 nmol/l) were

not associated with an increased level of developing preterm births (Mehta et al., 2010). Bodnar

et al. (2008) conducted a cohort study of 82,213 pregnant women with single live births and

found out that seasonal sunlight exposure and vitamin D are significant to preterm births. The

incidence of spontaneous preterm births among those women who conceive in summer season

and fall season were comparatively lower as compared to those women who were conceiving in

winters and spring season where the chances of developing the spontaneous preterm births were

higher (Bodnar et al., 2007). In the near future, there may be a study to reported the strongest

evidence regarding that vitamin D sufficiency may protect from developing preterm births and

this study will be reported by Hollis and his colleagues on their work on approximately 600

white, black, and Hispanic pregnant women and by giving external supplementation of 400 IU,

2000 IU, and 4000 IU/d of vitamin D3 respectively during early gestational age (Hollis and

Wagner, 2009).


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10.3 ROLE OF THYROID HORMONE IN PRETERM DELIVERY: ANATOMY AND

PHYSIOLOGY OF THYROID GLAND

Thyroid gland, a bi-lobed gland shaped as a “Butterfly”, is to be considered as one of the

main and largest glands of the endocrine glands whose weight is about 15-20 grams in a normal

adult that depend upon body weight, iodine, age and gestational status (Sari et al., 2003).

Thyroid gland is situated anteriorly to trachea just below the sternal notch. Right and left both

the lobes are approximately 4 cm in length from top to bottom and are wrapped around trachea,

they are attach superiorly to trachea and inferiorly to the larynx. The blood to the thyroid gland is

mostly supplied by the superior thyroid artery which is the branch of external carotid and also

from the inferior thyroid artery which is a branch of thyroid-cervical trunk of the sub-clavian

artery bilaterally and usually there is a fifth artery known as the thyroid eaima from which the

arch of the aorta enters the midline. From the blood ejected by the heart during cardiac output

thyroid gland blood supply covers up a total of 2% of total blood ejected none the less thyroid

gland accounts only 0.4% of total body weight. Inferiorly the blood drains into the

brachiocephalic vein and the internal jugular vein. The growth of thyroid gland is potentially

larger in amount and the term used for an enlarged gland is known as “GOITER”. The main

constituent of the thyroid glands are the follicular and the para- follicular cells which are also

known as the C-Cells of the thyroid glands. The functional unit for thyroid synthesis and storage

of the thyroid gland are the thyroid follicles (Conn et al., 2005). The follicular cells of the

thyroid glands produces certain follicles which vary in sizes, then thyroid hormones is secreted

from these follicles and then they are stored in the lumen.

The lumens have many homogenous colloids present in them known as the

THYROGLOBULIN (TG) and many other proteins also such as the ALBUMIN. C-cells are

present in between the follicles that secrete calcitonin in return to an increase level of calcium.

Calcitonin act directly on bone by preventing its reabsorption and hence lowering the level of

calcium in blood circulation. The first ever gland to be discovered in the 24th week of gestation is

the thyroid hormone (Trueba et al., 2005). Its formation is from the mesodermal layer of the

epithelial cells over median surface of the pharyngeal floor which is developing and it arises in

the first pharyngeal arch. From the neural crest cells the formation of C-cells occurs and from

here they are migrated towards the ultimo-brachial body which later on fuses with the thyroid

gland. Congenital hypothyroidism occurs when the above mentioned process fails to occur and


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thyroid hormone does not produced. The effects of congenital hypothyroidism is approximately

1 in 4000 newly born babies, thyroid dysgenesis is one of the cause (85%) while the rest 15% is

due to other causes including thyroid hormone synthesis (Trueba et al., 2005). Thyroid

dysgenesis occurs at irregular intervals having only 2% chances of family history (Castanet et

al., 2001).

The site for thyroid hormones synthesis is the thyroid gland. In the thyroid gland the

iodide is actively taken into from the diet by the help of sodium-iodide pump and is readily

converted to iodine. Pre-Thyroglobulin is formed by the coupling effect of tyrosine. In response

to binding with the thyroid stimulating hormone, thyroid hormones are secreted in the

circulation. Thyroid stimulating hormone (TSH) is a trans-membrane receptor that is linked to a

second messenger system G-protein (Hennemann and Visser.1997). As compared to the active

T3 more in-active T4 is secreted into the blood stream. In-active T4, by the help of peripheral

tissues is converted into the active T3 in the circulation. More than 99% of all the total T3 and

T4 in plasma are bounded to hormone binding protein and for cellular uptake only the unbound

form thyroid hormone is available (Friesema. 2001). Thyroid stimulating hormone (TSH) is the

key that regulates the above mentioned process and it is secreted by the pituitary gland. The

pituitary gland is stimulated by Thyroid Releasing Hormone which is situated in the

Hypothalamus. Thyroid Stimulating Hormone (TSH) and the Thyroid Releasing Hormone

(TRH) both are regulated by a negative feedback loop by the circulating T3 and T4 levels in the

circulation (Brent and Springer, 2010). For the synthesis of thyroid hormones several

components are required. The most necessary components for the synthesis of thyroid hormones

include Thyroid Peroxidase TPO, Hydrogen Peroxidase and Iodine.

Out of the total iodine in the body 70-80% is utilized by the thyroid, which is equal to a

roughly estimate of 15-20 mg. For an adequate thyroid hormone production thyroid gland must

extract approximately about 60 µg of iodine OR iodide from the circulation (Md Consult et al.,

2011). Sodium-Iodine symporter (NIS) transports inorganic iodine to the thyroid gland; Where

TPO-HPO system oxidizes it, and then uses it in the thyroglobulin to iodinate tyrosyl. By the

coupling effect of iodinated tyrosyl in the thyroglobulin T3 and T4 are produced. Then these

hormones are hydrolyzed and then from here are secreted into the circulation (Conn et al., 2005).

These above mentioned process are so linked to each other that if any disorder in the steps occurs

at a single point it may cause impairment in the production of thyroid hormones.


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10.4 THYROID FUNCTION DURING PREGNANCY

The net effect of pregnancy is an increase in the demand of thyroid hormones by the

thyroid gland (Glinoer et al., 1997). Pregnancy is a normal physiologic function that induces the

body normal volume and function hence affecting the rate of production of thyroid hormones.

During pregnancy a normal intake of 250μg iodine is recommended to compensate the

remarkable change in the iodine metabolism during pregnancy. This increased level of iodine is

not always achieved, so in order to prevent fetal damage (because of low iodine levels) iodine

supplementation may be required in areas of iodine depletion (Lazarus, 2011). In pregnancy, the

synthesis of thyroid hormones is increased up to 50% and this increase in the synthesis of thyroid

hormone is due to the increased concentration of estrogen induced in the thyroglobulin (Glinoer

et al., 1997; Kennedy et al., 2010).According to the assays used for thyroid hormones

measurements, the concentration of thyroid hormones in the circulation is to be decreased, or

increase or it can be unchanged (Brent and Springer, 2010). T4 assays measuring shows an

increase amount of T4 levels of 1.5 times greater as compared to non-gestational period. There is

a general agreement that there is a frequent rise in the release of free T4 due to the increase level

of circulating human chorionic gonadotropin hormone (hCG) during the first trimester and there

is a downfall in the levels of free T4 during the second and the third trimester, but it stays in their

normal ranges (Glinoer et al., 1997).

Changes in free tri-iodothyronine (fT3) concentration are also seen in which it parallels

the concentration of fT4, again within the normal range. The placenta secretes human chorionic

gonadotropin (hCG) in the first week after conception and the level is the highest at week 10

before it begins to decrease and reach a plateau at week 20 (Brent and Springer, 2010; Lazarus,

2011). Human Chorionic-Graphic hormone (hCG) acts as a agonist for the thyroid stimulating

hormone (TSH) to contribute the occurrence of transient hyperthyroidism due to its elevated

levels in circulations that accounts for almost 2% to 3% of all the total pregnancies (Lazarus,

2011). There is a similarity between the molecules of TSH and hCG, as in the case of both the

TSH and hCG receptors. Consequently, Thyroid gland stimulation is mainly controlled by the

weekly peak of the hCG hormone and the amplitude and the duration of hCG peak greatly

control the production of TSH. The first indicator regarding the thyroidal dysfunction in non-

gestational female is usually the TSH levels in the serum. It is due to the direct relation between

the TSH level and the free T4 levels (fT4) that a very minor change in T4 levels will greatly


87

affect in the levels of freeT4 levels in the serum. In early gestational age, there is a change in the

levels of TSH that it is suppressed to about 50-70% due to the over secretion of hCG in serum

and this level does not provide any information in controlling or treating this thyroidal

dysfunction (Lazarus, 2011). Hence, it is important to rely over T3 and T4 rather in bound or

unbound state as an indicator of thyroidal function in early gestational age, but in case of overt

hyperthyroidism there is a definite rise in the level of TSH. When gestational age reach to about

16 weeks TSH become the only indicator for thyroidal status (Brent and Springer, 2010).

