Female Infertility and Assisted Reproductive Technology

50 Female Infertility and Assisted Reproductive Technology S O N Y A K A S H Y A P , M D , F R C S (C) AND P A K C H U N G , M D , F A C O G Cornell Institute for Reproductive Medicine, Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY

I. Infertility

ovulation, sexual relations, sperm, normal sperm-cervical mucous interaction, an uninterrupted path for fertilization, normal fertilization, and a hospitable intrauterine environment for implantation. Failure at any of these stages may result in infertility. Indeed, interruption at any, several, or all of these stages provides the science for contraception. Approximately 50 to 60% of couple infertility is due to a female factor. Male factors account for another 40 to 50% of infertility. This chapter mainly addresses female infertility. We refer readers to the chapter by Hopps and Goldstein for male infertility. Ten to 15% of patients suffer from unexplained infertility (i.e., after all investigations, a specific cause for the infertility cannot be demonstrated). Taylor and Collins [1] suggested that that the incidence of unexplained infertility may decrease with increased sophistication in diagnostic abilities and time. For example, the incidence of unexplained infertility dropped from 22% before 1940 to 14% after 1980. Evers [1] reported that before 1900, virtually all infertility was "unexplained." Among female factors for infertility, anatomic reasons (uterine and tuboperitoneal diseases) account for about 40%. Another 40% can be caused by ovulatory dysfunction. Other factors may include immunologic factors, impenetrable cervical mucous, luteal phase defect, or medical conditions (e.g., diabetes mellitus), which may interfere with implantation.

Infertility is defined as the inability to achieve conception after 1 year of regular, unprotected intercourse. Healthy young couples having regular, unprotected intercourse may expect a 25% chance of pregnancy per cycle. Statistically, if cycle length is every 28 days, one would expect 98% of couples to achieve conception after 1 year. In fact, 85% of such couples achieve pregnancy in 1 year. Variability in cycle length, coitus, and perceived pregnancy (i.e., biochemical pregnancy) may account for the discrepancy. Fecundability is the percent chance of achieving a pregnancy per cycle, whereas fecundity is the percent chance of achieving a live birth from a pregnancy per cycle. Fertility clearly diminishes with age. This has been demonstrated repeatedly. Each female fetus is endowed with a fixed number of oocytes (6 to 7 million) by 20 weeks' gestational age. Through atresia, this number drops exponentially to 300,000 to 400,000 by menarche and 25,000 by age 37 to 38. Such changes are reflected in the average time to pregnancy. Assisted reproductive technology (ART) success rates also diminish drastically with age. Oocyte donation studies illustrate that in-vitro fertilization (IVF) success depends on the age of the donor rather than the recipient, and that oocyte age and health are stronger predictors of outcome than uterine senescence. Donor insemination programs also reflect the fact that female age predicts outcome. The percentage of couples presenting for infertility therapy has increased in the last 15 years, although the percentage of couples apparently suffering from infertility (10 to 15%) has not changed. The average age of marriage may be increasing, but even married couples tend to delay childbearing. Also, awareness of subfertility and potential therapeutic options have increased. Therefore, although the traditional definition of infertility has been failure to conceive after 1 year of unprotected intercourse, this definition should perhaps be amended for the current societal demographics. Older couples should perhaps be referred earlier for infertility workup and therapy because there is a more urgent need to "beat the biologic clock." Success rates, even with reproductive technology, are extremely limited in most centers for women older than age 40. Infertility may be further classified into primary and secondary infertility. The former describes patients who have never conceived, and the latter describes patients who have previously conceived but who have not been able to conceive the number of children wanted.

1. Uterine Factors

A normal uterine cavity facilitates implantation. Intrauterine filling defects including submucosal myoma, polyps, uterine synechiae (Asherman's syndrome), and uterine septae are recognized as deterrents to pre-embryo implantation. Uterine defects can be identified by hysterosalpingogram (HSG) (the gold standard), saline infusion sonography, or hysteroscopy. Although hysteroscopy is generally considered a surgical procedure requiring anesthesia, some centers offer office hysteroscopy for diagnostic or therapeutic purposes. 2. Tuboperitoneal Defects

