Sperm Function Tests

Beyond the Semen Analysis

Edmund Sabanegh, Jr., M.D.

Chairman, Department of Urology

Glickman Urological and Kidney Institute

Cleveland Clinic

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Routine Semen Analysis:

Gap in Knowledge

Parameters

1980

1987

1992

1999

Volume (mL) ND ≥ 2 ≥ 2 ≥ 2

Sperm count (106/mL) 20-200 ≥ 20 ≥ 20 ≥ 20

Total sperm count (106) ND ≥ 40 ≥ 40 ≥ 40

Motility (%) ≥ 60 ≥ 50 ≥ 50 ≥ 50

Progressive motility ≥ 2 ≥ 25% ≥ 25% (a) ≥ 25% (a)

Vitality (%) ND ≥ 50 ≥ 75 ≥ 75

Morphology (%) 80.5 ≥ 50 ≥ 30 >14*

Leukocyte count (106/mL) < 4.7 < 1.0 < 1.0 < 1.0

*Strict (Tygerberg) criterion

Evolution of WHO semen standards

Parameters

1980

1987

1992

1999

2010

Volume (mL) ND ≥ 2 ≥ 2 ≥ 2 ≥ 1.5

Sperm count (106/mL) 20-200 ≥ 20 ≥ 20 ≥ 20 ≥ 15

Total sperm count (106) ND ≥ 40 ≥ 40 ≥ 40 ≥ 39

Motility (%) ≥ 60 ≥ 50 ≥ 50 ≥ 50 ≥ 40

Progressive motility ≥ 2 ≥ 25% ≥ 25% (a) ≥ 25% (a) ≥ 32% (a+b)

Vitality (%) ND ≥ 50 ≥ 75 ≥ 75 ≥ 58

Morphology (%) 80.5 ≥ 50 ≥ 30 >14* ≥ 4*

Leukocyte count (106/mL) < 4.7 < 1.0 < 1.0 < 1.0 < 1.0

*Strict (Tygerberg) criterion

Evolution of WHO semen standards

Lower Higher fertile infertile

Overlap range

(cut off values) % of Overlap

Count (million) 27 99 62 37

Motility (%) 46 75 47 29

Morphology (%)

WHO 18 51 81 81

Strict criteria 4 15 61 84

Overlap Ranges of Sperm Characteristics

Nallella et al, Fertil Steril 85:629-34, 2006

Lower Higher fertile infertile

Overlap range

(cut off values) % of Overlap

Count (million) 27 99 62 37

Motility (%) 46 75 47 29

Morphology (%)

WHO 18 51 81 81

Strict criteria 4 15 61 84

Overlap Ranges of Sperm Characteristics

Nallella et al, Fertil Steril 85:629-34, 2006

Not helpful

Lower Higher fertile infertile

Overlap range

(cut off values) % of Overlap

Count (million) 27 99 62 37

Motility (%) 46 75 47 29

Morphology (%)

WHO 18 51 81 81

Strict criteria 4 15 61 84

Overlap Ranges of Sperm Characteristics

Nallella et al, Fertil Steril 85:629-34, 2006

More helpful

Lower Higher fertile infertile

Overlap range

(cut off values) % of Overlap

Count (million) 27 99 62 37

Motility (%) 46 75 47 29

Morphology (%)

WHO 18 51 81 81

Strict criteria 4 15 61 84

Overlap Ranges of Sperm Characteristics

Nallella et al, Fertil Steril 85:629-34, 2006

Most helpful

WHO 4th Edition, 1999

Variability in Sperm Concentration

• Time to conception correlates with total sperm numbers

and sperm concentration

– Zinaman (2000)- 200 couples off contraception for 12 months

– Longitudinal Investigation of Fertility and Environment (LIFE)

Study (2014)- 473 couples off contraception for 12 months

-Select morphology (head width) and sperm count were associated with

shorter time to pregnancy

-Overall strict morphology and kinetic parameters were not associated

with improved time to conception

Count and Conception

• Lacks information on sub cellular / molecular

changes in spermatozoa

• Need for tests to evaluate/stratify interventions

Routine Semen Analysis:

Gap in Knowledge

• Poor prediction of fertility since 50% of infertile men have

normal semen parameters

• High inter and intra observer variability

• Morphology in particular is poorly correlated with

outcomes

The dilemma

Men with abnormal semen analyses can be

fertile and infertile men can have normal

parameters

Limitations of Current Semen Testing

1. Specific diagnosis of sperm dysfunction

2. Prediction of fertilization/pregnancy rate

3. Suggest targeted therapy to reverse infertility

Ideal Sperm Function Test

– Sperm chromatin tightly packed due to cross linkages

– Sperm DNA fragmentation (DFI)- first described in 1993

– Correlates with poor semen parameters, leukocytospermia, elevated

oxidative stress

– Associated with unexplained infertility, recurrent miscarriage

– Sperm DNA integrity and morphology- poor correlation (Avendano, 2010)

– DNA sperm integrity correlates with reproductive success

– Elevated DFI- lower spontaneous pregnancy and assisted reproduction

success rates

Sperm DNA

DNA Damage

DNA repair Normal Normal

embryogenesis

Abnormal

Abnormal development

Failed implantation

Miscarriage

Abnormalities in offspring?

