1

Department of Obstetrics and Gynaecology,

Helsinki University Central Hospital,

University of Helsinki, Finland

UNEXPLAINED INFERTILITYStudies on aetiology, treatment options and obstetric outcome

RITA ISAKSSON

ACADEMIC DISSERTATION

To be presented by permission of the Medical Faculty of the University of Helsinki for

public criticism in the Auditorium of the Department of Obstetrics and Gynaecology,

Helsinki University Central Hospital, Haartmaninkatu 2, Helsinki,

on October 18th, 2002, at 12 noon.

Helsinki 2002

2

Supervised by

Docent Aila Tiitinen, M.D., Ph.D.

Department of Obstetrics and Gynaecology

Helsinki University Central Hospital

Docent Bruno Cacciatore, M.D., Ph.D.

Department of Obstetrics and Gynaecology

Helsinki University Central Hospital

Reviewed by

Docent Anne-Maria Suikkari, M.D., Ph.D.

The Family Federation of Finland,

Infertility Clinic, Helsinki

Docent Aydin Tekay, M.D., Ph.D.

Department of Obstetrics and Gynaecology

Oulu University Central Hospital

Offi cial opponent

Docent Hannu Martikainen, M.D., Ph.D.

Department of Obstetrics and Gynaecology

Oulu University Central Hospital

ISBN 952-91-5071-7 (print)

ISBN 952-10-0712-5 (PDF)

Yliopistopaino

Helsinki 2002

3

To my family

CONTENTS

LIST OF ORIGINAL PUBLICATIONS ....................................................................7

ABBREVIATIONS .................................................................................................. 8

ABSTRACT ............................................................................................................. 9

I INTRODUCTION .............................................................................................. 11

II REVIEW OF THE LITERATURE

1. Infertility ................................................................................................. 12

2. Unexplained infertility ............................................................................ 13

2.1. Defi nition and incidence ............................................................ 13

2.2. Speculations on possible underlying causes ...............................14

2.3. Treatment options ...................................................................... 17

3. Ultrasound examination .......................................................................... 20

3.1. Basic ........................................................................................... 20

3.2. Doppler ultrasound .................................................................... 21

3.3. Doppler ultrasound during natural and IVF cycles .................... 22

4. Assisted reproductive technology ........................................................... 28

4.1. Defi nition and rate of success .................................................... 28

4.2. Prognostic markers for successful outcome .............................. 31

4.3. Pregnancy outcome of ART-induced pregnancies ...................... 35

III AIMS OF THE STUDY ................................................................................... 41

IV PATIENTS AND METHODS ........................................................................... 42

1. Study populations and protocols ............................................................ 42

Study I ............................................................................................... 43

Study II ...............................................................................................43

Studies III and IV ............................................................................... 44

Study V .............................................................................................. 44

2. Hormonal assays ...................................................................................... 47

3. Ultrasound and Doppler assessment ....................................................... 47

4. Statistical analysis .................................................................................... 48

V RESULTS .......................................................................................................... 49

1. Uterine and spiral artery blood fl ow in unexplained infertility .............. 49

2. Prognostic signifi cance of uterine artery impedance ............................. 50

3. Comparison of ovulation induction combined with intra-uterine

......... 50 insemination, intraperitoneal insemination and timed intercourse

4. Course of ART-induced and spontaneous pregnancies ........................... 52

VI DISCUSSION .................................................................................................. 55

1. Uterine and spiral artery impedance among women with

55

unexplained infertility

2. Prediction of pregnancy outcome with uterine artery

57 impedance measurement

3. Treatment options of women with unexplained infertility ..................... 58

4. Long-term prognosis of couples with unexplained infertility ................. 59

5. Obstetric outcome of couples with unexplained infertility ................... 60

VII SUMMARY AND CONCLUSIONS ................................................................ 62

ACKNOWLEDGEMENTS .................................................................................... 63

REFERENCES ...................................................................................................... 65

......................................................................

............................................................................

7

LIST OF ORIGINAL PUBLICATIONS

I. Isaksson R, Tiitinen A, Reinikainen L and Cacciatore B. Comparison of the

uterine and spiral artery blood fl ow in women with unexplained and tubal

infertility.

II. Isaksson R, Tiitinen A and Cacciatore B. Uterine artery impedance to blood

fl ow on the day of embryo transfer does not predict obstetric outcome.

Ultrasound Obstet Gynecol 2000; 15: 527-530.

III. Isaksson R and Tiitinen A. Superovulation combined with insemination or

intercourse in the treatment of couples with unexplained infertility and

minimal endometriosis.

Acta Obstet Gynecol Scand 1997; 76: 550-554.

IV. Isaksson R and Tiitinen A. Obstetric outcome in patients with unexplained

infertility: Comparison of treatment-related and spontaneous pregnancies.

Acta Obstet Gynecol Scand 1998; 77: 849-853.

V. Isaksson R, Gissler M and Tiitinen A. Obstetric outcome among women with

unexplained infertility after IVF: a matched case-control study.

Hum Reprod 2002; 17: 1755-1761.

The orginal Publications are reproduced with permission of the copyright holders.

Ultrasound Obstet Gynecol, in press.

8

ABBREVIATIONS

ART assisted reproductive technology

CC clomiphene citrate

COH controlled ovarian hyperstimulation

CV coeffi cient of variation

DIPI direct intraperitoneal insemination

ET embryo transfer

FSH follicle-stimulating hormone

GIFT gamete intra-fallopian transfer

GnRHa gonadotrophin-releasing hormone agonist

hCG human chorionic gonadotrophin

hMG human menopausal gonadotrophin

HSG hysterosalpingography

HSSG hysterosalpingosonography

ICD international classifi cation of diseases

ICSI intracytoplasmic sperm injection

im intramuscular

IU international unit

IUI intra-uterine insemination

IUGR intra-uterine growth retardation

IVF in-vitro fertilization

LH luteinizing hormone

Mhz megahertz

MBR Medical Birth Register

OPU ovum pick-up

PIH pregnancy-induced hypertension

PI pulsatility index

PSV peak systolic velocity

RI resistance index

sc subcutaneous

SE standard error of mean

SD standard deviation

SGA small for gestational age

SPSS statistical package for social sciences

TI timed intercourse

US ultrasound

WHO World Health Organization

ZIFT zygote intra-fallopian transfer

9

ABSTRACT

Unexplained infertility, refering to the failure to conceive of a couple in whom

no defi nitive cause for infertility can be found, has an incidence of 10-20% in

all infertile couples. The purpose of this study was to examine the impedance

to uterine blood fl ow, different treatments and obstetric outcome in unexplained

infertility.

Main uterine and spiral artery impedance to blood fl ow was compared in 20

women with unexplained and 18 women with tubal infertility. Measurements were

taken both during the natural cycle (on days 11-12, 16-17 and 21-23) and during

the in-vitro fertilization (IVF) treatment cycle (on days 1, 5, 10, ovum pick-up

and embryo transfer). The mean uterine artery impedance was below three in

women with unexplained infertility during the natural and IVF cycles, and no

signifi cant differences were observed between the groups. The mean impedance

(± SD) in spiral arteareries was lower (pulsatility index 0.96 ± 0.25) in women

with unexplained infertility on day 5 of gonadotrophin stimulation compared with

women with tubal infertility (pulsatility index 1.24 ± 0.30; p<0.05).

The main uterine artery impedance to blood fl ow was studied among 102

women during 111 conceptional IVF cycles. The aetiology of infertility was

either unexplained, tubal, endometriosis or multifactorial. Predicting obstetric

complications with measurement of uterine artery impedance among these women

was not possible. The mean uterine artery pulsatility index (PI) was comparable in

pregnancies leading to spontaneous abortion (PI 2.71 ± 0.67), ectopic pregnancy

(PI 2.36 ± 0.54) and normal intra-uterine pregnancy (PI 2.69 ± 0.71).

Seventy women with unexplained infertility were treated with ovulation induction

combined with intra-uterine insemination, direct intraperitoneal insemination and

timed intercourse, and these treatments were equally effective.

The same seventy women were followed for six years after completed ovulation

induction treatment. In all, 64% of these couples conceived during the follow-up,

with the majority (60%) of pregnancies beginning spontaneously. In spontaneous

pregnancies, both the mean birthweight (3387 g vs. 3120 g) and the relative

birthweight were signifi cantly higher than in treatment pregnancies.

10

One hundred and seven women with unexplained infertility who conceived

with in-vitro fertilization treatment were studied. Two control groups (n=445

and n=2377) were chosen from the Finnish Medical Birth Register. The rate of

spontaneous abortion was 24% and the multiple pregnancy rate was 23%, including

one triplet pregnancy. No difference was found in mean birthweight, incidence

of low birthweight, gestational duration, umbilical artery pH and rate of major

congenital malformations in women with unexplained infertility as compared

with normally conceived or IVF pregnancies. The rate of pregnancy-induced

hypertension was signifi cantly lower among singletons in the study group compared

with other IVF singletons.

No difference was observed in uterine artery impedance when women with

unexplained infertility and women with tubal infertility were compared. Predicting

pregnancy outcome with measurement of uterine artery impedance during IVF

treatment was not possible. Ovulation induction was an effective treatment for

women with unexplained infertility. Obstetric outcome among women with

unexplained infertility was good, being similar to that of spontaneous pregnancies

and IVF pregnancies.

11

I INTRODUCTION

Infertility, defi ned as failure to conceive within one year despite normal cohabitation

(Zabrek 1996; Barbieri 1996) or within two years according to the European Society

for Human Reproduction and Embryology (Forti and Krausz 1998), presents in

10-15% of all couples. Typically, 80% of couples (aged 18-28 years) will conceive

over a one-year period and another 10% conceive during the following year. The

rate of infertility has slowly been increasing (Stephen and Chandra 1998; Silverberg

2000).

While many of these couples will conceive with assisted reproductive technology

(ART), about 20% will never conceive. The fi rst artifi cial insemination was reported

in 1799 in the United Kingdom. In-vitro fertilization (IVF) was developed more than

one century later, and the fi rst IVF baby was born in 1978 in the United Kingdom

(Steptoe and Edwards 1978). In Finland, the fi rst IVF baby was born in 1984 at the

Helsinki University Central Hospital (Koskimies et al. 1984). ART has subsequently

become an effective and widely used therapy for childless couples (Wikland et al.

1985). Approximately 7200 embryo transfers are performed in Finland each year,

and more than 10 000 IVF babies have been born since 1984 (Nygren and Nybo

Andersen 2001). Of all newborn babies, about 2.6% are born as the result of IVF

treatment compared with 1.3% in other European countries. More than half of all

IVF cycles world-wide are provided in Europe, mostly in France, United Kingdom

and Germany (Nygren and Nybo Andersen 2001). During IVF treatment trans-

vaginal ultrasound is an essential tool for monitoring follicular growth and

performing ovum pick-up. Since the mid-1980s, when transvaginal ultrasound

gained popularity among gynaecologists, its image quality and diagnostic accuracy

in infertility and many other gynaecological conditions have improved dramatically.

The Colour Doppler method has enabled non-invasive evaluation of pelvic and

foetal blood fl ow, which is useful in different pathological conditions.

IVF has revolutionalized the treatment of infertile couples, as well as profoundly

increasing the basic understanding of human reproduction. Treatment options and

results today are promising in most subgroups of infertility, including unexplained

infertility, where IVF is used both as a diagnostic and therapeutic tool.

12

II REVIEW OF THE LITERATURE

1. INFERTILITY

The incidence of infertility is approximately 10-15% (Lilford and Dalton 1987;

Templeton et al. 1991; Crosignani et al. 1996). Primary infertility refers to the

condition of an infertile woman who has never conceived, and secondary infertility

to one who has had a previous pregnancy. At present, almost 80% of infertile

couples can be helped to have a child. The incidence of voluntary infertility is very

low, approximately below 3% (Templeton et al. 1991).

Reasons for the increasing incidence of infertility include postponement of

childbearing, increased pelvic infections and, in some countries, deteriorating

sperm quality (Unkila-Kallio 2001). The reasons for infertility are related to female

factors, male factors, to a combination of the two or to unknown causes, each

comprising 25%. The most frequent causes for infertility are ovulatory disorder or

anovulation, tubal factor, abnormal semen parameters, endometriosis and uterine or

cervical factor (Table 1; Jaffe and Jewelewicz 1991; Collins et al. 1995). Less common

causes for infertility are immunological factors and genetic defects. Smoking also

decreases the ability to conceive (Stillman et al. 1986; Mueller and Daling 1989).

Infertility in many couples has multiple aetiologies; however, in approximately

15-17% of couples, no reason for infertility is found and the infertility is defi ned as

unexplained (Templeton and Penney 1982; Collins et al. 1995; Aboulghar et al. 2001).

Table 1. Most frequent causes of infertility (Jaffe and Jewelewicz 1991; Collins et al. 1995).

_______________________________________________

Cause of infertility %_______________________________________________

Ovulatory disorder or anovulation 27-30

Tubal factor 20-22

Abnormal semen parameter 25-40

Unexplained 15

Endometriosis 5

Uterine or cervical factor 4-5_______________________________________________

13

2. UNEXPLAINED INFERTILITY

2.1. Defi nition and incidence

Unexplained infertility, sometimes also called idiopathic infertility, refers to failure

to conceive in a couple for whom no defi nitive cause for infertility can be found.

Usually, the duration of infertility is more than two years (Jaffe and Jewelewicz

1991; Crosignani et al. 1993; Aboulghar et al. 2001). The incidence of unexplained

infertility varies in different studies but is usually 10-20% (Templeton and Penney

1982; Crosignani et al. 1993; Guzick et al. 1994, 1998; Lessey et al. 1995; Miller et al.

1999). Incidence depends on the selection and age of the study population and,

to some extent, on infertility evaluations. Unexplained infertility is not an absolute

condition but a relative inability to conceive, and while many of these couples may

conceive without treatment, treatment will often hasten conception (Fish et al.

1989).

The diagnosis is possible only after a complete infertility evaluation, including

a physical examination of both partners, has been performed (Templeton and

Penney 1982; Jaffe and Jewelewicz 1991). The basic infertility work-up includes

demonstration of 1) ovulation and normal luteal phase, 2) patent fallopian tubes and

3) normal fi ndings in semen analysis according to the World Health Organization

(WHO; Guzick et al. 1994; Forti and Krausz 1998). However, the recommended basic

infertility evaluation varies among different practitioners and no consensus exists

on which investigations are the most appropriate (Forti and Krausz 1998; Kelly et

al. 2001).

Ovulation is indicated by the existence of a regular menstrual cycle (26-32 days,

with the cycle length varying no more than 4 days from cycle to cycle), an ovulatory

progesterone rise at midcycle and a luteal phase of ≥12 days (Jaffe and Jewelewicz

1991). Different cut-off values indicative of ovulation are progesterone levels >16

nmol/l (Crosignani et al. 1993), >18 nmol/l at two assays (Forti and Krausz 1998) or

>25 nmol/l in midluteal phase (Miller et al. 1999).