Tuboperitoneal defects are common causes of infertility. Careful history may elicit a precipitating event. A history of sexually transmitted diseases, such as chlamydia or gonorrhea, suggests tubal disease. Westrom's [2] classic data delineates a dose-response effect. The incidence of infertility following one episode of laparoscopically documented pelvic inflammatory disease is 12%, two episodes are associated with a 23% infertility rate, and 54% of patients with three episodes of pelvic inflammatory disease will suffer from infertility. Pelvic inflammatory disease increases the risk of ectopic pregnancy (1% in the general population) in both natural and ART conceptions. (2- to

A. Cause and Evaluation

Fertility requires absence of pathology on both the male and female sides. Prerequisites to conception include normal Principles of Gender-Specific Medicine

550

Copyright9 2004by Elsevier. All rightsof reproductionin any formreserved.

CHAPTER 5 0 . FEMALE I N F E R T I L I T Y AND A S S I S T E D R E P R O D U C T I V E T E C H N O L O G Y

6-fold) [3]. Half of all patients with tubal factor infertility may not report antecedent pelvic infection. Patients should be questioned for a history of unexplained abdominal pain and fever. Elevated serum chlamydial IgG titers are often found in patients with tubal factor infertility; however, the reported sensitivity and specificity varies [4]. Tubal disease may result from alternative causes such as postpartum endometritis, diverticulitis, chronic or acute appendicitis, or pelvic adhesions from previous surgery. One recent epidemiologic study, however, disputed the traditional dictum that ruptured appendices may result in later tubal infertility because of adhesions and scarring. Interval sterilization, particularly with bipolar coagulation, if it fails is also likely to result in ectopic pregnancy [5]. Conditions such as endometriosis may also cause tuboperitoneal defects. Endometriosis affects 5 to 10% of the general population, but up to 25% of infertility patients have been diagnosed with endometriosis. Minimal to mild endometriosis may be associated with actively secreting lesions, which may impair tubal transport, oocyte quality, or ovum pick-up. Moderate to severe endometfiosis results in anatomic distortion, space-occupying ovarian lesions, extensive adhesions, and scarring. At least one well-conducted randomized controlled trial demonstrated that ablative treatment of minimal to mild endometriosis is associated with a modest increase in fecundability when surgery is followed by expectant management [6]. No randomized controlled trials are available regarding pregnancy outcomes following surgical correction of severe endometriosis versus IVF. However, medical management does not appear to increase reproductive potential, although it is associated with amelioration of symptoms. Hysterosalpingogram has traditionally been the gold standard for tubal evaluation. Laparoscopy, however, has the advantage of more precise observation and documentation and the potential to correct or improve anatomic abnormalities and ablate endometriosis. Laparoscopy does carry the minimal but real risks of surgery including anesthesia, infection, bleeding, and injury to surrounding structures. HSG has the advantage of being an outpatient procedure and has been documented to be of therapeutic benefit as well. Patients who undergo HSG have increased pregnancy rates in the first 6 months following the procedure [7]. If specific tubal information is not needed, as in preparation for IVF, a saline sonogram can replace the HSG, as discussed previously. A saline sonogram is a very sensitive test to detect intrauterine filling defects and is extremely specific for a normal cavity. 3. Ovulatory Disorders Ovulatory disorders are another cause of female infertility. Ninety-five percent of women who have regular menstrual cycles (24 to 32 days) are ovulatory. The most common anovulatory problem in young women is polycystic ovarian syndrome (PCOS). This is a syndrome characterized most frequently by the triad: oligo-/amenorrhea, obesity (although at least 10% of patients with PCOS are lean), and hirsutism. The diagnosis is clinical. However, characteristic biochemical abnormalities may exist that include hyperandrogenism (often with normal total testosterone but elevated free testosterone and/or dehydroepiandosterone sulfate [DHEAS]), increased estradiol levels, increased luteinizing hormone (LH) to follicle stimulating hormone (FSH) ratio greater than 2:1, an altered lipid profile, and