Exposure to scrotal heat stress

Age replication errors

Exposure to radiofrequency radiation Exposure to xenobiotics

(Lifestyle, meds, occupational)

Defects of spermatogenesis associated with

poor or aberrant protamination

IVF ICSI

Assisted

conception

Idiopathic

Causes of Impaired DNA Integrity

Sperm karyotyping

Sperm FISH analysis

Sperm DNA fragmentation tests

Sperm DNA Assessment

Methodology

Fluorescent labeling of

specific chromosome

(5+probes)

automated image analysis

Aneuploidy detection

noted in 7% of normal men

but much higher rates with

oligospermic men and those

with reciprocal or

Robertsonian translocations

Sperm FISH Analysis

Sperm FISH Analysis

Ramasamy, Fert Steril, Dec 2014

Sperm FISH Analysis

Ramasamy, Fert Steril, Dec 2014

Widely accepted indications

-Men with normal semen analysis-

unexplained infertility

-Partners with recurrent pregnancy

loss

-Repeated IVF failure

-Screening tool to counsel couples

regarding potential outcomes from

reproductive options

The use of the acridine orange test and the TUNEL assay to assess the integrity of

freeze-dried bovine spermatozoa DNA

C.F. Martins1,2, M.N. Dode1, S.N. Báo2 and R. Rumpf1; Gen Mol Research, 2007

Sperm with fragmented DNA Sperm with intact DNA

COMET Assay TUNEL Assay

Sperm DNA Testing

Natural Conception

7 times more likely to achieve pregnancy if DFI <30% (n = 362, P = 0.0001)

Evenson, 2006; Greco, 2005

Sperm DNA Fragmentation and Pregnancy

Natural Conception

7 times more likely to achieve pregnancy if DFI <30% (n = 362, P = 0.0001)

Intrauterine insemination

7.3 times more likely to achieve pregnancy if DFI was <30% (n = 518, P = 0.0001)

Evenson, 2006; Greco, 2005

Sperm DNA Fragmentation and Pregnancy

Natural Conception

7 times more likely to achieve pregnancy if DFI <30% (n = 362, P = 0.0001)

Intrauterine insemination

7.3 times more likely to achieve pregnancy if DFI was <30% (n = 518, P = 0.0001)

IVF/ICSI

Infertile couples - 2 times more likely to become pregnant if DFI was <30% (n = 381, P = 0.03)

Controversial since paternal genome not expressed until four-cell embryo

Varicocele repair prior to ICSI may improve outcomes (Esteves, 2010)

Evenson, 2006; Greco, 2005

Sperm DNA Fragmentation and Pregnancy

Bottom line- DFI>30% and reproductive failure-consider

ICSI and possible testicular sperm extraction (lower DNA

fragmentation in testis vs ejaculated sperm)

Osman,2015

DNA Fragmentation and IVF/ICSI Success

Meta-analysis of IVF and ICSI outcomes

IVF- High DFI- lower live birth rate

ICSI- High DFI- no difference in live birth rates

Higher spontaneous abortion rates in IVF and

ICSI with high DFI

– High DFI associated with oligospermia

and advanced paternal age

– No effect on intrauterine growth,

postnatal physicial development or

mental status of offspring of high DFI

sperm

– “Elevated sperm dna fragmentation

occurs regularly and the machinery of a

healthy oocyte might come to the

rescue”

Outcome of Pregnancies with

Elevated Sperm DNA Fragmentation

Palermo, Fert Steril, 2014

Unexplained infertility

Recurrent pregnancy loss

Prediction of ART outcome

Assessment of genetic integrity

– Post-chemotherapy

– Advanced paternal age

Indications for Sperm DNA Testing

Lack of standardization of assays

Significant intra-individual variation

Technology does not allow individual sperm assessment for ICSI and

sperm separation methods are not consistently effective in

separating “good DNA” sperm population from compromised

Predictive value for ART may improve if study DFI of motile portion

of ejaculate (Palermo, 2014)

ASRM (2013) and AUA Best Practice (2011)

guidelines- not enough data to recommend

routine sperm DNA testing

Sperm DNA Challenges

What is it :