Tubal patency is determined by ultrasound-assisted hysterosalpingosonography

(HSSG), hysterosalpingography (HSG) or, if necessary, laparoscopy and

chromopertubation (Heikkinen et al. 1995; Forti and Krausz 1998). The agreement

of HSSG with HSG is 84-94% (Spalding et al. 1998, 1999). When comparing HSSG

14

with laparoscopy in evaluation of tubal patency, results are consistent in 76-93%

of women (Allahbadia 1992, Stern et al. 1992; Spalding et al. 1997). There is also a

75% correlation with fi ndings in HSG and laparoscopy (Jaffe and Jewelewicz 1991).

Laparoscopy is indicated if the woman has suffered from pelvic infl ammatory

disease (PID), if endometriosis is suspected in cases of dysmenorrea or dyspareunia,

or if the woman has previously undergone many abdominal operations such as

complicated appendicectomy (Forti and Krausz 1998). Assessment of tubal patency

is unnecessary if IVF or intracytoplasmic sperm injection (ICSI) is indicated, as in

cases of severe male infertility (Forti and Krausz 1998).

Based on the WHO criteria for normality, a sperm concentration of ≥ 20 million/ml,

motility of ≥ 50% and normal morphology of ≥ 15% are required (World Health

Organization 1999).

The post-coital test (PCT) has been used to study the sperm-cervical mucus

interaction, but the predictive value of PCT is poor because of diffi culty in timing

the test. The test has mostly been abandoned due to poor standardization of the

technique and differences in interpretation of results (Glatstein et al. 1995; Forti and

Krausz 1998).

2.2. Speculations on possible underlying causes

Several theories have been tendered on the aetiology of unexplained infertility

(Table 2). Diminished ovarian reserve, as shown by elevated serum follicle-stimulating

hormone (FSH) level, may be one cause (Cahill et al. 1995; Blacker et al. 1997;

Luborsky et al. 2000) and can result in alterations to other hormonal levels. One

study revealed that 5% of women with unexplained infertility had elevated FSH

levels in the early follicular phase (Rodin et al. 1994). Changes in LH levels,

a low FSH/LH ratio and reduced LH concentration in follicular fl uid have also been

speculated to play a role in unexplained infertility (Cahill et al. 1995; Omland et al.

2001). FSH and LH abnormalities may refl ect a dysfunction on the pituitary-ovarian

axis (Leach et al. 1997; Omland et al. 2001). Both the serum estradiol levels in

follicular phase and the E2/P ratio have been shown to be elevated in women with

unexplained infertility, suggesting altered folliculogenesis (Blacker et al. 1997; Leach

et al. 1997) and an absent midcycle elevation of the hormone prolactin, which is

present in fertile women (Subramanian et al. 1997).

15

Table 2. Theories about the aetiology of unexplained infertility.

_________________________________________________________________________________________________Theory/Clinical fi nding Mechanism Study_________________________________________________________________________________________________

Pituitary and/or follicular dysfunction

FSH elevation diminished ovarian reserve Blacker et al. 1997LH fall impaired follicular development Omland et al. 2001poor ovulation impaired/ short luteal phase Rodin et al. 1994; Blacker et al. 1997oestrogen elevation altered folliculogenesis Blacker et al. 1997; Leach et al. 1997abnormal prolactin absent mid-cycle prolactin rise and Subramanian et al. 1997 impaired oocyte developmentpituitary-ovarian impaired follicular development Cahill et al. 1995dysfunction

impaired follicular impaired ovulation and Blacker et al. 1997development increased risk of LUF Omland et al. 1998

Gamete dysfunction

Oocyte dysfunction

decreased oocyte function reduced oocyte quality Blacker et al. 1997; Omland et al. 2001impaired oocyte transport impaired ovum pick-up and Ahmad-Thabet 2000 impaired tubal transport Omland et al. 1998impaired oocyte-sperm impaired fertilization Blacker et al. 1997;interaction Omland et al. 2001 Sperm dysfunction

impaired penetration of impaired fertilization Hull et al. 1998cervical mucus

impaired penetration of impaired fertilization Takeuchi et al. 2000;zona pellucida Hull et al. 1998 impaired penetration of impaired fertilization Takeuchi et al. 2000;ooplasmic membrane Hull et al. 1998

Alterations in endometrial function

subclinical infection impaired implantation Kundsin et al. 1987 suboptimal expression of impaired endometrial Omland et al. 2001integrins and pinopods development and switch in the window of Lessey et al. 1995 implantation

Immunological factors

elevated concentrations of:ovarian antibodies ovarian failure Luborsky et al. 2000anti-spermatozoal impaired fertilization Luborsky et al. 2000antibodiesanti-cardiolipin increased uterine artery Battaglia et al. 1998antibodies resistance_________________________________________________________________________________________________

16

Another possible explanation is an impaired luteal phase, with a shorter luteal

phase or a decreased peak serum progesterone level (Li et al. 1990; Guzic et

al. 1994; Blacker et al. 1997; Leach et al. 1997). Impaired luteal phase has been

demonstrated in about 30% of women with unexplained infertility. Among women

aged 40-45 years, the rate of impaired luteal phase is higher than among women

aged 20-29, as is also the incidence of unexplained infertility (Rodin et al. 1994;

Miller et al. 1999). Both increased and decreased follicular LH-pulse frequency and a

lower FSH/LH ratio or decreased midfollicular FSH have been postulated to induce

impaired luteal phase, which may primarily be due to a functional imbalance in the

hypothalamus (Nakajima and Gibson 1991).

Besides hormonal factors, gamete dysfunction may contribute to unexplained

infertility. Altered folliculogenesis, impaired oocyte maturation, reduced oocyte

quality and defects in gamete interaction have all been suggested (Gabrielsen et al.

1996; Blacker et al. 1997; Omland et al. 2001). A sperm dysfunction would impair

the ability of spermatozoa to penetrate the cervical mucus, the zona pellucida and

the ooplasmic membrane (Hull et al. 1998; Takeuchi et al. 2000). An insuffi cient

acrosome reaction could cause a fertilization failure, which is sometimes seen in IVF

(Esterhuizen et al. 2001). In addition, genetic defects may affect success rates of IVF

among women with unexplained infertility. Total failure of fertilization is reported

in 5-30% of the cycles in conventional IVF among these women, which is more

frequent than among other subgroups (Gürgan et al. 1995; Ruiz et al. 1997;

Takeuchi et al. 2000). One theory suggests that a failure in the natural ovum pick-

up mechanism by the fallopian tube may occur in these women, although this is

diffi cult to verify (Ahmad-Thabet 2000).

Altered endometrial function may be another cause of unexplained infertility

(Kleintzeris 1997). A subacute infl ammation may be present as differences have

been observed between fertile and infertile women in endometrial leucocyte

populations (Kundsin et al. 1987; Viniker 1999). Low endometrial progesterone

receptor concentrations, inadequate estrogenic induction of progesterone receptors,

decreased inhibin levels (Nakajima and Gibson 1991) and suboptimal expression

of integrins or pinopode formation in the endometrium have also been reported

in women with unexplained infertility (Kleintzeris 1997; Garcia-Velasco and Arici

1999; Nikas et al. 1999; Omland et al. 2001).

17

Ovarian antibodies are frequent (33%) in some reports among women with

unexplained infertility (Geva et al. 1997; Luborsky et al. 1999, 2000), as are

elevated antispermatozoal (Luborsky et al. 2000; Szczepanska et al. 2001) and

anticardiolipin antibodies; the antibody levels have been shown to correlate

with uterine artery resistance (Battaglia et al. 1998). Moreover, an inadequate

maternal immunosuppression, which might cause embryo rejection in women with

unexplained infertility, has been suspected (Kleintzeris 1997). The prevalence of

coelic disease is also higher among women with unexplained infertility compared

with both the general population and other infertile women (Meloni et al. 1999),

which may be due to the elevated antibody levels found in their tissues.

Unexplained infertility may also be functionally linked to endometriosis since many

women with unexplained infertility later develop endometriosis (Cahill et al. 1995;

Guzick et al. 1998; Hull et al. 1998). Furthermore, occupational exposure to noise,

chemicals, radiation, mercury and cadmium may be linked to unexplained infertility

(Mueller and Daling 1989). Women’s anxiety and stress levels may also lower the

chances of conception (Sarell et al. 1985).

Abnormal uterine artery blood fl ow might be associated with unexplained infertility

(Goswamy et al. 1988; Kupesic and Kurjak 1993; Steer et al. 1994). Increased uterine

artery impedance in midluteal phase or an abnormal fl ow in spiral arteries would

impair the implantation process, and have been suggested as possible mechanisms

for infertility (Kupesic and Kurjak 1993; Steer et al. 1994).

2.3. Treatment options

Different treatment options in unexplained infertility include: a) expectant

management, b) clomiphene citrate (CC), or c) gonadotrophins used for ovulation

induction, d) intra-uterine insemination (IUI), e) ovulation induction with CC or

gonadotrophins combined with IUI or direct intraperitoneal insemination (DIPI)

and f) in-vitro fertilization and its modifi cations.

18

Expectant management

Couples with unexplained infertility have a chance of spontaneous conception.

With expectant management, the pregnancy rate per cycle is estimated to be

0.9-4.1% (Collins et al. 1995; Guzick et al. 1998; Donderwinkel et al. 2000; Goverde

et al. 2000). Observation can be offered if the woman is under 28-30 years and the

infertility duration is less than 2-3 years (Crosignani et al. 1993; Forti and Krausz

1998). About 14-20% of all couples with unexplained infertility will conceive

without treatment within one year and 25-30% within two years (Eimers et al. 1994;

Collins et al. 1995). The cumulative three-year pregnancy rate without treatment is

30-80% (Hull et al 1985; Guzick et al. 1998) and the fi ve-year cumulative pregnancy

rate is 80% (Randolph 2000). Moreover, a life table analysis showed that as many

as 64% of primary and 79% of secondary unexplained infertility patients conceived

within nine years (Jaffe and Jewelewicz 1991).

Ovulation induction with clomiphene citrate

Clomiphene citrate is usually used for ovulation induction. CC has been shown to

increase pregnancy rate from 1-2% to 5% per cycle in women with unexplained

infertility (Hughes et al. 2001).

Ovulation induction with gonadotrophins

When gonadotrophins are used, a low starting dose (50-75 IU) for ovulation

induction is usually recommended (Goverde et al. 2000), and the dose is adjusted

according to ovarian response (Salha and Balen 2000). The ovarian response

and follicular count and size are monitored with serial ultrasound and serum E2

measurements (Nachtigall and Schwartz 1996; Sahakyan et al. 1999). Treatment

results of ovulation induction are shown in Table 3.

Intra-uterine insemination

Intra-uterine insemination in the spontaneous cycle is an alternative fi rst choice

of treatment of women with unexplained infertility (Goverde et al. 2000). After

standard swim-up or Percoll gradient preparation of the semen, a catheter is passed

through the cervical canal and the washed sperm suspension at volumes of 0.5-

19

2 ml is expelled into the uterine cavity (Nuojua-Huttunen et al. 1999a). This can be

combined with CC and/or gonadotrophins (Fisch et al. 1989; Forti and Krausz 1998;

Guzic et al. 1998; Lacin et al. 2001). DIPI in combination with ovulation induction

has also been shown to signifi cantly improve pregnancy rates (Crosignani et al.

1991). However, as DIPI is more invasive than IUI and the pregnancy rates are equal,

IUI is usually preferred (Hovatta et al. 1990).

In-vitro fertilization

In-vitro fertilization is recommended for treatment of couples with unexplained

infertility when other treatments have failed (Forti and Krausz 1998; Ola et al.

Table 3. Cycle fecundity rate in couples with unexplained infertility treated with ovulation induction combined with intra-uterine insemination or intercourse.

__________________________________________________________________________________________________________________________________________

Number of Number of Treatment protocol Pregnancy rate Study women treatment cycles (% per cycle)__________________________________________________________________________________________________________________________________________

155 564 CC/ CC+hMG/ hCG/placebo Fisch et al. 1989

241 COH + IUI 27.4 Crosignani et al. 1991 268 463 CC + hMG + IUI x 1-2 20.1 Aboulghar et al. 1993

50 130 GnRHa + FSH 150 IU + hCG 5000 21.8 Chung et al. 1995 IU + hCG 2000 IU (luteal support) + IUI x 2

50 110 GnRHa + FSH 150 IU + hCG 5000 7.7 Chung et al. 1995 IU + hCG 2000 IU (luteal support) + TI x 2 117 284 11.0 Tadokoro et al. 1997

40 40 30.0 Zayed et al. 1997

351 351 CC + hCG + IUI 10.0 van der Westerlaken et al. 1998 315 CC + IUI 6.7 Guzic et al. 1998 1133 hMG + IUI 18

119 119 CC 100mg + hMG/ FSH 75-150 33.6 Omland et al. 1998 or hMG/ FSH + hCG 10.000 IU + IUI x1-2

97 97 hMG/FSH 150-225 IU + hCG 10 Sahakyan et al. 1999 5-10.000 IU + IUI x 2

413 CC/ hMG + IUI 15.3 Nuojua-Huttunen __________________________________________________________________________________________________________________________________________

IU + hCG 10.000 IU + IUI x 1-2

hMG/ FSH + hCG

CC + FSH+ hCG + IUI + hCG

19 / 7.6 / 11 / 0

et al. 1999 a

20

2001). IVF is useful for detecting abnormalities in gametes and for evaluating the

fertilizing capacity of spermatozoa and oocytes. If no fertilization has occurred in

a previous IVF attempt, intracytoplasmic sperm injection (ICSI) is the treatment of

choice, as also in cases of low fertilization percentage with conventional IVF.

Gamete intrafallopian transfer (GIFT) and zygote intrafallopian transfer (ZIFT) are

variations of IVF. Their fi rst indication was unexplained infertility (Asch et al.

1986), and they were used in the 1980s and 1990s. These treatments required

similar stimulation as IVF, patent fallopian tubes and earlier also laparoscopic

approach (Crosignani et al. 1993). As both these treatments were more invasive and

expensive and offered no advantages compared with IVF, they have largely been

abandoned (Randolph 2000). The oviduct was thought to provide an optimized

environment for fertilization and fi rst cell divisions, and the embryo was thought

to enter the uterine cavity at a more physiological time in the luteal phase than

in IVF (Boatman 1997; Guzic et al. 1998). However, GIFT does not allow the

determination of fertilization and neither technique permits the assessment of

embryonic development (Randolph 2000).