10.5 MATERNAL THYROID FUNCTION AND FETAL BRAIN DEVELOPMENT

Before 12 to 14 weeks of gestational age the fetus is incapable of producing its own

thyroid hormone and hence its total relies on the thyroid hormone supplied by the mother. An

adequate amount of T3 and T4 are mandatory for the fetal brain for its normal growth and

nourishment. Until the third trimester the fetus is unable to produce thyroid hormone adequately

(Connors et al., 2010). It is not until full term that the normal levels of thyroid hormones are

achieved.

10.6 IMPACT OF THYROID AUTOANTIBODIES

Thyroidal antibodies measurement (i.e., thyroid peroxidase antibodies) do not give any

information regarding thyroidal status, their presence does have a remarkable inference over

pregnancy. Thyroid Per-Oxidase (TPO) antibodies are indicator for an increased risk of

miscarriage, preterm delivery and infertility (Lazarus, 2005). Thyroid Per-Oxidase (TPO)

antibodies are present in pregnant women in a ratio of 10% and are interlinked with the thyroidal

reserved dysfunction (Brent and Springer, 2010). In addition the presences of Thyroid Per-

Oxidase (TPO) antibodies have a 50% risk of producing postpartum thyroid dysfunction

(Browne-Martin et al., 1997; Stagnaro-Green, 2004). Most of the thyroidal disorders are

autoimmune disease and occurs due to the presence of auto-antibody. These antibodies attribute

to the pathogenesis of many thyroidal disorders as dis turn the thyroidal function by attacking the

thyroid gland (Larsen et al., 2003).

Pop et al. (1995) states that in a report about a strong relationship between the presence

of anti-Thyroid Oxidase and altered developmental stages in the offspring’s. Another study by

Kvetny and Hedvig in 2006 reported that those mothers having raised levels of anti-Thyroid

Oxidase during pregnancy will have neonates with congenital hypothyroidism. They postulates

that the neonates thyroid function was greatly affected by these auto-bodies in the form of


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cytotoxic, and brain development and other developmental factors goes beyond neonatal period,

Infant and neonatal development can be impaired by this transient hypothyroidism. In the same

case when these antibodies crosses the maternal placental barrier and attacks the fetal thyroid

gland, the transfer of these anti-bodies from the breast of the mother in milk to the neonates is

also a similar fashion. Tornhage and Grankvist (2006) conducted a research and reported that

mothers having positive Thyroid Stimulating Hormone (TSH) receptor antibody TRAb have

chances of developing thyroidal disorders in their infants and this may be worsen and can be

prolonged during the lactation phase through the breast milk. These reports shows that the

disorder caused in the fetal and neonatal development are caused by the transfer of auto

antibodies from the mother to the fetus through uterus or through the breast milk (Kvetny and

Hedvig, 2006; Tornhage and Grankvist, 2006).

10.7 PRETERM PREMATURE RUPTURE OF MEMBRANES (PPROM)

The spontaneous rupture of fetal membranes before the onset of labor contraction is

known as preterm premature rupture of membranes (PPROM).This occurs before the completed

37th week of gestational age (Simhan and Canavan, 2005). Approximately 2% to 3% off all the

pregnancies it PPROM complicates the normal pregnancies and about 25% to 30% off all the

preterm births (Mercer, 2003 and Goldenberg et al., 2008). Within 24 to 48 hours about 50% of

women goes into PPROM and those who go within seven days are about 70% to 90%and some

are also which deliver after few weeks and months (ACOG Committee, 2007 and Caughey et al.,

2008). Latency period is the time duration which starts from PPROM to the onset of labor

contractions. Gestational age is inversely proportional to the latency phase (Test et al., 2011).

When duration of PPROM exceeds above 72 hours is known as prolonged PPROM (Richardson

et al., 1974). In most of the cases of PPROM the mechanism of membrane rupture is still

unknown. In general the risk factors of PPROM are the same as listed in the case of preterm

births that include e.g. tobacco use, previous preterm delivery, vaginal bleeding and the most

important one is the infection (Minkoff et al. 1984; Harger et al., 1990; Ekwo et al., 1993;

Berkowitz et al., 1998; Silverman and Wojtowycz 1998 and Mercer et al. 2000). Against the

ascending infection fetal membranes form a barrier, there are chances of high risk of developing

intra-uterine infection after PPROM (Caughey et al., 2008). In prolonged PPROM chances of

developing infection increases (Test et al., 2011).


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10.8 COMMON BIOLOGICAL PATHWAYS TO PRETERM BIRTH

Spontaneous preterm births are sub-divided into mechanistic pathways such as intra-

uterine inflammation and infection, fetal stress, fetal anomalies, estrogen metabolism pathways

and extracellular matrix degradation.

10.9 INTRAUTERINE INFLAMMATION AND INFECTION PATHWAY

The alteration in maternal normal vaginal flora, which causes the normal flora

lactobacilli to be replaced by anaerobic bacteria (Hillier et al., 1993), is known bacterial

vaginosis (BV) and it is diagnosed by Gram-stain by the Amsel criteria. In asymptomatic

women, positive results of bacterial vaginosis are the markers of intra-uterine inflammation and

infection (Goldenberg et al., 2000) and there is an increase in two folds times of developing

spontaneous preterm birth when the presence of the bacterial vaginosis in the women serum is

detected (Meis et al., 1995 and Balu et al., 2003). The mechanism which is important to leading

preterm birth is the intra-uterine infection (Goldenberg et al., 2000). Numbers of cytokines are

produces by the activation of chorio-decidua due to the bacterial invasion and these markers are

tumor necrosis factor- [TNF-], C-reactive protein [CRP]) and Interleukins [IL] 1, 2, 6, and 8

have been estimated as biomarkers in ensuing preterm birth. Interleukin-6 (IL-6) is a pro-

inflammatory cytokine and is a major facilitator to response according to infection and

inflammation. It can be analyzed by the mother serum samples and the cervical fluid, one of the

most well studied bio-marker of preterm births is the IL-6 (Menon et al., 2011). In a case-control

study matched for parity, study center and race for asymptomatic women, the measurement of

IL-6 at 24th week of gestation were raised in those women who delivered at less than 32nd week

of gestation and at less than 35th week of gestational agein comparison to those women who

delivered at full term (Goepfert et al., 2001).

In a study of meta-analysis, at the 37th week of gestational age of asymptomatic women

shows that the high risk of preterm births were closely related to the raised levels of cervical IL-6

(Wei et al., 2010) . Paternoster et al. (2002) conducted a study and in that study he sustained that

among the pregnant women those who were asymptomatic cervical IL-6 was to be considered

the most powerful indicator for preterm births. Kramer et al.,(2010) found out that around the

24th week of gestation there was no association between the cervical IL-6 and preterm births,

while another case-control study conducted by Goldenberg et al., (2001) perceived that at 24th

week of gestation, there is a weak effect of cervical IL-6 over SPTB, and at less than 32nd week


90

and at less than 35th week of gestational period of asymptomatic women (Wei et al, 2010). In a

evaluated literature C-reactive protein (CRP) is considered as a potential marker in case of

preterm birth and is a protein which is involve in the maternal systemic inflammatory response.

Hvilsom et al., (2002) conducted a case-control study and observed that in early second trimester

(14 to 18 weeks) C-reactive protein (CRP) shows association to SPTB in asymptomatic pregnant

women. Ranges of C-reactive protein concentrations ranges from 5.6 g/mL to 16.4 g/mL, and

there is high risk of developing preterm births in those women who have high concentrations of

C-reactive protein in their serumin comparison to those women who have lower levels of C-

reactive proteins.