551

an elevated fasting insulin:glucose ratio resulting from insulin resistance. Patients with severe PCOS may present with hyperandrogenism, insulin resistance, acanthosis nigricans syndrome (HAIR-AN syndrome). This subset of PCOS is at increased risk for health sequelae other than infertility, such as cardiovascular disease, hyperlipidemia, diabetes mellitus type II, endometrial cancer (in the absence of regular withdrawal bleeds), and breast cancer. If they become pregnant, they may be at increased risk for pregnancy loss, gestational hypertension, and gestational diabetes. Fortunately, patients with PCOS in general have an excellent prognosis to achieve pregnancy with the use of ovulation induction techniques such as clomiphene citrate (CC) and controlled ovarian hyperstimulation using gonadotropins. Insulinlowering agents such as metformin have been shown to resume regular ovulation and menstruation in as many as 95% of patients [8]. Assisted reproduction, including IVF, can be used for extremely brittle PCOS patients who may tend to hyper-respond to controlled ovarian hyperstimulation. Hypothalamic anovulation and amenorrhea is common in anorexics, athletes, and at times of stress. Fortunately, hypothalamic amenorrhea also responds well to controlled ovarian hyperstimulation, although the underlying cause should be addressed. Thyroid disorders, particularly hypothyroidism, may be associated with anovulation. Thyroid-stimulating hormone (TSH) should be screened and thyroid supplements administered where appropriate. Hyperprolactinemia alone or in response to elevated TSH should be corrected in a patient with infertility. Agents such as cabergoline or bromocriptine may be used. All patients with anovulation should be questioned and examined for the presence of unusual headache, temporal visual field loss, and galactorrhea. Unexplained, elevated fasting prolactin levels may require a magnetic resonance imaging (MRI) to rule out micro/macro pituitary adenomas and more importantly any lesion outside the pituitary that may compress on the stalk. Microadenomas generally behave in a very benign fashion and rarely require intense follow-up. However, macroadenomas may require more sophisticated intervention with the appropriate endocrine and or neurologic consults. Medical treatment using bromocriptine is usually first-line management, but transsphenoidal resection may be used in certain cases. Euprolactinaemic patients who demonstrate galactorrhea and suffer infertility may also benefit from dopaminergic agents such as bromocriptine. The reason is that, although serum levels appear normal, prolactin has at least two isoforms. Several other sources of elevated prolactin and galactorrhea should be excluded that include psychotropic medications, excessive nipple stimulation, pregnancy, thoracotomy scars or other stimuli of the neural arc, ectopic prolactin production by tumors, and hyperestrogenic states (e.g., pregnancy or oral contraceptive medication) that can inhibit prolactin-inhibiting factor (dopamine). 4. Age and Diminished Ovarian Reserve Diminished ovarian reserve associated with age can obviously impede fertility. Nowadays, there are a higher number of women presenting with the complaint of infertility at an older age. Although age-related decline in ovarian reserve is usually not clinically perceivable, this very group of women deserves much more expeditious evaluations and an aggressive treatment plan.

552

SECTION 6 - REPRODUCTIVE BIOLOGY

To evaluate ovarian reserve, cycle day 3 FSH and estradiol levels and clomiphene challenge test can be used. An FSH level of less than 10 mlU/ml and estradiol level less than 60 pg/ml on cycle day 3 of the menstrual cycle suggest an optimal ovarian reserve. Patients with autoimmune processes (e.g., systemic lupus erythematosus, Hashimoto's thyroiditis) have a higher incidence of early ovarian failure. Gonadal dysgenesis should be considered in women with premature ovarian failure before age 30. These women require a karyotype. Women with ovarian failure before 40 are considered to have premature ovarian failure. Probability of conception decreases with age, although the fertile window (approximately 6 days before ovulation and 1 day after ovulation) does not change with age. Day specific probability of pregnancy drops 50% for women in their late twenties to their late thirties [9]. This statistic does not include the increased spontaneous abortion rate associated with sporadic genetic abnormalities associated with conception.

5. Cervical Factors Appropriate sperm-cervical mucus interaction is necessary for spontaneous conception. The usefulness of the postcoital test is the subject of much controversy. A properly timed postcoital test reveals that sexual relations have occurred, that ejaculation has been achieved, and that sperm is present in sufficient quantities. It may also give a clue to immunologic problems if the observed sperm are dead, nonmotile, or simply "shaking in place." Dead sperm may also result from the use of coital lubricants of which many are spermicidal. Ideally, intercourse is achieved periovulation, and the female patient presents for examination within 2 to 8 hours. The consistency of the cervical mucous is measured by the "spinnbarkeit" and a sample of mucus is observed microscopically for sperm. Intrauterine insemination has been demonstrated to improve pregnancy results when there are fewer than three sperm per high power field (suboptimal postcoital test).