Reported pregnancy rates with GIFT are 27-55%, and delivery rate per retrieval

29-34% per treatment cycle (Trounson and Wood 1993; Hull et al. 1994; Guzick

1998). With ZIFT, the pregnancy rates are 32-48% (Guzic et al. 1998; Randolph

2000). One reason for good pregnancy rates in GIFT as compared with IVF in some

studies may be the greater number of oocytes transferred, resulting in an increased

rate of multiple pregnancies (Cohen 1991). No prospective, large-scale, randomized

studies have proved GIFT and ZIFT to be superior to IVF.

3. Ultrasound examination

3.1. Basic

In vaginal ultrasound, the typical operating frequency is 5-7.5 MHz, which enables

good image quality within 8-12 cm from the transducer (Dickey 1997). In abdominal

probes, the operating frequency is generally 3.5 MHz, which gives deeper tissue

penetration at lower frequencies and enables examination of inner structures

(Dickey 1997). The pioneering investigators developed transvaginal ultrasound

21

to study follicular growth and enable easy oocyte retrieval (Dellenbach et al.

1984; Meldrum et al. 1984; Schwimer and Lebovic 1984; Wikland et al. 1985).

The use of transvaginal ultrasound later expanded to include other gynaecological

applications. Transvaginal ultrasound is an important tool for studying the anatomy

and pathology of the uterus and ovaries of infertile women. With ultrasound

examination of the uterus, uterine fi broids, endometrial polyps, Asherman’s syndrome

and different developmental uterine anomalies, such as bicornuate uterus and septate

uterine confi guration, can be detected (Allahbadia 1992; Zabrek 1996). When studying

the ovaries, polycystic ovaries, endometriomas, pyo- and hydrosalpinx and tubo-

ovarian abscesses can be recognized (Allahbadia 1992; Zabrek 1996). Ultrasound

has also increased our basic knowledge of follicular growth and endometrial

development, and is essential for monitoring both natural and induced cycles.

3.2. Doppler ultrasound

Because the evaluation of quantitative blood fl ow on the basis of blood fl ow

velocity waveform is diffi cult various qualitative indices have been introduced.

These are the systolic/diastolic (S/D) ratio, the pulsatility index (PI), the resistance

index (RI), also called Pourcelot’s ratio, and peak systolic velocity (PSV). Uterine

artery resistance is measured by S/D, PI and RI.

SD= systolic/diastolic ratio

PI= S/D

mean

RI= S/D

S

The measurement of PI requires computer-assisted calculation of mean velocity.

The RI approaches 1.00 when diastolic velocities are abnormally low. Both high PI

and RI refl ect impairment of fl ow and are independent of insonating angle and true

velocity. The PI and RI are highly correlated.

22

Colour Doppler sonography is useful in identifying blood vessels and enables study

of blood fl ow characteristics of small vessels within the endometrium and the ovary.

Colour Doppler sonography facilitates study of branching of the uterine vasculature

from main uterine arteries to radial and spiral arteries in the endometrium. Apart

from the location, each of the vessels can be recognized by a characteristic

waveform pattern. Colour Doppler sonography uses lower sound intensity than

grey-scale ultrasound. It is an indispensable tool for studying ectopic pregnancy,

molar pregnancy and gynaecological tumours. Power Doppler, also called Doppler

power spectrum analysis, uses echo amplitude (intensity) in the analysis and is

independent of angle (Harman 1995).

3.3. Doppler ultrasound during natural and IVF cycles

Doppler ultrasound during natural cycle

The highest uterine artery PI values occur on day 1 of the menstrual cycle, when

a mean PI of 4.6 has been shown (Steer et al. 1990; Sladkevicius et al. 1994). The

uterine artery PI is also high 2-3 days after the LH peak (Steer et al. 1990; Sladkevicius

et al. 1993). Many studies have shown that the lowest impedance-to-fl ow in the

uterine artery in the spontaneous menstrual cycle occurs in the luteal phase, 6-12

days after the LH peak, which coincides with the implantation window during

cycle days 19-25 (Steer et al. 1990; Sladkevicius et al. 1993; Tan et al. 1996;

Bloechle et al. 1997; Ziegler et al. 1999; Chek et al. 2000). Some studies have

reported a correlation between plasma estradiol and PI and an inverse correlation

between plasma progesterone and PI (Steer et al. 1990). Another study demonstrated

that a high PI in the follicular phase was associated with low estradiol and

progesterone, but no association was observed between serum estradiol and

progesterone levels and uterine artery PI in the luteal phase (Tinkanen et al. 1994).

Other studies have not found any correlation between serum estradiol and uterine

artery impedance (Steer et al. 1994; Zaidi et al. 1995; Ziegler et al. 1999).

In midluteal phase, uterine artery PI is typically below 3.0 and RI below 0.90

(Cacciatore 1996c). Fertility is usually affected when the uterine artery resistance

during midluteal phase is increased and when normal haemodynamic changes

in uterine artery impedance do not occur (Steer et al. 1994; Tinkanen et al.

23

1994; Coulam et al. 1999; Ziegler et al. 1999). Therefore, 20-40% of infertile

women have a high PI (>3.0) in luteal phase, which earlier was associated with

a poorer chance of conception (Steer et al. 1994; Cacciatore et al. 1996a;

Cacciatore 1996c). Uterine artery impedance refl ects the impedance of the whole

uterine vascular bed and may be affected by many factors such as fi broids,

adenomyosis and uterine contractility. The uterine artery resistance in the luteal

phase is also shown to be elevated in different infertile subgroups (Kurjak

et al. 1991; Tinkanen et al. 1994; Zaidi et al. 1998). The explanation for this fi nding

among women with anovulatory cycles is a subnormal hormonal concentration

(Kurjak et al. 1991, Tinkanen et al. 1994), among women with polycystic ovaries

(PCO) an elevated androgen concentration (Zaidi et al. 1998), and among women

with endometriosis or chronic PID peritoneal scarring (Tinkanen and Kujansuu

1992; Steer et al. 1994; Tinkanen et al. 1994). Besides these subgroups, it has

been suggested that abnormal uterine perfusion may also be a contributing factor

to unexplained infertility (Coulam et al. 1994; Steer et al. 1994). However, one

study showed that the mean uterine artery PI in the midluteal phase did not

differ signifi cantly when comparing women with unexplained infertility, women

with adhesions due to PID and women with moderate or severe endometriosis

(Tinkanen et al. 1994).

Besides infertility, some factors may also increase uterine artery impedance in fertile

women. Levonorgestrel intrauterine device (IUD) increases the uterine artery PI in

fertile women during the luteal phase, whereas the copper IUD has no effect on

uterine artery blood fl ow (Järvelä et al. 1998a, 1998b).

In addition, vascular perfusion of the uterus is known to decrease and uterine

artery impedance to increase during menopause, mainly as a result of declining

estradiol concentrations (Goswamy et al. 1988; Battaglia et al. 1995; Kurjak and

Kupesic 1995). About 50% of postmenopausal women have a decreasing PSV in

uterine arteries and 10% have an absent or reversed diastolic fl ow (Sladkevicius et

al. 1994). Furthermore, the uterine arteries become smaller, the shape of the blood

fl ow velocity waveform changes and the uterine artery becomes more diffi cult

to visualize (Sladkevicius et al. 1994). Many studies have shown that estradiol

treatment in postmenopausal women reduces uterine artery impedance (de Ziegler

et al. 1991; Pirhonen et al. 1993; Järvelä et al. 1997). However, synthetic progestins

may antagonize the vasodilatory effect in uterine arteries induced by estrogen

24

treatment (Pirhonen et al. 1993; Järvelä 1998c). Results of different studies have

been confl icting as to the effect of adding progesterone to HRT and its infl uence

on uterine artery impedance (de Ziegler et al. 1991; Bonilla-Musoles et al. 1995).

Interestingly, some studies report no decrease in uterine perfusion with age (Chek

et al. 2000).

Doppler ultrasound during IVF cycle

Transvaginal ultrasound is essential in monitoring ovulation induction cycles and

IVF cycles to determine the number and size of developing follicles during

gonadotrophin stimulation. The uterine artery impedance is usually lower in

gonadotrophin-stimulated cycles compared with natural cycles, and the PI of

uterine arteries on the day of ET is lower in stimulated than in spontaneous cycles

(Tekay et al. 1996a). Early studies showed promising results where uterine artery PI

on day of ET was lower in conception than in non-conception cycles (Steer et al.

1990, 1995; Cacciatore et al. 1996a). In a study by Steer et al. (1995), for example,

the median PI (range) was 2.85 (1.4-3.6) in conception and 4.15 (2.1-6.8) in non-

conception FET cycles (p<0.01). Similar fi ndings were found by Cacciatore et al.

(1996a), who reported a mean PI (± SD) of 2.45 (± 0.54) in conception and 2.66

(± 0.39) in non-conception cycles (p<0.05) during IVF treatment. Different cut-off

values have been suggested to predict non-conception in the IVF cycle. Coulam

et al. (1994) found a uterine artery PI>3.3, and Steer et al. (1994) and Cacciatore

(1996c) reported a uterine artery PI >3.0, whereas Favre et al. (1993) reported a

PI>3.55 to predict non-conception. Tekay and co-workers found a nonsignifi cant

increase in receptivity when the uterine artery PI was between 2.0 and 2.99 and

suggested that a cut-off value of PI>4.0 would predict a non-conception cycle

(Tekay et al. 1995, 1996b). Furthermore, the uterine receptivity is found to be poor

when no end diastolic fl ow in uterine artery is detected (Goswamy et al. 1988;

Kurjak et al. 1991; Steer et al. 1992; Coulam et al. 1994; Cacciatore et al. 1996a;

Tekay et al. 1996a; Tan 1999). Women with absent end diastolic fl ow or poor uterine

blood fl ow have been shown to also have an increased risk of spontaneous abortion

(Cacciatore 1996c). In natural cycles with FET, the implantation has been shown to

decrease when PI>3.5 or with absent end diastolic velocities (Cacciatore 1996c).

Estradiol levels are generally much higher during IVF cycle than natural cycles

25

(Tekay et al. 1996a). Gonadotrophin stimulation with elevated estradiol levels

has been shown to decrease uterine artery PI by approximately 20% (Cacciatore

1996c). During IVF an inverse correlation has been shown between uterine artery

PI values and estradiol levels (Weiner et al. 1993; Cacciatore and Tiitinen 1996b).

Nevertheless, according to current knowledge, it is not possible to distinguish

between conception and non-conception cycles with the sole measurement of

uterine artery PI and RI (Favre et al. 1993; Coulam et al. 1994; Bassil et al. 1995; Tekay

et al. 1995, 1996a). Many factors make comparison of the results diffi cult. Different

study populations, different stimulation protocols, small size of study populations,

an overlap of measured PI values when comparing conception and non-conception

cycles, diurnal variation in uterine artery blood fl ow in the periovulatory period,

showing a lower PI early in the morning, and different results when examining

uterine blood fl ow in women standing and in recumbent position (Coulam et

al. 1994; Dickey et al. 1994; Zaidi et al. 1995; Tekay et al. 1996b). In conclusion,

measurement of uterine artery impedance during IVF treatment to predict outcome

of treatment is not useful in routine clinical practice.

Uterine artery impedance during pregnancy

Efforts have been made to predict pregnancy complications by uterine artery

measurements. Blood fl ow velocity in the uteroplacental arteries increases and the

PI decreases linearly with advancing gestation, indicating an exponential increase

in uterine arteries and uterine perfusion during the fi rst trimester (Valentin et al.

1996). Some studies have shown that uterine and spiral artery resistance were

lower at the end of the fi fth week in blighted ovum pregnancies (Alfi revic and

Kurjak 1990) and the uterine artery RI was lower in missed abortion than in

women with normal pregnancies (Alfi revic and Kurjak 1990; Kurjak 1994). Another

study showed that uterine artery PI measurement at 12-13 weeks of gestation

could predict subsequent hypertensive disorders, small-for-gestational-age fetuses

and gestational diabetes during pregnancy (van den Elzen et al. 1995). In contrast

to these studies uterine artery RI measurements during the fi rst trimester of

pregnancy have not in other studies predicted miscarriage or normal delivery

(Frates et al. 1996).

26

The uterine artery mean fl ow velocity rises progressively from a non-pregnant

value nearly eightfold by week 36 (Palmer et al. 1992). By measuring uteroplacental

and umbilical artery Doppler S/D ratios during the 24th gestational week, it was

possible to predict intrauterine growth retardation and hypoxia (Newnham et al.

1990). Another study suggested that uterine artery measurement at 21-24 weeks

could predict PIH and IUGR in high-risk but not low-risk populations (Zimmermann

et al. 1997). Numerous other studies have also shown that measurement of uterine

artery blood fl ow is useful in predicting pre-eclampsia or fetal growth restriction

(Bewley et al. 1991; Strigini et al. 1995; Harrington et al. 1996; Kurdi et al. 1998).

With advancing gestation, the spiral artery RI decreases in normal pregnancies

(Murakoshi et al. 1996). An abnormal spiral artery RI has been proven to have

better diagnostic accuracy than the RI of uterine artery waveform for predicting

PIH and IUGR, and in one study with 43 pregnancies complicated with PIH and/ or

IUGR, 19 had abnormal spiral artery RI when examinations were performed during

gestational weeks 18-41 (Murakoshi et al. 1996).

Impedance in spiral artery

In healthy fertile women, a signifi cant decrease in spiral artery resistance occurs

from the proliferative to the luteal phase, and the changes are equivalent to those

seen in uterine arteries (Kurjak and Kupesic 1995). Because the sensitivity of colour

Doppler is limited, weak vascular signals in endometrial vasculature can be diffi cult

to observe (Yang et al. 1999). However, endometrial blood fl ow has been suggested

to be a more specifi c parameter for predicting uterine receptivity in IVF cycles

than uterine blood fl ow, and when endometrial blood fl ow is poor, implantation

is less likely to occur (Zaidi et al. 1995; Tan 1999). In 88.8% of infertile patients,

spiral arteries can be demonstrated during the natural cycle from day 10-12

onwards (Kupesic 1996b). However, not all studies have found predictive value

of measurements of subendometrial PI (Yuval et al. 1999). The absence of

subendometrial fl ow on day of HCG administration during the IVF cycle has

been shown to predict failure of implantation irrespective of the morphological

appearance of the endometrium (Zaidi et al. 1995). Spiral artery impedance

measurement has greater inter-observer variability compared with that of uterine

arteries (Dickey et al. 1994). Levonorgestrel IUD has been shown to suppress the

27

formation of spiral arteries (Järvelä 1998c). Age also affects spiral artery blood fl ow.

With advancing age, blood fl ow to the endometrium decreases and blood fl ow in

spiral arteries is detected in only 30% of postmenopausal women within 1-5 years

of cessation of the menstrual cycle (Kurjak and Kupesic 1995).