Catov et al., (2007) described an increasing menace of early to moderate spontaneous

preterm births at 34th weeks of gestational age (OR = 2.8, 95% CI: 1.1, 7.5) and later

spontaneous preterm births which is at 34th week to 37th week of gestational age (OR = 2.6, 95%

CI: 1.3, 5.5) in those women who are asymptomatic. Riboni and his colleagues observed that in

mid-second trimester there is an increase in the risk of spontaneous preterm births (OR = 3.1,

95% CI: 1.4, 6.8) in those women who have elevated concentrations of serum C-reactive protein

(8.4 g/mL) (Riboni et al., 2012). Another study shows no association to C-reactive protein in

envisaging preterm births in asymptomatic pregnant women; hence not validating the previous

studies results (Kramer et al., 2010). The assessment of C-reactive protein and IL-6 in

calculating succeeding preterm birth, many studies also studied the association of other cytokines

(IL-1, IL-2, IL-8, and TNF) but no evidence suggested the raised cytokine standards were related

to a high risk of spontaneous preterm births (Goldenberg et al., 2001; Paternoster et al., 2002;

Kramer et al., 2010 and Riboni et al., (2012).

10.10 EXTRACELLULAR MATRIX DEGRADATION PATHWAY

So far, the presence of fibrinoectin is the most effective marker for predicting preterm

birth (Goepfert et al., 2000 and Goldberg et al., 2001). Fetal fibronectin is a glycoprotein which

is formed by the fetal membranes and it follows the fetal membranes as well as placenta to the

uterine lining (lockwood et al., 1991 and Goldberg et al., 2005) and plays a vital part after

delivery in separating the placenta from the uterus (Conde-Agudelo et al., 2011). Between 16th

week to 22nd week, there is absence of fetal fibrinoectin at the level of 50 ng/mL, whereas

previous studies had proved that from 22nd week of gestation there is presence of fetal

fibrinoectin at the level of 50 g/mL in the cervix and vagina and also observed that it is a


91

powerful predictor of spontaneous preterm birth (Morrison et al., 1996; Goldberg et al., 1996;

Goldberg et al., 2000; Goldberg et al., 2001 and Roman et al., 2005). In an another study a test

of was conducted in which they show that 29% women were positive result with cervical and

vaginal fibrinoectin if the last fetal fibronectin test done was positive, and other 42% women

were those who had a ensuing positive result if the last two tests done were positive, only 3%

women were those who have fibronectin tests positive if the last fetal fibronectin test performed

was negative (Goldberg et al.1997).

Data from this study provide the evidence that greater numbers of positive fetal

fibronectin result are associated with an increased risk of sPTB at 35th week to 37th week of

gestational age. Morrison and his coworkers also verified the efficacy of cervical fetal

fibronectin for assessing different tools in expecting spontaneous preterm birth in between who

were high risk asymptomatic women (Morrison et al., 1996). Regardless of very small sample

size, the data from a cohort prospective study shows that those women with positive fetal

fibrinoectin results (50 ng/mL) were at higher risk of developing spontaneous preterm births

earlier to 34th week of gestational period in comparison to those women having fetal fibronectin

negative results, spontaneous preterm risks were additionally exaggerated when positive uterine

contraction were coupled with positive fetal fibrinoectin. In the same way, at 24th week of

gestational period positive cervical fetal fibronectin (50ng/mL) was observed to have association

with spontaneous preterm birth at 32nd week to 35th week of gestation as compared to those

women having results negative (Paternoster et al., 2002). Roman et al. (2005) conducted a study

on asymptomatic women and found out high negative prognostic values and specification for

vaginal fetal fibronectin in predicting spontaneous preterm births in between 14 to 21 days of

assessment and also for spontaneous preterm births at the 34th week of gestation. Honest et al.

(2002) conducted a meta-analysis and found out the association between the asymptomatic

women and the presence of fetal fibronectin.

10.11 FETAL STRESS PATHWAY

Corticotrophin-releasing hormone (CRH) is a hormone which is uttered by the human

placenta and also by the fetal membranes, and during the third trimester the highest peak of this

hormone secretion is seen (Yeast and Lu, 2005). Berkowitz et al. (1996) conducted a

retrospective study and examined the plasma Corticotrophin-releasing hormone (CRH) levels at

four gestational age intervals and found out nil differences in complete mean Corticotrophin-


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Releasing hormone (CRH) between the asymptomatic women who delivered at preterm and the

mean of Corticotrophin-releasing hormones (CRH) amongst those women who delivered at full

term. Gestational age related corticotrophin-releasing hormone (CRH) values were not

associated with envisaging succeeding spontaneous preterm births. Analysis done through a

case-control study of 254 asymptomatic women shows that raised levels of maternal plasma

Corticotrophin-Releasing Hormone (CRH) at the 28th week of gestation shows association with

spontaneous preterm births at the 35th week but higher concentrations of Corticotrophin-

Releasing Hormone (CRH) at 24th to 28th week of gestation do not foresee spontaneous preterm

births at the 32nd week of gestational age or less than 37th week of gestation (Moawad et al.,

2002). Goldenberg et al. (2001) performed a study and additionally established similarities in

finding association among Corticotrophin-Releasing Hormone (CRH) levels with successive

preterm birth.

10.12 FETAL ANOMALIES PATHWAY

Several studies have recognized raised concentrations of human-chorionic gonadotropin

(hCG) and fetoprotein association with increased risk for developing spontaneous preterm births

in cervical and vaginal fluids between pregnant women who were asymptomatic (Goldenberg et

al., 2002; Sanchez-Ramos et al., 2003 and Goldberg et al., 2005 ). Early and late preterm births

were associated with raised concentrations of fetoprotein. Moawad et al. (2002) performed a

case-control study which includes 127 asymptomatic women and found out that there is an

increase in the risk of developing spontaneous preterm birth (SBP) at less than 32nd week of

gestation and for spontaneous preterm birth at 35th week. At 28th week of gestation levels of

fetoprotein measured shows association with following spontaneous preterm birth.

10.13 ESTROGEN METABOLISM PATHWAY

The major form of circulating estrogen is the estriol (Goodwin, 1999). Some earlier

studies shows that raised salivary estriol (2.1ng/mL) was a well indicator for late spontaneous

preterm birth in those pregnant women who were asymptomatic and use of estriol from saliva

testing has multiple benefits, the use of saliva as a bio marker has one limitation (McGregor et

al., 1995 and Heine et al., 2000) and that is confusing by issues such as, posture, food

consumption, diurnal variations and patient activity in estriol levels (Ramsey and Andrews, 2003

and Yeast and Lu, 2005). Though medical advances have enhanced the endurance of preterm

infants and also treatment of short and long terms morbidities, a very little success has been


93

achieved in accepting and stopping preterm births. Great efforts have been done in explaining the

usage of biologic fluids from different sources in expecting spontaneous preterm births. Not only

the identification of biochemical indicators which shows association with spontaneous preterm

births allows the researchers to understand much better about the fundamental mechanisms or

pathways which lead to preterm birth but also helps in guiding the most active targeted

intervention approaches aimed for women who were at risk for preterm birth. There are many

studies which evaluate the association between the different biochemical indicators and

spontaneous preterm births between the asymptomatic women. Understanding spontaneous

preterm births is a challenge for the researchers because of its multiple causes and

pathophysiologic different characters. Moreover, maternal and fetal risks factors are also of great

worry. Fetal fibronectin (50ng/mL at 24th-26th week of gestation), so far, is the most effective

indicator in predicting spontaneous preterm births in asymptomatic women.


94



95

11.0 MATERIALS AND METHODS 11.1 SOURCE OF DATA

The present study was designed to investigate the key processes involved in thyroid

hormone and their related auto-antibodies, oxidative stress markers and antioxidant capacity in

the development of acute myocardial infarction. All the selected patients were screened at Gulab

Devi Chest Hospital, Lahore. Informed consent was obtained before being included in this study.

Twenty clinically apparently healthy individuals were included as controls. The experimental

protocol was approved by the Research Ethical Committee of The Institute of molecular biology

and biotechnology, The University of Lahore. Five ml of venous blood sample were taken from

the anti cubital vein of each participant. The sample bottles were centrifuged within one hour of

collection, after which the serum were separated and stored at -70°C until assayed.