6. Luteal Phase Defect Adequacy of the luteal phase can be measured by the sum of three postovulatory progesterone levels (days 5, 7, and 9 postovulation) greater than 30 ng/ml or a single level greater than 10 ng/ml. An endometrial biopsy is the gold standard. It should be scheduled as close to the expected menstrual period as possible. The pathologic dating of the biopsy is compared to the actual date of the biopsy, which is calculated from the first day of the subsequent menses (assigned as day 28). If there is a lag of more than 2 days in two consecutive biopsies, luteal phase defect is diagnosed. History of the male partner and semen analysis should also be obtained. For more detail about male factor infertility please refer to the chapter by Hopps and Goldstein.

B. Treatment for lnfertility Infertility treatment should be targeted at the specific cause.

1. Ovulatory Dysfunction If there is an underlying cause for anovulation or oligo-ovulation, such as thyroid dysfunction, treatment should be directed toward

that specific cause. Otherwise ovulation induction medications such as CC or gonadotropin should be considered. Clomiphene citrate can be used for ovulation induction in anovulatory or oligo-ovulatory patients or for superovulation in those who regularly ovulate. Patients who best respond to CC tend to be relatively well estrogenized and have intact pituitary function/ normal gonadotropin levels. Other patients who might benefit from CC are patients with luteal phase defects or unexplained infertility. Clomiphene citrate has both estrogen antagonist and agonist effects. It deceives the pituitary gland to produce more gonadotropins to stimulate follicular growth. Because of its estrogen antagonistic effects, it can potentially thin the endometrium lining and decrease cervical mucus production. The usual starting dose is 50 to 100 mg per day from day 3 to 7 or day 5 to 9. Once ovulation is attained with a certain dose, that dose should be maintained for several cycles before more aggressive treatment is attempted. Maximum daily dose used is 200 to 250 mg. The use of CC is not usually associated with intensive monitoring. If timed intercourse is anticipated, patient can monitor urine LH from day 10 (given a 28-day cycle). Intercourse is recommended for three consecutive nights starting the evening of the surge. If there is no LH surge detected by urine by day 14, vaginal ultrasound examination should be performed to determine follicular sizes. Human chorionic gonadotropin (hCG) triggering is then performed when the lead follicle is 18 mm or larger in diameter. If in utero insemination (IUI) is to be performed, patients can either monitor urine LH as described previously or be monitored starting day 10 by ultrasound examination. Once an LH surge is detected or lead follicle reaches 18 mm when hCG is given, patients are then scheduled for IUI for the following morning. The common side effects of CC include hot flushes, headaches/mood changes, and visual changes. Visual changes are an indication to discontinue treatment. Every patient who undergoes CC treatment should be counseled that there is a 12 to 15% multiple pregnancy rate, largely consisting of twins. If a patient does not respond to maximal doses of CC, prolonging the duration of CC treatment from 5 to 8 or 10 days can be considered. In PCOS patients, the use of metformin (Glucophage) may enhance the responsiveness of the ovaries to CC. Failure with the use of CC should be defined as to whether it is ovulation failure or conception failure. If a patient does not ovulate even with the highest dose of CC (200 to 250mg per day), the next step is injectable gonadotropins. However, if it is conception failure, the patient and physician should be aware that each treatment cycle with CC/IUI is only associated with an 8 to 12% pregnancy rate. Clomiphene citrate/IUI should be repeated up to three to six cycles (depending on the age and the anxiety level of the patient) before treatment should be escalated. The next step in ovulation induction is the use of gonadotropins: FSH and LH. They are either urine derived or manufactured using recombinant DNA technology. The starting dosage of gonadotropins is based on the patient's age, ovarian reserve, body mass index (BMI), and past stimulation history (if any). Because of its increased risk of hyperstimulation, patients have to be monitored very closely by ultrasound examination and estradiol level examination. Human chorionic gonadotropin trigger for ovulation should occur when follicles are 16 to 18 mm in size.