A surprising fi nding is that better perfusion of the uterus, measured as lower PI

values both in the radial and spiral arteries in the natural cycle compared with

corresponding values in ovulation induction cycles, suggests that receptivity of the

uterus would be better in natural cycles (Kupesic and Kurjak 1993).

Most authors agree that treatment cycles with absent spiral arteries have poor

implantation rates. Despite some initially promising results in predicting conception

and non-conception cycles with measurement of spiral artery impedance in women

with detectable spiral arteries, some studies show no difference in spiral artery

impedance measurement in conception and non-conception cycles; therefore,

further research is clearly needed.

Impedance in ovarian artery

In ovarian arteries, the cyclic variations are smaller than in uterine arteries (Tekay

et al. 1995). In natural cycles, the ovarian artery RI has been shown to decline on

the day before ovulation and to remain low in the luteal phase both in healthy

and infertile women (Kurjak et al. 1991; Tinkanen et al. 1994; Kupesic et al. 1995).

Intrafollicular PSV has also been demonstrated to increase on the day of ovulation

and remain high for at least 72 hours (Campbell et al. 1993). This increases blood

supply to the corpus luteum, which is important for steroidogenesis and favours

implantation and early embryo development (Kupesic 1996a). The ovarian PI on

the dominant side is signifi cantly lower and PSV signifi cantly higher in the luteal

phase in the ovarian stroma and hilus (Sladkevicius et al. 1993). An increased intra-

ovarian impedance has been found in patients with out-of-phase endometrium

biopsies thoughout the luteal phase, since among menopausal women and later in

menopause, no blood fl ow can be detected in the ovaries (Sladkevicius et al. 1994;

Kurjak and Kupesic 1995).

28

Cyclic alterations in ovarian artery blood fl ow seen in natural cycles do not occur

in gonadotrophin-stimulated cycles, where arteries seem maximally dilated (Tekay

et al. 1995). In IVF cycles, no difference has been found in ovarian artery impedance

when comparing conception and non-conception cycles (Sterzic et al. 1989; Tekay

et al. 1995). According to a recent study, a follicle with high PSV in a natural or IVF

cycle will most likely have a good oocyte with high implantation potential (Kelly et

al. 2001). However, the role of ovarian blood fl ow in successful IVF treatment has

yet to be studied in larger study populations.

4. Assisted reproductive technology

4.1. Defi nition and rate of success

Assisted reproductive technology (ART) includes controlled ovarian stimulation

alone or combined with intra-uterine insemination or direct intraperitoneal

insemination and in-vitro fertilization and its modifi cations. The success rates of

treatment vary considerably with patient selection, infertility duration, indication

for treatment and type of treatment.

Ovarian stimulation and/or artifi cial insemination (IUI/DIPI)

Ovarian stimulation with CC or gonadotrophins increases the levels of gonadal

steroids as a result of development of multiple pre-ovulatory follicles and may

overcome subtle ovulatory defects, such as luteinized unruptured follicle syndrome,

not detected in routine diagnostic studies; pregnancy rate may increase with the

larger number of oocytes available for fertilization (Fish et al. 1989; Guzic et al. 1998;

Aboulghar et al. 1999; Sahakyan et al. 1999). Swim-up of sperm before insemination

may enhance the fertilizing capacity of sperm, and increase the density of motile

sperm in the uterus and fallopian tubes available for fertilization and implantation

(Aboulghar et al. 1999). By IUI or DIPI, problems in cervical mucus, mild defects in

sperm motility and function are overcome (Crosignani and Rubin 1996; Wolf 2000).

By bringing together multiple mature oocytes and highly motile sperm in the fallopian

tube, the likelihood of pregnancy is increased (Fish et al. 1989).

29

Although couples with unexplained infertility usually have ovulatory cycles even

without treatment, ovulation induction may overcome subtle defects in ovulatory

function not detected in conventional tests, and increase the number of oocytes

available for fertilization and implantation (Guzic et al. 1998; Aboulghar et al.

1999; Sahakyan et al. 1999). CC has also been shown to reduce the incidence of

luteal-phase defi ciency from 31% to 3% among women with unexplained infertility

(Rodin et al. 1994).

IUI in the spontaneous cycle among women with unexplained infertility yields

pregnancy rates of 2.4-7.4% per cycle (Guzic et al. 1998; Goverde et al. 2000);

which is similar to pregnancy rates of expectant management (Goverde et al. 2000).

If CC is used without IUI, the pregnancy rate per cycle is 4-5.6% among women

with unexplained infertility (Guzic et al. 1998; Hughes et al. 2001). The use of

gonadotrophins (hMG) alone is associated with a pregnancy rate of 7.7% (Guzic

et al. 1998). If CC is used in combination with IUI, the pregnancy rate per cycle is

approximately 8.3% (Guzic et al. 1998). If CC or hMG is used in combination with

IUI, the pregnancy rate per cycle increases to approximately 10-17.1% and 29.8%,

respectively, per patient after three treatment cycles (Guzic et al. 1998; Aboulghar

et al. 1999; Sahakyan et al. 1999).

In-vitro fertilization

IVF is now recommended for essentially all infertility conditions that have not been

successfully treated by other modalities (Dor et al. 1996; Forti and Krausz 1998;

Unkila-Kallio 2001). IVF is useful for recognizing abnormalities in gametes and for

evaluating the fertilizing capacity of spermatozoa and oocytes, i.e. we know whether

the sperm penetrate and fertilize the oocytes. This is especially important among

couples with unexplained infertility or male infertility. Figure 1 shows the treatment

protocol for IVF.

30

Figure 1. A model for ovarian hyperstimulation for IVF with long pituitary downregulation with a GnRH agonist. The squares in the fi gure represent days of stimulation.

The reason for the favourable success rate in IVF is partly the same as in COH, i.e.

it increases the production of a superabundance of oocytes with superovulation

which are then available for fertilization, overcomes ovulatory defects and

enhances fertilizing ability (Guzic et al. 1998; Hull et al. 1998). Furthermore, IVF is

useful for documenting the occurrence of fertilization and for evaluating embryo

quality.

The pregnancy rates are almost equally good in all indications. Couples with

unexplained infertility treated with IVF usually have the same oocyte recovery as

other groups, but they have been shown to have reduced fertilization and cleavage

rates, and a complete failure of fertilization has been reported in 5-30% of the cycles

(Hull et al. 1998; Takeuchi et al. 2000). However, the complete fertilization failure is

usually not repetitive (Hull et al. 1998). New evidence points to the possibility of

oocyte metaphase I maturation block, which is responsible for at least part of the

repetitive fertilization failures (Harrison et al. 2000; Bergere et al. 2001).

Among women with unexplained infertility treated with IVF, clinical pregnancy

rates per oocyte pick-up (OPU) are 21-37% (Crosignani et al. 1991; Tanbo et al. 1995;

Guzic et al. 1998; Aboulghar et al. 1999). The overall reported cumulative pregnancy

rates among unselected patients undergoing IVF treatment are 22-32% after one,

36-57% after two, 47-71% after three and 53-81% after four IVF attempts (Rowlands

et al. 1994; Stolwijk et al. 2000). The reported corresponding cumulative birth rates

are 29%, 52%, 63% and 75%, respectively (Rowlands et al. 1994).

31

4.2. Prognostic markers for successful outcome

Many investigators have tried to fi nd predictive markers for successful outcome of

IVF treatment. Traditionally, the woman’s age and early follicular phase serum FSH

are the most useful parameters for prediction of ovarian reserve (Chek et al. 1994;

Kupesic and Kurjak 2002). The most important predictive factor for success in IVF

treatment in many studies is age of the woman. The highest livebirth rates after IVF

are equivalent in both age group 25-30 years (Templeton et al. 1996) and in age

group below 35 years (Abdelmassih et al. 1996; Engmann et al. 2001). Conception

rates begin to decline at 30-33 years due to oocyte and endometrial aging

(Battaglia et al. 1996; Dor et al. 1996; Lim and Tsakok 1997). Another study reported

declining pregnancy rates after IVF-ET with increasing female age as follows: in

women aged <30 years, clinical pregnancy rate 29.4%; in women 30-35 years,

19.8%; in women 36-39 years, 17.1%; in women >40 years, 12.8% (Szamatowicz and

Grochowski 1998). With increasing age, the number and quality of oocytes decline,

a larger proportion of the oocytes remain unfertilized in IVF and the pregnancy

rates tend to be lower (Lim and Tsakok 1997). After the mid-thirties, follicles also

have fewer granulosa cells, thus producing less steroids and glucoproteins, and

decreased mitosis and increased apoptosis appear (Nikolaou et al. 2002). Female

age is also of important prognostic value in gonadotrophin-induced COH-IUI cycles

(Buyalos et al. 1997; Sahakyan et al. 1999). Increased early follicular phase FSH and

E2 values have been shown to correlate with poor ovarian reserve and reduced

fertility potential in COH treatment cycles (Magarelli et al. 1996; Buyalos et al.

1997). Elevated FSH has been suggested to induce enhanced E2 production by

granulosa cells (Buyalos et al. 1997).

Besides female age, the cause of infertility may also infl uence the outcome of

ART treatment; couples with unexplained infertility are reported to have a better

prognosis than couples with tubal infertility or endometriosis (Templeton et al.

1996; Omland et al. 1998; Nuojua-Huttunen et al. 1999a; Sahakyan et al. 1999).

Women with tubal infertility have also been reported to have lower cumulative

delivery rates than other subgroups of infertility (Sundström et al. 1997). Among

women with tubal infertility, a relationship exists between grade of tubal damage

and pregnancy rate following IVF. Patients with milder tubal damage have higher

take-home baby rates per started IVF cycle (48%) than patients with severe tubal

damage (6%; Csemiczky et al. 1996). The pregnancy rates of infertile women with

32

hydrosalpinx are signifi cantly lower (19.7%) than those of women with tubal

infertility without hydrosalpinx (31.2%; Camus et al. 1999). One study showed that

women with endometriosis had a higher and couples with male factor infertility a

lower pregnancy rate after IVF than in other aetiologies of infertility (Minaretzis et

al. 1998). In addition, women with ovulatory disorders or endometriosis have also

been reported to have good pregnancy rates compared with male and tubal factor

infertility in COH-IUI treatment cycles (Sahakyan et al. 1999). However, some other

studies have not shown any difference in pregnancy rate after IVF when comparing

different subgroups of infertile patients (Tan et al. 1992; Dor et al. 1996).

The duration of infertility and parity of women infl uence pregnancy rates in ART.

An increase in duration of infertility decreases the chance of success after IVF

treatment (Templeton et al. 1996) and COH-IUI cycles (Nuojua-Huttunen et al.

1999a). In COH-IUI cycles, an infertility duration of over six years has been shown to

decrease the chance of success (Tomlinson et al. 1996; Nuojua-Huttunen et al. 1999a).

A previous pregnancy and delivery, especially if the earlier pregnancy was achieved

after IVF treatment, increases the chance of success in a subsequent IVF treatment

(Templeton et al. 1996; Engmann et al. 2001). Patients with secondary infertility

have been reported to have nearly double the pregnancy rate of women with

primary infertility (Lessing et al. 1988; Collins and Rowe 1989). On the other hand,

the highest livebirth pregnancy rate is reported in the fi rst three treatment cycles

(Engmann et al. 2001). Another study showed that the clinical pregnancy rate

remained similar during the fi rst six IVF cycles (Dor et al. 1996). A recent study

revealed that the following factors increased the chance of success in the IVF cycle:

age of woman (<35 years), previous IVF live birth, other than tubal infertility, large

number of embryos created and fewer than three previous unsuccessful IVF cycles

(Engmann et al. 2001). All of these factors increase the chance of both singleton

and multiple births (Engmann et al. 2001).

An ideal method to predict uterine receptivity has yet to be established (Friedler

et al. 1996). Furthermore, the overall success rate of the clinic is very important

when predicting success for a particular couple (Templeton et al. 1996). According

to recent studies, major determinants of successful in-vitro fertilization besides

woman’s age are embryo quality, number of embryos transferred and endometrial

thickness (Goverde et al. 2000; Bassil 2001).

33

Embryo quality is largely based on cleavage rate and the degree of fragmentation,

which are important factor in predicting implantation (Ziebe et al. 1997; Alikani

et al. 1999). Lundin et al. (2001) showed that embryos which cleave early, i.e.

complete the fi rst mitotic division within 25-27 hours post-insemination, provide

higher pregnancy per transfer rate (40.5% vs. 31.5%; p=0.0049) than slower-cleaving

embryos (van Royen et al. 1999). Furthermore, the rate of twin pregnancies has

been found to be higher (33.3% vs. 22.3%; p=0.036), and the rate of spontaneous

abortion to be lower in the early-cleaving group (Lundin et al. 2001). Unevenly

cleaved embryos with large differences in blastomere size have higher rate

of aneuploidy and lower implantation rates than embryos with equally sized

blastomeres (Hardarson et al. 2001).

The presence of large fragments is detrimental to the developing embryo and

these embryos have signifi cantly lower pregnancy rates, whereas small fragments

do not affect implantation (Alikani et al. 1999). It is possible to score embryos

in IVF cycles, and according to this scoring, decide how many embryos should

be transferred. A top-quality embryo is characterized by the presence of 4 or 5

blastomeres on day 2 and at least 7 blastomeres on day 3 after insemination, the

absence of multinucleated blastomeres and <20% cellular fragments on day 2 and

day 3 after fertilization (Gerris and van Royen 2000). By transferring only one top-

quality embryo in the fi rst IVF/ICSI cycle of women <34 years of age, a prospective

study showed that the implantation rate was 42.3%, and thus, the pregnancy rate

was similar or even better compared with normal fertile couples (Te Velde and

Cohlen 1999). Moreover, this procedure limited the twin pregnancy rate to <1%

of pregnancies (Gerris and van Royen 2000). The number of normally developed

embryos, i.e. the number of embryos available for transfer, is more important

in determining the probability of birth than the number of embryos actually

transferred (Engmann et al. 2001). In contrast, the risk of multiple gestation

increased both with the number of embryos available and the number of embryos

transferred (Engmann et al. 2001). Also, if several embryos are created, it increases

the likelihood of selecting good-quality embryos for transfer, thus, the number of

embryos available may be an indirect measure of embryo quality (Devreker et al.

1999; Engmann et al. 2001). The cell stage at transfer of the embryo is also important

in predicting success of treatment (Yang et al. 1999; Bassil 2001).

34

The type of embryo transfer catheter has also been shown to infl uence pregnancy

rates, which in a recent study were signifi cantly higher with a soft catheter

compared with a stiff catheter (27.1% vs. 20.5%; van Weering et al. 2002).