11.2 INCLUSION CRITERIA

The AMI disease patients included were based on the following criteria: 1) patients with Age

20-70years were included in study 2) All patients of AMI were included in this study.

11.3 EXCLUSION CRITERIA

The subjects with the history of taking drugs (including alcohol and cigarette), pre-

diagnosis medications (e.g. antiparkinsonian/antipsychotic), were excluded from this study.

None of the controls were on any medication, history of chronic infections, malnutrition

syndrome, depression, psychosis or metabolic dysfunction (Such as diabetes mellitus, liver

diseases, cancer) that could interfere with their oxidative metabolites and thyroid hormone

status.

11.4 CHEMICALS

All chemical reagents of analytical grades were purchased from sigma/Invitrogen

Chemical Co. (St. Louis, Mo, USA).

11.5 FOLLOWING PARAMETERS WILL BE ESTIMATED

I- THYROID BIOMARKERS

II- STRESS BIOMARKERS

III- ANTI-OXIDANT


96

11.5.1 ESTIMATION OF THYROID PROFILE IN SERUM SAMPLES

11.5.2 ESTIMATION OF T3 (Tri-iodothyronine) ELISA

Were estimated by using human ELIZA kit (BioVendor)

11.5.3 ESTIMATION OF T4 (Thyroxine) ELISA


11.5.4 ESTIMATION OF TSH (Thyroid stimulating hormone) ELISA


11.5.5 ESTIMATION OF TPO (Thyroid peroxidase) IgG ELISA


11.5.6 ESTIMATION OF Thyroglobulin IgG ELISA


11.5.7 ESTIMATION OF STRESS BIOMARKERS IN SERUM SAMPLES

11.5.8 ESTIMATION OF THIOBARBITURIC ACID REACTIVE SUBSTANCES

(TBARS)

Lipid peroxidation in blood samples was estimated calorimetrically by measuring

Thiobarbituric acid reactive substances (TBARS) by the method of Ohkawa et al., 1979. To

0.2ml of sample, 0.2ml of 8.1% Sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid and

1.5 ml of 0.8% TBA was added. After centrifugation at 3000 rpm for 10 min the upper organic

layer was taken and its OD was read at 532 nm against an appropriate blank without the sample.

The levels of lipid peroxides were expressed as milimoles of Thiobarbituric acid reactive

substances (TBARS)/g of liver tissue using standard curve.

11.5.9 ESTIMATION OF SUPER OXIDE DISMUTASE (SOD)

Superoxide dismutase (SOD) activity was determined by the method of Kakkar, 1972.

The assay mixture contained 0.1ml of sample, 1.2ml of sodium pyrophosphate buffer (pH 8.3,

0.052 M), 0.1 ml of Phenazine methosulphate (186μm), 0.3 ml of nitro blue tetrazolium (300μ

m), 0.2 ml of NADH (750 μmol). Reaction was started by addition of NADH. After incubation at

300 C for 90 sec, the reaction will be stopped by the addition of 0.1 ml of glacial was allowed to

stand for 10 min; centrifuged and n-butanol layer will be separated. The color intensity of the

chromogen in butanol layer will be measured at 560 nm against n-butanol and concentration of


97

SOD will be expressed as units/g of liver tissue. Absorbance values were compared with a

standard curve generated from known SOD.

11.5.10 ESTIMATION OF GLUTATHIONE (GSH)

Blood GSH was estimated according to the method of Moron et al., 1979.GSH reacts

with Ellman’s reagent (5, 5-dithio bis (Nitrobenzoic acid) or DTNB) to produce a chromophore

TNB with maximal absorbance at 412 nm and oxidized glutathione GSSG. The amount of

glutathione measured represents the sum of reduced and oxidized glutathione in the sample

([GSH]t = [GSH] + 2 x [GSSG]). The rate of absorbance change (ΔA 412nm/min) is made to be

linear for the convenience and consistence of measurement, and is linearly proportional to the

total concentration of GSH. The concentration of an unknown (sample) is determined by

calculating from the linear equation generated from several standards of glutathione (Tietze,

1969).

11.5.11 ESTIMATION OF CATALASE (CAT)

Catalase was assayed according to the method of Aebi, 1974. The estimation was done

spectra photo metrically following the decrease in absorbance at 230 nm. The blood sample

was homogenized in M/150 phosphate buffer (pH 7.0) at 1-40C and centrifuged at 5000 rpm.

The reaction mixture contained 0.01 M phosphate buffer (pH 7.0) 2mM H2O2 and the enzyme

extract. The specific activity of Catalase was expressed in terms of units/gram. Absorbance

values were compared with a standard curve generated from known Catalase.

11.5.12 DETERMINATION OF NITRIC OXIDE

NO is produced by various cell types in picomolar to nanomolar range and has a very

short half-life (t1/2 <5s) in biological fluids; therefore, a direct measurement of its production is

difficult and the analysis of NO2- and NO3

-, the stable products of NO oxidation, is often

performed to estimate NO level in biological fluids and cell culture medium (Moncada et al.,

1991). Nitrite concentration is typically measured by a well-known method such as colorimetric

Griess assay (Moshage et al., 1995).

11.5.12.1 PRINCIPLE

Sulfanilic acid is quantitatively converted to a diazonium salt by reaction with nitrite in

acid solution. Thediazonium salt is then coupled to N-(1-naphthyl) ethylenediamine, forming an

azo dye that can be spectrophotometrically quantitated based on its absorbance at 548 nm.


98

11.5.12.2 CHEMICALS REQUIRED

N-(1-naphthyl) ethylenediamine dihydrochloride (Component A)

Sulfanilic acid (Component B)

Nitrite standard solution (Component C)

11.5.12.3 PROTOCOL

Mixed together equal volumes of N-(1-naphthyl) ethylenediamine (Component A) and

sulfanilic acid (Component B) to form the Griess Reagent. Mixed the following in a

spectrophotometer cuvette:

100 μL of Griess Reagent

300 μL of the nitrite-containing sample

2.6 mL of deionized water

The mixture was incubated for 30 minutes at room temperature. A photometric reference sample

was prepared by mixing 100 μL of Griess Reagent (above) and 2.9 mL of deionized water.

Measured the absorbance of the nitrite-containing sample at548 nm relative to the reference

sample.Absorbance values were compared with a standard curve generated from known NO.

11.5.13 ESTIMATION OF INTERLEUKIN-6 (IL-6) (BY ELISA KIT)

11.5.13.1 Procedure

All the solutions present in the kit were mixed and reagents were prepared according to

the instructions and allowed to reach at room temperature for at least 30 min.

At the end of the assay, store immediately the reagents at 2-8ºC: avoid long exposure to

room temperature.

Unused coated micro well strips should be released securely in the foil pouch containing

desiccant and stored at 2-8ºC.

To avoid potential microbial and/or chemical contamination, unused reagents should

never be transferred into the original vials.

As it is necessary to perform the determination in duplicate in order to improve accuracy

of the test results, prepare two wells for each point of the calibration curve (C0-C5), two

for each sample, and one for blank.


99

Reagent Calibrator Sample/control Blank

Sample 50μL

Calibrator (C0-C5) 50Μl

Diluted conjugate 100Μl 100μL

Incubate 1h at room at room temperature (22-28ºC).

Remove the content from each well; wash the wells 3 times with 300μL of diluted Wash

Solution.

Important note: during each washing step, gently shake the plate for 5 seconds and

remove excess solution by tapping the inverted plate on an absorbent paper towel.

TMB Substrate 100μL 100μL 100μL

Incubate 15 min in the dark at room temperature (22-28ºC).

Stop Solution 100μL 100μL 100μL

Shake the micro plate gently.

Read the absorbance at 450nm against a reference wavelength of 620-630nm or against blank

within 5 min.