C H A P T E R 5 0 . F E M A L E I N F E R T I L I T Y AND A S S I S T E D R E P R O D U C T I V E T E C H N O L O G Y

About 36 hours after hCG administration, IUI can be performed. Luteal support with progesterone should be started 2 days after IUI. Gonadotropins are more potent stimulants to the ovaries than CC. Patients have to be counseled on the risk of multiple births and hyperstimulation. If more than three to four mature follicles are detected in any gonadotropin treatment cycle, consideration should be given to cancel the cycle or convert to IVF if it is an option. 2. Tubal Factors Tubal obstruction as a cause for infertility can be managed by tuboplasty or bypassing the tubes in IVF. Tuboplasty is usually accomplished laparoscopically. However, in any subsequent pregnancy, there is an increased risk of ectopic pregnancy. Tubal surgery is considered only in the absence of male and ovarian reserve factors. Otherwise, IVF is the option for treatment. In-vitro fertilization is discussed in a subsequent section of this chapter.

553

The evolving progression of using vaginal ultrasound-guided retrieval, controlled ovarian hyperstimulation for multiple follicular recruitment, optimal laboratory conditions, and micromanipulation techniques such as intracytoplasmic sperm injection (ICSI) to overcome male factor infertility have greatly improved the efficiency of ART. More recently, improved outcomes after embryo cryopreservation and blastocyst culture prevent embryo wastage and hold the promise of reducing the incidence of multiple births secondary to IVF. Preimplantation genetic diagnosis (PGD), as opposed to prenatal genetic diagnosis, allows pre-embryos to be biopsied and evaluated for a multitude of heritable diseases before they are transferred back to the uterus. Oocyte donation allows women with ovarian failure, poor oocyte health, or multiple failed IVF attempts to carry and deliver a pregnancy that carries the genetic contribution of the male partner. Gestational surrogacy offers the potential of women who have major uterine factor (e.g., congenital absence of the uterus) an opportunity to have their genetic children.

B. Indications for In-vitro Fertilization 3. Male Factors Please refer to the chapter addressing male factors in this book. 4. Cervical Factor If a hostile cervix is encountered as in the case of anti-sperm antibodies, bypassing the cervix with IUI is recommended. 5. Luteal Phase Defect The use of progesterone supplementation in the luteal phase can be considered. Some patients may benefit from CC to augment folliculogenesis. II. In-vitro Fertilization

A. History of In-vitro Fertilization The first attempts at mammalian IVF dated back to the 1800s. In 1930, Pincus documented the first embryo transfer in a rabbit. The first recorded successful IVF was also by Pincus in 1934 [10]. For several reasons, this record was considered controversial. Before the advent of phase microscopy, it would have been impossible by standard techniques to document fertilization. Also, parthogenic cleavage is possible in rabbit oocytes. The true proof of successful IVF is the live birth of an in-vitro fertilized pre-embryo. This did not unequivocally occur until 1954 under the direction of Thiabault [ 11 ]. The first attempts at human IVF occurred in the 1960s and were initially limited by timing of ovulation, single oocyte retrieval, and low fertilization rates in vitro. The birth of Louise Brown in July, 1978 marked the first live human birth from a human IVF conceptus. The second and third births occurred in Australia and the United States, respectively. The initial two births were the result of laparoscopic, single oocyte retrieval and IVF. Jones etal, however, used human menopausal gonadotropin (hMG) to stimulate multiple follicular maturation to increase the chances of having a viable pre-embryo for transfer [ 12].

1. Tubal Disease In-vitro fertilization was originally intended to treat tubal infertility. Previously, such patients had very poor prognosis. Complete tubal obstruction prevents spontaneous conception. Partial obstruction is often associated with bilateral disease and macro or microscopic defects in tubal anatomy and ciliary function. In these patients, attempts at conception via non-IVF route carry a significant risk (-~5 to 15%) of ectopic pregnancy. The risk of ectopic pregnancy in the general population is approximately 1%. In fact, the first efforts at assisted reproduction in mammals involved intrauterine, cornual ovarian transplantation to the site of the removed obstructed tube [13]. Patients with tubal infertility are generally considered very good prognosis IVF candidates, the reason being that they tend to be younger and have normal ovarian reserve and function. Tubal obstruction is a mechanical factor that is relatively easy to overcome by IVF. Nevertheless, recently we have recognized that the degree of tubal disease may affect conception rates with IVF. Several randomized controlled trials and at least one systematic review (of three randomized controlled trials) [15-18] indicated that IVF outcome can be affected by the presence of hydrosalpinges. It is generally believed that the fluid of the dilated tubes may be inflammatory in nature and therefore detrimental to early embryonic growth if it is effiuxed back to the uterine cavity after embryo transfer. Therefore, patients with hydrosalpinges can be optimized by either a salpingectomy or interruption of the tube (clip or cautery) via laparoscopy before IVF. Ectopic pregnancies are not completely avoided by IVF. Post embryo transfer, the embryo remains "floating" in the endometrial cavity for approximately 2 days. During this time, the embryo may travel and implant in the fallopian tube, cervix, or in the peritoneum of the abdominal cavity. The incidence of ectopic pregnancy after IVF may be as high as 4%. We recently reviewed the results at our center and found the incidence of ectopic pregnancy after fresh embryo transfer to be 0.868%. However, this incidence appears to increase with a history of tubal disease. In our study, 46% of these ectopic pregnancies