Studies have shown that a small ovarian volume, reduced stromal area and decreased

number of antral follicles predict poor response in controlled ovarian stimulation

(Chek et al. 1994; Syrop et al. 1995; Pellicer et al. 1998; Kupesic and Kurjak

2002). A strong association between ovarian volume and ovarian reserve has been

demonstrated, and measurement of ovarian volume was recommended as an

essential part of infertility evaluation (Lass et al. 1997). A cut-off value of less than

3 cm3 may refl ect failure to respond to gonadotrophin stimuli (Lass et al. 1997).

However, when measured with three dimensional ultrasound, it was shown that

some patients may have diminished ovarian reserve without changes in ovarian

volume (Kupesic and Kurjak 2002). Other authors suggested that predicting ovarian

response was possible by counting the number of antral follicles (Tomas et al.

1997). The best predictive factor for ovarian response to stimulation is in fact

assessment of the total number of antral follicles by ultrasound (Ng et al. 2000;

Kupesic and Kurjak 2002). The number of antral follicles is clearly related to

reproductive age and refl ects the remaining primordial follicle pool (Kupesic and

Kurjak 2002). Measurement of ovarian stromal PSV in early follicular phase may also

predict follicular response (Zaidi et al. 1995). Poor responders have been shown to

have a poor ovarian stromal PSV (Zaidi et al. 1995).

Measurements of the intra-endometrial power Doppler area (Yang et al. 1999) have

been postulated to predict implantation. According to another study, however,

the three-dimensional power Doppler measurement of the endometrial area is not

helpful in predicting implantation (Kupesic et al. 2001). Nevertheless, in a study

of 56 infertile women, statistically signifi cant differences were shown in the antral

follicle number, ovarian volume, ovarian stromal area and ovarian stromal fl ow

index in conception and non-conception cycles when ovaries were studied after

pituitary suppression during IVF treatment (Kupesic and Kurjak 2002).

Uterine artery impedance has previously been demonstrated to predict conception

in IVF cycles, but it is an inaccurate method and no consensus on a cut-off rate for

good pregnancy rates exists. However, most authors agree that an absent end diastolic

fl ow on day of ET predicts non-conception during the present IVF cycle (Goswamy

35

et al. 1988; Kurjak et al. 1991; Steer et al. 1992; Coulam et al. 1994; Cacciatore

et al. 1996a; Tekay et al. 1996b; Tan 1999).

The endometrium is thicker in ART cycles compared with natural cycles mainly

due to higher E2 levels in ART cycles (De Geyter et al. 2000). No differences were

found in endometrial thickness between conception and non-conception cycles

during ART treatment (Coulam et al. 1994; Bohrer et al. 1996; De Geyter et al.

2000), although cut-off values of 6-8 mm have been suggested as the minimum

for embryo implantation (Steer et al. 1992; Coulam et al. 1994; Zaidi et al. 1995).

A multilayer endometrial pattern has also been shown to predict conception in

ovulation induction cycles (Check et al. 1991; Coulam et al. 1994; Serafi ni et al. 1994;

Hock et al. 1997). In addition, a conversion of the endometrial pattern from the

homogeneous pattern seen in the early proliferative phase to the trilaminar pattern

must occur to produce optimal receptivity (Zaidi et al. 1995; Hock et al. 1997).

A pre-ovulatory triple-line endometrium combined with end diastolic velocity in

the fi rst branch of uterine artery was the most important predictor of conception in

one study (Serafi ni et al. 1994). However, endometrial thickness and morphological

appearance of the endometrium are rather inaccurate methods for predicting

conception, and evaluation of morphological appearance is quite subjective (Tan

1999; Kupesic et al. 2001). Furthermore, measurements of spiral artery impedance

to predict conception have given inconsistent results (Zaidi et al. 1995; Schild

et al. 2001). Most authors do, however, agree that absent spiral arteries predicts

non-conception during the present IVF cycle (Zaidi et al. 1995; Yang et al. 1999).

4.3. Pregnancy outcome of ART-induced pregnancies

The rate of spontaneous abortions is approximately 19-25% after IVF, and this rate

is similar to that of women with normal fertility (Wilcox et al. 1999; Pezeshki et

al. 2000; Gissler and Tiitinen 2001). No association has been shown between the

aetiology of infertility and the risk of spontaneous abortion after IVF (Pezeshki et

al. 2000). The risk of spontaneous abortion is similar in IVF and ICSI pregnancies

(Govaerts et al. 1996; Gissler and Tiitinen 2001), but results of other studies are

confl icting (Aytoz et al. 1999; Griffi ths et al. 2000; Ola 2000; Bonduelle et al. 2002).

Bleeding, particularly in the fi rst trimester, is more common in IVF pregnancies

than in spontaneous pregnancies (Olivennes et al. 1993; Verlanen et al. 1995;

36

Wennerholm et al. 1996; Koudstaal et al. 2000a).

The rate of ectopic pregnancies after IVF is reported to be 2-4%, and the

corresponding fi gure after natural conception is 2% (Marcus et al. 1995; Strandell

et al. 1999; Adamsson and Brzyski 2000). Tubal infertility, tubal surgery and PID

increase the risk to 7-10% after IVF treatment, while male factor infertility has

a protective effect against ectopic pregnancy (Verlust et al. 1993; Strandell et

al. 1999). Among IVF pregnancies, the rate of heterotopic pregnancies is <1.0%

(Ludwig et al. 2001).

A period of infertility of more than 12 months signifi cantly increases the woman’s

risk of preterm birth (1.6- to 1.8-fold) and a low birth weight baby (1.9-fold) even

in the absence of infertility treatment (Williams et al. 1991; Henriksen et al. 1997).

COH-IUI treatment has been reported to reduce the mean birthweight of newborns

compared with spontaneous pregnancies (Nuojua-Huttunen et al. 1999b). Another

study showed increased risk of non-insulin-dependent diabetes mellitus (12.7%

vs. 6.9%), pregnancy-induced hypertension (9.3% vs. 6.2%) and Caesarean section

(18.4% vs. 13.1%) after COH treatment compared with spontaneous pregnancies

(Maman et al. 1998). Moreover, a small increase in the rate of preterm birth after

COH-IUI or donor insemination has been reported (Wang et al. 2002).

The risk of pregnancy-induced hypertension, pre-eclampsia, placenta previa and

non-insulin-dependent gestational diabetes increase in IVF-pregnancies, also among

singletons (Wennerholm et al. 1991; Doyle et al. 1992; Tan et al. 1992; Wang et

al. 1994; Tallo et al. 1995; Tanbo et al. 1995; Maman et al. 1998; Minakami et al.

1998; Hansen et al. 2002), but some authors have suggested that these increased

risks are associated with patient characteristics, such as increased maternal age and

lower parity (Doyle et al. 1992; Lansac 1995; Wennerholm et al. 1996). Pregnancies

achieved after IVF also increase the risk of premature deliveries and low birthweight

also in singleton pregnancies (Bergh et al. 1999; Tarlatzis and Grimbizis 1999;

Westergaard et al. 1999; Hansen et al. 2002; Schieve et al. 2002; Wang et al.

2002). On average, IVF singletons were born 5.1 days earlier and weighed

214 grams less than spontaneously conceived infants (Koudstaal et al. 2000a).

A signifi cant increase in rate of low birthweight babies has been shown both among

preterm and full-term singletons (Schieve et al. 2002). The risk of low birthweight

37

infants is estimated to be 2.6-fold that of the general population (Schieve et al.

2002). In singleton pregnancies, the rate of preterm delivery is approximately 9-15%

in IVF vs. 3-6% in spontaneously conceived pregnancies (Maman et al. 1998; Tarlatzis

and Grimbizis 1999), the rate of low birthweight newborns (<2500 g) is 13.4% vs.

7.3% (Tarlatzis and Grimbizis 1999; Koudstaal et al. 2000a) and the rate of very low

birthweight newborns (<1500 g) is approximately 2.6% (Tan et al. 1992; Olivennes

et al. 1993; Schieve et al. 2002) vs. 1% (Hansen et al. 2002). Low birthweight is

less common in infants conceived with ICSI treatment due to male factor infertility

compared with other subgroups (Schieve et al. 2002). In comparing IVF and ICSI

pregnancies, statistically signifi cant differences have been seen in the rate of very

low birthweight infants (IVF 5.6% vs. ICSI 4.4%), whereas the rate of preterm birth

was signifi cantly higher in ICSI pregnancies (Bonduelle et al. 2002). Many reports

also show an elevated rate of Caesarean section among both singleton and multiple

pregnancies after IVF, as well as increased induction of labour in unselected IVF

patients (Verlanen et al. 1995; Maman et al. 1998; Westergaard et al. 1999).

Studies among women with unexplained infertility have shown an especially high

risk of IUGR after ART compared with other subgroups (Wang et al. 1994; Dawood

1996). Also signifi cantly higher incidences of pre-eclampsia, placental abruption,

preterm labour, induction of labour and emergency Caesarean section have been

demonstrated among women with unexplained infertility compared to the general

population (Pandian et al. 2001). In addition, spontaneous pregnancies among these

women had elevated rates of obstetric complications.

Pregnancies achieved after IVF bear an increased risk of multiple pregnancy, the

rate of multiple pregnancy being approximately 25-35% (Koudstaal et al. 2000b;

Nygren and Nyboe Andersen 2001; Tiitinen et al. 2001), compared with only 1%

in naturally conceived pregnancies (Addor et al. 1998). Multiple pregnancy is,

according to many authors, the most serious complication of ART (Vilska et al.

1999; Tiitinen et al. 2001; Koivurova et al. 2002). The risk of multiple pregnancy

is related to the number of embryos transferred and not to IVF per se. Multiple

pregnancies, either conceived spontaneously or after IVF, increase the risk of many

complications, the most important being low birthweight infants (Koudstaal et

al. 2000b). IVF twins have also higher rates of very low birthweight, discordant

birthweight, prematurity, neonatal morbidity and mortality and women with IVF

twins have an elevated risk of vaginal bleeding compared with women with

38

spontaneously conceived twins (Bernasko et al. 1997; Moise et al. 1998; Koudstaal

et al. 2000b). However, in some studies, twins conceived with ART did not have an

increased rate of low birthweight compared with spontaneously conceived twins

(Westergaard et al. 1999; Schieve et al. 2002).

In most earlier studies, the incidence of congenital and chromosomal anomalies

after IVF and ICSI has been comparable to that in the general population,

approximately 1.8-4.5% (Lancaster et al. 1995; Govaerts et al. 1996; Palermo et

al. 1996; Wennerholm et al. 1996; Johnson 1998; Tarlatzis and Grimbizis 1999;

Westergaard et al. 1999; Ludwig et al. 2001; Bonduelle et al. 1996, 1999, 2002).

No statistically signifi cant difference has been observed in major malformation

rates when ICSI and IVF children were compared (Bonduelle et al. 2002). In some

studies higher rates, such as 7.6% congenital malformations after ICSI, have been

reported (4.1% were classifi ed as major; Wennerholm et al. 2000). And a recent

report from Australia showed an increased rate of major congenital anomalies after

IVF (9.0%) and ICSI (8.6%), compared with 4.2% for naturally conceived infants

(Hansen et al. 2002). Signifi cantly increased rates of cardiovascular, urogenital,

musculoskeletal and chromosomal defects were demonstrated among infants

conceived with IVF, and musculoskeletal and chromosomal defects among infants

conceived with ICSI. The reason for these fi ndings may be the relatively small

number of patients included in the latter study as well as the follow-up of newborn

being extended to one year of age. Furthermore, infants conceived with ART had

more multiple major congenital anomalies compared with their naturally conceived

counterparts (Hansen et al. 2002). Other studies have also shown a slightly

increased rate of neural tube defect, alimentary atresia, omphalocele, hypospadia

and congenital heart anomalies (ASD, VSD or transposition of the great vessels)

among infants conceived with IVF or ICSI (Lancaster 1987; Bergh et al. 1999; Silver

et al. 1999; Ericson and Källén 2001; Koivurova et al. 2002).

Malformation rates in both IVF and ICSI multiples are signifi cantly higher than in

IVF and ICSI singletons (Wennerholm et al. 2000; Bonduelle et al. 2002). In addition,

a signifi cantly higher malformation rate has been shown in IVF twins compared

with ICSI twins (Bonduelle et al. 2002). It should be noted that follow-up of

children conceived with ART is often more detailed, and thus anomalies that would

likely have gone unnoticed in the general population might be detected in the

ART population, elevating congenital anomaly rates in some studies (Tarlatzis and

39

Grimbizis 1999; Hansen et al. 2002).

In a recent study, no difference was observed in the rate of abnormal foetal karyotype

among ICSI (2.9%) and IVF (3.0%) foetuses (Bonduelle et al. 2002). However,

a small increase in sex-chromosomal abnormalities (1%) has been reported in

many studies, which could be explained by ICSI bypassing the natural selection

of spermatozoa and sub-optimal male gametes being used in ICSI (Bonduelle et

al. 1996; Govaerts et al. 1996; Tarlatzis and Grimbizis 1999; Macas et al. 2001; Ola

et al. 2001). A follow-up of 1987 children born with ICSI reported 0.83% sex-

chromosomal abnormalities, which is four times greater than the expected rate

of 0.19% (Bonduelle et al. 1999); these results are in concordance with several

other studies (Wisanto et al. 1996; Johnson 1998; Bonduelle et al. 1999; Tarlatzis and

Grimbizis 1999; Macas et al. 2001; Ola et al. 2001). A small increase in autosomal

aberrations has also been reported (Bonduelle et al. 1999). ICSI does not increase

the risk of chromosomal anomalies or congenital malformations, unless parents

have an abnormal karyotype or family history of some disease (Johnson 1998),

but the possibility of transmitting fertility problems of genetic origin to the male

offspring cannot be excluded (Tarlazis and Grimbizis 1999), although the risk is

marginal (Bonduelle et al. 1999, 2002).

In conclusion, the absolute risk for a serious malformation to occur after IVF

is, according to several studies, small; however, the results of some studies are

confl icting. ICSI may marginally increase the risk of sex-chromosomal abnormalities,

which are diffi cult to detect in routine examination of newborns (Ericson and

Källén 2001). The slightly increased risk for a congenital malformation after IVF

may partly be explained by maternal characteristics. Increased risk of hypospadia

may be explained genetically as due to paternal subfertility, while increased risk

of alimentary atresia might be connected to the monozygotic twinning process

(Ericson and Källén 2001). Multiple births can increase malformation rate to some

extent, and some malformations, such as persistent ductus arteriosus, may be related

to premature birth (Bergh et al. 1999).