11.5.14 DETERMINATION OF TUMOR NECROSIS FACTOR BY ABCAM’s TNF

ALPHA HUMAN ELISA KIT

Sample type: cell culture supernatant, serum, plasma

Assay type: sandwich (quantitative)

Sensitivity: <10pg/ml

Range: 25pg/ml-800pg/ml

Recovery: 107%

Assay time: 3h 40 min

Assay duration: multiple steps standard assay

Species Reactivity: reacts with human


100

Abcam’s TNF alpha human in vitro ELISA (enzyme linked immunosorbent assay) kit is

designed for the quantitative measurement of TNF alpha in supernatants, serum, plasma samples

and buffered solutions. A monoclonal antibody specific TNF alpha has been coated onto the

wells of the microtiter strips provided. Samples including standards of known TNF alpha

concentrations, control specimens or unknown samples were pipette into these wells. During the

first incubation, the standards or samples and a biotinylated monoclonal antibody specific for

TNF alpha were simultaneously incubated. After washing, the enzyme Streptavidin-HRP that

binds the bio-tinylated antibody was added, incubated and washed. A TMB substrate solution

was added which acts on the bound enzyme to induce a colored reaction product. The intensity

of this colored product is directly proportional to the concentration of TNF alpha present in the

samples. This kit recognized both endogenous and recombinant Human TNF alpha. The platform

used was microplate.

11.5.15 DETERMINATION OF GLUTATHION PEROXIDASE (GPx)

Glutathione peroxidase was determined by the method of spectrophotometer with the

help of buffer/ enzyme reagent (Aebi Bergmeyer 1983).

BUFFER/ENZYME REAGENT

9.20 sodium azide solution

NADPH

Glutathione

0.1 GPx solution

48mM sodium phosphate

0.38mM ethylene diaminetetra acetic acid

HCL

ASSAY

Wavelength 340nm

Optical path 1cm

Temperature 25°C

Ph 7.0

Measurement against blank


101

11.5.15.1 PROCEDURE

The GPx concentration can be measured spectra photo metrically. The 9.02 sodium azide

solution had taken 0.1 GPx solution and glutathione into β-NADPH vial. Mixed by inversion and

pH was adjusted upto 7.0 at 25°C with 1M HCL. In the 3.05 reaction mixture, final

concentration at 48mM sodium phosphate, 0.38mM ethylene diaminoteraacetic acid, 0.12mM β-

NADPH, 0.95mM sodium azide.3.2 units of glutathione peroxidase and 0.075 to 0.15 unit of

glutathione, 0.02mM DL-dithiothreitol , hydrogen peroxide. Absorbance was measured at

340nm after 5 minutes.

11.5.16 ESTIMATION OF GLUTATHIONE REDUCTASE (GR)

Glutathione reductase was evaluated by using method of David and Richard (1983). The

amount of NADPH employed is a direct measure of enzyme activity. Reagents used are PBS

(0.12 M), EDTA -15 milli mole, Sodium azide (0.1ml), Oxidized Glutathione-6.3mili mole and

NADPH. The phosphate buffer (1ml),EDTA (0.1ml), sodium azide (0.1ml),oxidized glutathione

(0.1ml) and sample (0.1ml) were added into the cuvette and distilled water (600 micro litre) was

also added to make the volume upto 2ml,then incubated for 3 minutes and NADPH (0.1ml) was

added. The absorbance was read at 340nm in a spectrophotometer at every 15 seconds interval

for 2-3 minutes. For each series of measurement, controls were set up that contained water

instead of oxidized glutathione. The activity of GR represented as micro moles of NADPH

oxidized/minute/g of sample.

11.5.17 DETERMINATION OF AOPPs

AOPP were determined in the plasma using the semi-automated method previously

devised in our laboratory. Briefly, AOPP were measured by spectrophotometry on a microplate

reader (model MR 5000, Dynatech, Paris, France) and were calibrated with chloramine-T

(Sigma, St. Louis, MO) solutions that in the presence of potassium iodide absorb at 340 nm (27).

In test wells, 200 ml of plasma diluted 1/5 in PBS was placed on a 96-well microtiter plate

(Becton Dickinson Lab ware, Lincoln Park, NJ), and 20ml of acetic acid was added. In standard

wells, 10 ml of 1.16 M potassium iodide (Sigma) was added to 200ml of chloramine-T solution

(0-100mmol/liter) followed by 20ml of acetic acid. The absorbance of the reaction mixture is

immediately read at 340 nm on the microplate reader against a blank containing 200ml of PBS,

10 ml of potassium iodide, and 20ml of acetic acid. The chloramine-T absorbance at 340 nm


102

being linear within the range of 0 to 100mmol/liter, AOPP concentrations were expressed as

micromoles per liter of chloramine-T equivalents.

11.5.18 DETERMINATION OF VITAMIN A

Measured 1 ml of the analyzed liquid to the test-tube I (centrifugal) with a tight stopper

and added 1 ml of the KOH solution, plug the tube and shake vigorously for 1 minute- heated the

tube in a water bath (60oC, 20 minutes), then cooled it down in cold water-add 1 ml of xylene,

pluged the tube and shaked vigorously again for 1 minute- centrifuge the tube (1500×g, 10

minutes), collected the whole of the separated extract (upper layer) and transferred it to the test

tube II made of “soft” (sodium) glass-measured the absorbance A1 of the obtained extract at 335

nm against xylene-irradiate the extract in the test tube II to the UV light for 30 minutes, then

measured the absorbance A2-calculate the concentration cx of vitamin A (µM) in the analyzed

liquid, using the formula:

cx = (A1 - A2) · 22.23

Where: 22.23 multiplier received on basis of the absorption coefficient of 1% solution of vitamin

A (as the retinol form) in xylene at 335 nm in a measuring cuvette about thickness = 1 cm.

11.5.19 ESTIMATION OF VITAMIN-E IN SAMPLE

Vitamin E was evaluated in samples by the Emmerie-Engel reaction as reported by

Rosenberg (1992).

11.5.19.1 PRINCIPLE

The Emmerie-Engel reaction is based on the reduction of ferric to ferrous ions by

tocopherols, which, with 2, 2’-dipyridyl, forms a red colour. Tocopherols and carotenes were

first extracted with xylene and read at 460nm to measure carotenes. For correction, ferric

chloride solution is added and absorption ia taken at 520nm.

11.5.19.2 REAGENTS

1. Absolute alcohol

2. Xylene

3. 2, 2-dipyridyl (1.2g/L in n-propanol)

4. Ferric chloride (1.2g/L in ethanol)

5. Standard solution

6. H2SO4 (0.1N)


103

11.5.19.3 PROCEDURE

1.5ml of liver extract, 1.5ml of the standard and 1.5ml of water were pipetted out

separately. To all the tubes, 1.5ml of ethanol and 1.5ml of xylene were added, mixed well and

centrifuged. Xylene (1.0ml) layer was transferred into another stopper tube. To each tube, 1.0ml

of dipyridyl reagent was added and mixed well. The mixture (1.5ml) was pipetted out into a

cuvette and the extinction was read at 460nm. Ferric chloride solution (0.33ml) was added to all

the tubes and mixed well. The red colour developed was read exactly after 15 min at 520nm in a

spectrophotometer. The concentration of tocopherols in the sample was calculated using the

formula,

Sample A520 - A460

Tocopherols (µg) = --------------------------------- × 0.29 × 0.15

Standard A520

11.5.20 ESTIMATION OF VITAMIN-C IN LIVER SAMPLE

Vitamin- C was estimated by using the method of Roe and Keuther (1943).

11.5.20.1 PRINCIPLE

Ascorbate is converted into dehydroascorbate on treatment with activated charcoal,

which reacts with 2, 4-dinitrophenyl hydrazine to form osazones. These osazones produce an

orange colored solution when dissolved in sulphuric acid, whose absorbance was measured by

spectrophotometer (540nm).

11.5.20.2 REAGENTS

1. TCA (4%)

2. 2,4-dinitrophenyl hydrazine reagent (2%) in 9N H2SO4

3. Thiourea (10%)

4. Sulphuric acid (85%)

5. Standard ascorbic acid solution: 100µg / ml in 4% TCA

11.5.21 EXTRACTION OF VITAMIN-C

Ascorbate was extracted from 1g of the liver sample using 4% TCA and the volume was

made up to 10ml with the same. The supernatant obtained after centrifugation at 2000rpm for 10

min was treated with a pinch of activated charcoal, shaken vigorously using a cyclomixer and

kept for 5min. The charcoal particles were removed by centrifugation and aliquots were used for

the estimation.