• When oxidants outnumber antioxidants Reactive oxygen species (ROS)

• Generated by seminal leukocytes and sperm

• React with lipids, proteins and nucleic acids

Physiological Role:

• Induces acrosome reaction

• Mediates activation and capacitation

• Needed for membrane fluidity

Spermatozoa are susceptible to oxidative stress:

• High concentration of polyunsaturated fatty acids

• Little capacity for membrane repair

• Limited cytoplasmic defensive enzymes

Macrophage Neutrophils

Oxidative Stress

• Potential common pathway of injury- cryptorchidism, infection,

aging, environmental exposures, cancer (Ko, 2014)

• Varicocele

Increased ROS levels in serum, testes, semen (Sabanegh, 2009)

Varicocelectomy increases seminal antioxidant levels and improves bulk semen

parameters (Mostafa, 2001)

• Tobacco

Smoking associated with poorer sperm quality (Kunzle, 2003; Raghed, 2009)

Infertile smokers- higher levels of seminal oxidative stress than infertile non-

smokers (Saleh, 2002)

Smoking cessation- improve semen quality, lowers ROS levels

Clinical Implications of ROS

Antioxidant therapy

Scavenger: vitamins E and C, lycopene, carnitine, etc.

Fertility outcomes

Vit E+C supplementation- decreased semen ROS (DeFoor, 2004)

Vit C (200-1000mg/day)- increased sperm counts in infertile males (Dawson, 1987)

Randomized crossover study of Vit E (600IU q day)-improved zona binding

– Microarray

–Analyzes transcriptome of cells- comparison at different stages of

spermatogenesis

– Proteomics

–Seminal fluid has >1000 different proteins and at least 20 have had altered

expression in infertile men (Pilch,2006; Pixton, 2004)

– Genetics and Epigenetics

–Spermatozoal epigenetic disorders (Boissonnas, 2013; Gannon, 2014;

Klaver, 2014)

–RNA defects (De Mateo, 2014; Jodar, 2013)

Emerging Technologies

Proteomics and Male Infertility

2 DE = 2 dimensional gel electrophoresis; MALDI-TOF = matrix assisted laser; LC-MS liquid chromatography mass spectrometry; ROS = reactive

oxygen species; 2-DIGE = 2 dimensional in gel electrophoresis

Study Subjects Sample size Source Technique Main finding Reference

Varicocele Varicocele

Control

(n = 20)

(n=20)

Spermatozoa 2 DE; MALDI-TOF

MS

15 proteins; 4 reduced in varicocele group; 11 overexpressed ;

HSP60 and HSP 90 upregulated in varicocele group

Chan et al. 2013

Varicocele Pre-varicocelectomy

Post-varicocelectomy

(n = 18) Seminal plasma LC/MS 316 proteins; 53 in pre- and 38 in post varicocelectomy;

68 in both groups

Camargo et al. 2012

Oxidative

stress

Controls

Infertile men

ROS + and ROS -

( n = 20)

( n = 32)

spermatozoa 2-DIGE; LC-MS 1,343 spots in ROS – and 1,265 spots in ROS+ samples. 31

spots differentially expressed; 6 spots increased and 25

decreased in ROS- samples.

Hamada et al. 2013.

Oxidative

stress

Controls

Infertile men

ROS + and ROS -

( n = 20)

( n = 32)

Seminal fluid LC-MS 14 proteins; 7 common and 7 unique in both ROS+ and ROS –

grp.

Sharma et al. 2013

Idiopathic

astheno-

zoospermia

Donors

Patients

(n = 30)

(n= 30)

2DE; Tandem mass

spectrometry;

MALDI-TOF

33 DEP; 15 unique proteins Shen et al. 2013

Sperm

motility

proteins

Fertile men

Asthenozoospermic men

(n = 8)

(n = 8)

spermatozoa 2 DE; MALDI-TOF 14 spots; 10 proteins;

Energy metabolism proteins

Zhao et al. 2007

NOA Control

NOA

Post vasectomy

(n =5)

(n =5)

(n = 5)

Seminal plasma Spectral counts 2048 proteins in NOA; Batruch et al. 2011

Astheno-

zoospermia

Controls

Asthenozoospermic men

(n = 10)

( n= 20)

Spermatozoa 2 DE; 17 protein spots; 8 decreased; 9 increased in

asthenozoospermic men

Martınez-Heredia et

al. 2008

Astheno-

zoospermia

Controls

Asthenozoospermic men

(n = 20)

(n = 38)

Seminal plasma SDS PAGE; LC-

MS/MS

741 proteins; 45 upregulated; 56 downregulatedDJ-11

downregulated in asthenozoospermic gp.

Wang et al. 2009

Complexity of Proteomics

Sharma, 2015