Children born after IVF in Sweden have in one large study been shown to have an

increased risk of neurological disability, such as cerebral palsy, and an increased risk

of developmental delay (Strömberg et al. 2002). These risks are elevated mainly due

to the high frequency of twin pregnancies, low birthweight infants and premature

40

births among IVF children. However, long follow-up of children aged 6-13 years

conceived with IVF revealed no difference in school performance; 92% of these

children presented an encouraging outcome (Olivennes et al. 1997), and no large

differences in IVF children’s psychosocial development were shown compared

with naturally conceived children (Colpin and Soenen 2002).

The need for treatment in the neonatal care unit among IVF children is higher

primarily due to the increased rate of preterm and multiple births (Ericson et

al. 2002; Koivurova et al. 2002), and an increased rate of hospital care was also

present among full-term and singleton IVF children up to an age of six years

(Ericson et al. 2002). IVF twins did not have an excessive hospitalization rate

compared with other twins (Ericson et al. 2002). The reported stillbirth rate is

1.7% in ICSI and 1.3% in IVF pregnancies (Bonduelle et al. 2002). The neonatal

death rate was, in another report, 1.0% after IVF treatment (Bustillo and Yee

1999), but the Finnish study showed a perinatal mortality rate which was twice

as high in IVF children compared with national fi gures for the general population

(Koivurova et al. 2002).

The postnatal growth of children born with IVF is usually normal if the neonatal

period is uncomplicated (Wennerholm et al. 1991; Tarlatzis and Grimbizis 1999;

Ludwig et al. 2001). No excessive risk of childhood cancer has been found after

IVF treatment (Ericson et al. 2002). In a follow-up study of pregnancy outcome

following cryopreserved ET, fresh IVF-ET and spontaneous pregnancies until the

age of 5.5 years of the child, no differences were found when length, weight,

head circumference and chronic illnesses (at 18 months of age) were compared

(Olivennes et al. 1997; Wennerholm et al. 1998).

41

1. To assess uterine and spiral artery impedance in natural and IVF cycles

among women with unexplained infertility compared with women with tubal

infertility and to evaluate the usefulness of uterine artery impedance in the

prediction of outcome of treatment and pregnancy.

2. To compare the effi cacy of ovulation induction combined with intra-uterine

insemination, direct intraperitoneal insemination and timed intercourse in

treatment of unexplained infertility.

3. To study the long-term prognosis of couples with unexplained infertility.

4. To compare the obstetric outcome of treatment and spontaneous pregnancies.

III AIMS OF THE STUDY

42

IV PATIENTS AND METHODS

1. Study populations and protocols (Studies I, III, IV and V).

All patients had infertility of 2-12 years in duration, normal tubal patency, normal

semen parameters and documented ovulation. Women with minimal endometriosis

were included in the group with unexplained infertility in studies III and IV.

The study population and study design are shown in Tables 4 and 5.

Table 4. Study population.

Original I II III IV Vpublication

Mean age 31.9 (24-38) 34.4 ± 3.8 31.3 ± 3.3 31.3 ± 3.3 33.8 ± 3.2of women

Aetiology of unexplained tubal unexplained unexplained unexplainedinfertility endometriosis unexplained multifactorial No. of women 20 102 70 70 107

Control group 18 women with - - same women I: 445 spontanous tubal infertility with spontaneous pregnancies pregnancies II: 2377 IVF pregnancies

Treatment offered IVF IVF/ FET COH+ IUI/ COH + IUI/ IVF/ ICSI DIPI/ TI DIPI/ TI IVF, ZIFT

No. of cycles 76 140 233 83 118

No. of cycles 38 natural and 111 IVF COH+130 IUI, 83 118studied 38 IVF cycles 29 FET 37 DIPI, and 66 TI

Table 5. Study design and main outcomes in the original publications.

Original I II III IV Vpublication

Purpose of To compare uterine To evaluate To compare To study long- To study the study and spiral artery predictive value ovulation term fecundity and obstetric outcome impedance in natural of uterine induction + compare outcome after IVF among and IVF cycles artery impedance IUI, DIPI and TI in spontaneous and women with in two different during IVF on treatment unexplained populations obstetric outcome pregnancies infertility

Study design prospective prospective prospective retrospective register-based comparative follow-up randomized follow-up case-control study study study study study

Mean methods Doppler Doppler Clinical Clinical Register study measurement measurement study study

Main results No difference in Uterine artery Pregnancy rates: 64% conceived Obstetric uterine artery impedance did IUI: 7.7% during the study. outcome was impedance not predict DIPI: 8.1% The mean good. An between the pregnancy TI: 10.7% birthweight elevated rate groups outcome per cycle was lower in of breech ART pregnancies presentation was found

43

Study I

Twenty women with unexplained infertility and 18 women with tubal infertility,

all scheduled for treatment with IVF (or ICSI) were recruited to participate. The mean

age of women in the group with unexplained infertility was 32.6 (range 26-39) years,

and in the control group 31.9 (range 24-38 years; NS). The mean (± SD) duration

of infertility was 69 (± 32.4) months among women with unexplained infertility

and 44 (± 16.5) months (p<0.01) among women with tubal infertility. Infertility

was primary in 13 (65%) of the women with unexplained and in 7 (38.9%) of

women with tubal infertility (NS). We prospectively longitudinally measured the

uterine and spiral artery pulsatility index (PI) and peak systolic velocity (PSV) on

cycle days 11-12, 16-17 and 21-23 in the natural cycle and on days 1, 5, 10, OPU

and ET during the subsequent IVF cycle. The reproducibility of uterine artery PI

measurement was assessed by calculation of the coeffi cient of variation (CV). CV

was calculated by dividing the within-subject variance by the population mean.

Variances required for the calculation of CV were estimated by ANOVA.

Study II

A total of 102 infertile women were studied on the day of ET during the IVF

cycle or replacement cycle for frozen-thawed embryo transfer. Of these women, 56

(55%) had tubal infertility, 13 (13%) endometriosis, 23 (22%) unexplained and 10

(10%) were multifactorial. The mean age (± SD) of women was 34.4 (± 3.8) years.

The uterine artery impedance (PI and RI) was prospectively evaluated one hour

before ET and the impedance was compared with obstetric outcome in the

conceptional cycles. Of the 111 cycles studied, 82 were fresh (IVF-ET) and 29

frozen-thawed embryo transfers. Of these frozen-thawed ET, 20 were performed in

natural cycles and nine in substitution cycles. In the substitution cycles, goserelin

acetate (Zoladex; Zeneca, Macclesfi eld, UK), estradiol valerate (Progynova; Schering

AG, Berlin, Germany) and natural micronized progesterone were administered.

The predictive value of uterine artery impedance measurement for spontaneous

abortion, ectopic pregnancy, multiple pregnancy, birthweight, relative birthweight

and obstetric complications (IUGR, pregnancy-induced hypertension, preterm

birth) was studied. Relative birthweight was calculated as the difference between

actual and expected birthweight (according to sex and pregnancy duration)/ SD of

expected birthweight.

44

The uterine artery blood fl ow was defi ned as good if the uterine artery PI was < 3.0

(Cacciatore et al. 1996a).

We studied 70 couples with unexplained infertility of over 2 years in duration

during 1988-1992, including 23 women with minimal endometriosis. The mean age

(± SD) of women was 31.3 (± 3.3 years, range 23-38). The average duration of

infertility was 4.8 (range 2-12) years. Couples were offered superovulation treatments

combined with IUI, DIPI or timed intercourse (totally 233 treatment cycles) and

most couples received 1-4 treatment cycles. The stimulation protocol, pregnancy

rate, pregnancy outcome and complications of treatment were analysed.

A follow-up study was carried out 6 (range 4-7) years later. Information regarding

all treatment and spontaneous pregnancies was collected. Obstetric outcome

(birthweight, rate of SGA (<-2 SD) infants and preterm births (<37 weeks), Apgar

scores, obstetric complications and congenital malformations) was compared in

spontaneous and treatment pregnancies. The long-term prognosis of fecundity was

determined.

Study V

Altogether 107 women with unexplained infertility and 118 clinical pregnancies

after treatment with IVF or ICSI during 1.1.1993-30.3.1999 were studied. These

pregnancies resulted in 90 deliveries, of which 69 were singleton, 20 twin and

one triplet. Twenty-eight pregnancies (23.7%) resulted in a spontaneous abortion.

The multiple pregnancy rate was 23.3%. Two different control groups for the

deliveries and infants were chosen from the Finnish Medical Birth Register (MBR).

One control group included spontaneous pregnancies leading to birth (comprising

445 women and 545 children), which was matched according to year of delivery,

woman’s age, parity, number of foetuses and area of residence (Southern Finland).

The other control group included all pregnancies after ART treatment leading to

birth in Southern Finland during the study period (2377 women and 545 children).

The following factors were studied: maternal background, use of antenatal care,

gestational age at birth, birthweight, pregnancy and delivery complications, mode of

delivery, major congenital anomalies, infant’s outcome and need for intensive care.

Studies III and IV

45

Ovarian stimulation and preparation of spermatozoa for IUI and DIPI

(Study III)

Clomiphene citrate 50-100 mg (on cycle days 3-7) and human menopausal

gonadotrophin 75-150 IU (on cycle days 8-10 or 5-8) was used for ovarian

stimulation. The number of follicles was examined on day 10-12 during stimulation

with transvaginal sonography. Typically hCG (Pregnyl) 5000-10000 IU was also

given when the diameter of the leading follicle was at least 17 mm. Semen was

prepared by swim-up using the husband’s semen. Sperm was gently centrifuged

in Ham’s F 10 medium (HF 10, Gibco, Grand Island, NY, USA) supplemented with

human serum albumin. The spermatozoa were injected in 0.5 ml of medium into

the uterine or peritoneal cavity. Insemination was usually performed on cycle day

13 or 14.

Ovarian stimulation for IVF and ICSI (Studies I, II, and V)

Most patients were treated with the long protocol for ovarian stimulation.

A standardized ovarian stimulation protocol for IVF was used in all cycles. After

pituitary down-regulation with a gonadotrophin-releasing hormone analogue,

buserelin (Suprecur, Hoechst) or nafarelin (Synarela, Syntex), administered

intranasally usually beginning on cycle day 21, suppression was confi rmed by

transvaginal sonography. Ovarian stimulation commenced with daily injections

of gonadotrophins/FSH (Puregon, Organon, Oss, The Netherlands, or Gonal-F,

Laboratories Serono, Aubonne, Switzerland). The cycle was monitored by repeated

serum oestradiol assays and vaginal ultrasounds. When two or more follicles were

≥17 mm in diameter, human chorionic gonadotrophin (hCG, Pregnyl, Organon or

Profasi, Laboratories Serono) was administered and OPU was performed 36-38 hours

later. Embryo transfer was performed two days after follicle aspiration and luteal

phase was supplemented with vaginally administered progesterone (Lugesteron,

Leiras, Tampere, Finland).

In Study V, the starting dose of FSH was 100 IU in 1.7%, 150 IU in 66.1%, 200 IU in

11.6%, 225 IU in 19.5% and 300 IU in 1.7% of cycles.

46

Follow-up of IVF and ICSI pregnancies (Studies I, II and V).

If the quantitative hCG in serum was found to be positive 12 days after ET, the fi rst

ultrasound examination was performed fi ve weeks after ET. A clinical pregnancy

was defi ned as a pregnancy showing a visible gestational sac upon the ultrasound

fi ve weeks after ET; no biochemical pregnancies were included in the studies. All

pregnancies were followed at antenatal care units. Women attended the screening

ultrasound for examination of foetal morphology in weeks 16-18. The pregnancies

were followed at the delivery hospital and included repeated clinical examinations,

measurement of maternal blood pressure, urinary analysis, cervical status, foetal

cardiotocography (usually after 30 weeks of pregnancy) and repeated ultrasound

examinations to assess foetal growth and bioprofi le.

Preterm delivery was defi ned as delivery before 37 completed weeks. Small for

gestational age was defi ned as a birthweight below -2 SD according to Finnish

birthweight standards adjusted for gestational age and sex (Pihkala et al. 1989).

Pregnancy-induced hypertension was defi ned as a blood pressure of ≥140/90

mmHg (after 20 weeks of gestation), an increase in systolic blood pressure of ≥30

mmHg or an increase in diastolic blood pressure of ≥15mmHg (Gifford et al. 1990).

A major congenital anomaly is defi ned as a signifi cant congenital structural

anomaly, chromosomal defect or congenital hypothyroidism involved in a birth

with congenital anomalies. This does not include hereditary diseases and other

diseases not associated with congenital anomalies, dysfunction of organs or

tissues, developmental disabilities, congenital infections, isolated minor dysmorphic

features, normal variations and common less signifi cant congenital anomalies, which

are included in the exclusion list utilized by the Finnish Malformation Register

(Stakes 2001).

The Finnish Medical Birth Register (MBR) is a statutory nation-wide register, which

includes information on maternal background, pregnancy, delivery and perinatal

outcome. The MBR was used to get information on pregnancies and deliveries

and to select the two control groups (Study V). Diagnoses of malformations were

verifi ed from the Finnish Malformation Register.

47

2. Hormonal assays

Estradiol was quantitated using the Immuno 1 analyser (Tarrytown, NY, USA).

The detection limit of the assay is 37 pmol/l. Total variation is <6% at

concentrations of 270-5300 pmol/l. For progesterone, Spectria Progesterone (125 I)

Radioimmunoassay (Farmos Diagnostica, Turku, Finland) was used. The inter-assay

CV is 7-9% at concentrations of 1.6-27 nmol/l.

In FET cycles, the luteinizing hormone (LH) surge was determined with home

urinary kits (Clearplan, Unipath Limited, Bedford, England) to detect ovulation,

and women began testing 3-4 days before expected ovulation.

HCG was analysed with an immunofl uorometric assay (DELFIA, Wallac, Turku,

Finland) 12 days after ET. Inter-assay CV is <10% in the concentration range of

1-5000 IU/l.

3. Ultrasound and Doppler assessment (Studies I and II).

The examination began with inspection of the uterus and measurement of

endometrial thickness in the antero-posterior dimension. The transducer was then

angled to the right and left of midline in the sagittal plane to examine the number

and size of follicles in the ovaries. The uterine vessels were located with colour

Doppler sonography and the ascending branch was identifi ed lateral to the cervix,

at the level of the internal os. A gate was placed over the vessel, the width of the

sample gate (2-3 mm) was adjusted according to the vessel diameter and a blood

velocity waveform was obtained. When the signals passing the gate returned, the

Doppler frequency shift echoes were converted electronically by a mathematical

technique and displayed as a Doppler shift versus time waveform. The PI was

calculated using three consecutive uniform heart beats and averaged. The mean

value of uterine artery PI and RI/PSV from both ascending uterine artery branches

was used in calculations.