104

11.5.21.1 PROCEDURE

Standard ascorbate ranging between 0.2-1.0ml and 0.5ml and 1.0ml of the supernatant

were taken. The volume was made up to 2.0ml with 4% TCA. DNPH reagent (0.5ml) was added

to all the tubes, followed by 2 drops of 10% Thiourea solution. The contents were mixed and

incubated at 37°C for 3 hours resulting in the formation of osazones crystals. The crystals were

dissolved in 2.5ml of 85% sulphuric acid, in cold. To the blank alone, DNPH reagent and

Thiourea were added after the addition of sulphuric acid. The tubes were cooled in ice and the

absorbance was read at 540nm in a spectrophotometer. A standard graph was constructed using

an electronic calculator set to the linear regression mode. The concentration of ascorbate in the

samples were calculated and expressed in terms of mg/g of sample.


105

RESULTS


106

12.0 RESULTS

A total of fifty preterm mothers and twenty control subjects were included in the study.

Thyroid biomarkers, circulating stress biomarkers, inflammatory biomarkers and antioxidants

were assessed in array to set their effective roles in causing preterm delivery and also to

underline the consequence of these markers as valuable PTD prognostic markers.

12.1 THYROID BIOMARKERS PROFILE OF PRETERM MOTHER PATIENTS Vs

CONTROL

The levels thyroid hormone and its associated antibodies portray a high contribution of

this hormone in causing preterm delivery. Statistically Significant (p≤0.036, p≤0.008) higher

level of FT4 (16.35±3.11pmol/L) and TSH (4.98±1.03IU/L) were observed in preterm mothers

in comparison with normal delivering mother’s FT4 (11.25±2.25pmol/L) and TSH

(3.26±1.005IU/L). On the other hand the mean FT3 of controls and patients is 5.26μg/dl and

3.58μg/dl respectively. An overall significant decreasing trend was also observed in GSH levels

between PTD patients and controls (p=0.019). As far as levels of auto antibodies related with

thyroid hormone is concerned, a persistent higher pattern was observed in preterm mothers TgAb

(47.16IU/L),TPOAb (9.28IU/L) as compared to control group’s TgAb (31.25IU/L) and

TPOAb(6.35IU/L) and is statistically highly significant (p=0.032, p=0.004)respectively.

12.2 CIRCULATING STRESS BIOMARKERS PROFILE OF PRETERM MOTHERS VS

CONTROL

The results regarding stress biomarkers MDA, GSH, NO, AOPPs IL-6 and TNF-α shown

in table 1 demonstrate the critical role of stress markers in the consequences of causing preterm

labour. The mean MDA of control subjects is 0.951μmol/L and that of oral preterm mothers is

2.06μmol/L. An overall increasing trend was observed in MDA levels between PT mothers and

controls. On the other hand the mean GSH of controls and patients is 8.06μmol/L and

6.28μmol/L respectively. An overall decreasing trend was also observed in GSH levels between

PTD patients and controls. The nitrosative stress biomarker, NO, in control and preterm delivery

patients showed a mean value of 23.26μmol/L and 40.26μmol/L respectively, scrutinizing an

increasing level of NO in patients. Likewise the mean value of AOPPs in controls was

99.26±4.23μmol/L and in preterm mothers 131.26±1.06μmol/L respectively. An increasing drift

was observed in AOPPs levels between diseased patients and controls. The inflammatory stress

markers (IL-6, TNF-α) depicted that mean value of IL-6 in control and diseased is 3.73 pg/ml


107

and 6.29 pg/ml respectively and the mean value of TNF-α in healthy and diseased individual is

21.26 pg/ml and 39.26 pg/ml respectively. Hence, pointing towards the existence of increased

trend among preterm mothers. It is apparent from our results that the stress biomarker MDA,

GSH, NO, AOPPs IL-6 and TNF-α had an increasing trend in patients vs. controls while GSH

showed a decreasing trend in patients when compared to healthy subjects. All stress biomarkers

exhibited statistically significant values (p<0.05).

12.3 ANTIOXIDANT BIOMARKERS PROFILE OF PRETERM MOTHER PATIENTS

Vs CONTROL

The enzymatic antioxidant biomarkers SOD, Catalase, GPx and GRx and non-enzymatic

antioxidant biomarkers vitamin E, vitamin A, vitamin C, vitamin B6, vitamin B9 AND vitamin

B12 results fluctuations clearly illustrated their role at the time of preterm labour. As compared

to normal, all foresaid variable showed significant (p=0.025, 0.001, and 0.015 respectively) low

levels of SOD (0.14±0.0011μmol/L vs. 0.625±.0011μmol/L), CAT (3.19±0.15U/L vs.

4.26±0.016U/L), GPx (7.18±1.09 μmol/L vs. 6.26±1.22 μmol/L), GR (2.29±1.28 μmol/L vs.

4.26±0.0015 μmol/L) in preterm mothers group were recorded. While the mean value for GPx in

controls was found to be 6.26±1.22 μmol/L whereas in diseased it was observed to be 7.18±1.09

μmol/L. An overall increasing trend was seen between both PTD patients and healthy subjects.

The mean value of antioxidant vitamin A in controls was 6.01±1.76nmol/L whereas in preterm

delivery females the mean value was 4.29±1.09nmol/L, hence a decreasing trend was seen in

vitamin A levels between diseased and controls. The vitamin C mean value in controls was 0.65

nmol/L while in diseased individuals it was 0.462 nmol/L. The mean value for GPx in controls

was found to be 1.59 U/g Hb whereas in diseased it was observed to be 6.51 U/g Hb. An overall

decreasing trend in vitamin E was seen between both preterm mothers (0.36±0.0019nmol/l) and

normally delivering subjects (0.498±0.001nmol/L). The low levels of vitamin B6

(61.28±4.26nmol/L), vitamin B9 (2.18±0.168nmol/L) and vitamin B12 (206.15±4.26pmol/L)

were observed in preterm delivering mothers as compared to vitamin B6 (77.26±6.25nmol/L),

vitamin B9 (3.26±0.0018nmol/L) and vitamin B12 (226.25±8.26pmol/L) respectively. All

antioxidant showed statistical significance with the condition (p≤0.05).


108

Table 03: THYROID BIOMARKERS PROFILE OF PRETERM DELIVERY FEMALE

PATIENTS Vs CONTROL

PARAMETERS CONTROL (20) PRETERM MOTHERS(40) P VALUE

T4 (pmol/L) 11.25±2.25 16.35±3.11 0.036

T3 (μg/dl) 5.26±1.25 3.58±0.94 0.019

TSH (IU/L) 3.26±1.005 4.98±1.03 0.008

TgAb (IU/L) 31.25±3.26 47.16±5.26 0.032

TPOAb (IU/L) 6.35±1.05 9.28±1.28 0.004

Table 04: STRESS BIOMARKERS PROFILE OF PRETERM DELIVERY FEMALE

PATIENTS Vs CONTROL

PARAMETERS CONTROL (20) PRETERM MOTHERS(40) P VALUE

MDA (μmol/L) 0.951±0.001 2.06±0.056 0.016

NO (μmol/L) 23.26±4.26 40.26±3.68 0.000

GSH (μmol/L) 8.06±1.26 6.28±1.88 0.012

AOPPs(µmol/L) 99.26±4.23 131.26±1.06 0.016

IL-6 (pg/ml) 3.73±2.76 6.29±1.44 0.041

TNF-α (pg/ml) 21.26±7.26 39.26±3.29 0.011


109

Table 05: ANTIOXIDANT BIOMARKERS PROFILE OF PRETERM MOTHERS Vs

CONTROL

PARAMETERS CONTROL(20) PRETERM MOTHERS(40) P VALUE

SOD (U/ml) 0.625±.0011 0.14±0.0011 0.025

CAT (U/L) 4.26±0.016 3.19±0.15 0.001

GPx (μmol/L) 6.26±1.22 7.18±1.09 0.032

GRx (μmol/L) 4.26±0.0015 2.29±1.28 0.015

Vit A(nmol/L) 6.01±1.76 4.29±1.09 0.033

Vit C(nmol/L) 0.65±0.0016 0.462±0.0016 0.016

Vit E(nmol/L) 0.498±0.001 0.36±0.0019 0.035

Vitamin-B6 (nmol/L) 77.26±6.25 61.28±4.26 0.011

Vitamin-B9 (nmol/L) 3.26±0.0018 2.18±0.168 0.000

Vitamin-B12 (pmol/L) 226.25±8.26 206.15±4.26 0.045


110

DISCUSSION


111

13.0 DISCUSSION

Because of the high mortality and high morbidity rate preterm labor is one of the most

common and important obstetrical and perinatology disorder which is responsible for the

inhibition of the body organs and system. Hence, in term of those whose early detection has been

diagnosed must be given preventive steps. The prevalence of preterm labor is relatively higher in

developing countries as compared to developed countries. Rate of preterm birth in year 2005 was

calculated to be about 9.6%. In which approximately the incidence of preterm labor in Africa and

Asia continent was 85%. The reason or the causes which are responsible for the preterm labor

are not yet fully known. Still, there are many mechanisms which are responsible for preterm

labor: maternal and fetal H-P-A (Hypothalamic- Pituitary- Adrenal) axis activation,

inflammation and other infections, excess enlarging of uterus and decidua bleeding, which

produces certain clinical signs and also shows a synchronize movements between the

myometrium contractions, rupture of the fetal membranes and opening of the cervix also known

as the cervical ripening. Reactive species (ROS) may also cause the activation of myometrium

muscle contraction. Hence, a study was performed to assess the oxidative markers in the body.

One function is also the evaluation of relation between the thyroid hormones and the anti-bodies

which are responsible for its aggravation. Inflammatory markers have certain characteristics

which are responsible for the pathophysiology of term and preterm labor. Preterm labor is said to

be a state in the female pregnancy that intends to inhibit the uterine physiology that is

responsible for the organ maturity of the fetus. About 50% off all the preterm births, Intra-

amniotic infection (IAI) and inflammation are associated with preterm labor. Cytokines that are

associated with the labors are characterized by the immune penetrations and also by expressing

tumor necrosis factor alpha (TNF-α), IL-8, IL-, interleukin (IL)-1β and monocyte chemo-

attractant protein (MCP-1), amniotic fluid, cervix, placenta and in the fetal membranes.

Expressing these pro-inflammatory mediators results in

(i) There is an increase in the prostaglandins levels which are responsible for increase

uterine contractions;

(ii) Chorio-amnion degradation in the extracellular matrix (ECM); (iii) Cervix ripening

An important pro-inflammatory cytokine and a basic organizer that respond to

inflammation and infection is the Interlueiken-6. In mother serum samples interlueiken-6 can be


112

assessed, IL-6 is one of the most important preterm bio-marker that is studied until now (Menon

et al., 2011). In fetal membranes, placenta and uterine epithelial cells activation of TLRs along

with their ligands tends to induce the release of pro-inflammatory cytokines. These pro-

inflammatory cytokines may be responsible for the link between the PPROM, uterine contraction

and cervical ripening (Schaefer et al., 2004; Noguchi et al., 2010). In a study, there is an

increased in the levels of IL-6 in mothers with preterm labor in comparison to control healthy

group which are simultaneous with another study of Paternoster et al. (2002) in which he shows

that in asymptomatic pregnant women levels of cervical IL-6 was well thought out to be the

most strong parameter regarding preterm births. Wei et al., (2010) conducted a study and

sustained that at the 37th week of pregnancy of women with no symptoms have higher risk of

preterm births association with elevated values of cervical IL-6.

Macrophages, VSMCs, foam cells, fibroblasts, inflammatory cells and monocytes are

various cells that are responsible for the production of the TNF-α and it increases the synthesis of

protein, macrophages phagocytosis and growth at cellular level and discernment by enhancing

response to inflammation. In case of preterm birth there is an up-regulation of TNF-α. In our

conducted study, elevation of TNF- α was observed in women with those who were delivering

preterm births in comparison to those mothers delivering normal births and this was similar to a

study done by Lyon et al. (2010) in which he observed a relationship between preterm birth and

the increased values of IL-6, IL- Beta and TNF- α in the circulation. Those women having both

TNF- α polymorphism and also bacterial vaginosis in them tend to have higher risk of

developing preterm births as compared to those women having only TNF- α or bacterial

vaginosis alone in them (Macones et al., 2004).

Reactive Oxygen Specie aids as important molecules that regulate the normal procedures

but it also have involvement in the pathological procedures that happens in the female

reproductive system by affecting numerous physiological procedures ranging from maturation of

oocyte to fertilization, development of embryo and even pregnancy. It has also a role in the

ignition of labor. Reactive RNS (Reactive Nitrogen Specie) has affect over vasodilation by

showing effects on nitric oxide signaling and also it causes DNA damage. Vasodilation ability in

the body will be damaged and get depressed by the over presence of the free radicals in the

blood; hence, flow of blood will get low due to the activation of vasoconstriction, which

ultimately leads to IUGR (Intra-Uterine Growth Retardation), pre-eclampsia and preterm labor


113

(Stein et al., 2008). Raised levels of NO were observed in our study in preterm births apart from

those mothers who were delivering at full term due to the presence of nitric oxide which get

stimulated by the inflammatory cytokines which may include TNF-α, IL-1 and lipo-

polysaccharides. During the follicular development granulose cells, theca cells and the oocytes

expressed the endothelial NO synthase. Inducible NO in cases of pathological state is sole

responsible for the production of Nitric Oxide NO. In response to immunologic cases inducible

NO synthase is expressed in different organs.

During the oxidative stress proteins are at high risk of being affected by the free radicals.

AOPPs (Advanced Oxidation Protein Products) are those biomarkers that contain di-tyrosine that

are produced due to the stressed state and it initiates inflammatory process (Piwowar et al.,

2010). In our study, raised levels of AOPPs were observed in the serum of women with preterm

labor in comparison to control group in which there is an increase in the production of hypo-

chlorous acid from an enzyme known as the myeloperoxidase under the circumstances of stress

caused by oxidation and carbonyl that react with albumin, in human blood it is the most plentiful

proteins present and which oxidizes it to AOPPs that triggers NADPH oxidase by the RAGE

receptor. Ultimately, there is an increase in inflammatory process and oxidative stress. There are

many studies that show that significant relationship between the decrease levels of antioxidants

and preterm labor. Antioxidants may be endogenous or they may be exogenous that may be

helpful in the prevention of formation of dangerous oxidants and it also in activates the already

present oxidants that tends to blocks the proliferation reactions that are due to these toxic agents.

Enzymatic antioxidants may include catalase; superoxide dismutase (SOD), glutathione

reductase (GR) and glutathione peroxide (GPx) whereas non-enzymatic antioxidants are

Vitamins A, C and E and reduced glutathione (GSH). The human myometrium soft muscles are

gifted with the enzymatic hunting system that tends to lower the possible destruction caused by

ROS (Telfher et al. 1997, Matharoo-Ball & Khan 2003). A large number of studies suggest that a

low level of enzymatic antioxidants is the basic reason for the onset of preterm labors.

The major anti-oxidant defense system against the reactive O2- is the superoxide

dismutase (SOD), and in mammals there are three forms of SOD present: cytoplasmic Cu/Zn

SOD (SOD1), mitochondrial Mn/SOD (SOD2) and extracellular Cu/Zn SOD (SOD3). All of

which catalytic metals (Cu or Mn) are mandatory for their stimulation. Relative studies show that

in each sub-cellular region conversion of O2- and H2O2 is catalyzed by the SOD, which may


114

contribute in the cell signaling process. Furthermore, inhibition of oxidative nitric oxide is

carried out by the SOD; hence formation of per-oxy nitrite is prevented (Fukai and Ushio-Fukai,

2011). In our study levels of SOD were decreased to a low level in preterm births women as

compared to those women who delivered at full term. A protein that uses Fe as a co-factor and is

encoded on chromosome 11 by a gene is the catalase. Low catalase levels may happen due to the

mutation of this gene and relatively high oxidation state known as acatalasemia. Furthermore to

SOD activity, catalase hydrolyses free radicals and peroxidase into oxygen and water. By

breaking the oxidative chain reaction these enzymes helps to inhibit the formation of free

radicals and convert them into more stable products known as the anti-oxidants. The levels of

catalase were reduced in our study as compared to the healthy women groups and it was

simultaneous to another study conducted by Negara et al. 2010 in which he reported a decrease

in the values of catalase in women delivering preterm births.