Blood vessels in the subendometrial region were located using the Power Doppler

and colour Doppler techniques. Areas of maximum colour intensity were selected,

a Doppler range gate applied and the pulsed Doppler function activated to assess

48

blood fl ow velocity. Peak systolic blood fl ow velocity waveforms were detected. No

correction was made for insonation angle since the angle of insonation for small

vessels cannot be determined (Zaidi et al. 1995).

All measurements in Studies I and II were performed between 7 and 9 a.m.

using US equipment (model ATL HDI 5000, Seattle WA, USA, model 515A; Hitachi,

Tokyo, Japan, or model SS-2000; Aloka, Tokyo, Japan) with 5.0-6.5 MHz transvaginal

transducer. The high-pass Doppler fi lter was set at 100 Hz. The average duration of

the procedure was 30 minutes.

4. Statistical analysis

Statistical analysis of the data was performed using the Statistical Package for Social

Sciences (SPSS 8.0.1., Chicago, IL, USA) in Studies I-IV. The SAS statistical package

was used in Study V.

The shape of the frequency distribution of continuous variables was evaluated by

means of histograms to determine whether or not it had a normal (symmetrical)

or skewed distribution, and by using the Kolmogorov-Smirnov normality test. In

normal distributed data, differences between means of the variables were assessed

by Student’s t-test, and in data without normal distribution, differences between

medians were assessed by the Mann-Whitney U-test. In cases of categorical data,

statistical signifi cance of differences in frequency between groups or comparisons

between proportions were assessed using the Chi-square test. The Wilcoxon signed-

rank test and Pearson’s correlation coeffi cient were used to analyse associations

between variables measured from the same patient on different days. Group

comparisons were performed with Fisher’s exact test, test for relative proportions

and Chi-square test, which were also adjusted by the Bonferroni method.

The level of statistical signifi cance in all studies was defi ned as p<0.05.

49

RESULTS

1. Uterine and spiral artery blood fl ow in unexplained infertility (Study I)

The mean resistance to blood fl ow in uterine arteries measured as PI was below

three in all women with unexplained infertility studied during both natural and IVF

cycles, and no differences were found when comparing their results with those

of women with tubal infertility. Uterine artery PI measurements had a mean CV

of 7.8%. With regard to uterine artery PSV, no difference was observed between

the groups. No difference was found in mean PI or PSV in spiral arteries when

comparing women with unexplained infertility and those with tubal infertility,

except on day 5 of the IVF cycle (Figure 2), when mean PI (±SD) was signifi cantly

lower (p<0.05) in the former (0.96 ± 0.25) than the latter (1.24 ± 0.30) group.

Figure 2. Comparison of spiral artery pulsatility index (PI) in women with unexplained and tubal infertility during spontaneous and IVF-cycles.

(n)

Values are mean ± SE. *p<0.05.

50

2. Prognostic signifi cance of uterine artery impedance (Study II)

No differences were found in the mean (±SD) uterine artery PI and RI between

normal intra-uterine pregnancies (PI 2.69 ± 0.71 and RI 0.88 ± 0.06) and those

resulting in spontaneous abortion (PI 2.71 ± 0.67 and RI 0.88 ± 0.05) or ectopic

pregnancy (PI 2.36 ± 0.54 and RI 0.85 ± 0.06). Nor were there any differences in

uterine artery PI and RI between women with obstetric complications (including

IUGR, pregnancy-induced hypertension or preterm birth) with a mean PI of 2.54 ±

0.47 and RI of 0.87 ± 0.04 compared with those with uncomplicated pregnancies

with a mean PI of 2.74 ± 0.78 and RI of 0.88 ± 0.06. No correlation was found with

uterine artery PI and RI and absolute or relative foetal birthweight. In singleton

pregnancies, no difference was observed for any obstetric outcome parameters

between cycles with PI>3.0 and PI≤3.0 (Table 6).

Table 6. Outcome of singleton pregnancies in relation to uterine artery blood fl ow. Data presented as mean (range), mean ± SD or actual number (% of all). Relative birthweight was calculated as the difference between actual and expected birthweight (according to sex and pregnancy duration)/ SD of expected birthweight.Intergroup differences not statistically signifi cant.

_________________________________________________________________

PI≤3 PI>3 (n=49) (n=14)_________________________________________________________________

Gestational age 276.9 (249-295) 277.4 (260-288)(days)

Birthweight 3365 ± 586.9 3369 ± 493.6(grams)

Relative birthweight -0.50 ± 1.1 -0.47 ± 1.1

Pregnancy complications 8 (16.3%) 3 (21.4%)

_________________________________________________________________

3. Comparison of ovulation induction combined with intra-uterine insemination, intraperitoneal insemination and timed intercourse (Study III)

The pregnancy rate per couple was 28.5% and 8.6% per cycle. The pregnancy

rates per cycle were equally good with superovulation combined with IUI (7.7%),

DIPI (8.1%) and TI (10.7%), as shown in Table 7. No differences in pregnancy rates

were found when comparing women with primary and secondary infertility. In

conceptional cycles, luteal phase progesterone was signifi cantly higher than in non-

conceptional cycles. The serum estradiol concentration, number of follicles before hCG

and concentration of sperm after swim-up were comparable in conception and

non-conception cycles. The duration of infertility did not infl uence the chance of

conception. Of the pregnancies, 85% occurred during the fi rst four treatment cycles

(Table 8). The following pregnancy complications were found in the study group:

two cases of ovarian hyperstimulation (0.9% of the cycles), one ectopic pregnancy

(5% of the pregnancies), seven multiple pregnancies (40% of the deliveries),

including one quintuplet pregnancy (5% of the pregnancies). In this pregnancy,

a selective fetal reduction was performed, and the pregnancy continued until week

34 of gestation when healthy twins were born.

Table 7. Outcome of treatment with superovulation in combination with insemination or timed intercourse.

_______________________________________________________________________

Intrauterine Direct Timed insemination intraperitoneal intercourse insemination_______________________________________________________________________

Number of cycles 130 37 66

Number of pregnancies 10 3 7

Delivery 7 3 5

singleton 4 2 3

twins 3 1 2

Spontaneous abortion 2 - 2

Ectopic pregnancy 1 - -

Pregnancy rate per 7.7 8.1 10.7treatment cycle (%) _______________________________________________________________________

Table 8. Relation between number of treatment cycle and pregnancy rate.

_______________________________________________________________________________

Cycle number No. of No. of Total no. of patients pregnancies pregnancies_______________________________________________________________________________

1 62 5 5

2 52 6 11

3 42 4 15

4 32 2 17

5 22 1 18

6 15 1 19

7 5 1 20

8 2 0 20

9 1 0 20_______________________________________________________________________

52

4. Course of ART-induced and spontaneous pregnancies(Studies IV and V)

The treatment pregnancies in Study IV included pregnancies achieved with

ovulation induction alone or in combination with IUI/ DIPI or IVF. Table 9

shows the respective rates of spontaneous abortion, ectopic pregnancies and

multiple pregnancies. Among singletons, the mean gestational duration was 39.6

weeks in treatment-related pregnancies, which was similar to that of spontaneous

pregnancies. The mean birthweight (±SD) of singletons was 3120 g (± 488) in

treatment-related pregnancies and 3387 g (± 470) in spontaneous pregnancies

(p<0.05). Table 10 shows a comparison of perinatal outcome in spontaneous and

treatment-related singleton pregnancies. In singletons, no difference was observed

in the rate of preterm delivery or small-for-gestational-age babies. Among women

with treatment-related twins and those with spontaneous twins, 66.7% and 100%,

respectively, were delivered preterm (NS). The rates of congenital anomalies were

18.2% in treatment related and 10% in spontaneous pregnancies (NS).

After IVF/ ICSI treatment, the rate of spontaneous abortion was 23.7% (Study V).

The multiple pregnancy rate was 23.3%, and all but one were twin pregnancies.

One triplet pregnancy that resulted from a two-embryo transfer occurred, and the

children were born healthy in week 33 of gestation.

Among singleton pregnancies, no difference was found in the mean birthweight, rate

of low birthweight (<2500 g) or very low birthweight (<1500 g) when comparing

the study group and control groups, as shown in Table 11. Nor were differences

found in gestational duration, major congenital malformations or perinatal mortality

among the groups studied. However, among singletons in the study group, more

term breech presentations (10.1%) occurred as compared with both spontaneously

conceiving women and all IVF women (p<0.01). The rate of pregnancy-induced

hypertension was signifi cantly lower among singletons in the study group (p< 0.05)

compared with other IVF singletons. Among study group singletons, the rate of

Caesarean section was 24.6%, which was comparable with the rates of control

groups.

53

Table 9 . Comparison of pregnancy outcome in spontaneous and treatment-relatedpregnancies in women with unexplained infertility. Data presented as % of all.

_______________________________________________________________ ART Spontaneous group pregnancy group_______________________________________________________________

Multiple pregnancies 25.0 2.8 *

Spontaneous abortion 21.2 10.0 NS

Ectopic pregnancies 6.1 8.0 NS_______________________________________________________________

* p< 0.05

Table 10. Comparison of perinatal outcome in spontaneous and treatment-related singleton pregnancies.

_____________________________________________________________________________ Spontaneous Treatment-related pregnancies pregnancies (n=35) (n=18)_____________________________________________________________________________

Mean gestational duration (days) 276.4 277.4

Preterm birth 1/35 (2.9%) 1/18 (5.5%)(<37 weeks of gestation)

Mean birthweight ± SD (grams) 3387 ± 470 3120 ± 488 *

Relative birthweight -0.33 -0.97 **

Mean 1 min Apgar score 8.7 8.7

Small for gestational age (<- 2 SD) 1/35 (2.9%) 2/18 (11.1%)

Perinatal mortality (%) 0.0 0.0

Congenital anomalies of newborn (%) _____________________________________________________________________________

*p<0.05 **p<0.01.

5.7 5.5

54

Table 11. Outcome of singleton IVF pregnancies in women with unexplained infertility. Intergroup differences not statistically signifi cant.

_____________________________________________________________________________

Study group Control Control group I group II (n=69) (n=345) (n=1901)_____________________________________________________________________________

Mean duration of gestation 39.3 39.7 39.4(weeks)

Preterm birth (< 37 weeks ; %) 5.8 10.1 6.8

Mean birthweight 3425 3438 3468(grams + SD) (± 621) (± 572) (± 593)

Birthweight<1500g (%) 1.5 0.0 1.2<2500g (%) 2.9 5.8 5.0

SGA (%) 2.9 2.9 2.4

Mean 1 min Apgar score 8.7 8.8 8.7

Apgar score 0-3 (%) 4.3 1.4 1.4 4-6 (%) 0 1.4 2.3 7-10 (%) 95.7 97.1 96.2

Mean umbilical arterial pH 7.24 7.24 7.25ph< 7.10 (%) 1.5 4.3 1.9pH< 7.20 (%) 10.1 11.6 11.9

Perinatal mortality (%) 1.5 1.5 0.7

Congenital anomalies 7.2 3.5 4.4of newborn (%)_____________________________________________________________________________

The obstetric outcome of the IVF twins among women with unexplained infertility

was similar as in both control groups. The rates of major congenital anomalies were

7.2% among singletons and 2.5% among twins, which were comparable with both

control groups.

55

VI DISCUSSION

1. Uterine and spiral artery impedance among women with unexplained infertility

Evidence suggests that uterine artery blood fl ow may be a factor affecting

successful implantation. Studies conducted among unselected infertile women both

in spontaneous and IVF cycles have shown poor implantation rates when the

uterine artery pulsatility index is over 3.0-3.3 or when there is an absence of end

diastolic velocities in the uterine artery Doppler velocity waveform (Coulam et al.

1994; Cacciatore et al. 1996a). All patients suffering from unexplained infertility

enrolled in our study had normal resistance to blood fl ow, i.e. PI below 3, at

all points during the spontaneous and IVF cycles. No differences in uterine

artery impedance were observed during the spontaneous or the IVF cycle when

women with unexplained and tubal infertility were compared. Therefore, under the

conditions of our study, uterine blood fl ow was not reduced among women with

unexplained infertility.

Some earlier studies have postulated that abnormal uterine perfusion might be

a contributing factor in unexplained infertility (Goswamy et al. 1988; Kupesic and

Kurjak 1993; Steer et al. 1994). Increased uterine artery impedance in midluteal

phase (Steer et al. 1994) and abnormal fl ow in spiral arteries have been suggested

as possible mechanisms for infertility in these women (Kupesic and Kurjak 1993).

But very few earlier studies have been conducted on uterine artery blood fl ow

among women with unexplained infertility. Steer and co-workers studied uterine

artery pulsatility index in 35 women with unexplained infertility as well as in

other subgroups of infertility. Limitations of their study were, however, that Dopper

assessments were only performed on day 21 of the unstimulated cycle and that

no evaluation of spiral artery impedance was conducted. The main results of their

study were in accord with our fi ndings; no signifi cant differences were observed in

the median PI for patients with different causes of infertility, although women in

all categories of infertility had signifi cantly higher PI than healthy women. Kupesic

and Kurjak (1993) investigated both natural and stimulated cycles, and also studied

spiral artery impedance, in contrast to the above-mentioned study. The spiral artery

PI was shown to be signifi cantly lower in unstimulated cycles than in hormonally

stimulated cycles. Limitations of this study were, however, that only patients with

male factor infertility were included as a study group and that ovarian stimulation

56

was performed with three different stimulation protocols: CC, CC combined with

hMG and hMG alone. Therefore, no direct comparison of results can be made

between their study and ours.

However, the results of Tinkanen and co-workers (1994) are in accord with our

results. In their study, the mean uterine artery PI was also normal among women

with unexplained infertility, both in the pre- and post-ovulatory period. In contrast

to our study, they showed that 15% of these women had a uterine artery PI over 2.8,

which was higher than among healthy women with no history of infertility. The use

of clomiphene citrate has also been shown to decrease the uterine blood fl ow in

the peri-implantation period among women with unexplained infertility (Hsu et al.

1995). In addition, another earlier study (Groutz et al. 1997) showed that the uterine

artery RI on day of hCG of the IVF cycle was signifi cantly higher in this patient

group as compared with women with tubal infertility. Our fi ndings differ slightly

from these studies. However, it is diffi cult to compare results because these studies

had different stimulation protocols and different days of Doppler measurement.

Furthermore, the information obtained from the study by Ziegler and co-workers

(1999) is limited because Doppler measurements were performed only among

healthy nulliparous women during natural cycles and they failed to measure spiral

artery impedance. Their study population also included both white and Asian

women. The study by Coulam and co-workers (1994), in addition to including IVF

cycles, also examined FET, COH, natural cycle-IUI and ovum donation cycles, and

therefore, their results are diffi cult to interpret. By contrast to the above studies,

our study protocol was new, with longitudinal evaluation of both uterine and spiral

artery impedance in women with unexplained infertility, with women with tubal

infertility serving as a control group.