H2O2 is catalyzed by the catalase and glutathione by sharing same nature of catalyzing.

Regardless GPx has more affinity of against H2O2 when compared with catalase. Difference is

between there mode of kinetics. In accordance to concentration of H2O2 catalase catalyzes it, and

in case of GPx it becomes saturated when concentration of H2O2 falls below 10-5 mol/L. GPx is

more potent in catalyzing H2O2 in comparison to catalase when the concentration falls to a lower

level or normal value Kobe et al., 2012. As compared to healthy groups levels of GPx falls in

diseased groups was stated in the present studies. Reduction in the levels of SOD and CAT

shows that the increased levels of GPx are due to this reduction and this enhancement in the GPx

is to respond against stress state and removal of H2O2. Production of GPx may be also due to the

increased formation of lipo-peroxidase. Conversion reaction to reduced form of GSH from

oxidized glutathione is catalyzed by the glutathione reductase. Present studies shows a fall in the

levels of GR in pregnant women delivering preterm births when compared to normal healthy

women

Pituitary gland secretes a hormone through thyroid stimulating hormone which is the

main regulator of the above mentioned literature. In the hypothalamus, thyroid releasing

hormone is present which is responsible for the stimulation of thyroid hormone. In the

circulation, regulating T3 and T4 are present which regulates the production of thyroid hormone

by the TRH and TSH from a mechanism known as the negative feedback mechanism (Brent and

Springer, 2010). Several components are in demand for the synthesis of thyroid hormone. Iodine,


115

Hydrogen Per-oxidase and Thyroid Per-oxidase are the most important components which are

required for the synthesis of thyroid hormones. In some studies preterm birth is associated with

clinical hypothyroidism and increased levels of TRH (Allan et al., 2000; Casey et al., 2005;

korewaar et al., 2013). In preterm mothers at the time of delivery there is an increase in the

levels of thyroid hormone in comparison to healthy group delivering and this study shows

similarity with a study which was conducted by Johns et al. (2017) which reported that changes

in the levels of thyroid hormones increases the risk of preterm births. There are many

physiological mechanisms which are regulated by tri-iodothyronine (T3) that include

differentiation, proliferation, metabolism, inflammation and cellular growth through binding with

thyroid hormone response elements (TREs) by regulating the target genes. In our study there is a

decrease in the levels of T3 when it was compared with healthy women. This fall in the level of

thyroid hormone triggers the hypothalamic-pituitary-thyroid axis; there is an increase in the

levels of human chorionic gonadotropin (hCG), this hormone share a structural similarity

between the TSH, and it also stimulates and increase in the levels of T3 and T4.

Apart from its various functions Vitamin A which is actually stored in the liver is considered

as a good antioxidant, in immune functions it has a vital role. A form of vitamin A known as the

retinoic acid is formed by the dendritic cells and the macrophages of lymph nodes and mucosa.

When state of inflammation is achieved, conversion of CD4 T lymphocytes into T-helper cells

leads to formation of inflammatory cytokines (Green and Mellanby, 1928 and Raverdeau and

Mills, 2014). In our study levels of vitamin A were decreased in case groups and these results

show correlation with a study conducted by Azaïs-Braesco and Pascal, 2000; Mactier and

Weaver, 2005. Low levels of vitamin A in this group shows that they have more affinity towards

catching infection. Furthermore low levels of vitamin A induce pro-inflammatory cytokines to

secrete that may lead to inflammation and triggering oxidative stress through NADPH.

The damage caused by the hydroxyl radicals are eliminated actively by the non-toxic

vitamin E. Initially this antioxidant is found in the lipoproteins and cellular membranes where its

most important function is to delay the lipid peroxidation process. Inflammation has been

associated with decrease levels of vitamin E. To minimize the wound healing time is one of the

functions of vitamin E and maintaining the stability and normal physiology of membranes.

Vitamin E normal levels in the body prevent it from the onset of disease. In our study we

observed low levels of vitamin E which is responsible for more growth of hydroxyl radicals,


116

resulting in more lipid peroxidation leading in the disturbance of normal cell integrity and

eventually leading to increase chances of preterm births. For the synthesis and degradation of

collagen vitamin C has an important role and is also essential for maintaining chorio-amniotic

fluids. Decrease presence of vitamin C in pregnancy leads to an increase risk of premature

rupture of chorio-amniotic membranes. In a study conducted earlier we showed that PROM can

be a standard protocol for the estimation of vitamin C throughout the pregnancy (Casanueva et

al., 1998). There was a fall in the levels of vitamin C in our study in those women who were

delivering preterm births and this study was in accordance to a study which was conducted by

Casanueva et al., 2005. In our study vitamin C was derived in accordance to the collagen

metabolism and that of collagen in maintaining the integrity of membranes throughout the

pregnancy (Bryant-Greenwood, 1998; Tejero et al., 2003). For collagen post-transcriptional

modification vitamin C was considered as an important co-factor and a down regulator for the

gene 72-kDa type IV collagenase, matrix metalloproteinase (MMP-2), in amnion cells; it also

controls the catabolism of collagen (Pfeffer et al., 1998).

An observation was made in the increase levels of homo-cysteine supports the role of FA

in placenta on which induces a cytotoxic as well as oxidative stress on placental circulation and

function. Apoptosis may increase when homo-cysteine are introduced with trophoblastic cells

(Clarke, 2000; Steegers-Theunissen and steegers, 2003; Parazzini et al., 2011). Recent studies

shows an association between the folate metabolizing genes and incidence of spontaneous

preterm birth (johnson et al., 2005; Zafarghandi et al., 2013) which is similar to our finding.

Present study shows that levels of vitamin B6 and B12 fall to a low level in those females who

were delivering preterm births when compared to normal healthy delivering births and this study

was similar to another study conducted by Van Sande et al., 2013.

CHAPTER FIVE

FIGURE 0several causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes the neutrophilOxygen Specie) +and results in the lipid peroxidation due to decrease in the antithe maternal and fetal HPA axis to activate aimbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Cacontraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.

CHAPTER FIVE

FIGURE 02: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes the neutrophils to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the antithe maternal and fetal HPA axis to activate aimbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Cacontraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.

: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes

s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the antithe maternal and fetal HPA axis to activate aimbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Cacontraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.


s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the antithe maternal and fetal HPA axis to activate and secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Cacontraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.

117


s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the anti

nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Ca++ ions influx activating actin myosin cascade causes the muscles contraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.


s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the anti

nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in PROM and preterm labor. Oxytocin activates

ions influx activating actin myosin cascade causes the muscles contraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.


s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the anti-oxidants. This mechanism causes

nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of T

PROM and preterm labor. Oxytocin activates ions influx activating actin myosin cascade causes the muscles

contraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.



s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive oxidants. This mechanism causes

nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of T


contraction resulting in preterm labor. Other factors like placental abruption and pregnancies anomalies causes decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.


: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTH There are

several causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes


nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of T3 through


anomalies causes


There are

several causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes


nd secretes cortisol and oxytocin. Decreased cortisol levels causes the through


anomalies causes

CHAPTER SIX CONCLUSION MISBAH, 2017

118

CONCLUSION

CHAPTER SIX CONCLUSION MISBAH, 2017

119

14.0 CONCLUSION

Current work concluded the facts and figures that may contribute as the reason behind

preterm delivery involves both the endocrinal and immunological responses that may lead to the

activation of inflammatory markers which in term are responsible for increased oxidative stress,

may be involved in the over activity of thyroid gland. Hormones such as oxytocin and estrogen

were increased while progesterone remained decreased which may initiates preterm labor. The

levels thyroid hormone and its associated antibodies portray a high contribution of this hormone

in causing preterm delivery. Statistically Significant higher level of free T4 and TSH were

observed in preterm mothers. On the other hand the level of free T3 falls when compared to

normal healthy pregnant women.

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IN PARTIAL FULFILLMENTS FOR THE DEGREE OF ...prr.hec.gov.pk/jspui/bitstream/123456789/14886/1/Misbah...iii EXAMINATION COMMITTEE The thesis viva MISBAH SULTANA (REG.# DBC -0212300