Poor endometrial blood fl ow has in some studies predicted poor implantation

rate (Zaidi et al. 1995; Yang et al. 1999). Impedance to blood fl ow in the uterine

or spiral artery did not differ in women conceiving with IVF-ET compared with

the other women in our study. We did show that spiral artery PI on the 5th

day of gonadotrophin stimulation was signifi cantly lower among women with

unexplained infertility than among those with tubal infertility; however, on all

other days, the spiral artery impedance was comparable with the control group.

In addition, clinical pregnancy rates were comparable between these two groups:

57

40% for women with unexplained infertility and 33.3% for women with tubal

infertility, and therefore, it seems unlikely that the difference observed in spiral

artery impedance at the beginning of the IVF cycle would have clinical signifi cance.

We cannot exclude the possibility that this is a purely chance fi nding. As far as

we are aware, no earlier studies have focused on spiral artery impedance among

women with unexplained infertility. In conclusion, increased uterine or spiral

artery impedance is likely not an important factor in unexplained infertility since

no differences were found between the two study groups, neither during the

spontaneous nor during the IVF cycle, with the exception of day 5 of stimulation.

2. Prediction of pregnancy outcome with uterine artery impedance measurement

Comparing unexplained infertility and tubal infertility, we found no difference in

implantation rates during IVF treatment. Furthermore, no differences were present

in uterine or spiral artery impedance when comparing women who conceived with

IVF-ET with those who did not. Earlier studies are in concordance with this fi nding

(Tekay et al. 1995). However, this observation is in contrast to earlier fi ndings by

ourselves and other research groups, which show signifi cant differences in uterine

artery impedance between conception and non-conception cycles (Coulam et al.

1994; Tinkanen et al. 1994; Cacciatore et al. 1996a, 1996c). However, as all women

in our study had haemodynamic parameters within the normal range, this is not

a surprise. One reason for these fi ndings may be the limited size of the study

population.

This is the fi rst study in which preconceptional uterine blood fl ow and subsequent

pregnancy outcome have been related. Uterine artery PI and RI were unrelated to the

risk of spontaneous abortion or ectopic pregnancy. It seems that pre-implantation

uterine artery impedance does not infl uence trophoblast formation or placental

development. Uterine artery impedance was of no value in prediction of IUGR,

PIH or preterm birth among unselected infertile patients. Some studies have shown

that uterine and spiral artery resistance were lower at the end of the 5th week

in blighted ovum pregnancies (Alfi revic and Kurjak 1990), and the uterine artery

RI was lower in missed abortion compared with women with normal pregnancies

(Alfi revic and Kurjak 1990; Kurjak 1994). Uterine artery PI measurement at 12-13

weeks of gestation has been shown to predict subsequent hypertensive disorders,

58

SGA fetuses and gestational diabetes during pregnancy (van den Elzen et al. 1995).

However, uterine artery impedance measurements during the fi rst trimester in other

studies have not predicted pregnancy outcome (Frates et al. 1996). By contrast,

several studies show that the measurement of uterine artery blood fl ow in the

second and third trimesters may be valuable in predicting obstetric complications

(Strigini et al. 1995; Hofstaetter et al. 1996).

3. Treatment options of women with unexplained infertility

Ovulation induction in combination with insemination or timed intercourse was

an effective treatment for couples with unexplained infertility. The pregnancy rates

were equally good (7.7-10.7%) per cycle in all three treatment groups. Our results

are comparable with other studies reporting pregnancy rates of 8.7-15.3% per

cycle (Mascarenhas et al. 1994; Nuojua-Huttunen et al. 1999a; Sahakyan et al. 1999;

Goverde et al. 2000). Because the spontaneous pregnancy rate is 1-2% per cycle

among couples with unexplained infertility (Hughes et al. 2001), our ovulation

induction treatment must be considered satisfactory. No pregnancies began with

ovulation induction in the group of women aged over 36 years. The duration of

infertility did not infl uence the chance to conceive with this treatment. The majority

(85%) of the pregnancies began during the fi rst four treatment cycles, which is

in concordance with other studies (Nuojua-Huttunen et al. 1999a; Aboulghar et al

2001), reporting most pregnancies to occur within the fi rst three or four treatment

cycles. Several studies have demonstrated an improvement in pregnancy rates with

the addition of IUI to the ovulation induction protocol (Chaffkin et al. 1991; Guzick

et al. 1998), in contrast to our results. However, according to our fi ndings and other

studies no more than three to four ovulation induction cycles can be recommended

before proceeding to IVF (Aboulghar et al. 1999; Nuojua-Huttunen et al. 1999a;

Sahakyan et al. 1999; Goverde et al. 2000). The age of the woman is important

when deciding on the number of ovulation induction cycles and when selecting

the treatment schedule, consistent with other studies (Figure 3; Guzick et al. 1998;

Aboulghar et al. 1999; Sahakyan et al. 1999; Goverde et al. 2000). In the ovulation

induction cycles studied, the serum estradiol concentration, the number of follicles

and the concentration of sperm after swim-up were similar in conception and non-

conception cycles. The only predicting factor for conception appeared to be the

luteal phase progesterone concentration, which was signifi cantly higher in these

cycles.

59

The clinical pregnancy rate was 40% per cycle with IVF treatment among women

with unexplained infertility. This pregnancy rate is higher than usually reported in

Europe (Nygren and Nyboe Andersen 2001). Therefore, the prognosis for women

with unexplained infertility to achieve a pregnancy is good, both with ovulation

induction and with IVF. We recommend three cycles with ovulation induction

before proceeding to IVF. In addition, a cautious low-dose FSH treatment schedule

is recommended in ovulation induction.

4. Long-term prognosis of couples with unexplained infertility

Long-term fertility prognosis in unexplained infertility is favourable. Of the 70

couples studied, 45 conceived and 83 pregnancies began. The long-term pregnancy

Figure 3. Management of couples with unexplained infertility after completed infertility evaluation.

60

rate was 64% per couple during the follow-up period and as many as 60% of the

pregnancies began spontaneously after completed ovulation induction treatment.

Of the women studied, 31% achieved two pregnancies or more during the follow-

up. Infertility treatment that resulted in pregnancy included IVF (n=10), CC (n=1),

ovulation induction combined with IUI (n=12), DIPI (n=3) and TI (n=7). These

fi ndings are in agreement with earlier life table analyses, which have shown that

many couples with unexplained infertility will ultimately conceive also without

treatment (Collins et al. 1983; Fish et al. 1989; Jaffe and Jewelewicz 1991). Duration

of infertility of more than three years has in several earlier studies been the

major negative prognostic factor (Levene et al. 1992; Crosignani et al. 1993), but

in our study, the duration of infertility did not infl uence the couple’s chances of

conception. The pregnancy rates were also equally good in women with primary

and secondary infertility. Therefore, many couples with unexplained infertility

do ultimately conceive but do so over a longer period of time than the fertile

population.

5. Obstetric outcome of couples with unexplained infertility

The rate of spontaneous abortion was similar with ovulation induction (20%) and

IVF (23.7%). The ectopic pregnancy rate was 5% after ovulation induction and 0%

after IVF, these fi gures being within the normal variation of rates reported earlier.

Multiple pregnancy rate was slightly higher after COH (30%) than after IVF (23.3%).

Our multiple pregnancy rate after COH was higher than in another report from

Finland, which claimed a multiple pregnancy rate of only 17% (Nuojua-Huttunen

et al. 1999b). However, the multiple pregnancy rate with IVF was similar to

other reports from Europe (Nygren and Nyboe Andersen 2001). The intensity

of stimulation used for COH is related to the multiple pregnancy rate. Our

treatment protocol for COH included CC combined with hMG, yielding mostly

5-6 preovulatory follicles. Today, low-dose FSH is preferred, which results in 1-2

preovulatory follicles. Milder stimulation protocols can achieve similar success rates

with a lower proportion of twins. Therefore, stimulation has become more cautious

in recent years, using lower doses of gonadotrophins. In our clinic, the multiple

pregnancy rate has also decreased since this study was conducted.

Only a few studies have been carried out to evaluate possible differences in

pregnancy complications in different infertility subgroups. Pregnancies achieved

61

with superovulation did not carry an increased risk of obstetric complications,

other than a slightly reduced birthweight and a relatively high rate of multiple

pregnancies. The malformation rate after COH seems higher than in another Finnish

report (Nuojua-Huttunen et al. 1999b). One reason for this difference might be

a different classifi cation of malformations. After IVF, no difference was observed

when comparing the malformation rates with the control groups. Moreover,

obstetric outcome of IVF pregnancies after unexplained infertility was good, with

no differences in mean birthweight, gestational duration, Caesarean section or

perinatal mortality when compared with the control groups. IVF twins had a

similar obstetric outcome as spontaneously conceived twins. This is in contrast

with a recent study showing that IVF twins had lower average birthweights than

spontaneously conceived twins (Koudstaal et al. 2000b). We found signifi cantly less

pregnancy-induced hypertension (0%) among singletons of the study group than

among other IVF pregnancies, and signifi cantly more term breech presentation

(10%) compared with both control groups; as far as we are aware, such fi ndings

have not been reported earlier. Another recent report showed an increased rate

of pre-eclampsia, placental abruption and preterm labour among women with

unexplained infertility, which is also in contrast to our results (Pandian et al. 2001).

Obstetric interventions, including Caesarean section and induction of labour, were

more common in their study. In addition, while an increased rate of IUGR has

been shown among women with unexplained infertility (Wang et al. 1994; Dawood

1996), our fi ndings do not support this. However, in pregnancies after ovulation

induction, the mean birthweight was lower than in spontaneous pregnancies. No

differences were observed after IVF treatment when mean birthweight, incidence

of low birthweight or very low birthweight babies among women with unexplained

infertility were compared with those of spontaneously conceived pregnancies.

Therefore, according to our results, the obstetric outcome of pregnancies among

women with unexplained infertility is promising and generally comparable with

spontaneous and ART pregnancies. The only factor affecting pregnancy outcome

adversely is the increased risk of multiple pregnancies after ART treatment, which

can be reduced by low-dose gonadotrophin stimulation and by the transfer of

a single embryo in IVF cycles.

62

VII SUMMARY AND CONCLUSIONS

1. Uterine artery and spiral artery impedance were low during both natural

and stimulated cycles in women with unexplained infertility and were

comparable with the results of women with tubal infertility. Therefore, under

the conditions of our study, uterine artery impedance does not appear to

be an important factor in the aetiology of unexplained infertility. Predicting

obstetric complications and outcome with measurement of uterine artery

impedance during IVF treatment was not possible. Pre-implantation uterine

artery impedance evidently has no infl uence on trophoblast formation or

placental development.

2. Treatment with superovulation combined with IUI, DIPI and TI were all good

treatment options for unexplained infertility, with similar pregnancy rates of

approximately 9% per treatment cycle and 28.5% per couple and limited

treatment complications.

3. The long-term pregnancy rate was good, with 64% of women conceiving;

the majority of pregnancies during follow-up began spontaneously. Expected

management can therefore be considered if the woman is young (<30 years)

and duration of infertility has been short (<2 years).

4. Obstetric outcome of IVF pregnancies, both singletons and twins, was good

among women with unexplained infertility. Birthweight, gestational duration

and major congenital malformation rates were comparable with those of

spontaneous pregnancies. Therefore, IVF treatment in this patient group seems

to be a good option when treatment with ovulation induction has failed.

The only factor affecting pregnancy outcome is an increased risk of multiple

pregnancies, which can be reduced by the transfer of a single embryo in

selected cases.

63

ACKNOWLEDGEMENTS

All studies of this thesis were carried out at the Department of Obstetrics and

Gynaecology, Helsinki University Central Hospital, during the years 1995-2002.

I wish to express my sincere gratitude to the Head of the Department of Obstetrics

and Gynaecology, Professor Olavi Ylikorkala, MD, PhD, to the Administrative Head

of the Department, Docent Maija Haukkamaa, MD, PhD, and to the former Head of

the Department, Professor Markku Seppälä, MD, PhD, for providing me with excellent

working facilities and for their support and interest in my research.

I am deeply grateful to Professor Olavi Ylikorkala for suggesting the topic of this

study and his generous advice. His support and encouragement have been essential

for fi nishing this study.

My sincere gratitude is due to my two supervisors:

Docent Aila Tiitinen, MD, PhD, for guiding me in scientifi c research and introducing

me to the fascinating fi elds of infertility and endocrinology, and for her insightful

comments and constructive criticism in preparing these manuscripts.

Docent Bruno Cacciatore, MD, PhD, for teaching me how to perform Doppler

measurements of uterine and spiral arteries, introducing me to ultrasound research

and providing valuable comments on my work.

Professors Jorma Paavonen, MD, PhD, and Carl Gustav Nilsson, MD, PhD, are warmly

acknowledged for continuous support and interest in my study.

I am very grateful to Professor Outi Hovatta, MD, PhD, Department of Obstetrics and

Gynaecology, Karolinska Institutet, Huddinge University Hospital, Stockholm, for

familiarizing me with the fi elds of infertility and IVF, for giving me the opportunity

to work at the IVF unit at Huddinge University Hospital in 1998, and for teaching

me about hormonal stimulations, oocyte pick-ups and embryo transfers in IVF.

I am most grateful to Docent Anne-Maria Suikkari, MD, PhD, and Docent Aydin Tekay,

MD, PhD, for carefully reviewing the fi nal manuscript.

64

My warmest thanks are due to Docent Mika Gissler, PhD, Leena Reinikainen, MD,

and Jarna Moilanen, MD, PhD, for helping me with the statistical analysis. Their

expertise and friendly co-operation are very much appreciated.

Docent Erja Halmesmäki, MD, PhD, is acknowledged for her valuable comments on

one of the manuscripts.

Laila Selkinen, Nina Hedkrok, Raili Alanne, Leena Vaara and Teija Karkkulainen are

thanked for their kind assistance in all manner of practical things.

All patients are gratefully acknowledged for volunteering for the time-consuming

ultrasound examinations that made this research possible.

I also wish to express a special thanks to all my friends and colleagues at the

Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, for

companionship, friendly interest and support of my study. I thank all the midwives

and nurses at the Department for pleasant collaboration. I also thank my friends

outside the Clinic for their interest in my work.

Finally, my heartfelt thanks to my parents Mirjami and Erik Isaksson for standing

behind my decision to study medicine. I am very grateful to them and my sister

Kristina for ongoing support and love.

This study was supported by grants from the Helsinki University Central Hospital

Research Fund, Finska Läkaresällskapet, the Paulo Foundation for Medical Research,

the Clinical Research Institute of Helsinki University Central Hospital, the Perklen’s

Foundation for Medical Research, the Research Foundation of Orion Corporation

and the Finnish Foundation of Gynaecology and Obstetrics.

Helsinki, September 2002

Rita Isaksson

65

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