554

SECTION 6 - REPRODUCTIVE BIOLOGY

had a history of tubal disease. Of those patients with pre-existing tubal disease 67% had their ectopic pregnancy on the same side as the tubal disease [14].

2. Endometriosis

As discussed previously, endometriosis affects approximately 25% of infertile women as compared with 5 -to 10% of women in the general population. Pathologic diagnosis of endometriosis requires biopsy of lesions and demonstration of glands, stroma, and hemosiderin by histology. There are several staging systems to describe the severity of endometriosis (minimal, mild, moderate, and severe and stage I to IV). Recently, the American Society for Reproductive Medicine revised the classification system, based on observed findings at the time of surgery, to allow more consistent and accurate staging both for purposes of description and for following outcomes post treatment. Endometriotic lesions occur typically first on the ovarian surfaces, followed by the broad ligament and posterior cul-de-sac. In cases of severe endometriosis, lesions may be old and scarred. Therapeutic, options for women with minimal to mild endometriosis include expectant management, surgery and controlled ovarian hyperstimulation, and intra-uterine insemination. Unfortunately, as in many areas of reproductive medicine there is again little information available in the form of randomized controlled trials to suggest optimal management. In cases of minimal to mild endometriosis, Marcoux et al [6] demonstrated, in a well-conducted randomized controlled trial that surgical ablation of laparoscopically documented minimal to mild endometriotic lesions resulted in higher cumulative pregnancy rates (30% vs 17%) over a period of 36 weeks, versus expectant management without ablation. Treatment of advanced stage endometriosis remains challenging. No randomized controlled data are available comparing surgery with IVF, and such types of studies would be difficult to undertake. Most of the available data is in the form of less robust, retrospective, case-controlled, cohort, and case-series studies. Some authors have suggested conservative surgical therapy at the time of diagnosis to restore anatomy, followed either immediately, or after a period of observation, by IVF. We would recommend prompt ART in severe endometriosis, because endometriosis has been documented to recur at a rate of 10 to 20% per year post conservative laparoscopy. There is clearly an absence of robust data to delineate the effects of endometriosis on assisted reproduction. Endometriosis may potentially affect assisted reproduction in several ways. First, space-occupying lesions in the ovaries and anatomic distortion of the adnexa may interfere with both the number and quality of oocytes produced. There have been suggestions that the ability of pre-embryos to successfully implant in a hospitable endometrial environment can be affected in endometriosis. Immunologic factors such as cytokines, intedeukins, and natural killer cells also may interfere with host acceptance of the conceptus and such factors, if directed toward pathologic, ectopically growing endometrium may also attack normal uterine endometrium. In an effort to assess for a possible detrimental impact on the endometrium, Diaz et al [ 15] undertook a study in which donor oocytes were split between recipient patients with and without endometriosis. Here, pregnancy rates were similar; however

this study did not have sufficient power (57%) to exclude a difference. Other authors have suggested that oocyte quality may be affected in women with endometriosis and embryotoxic factors from endometriosis may affect implantation [16]. A large retrospective study suggested that outcome was as good for patients with endometriosis as for patients with tubal infertility and did not vary appreciably by stage. SART data supports the latter concept [17,18]. However, these studies may have lacked sufficient multivariate analysis to exclude the impact of confounding factors. In 2002, Barnhart et al [ 19] published a systematic review about the impact of endometriosis on IVF. They concluded that, after careful analysis of multiple confounding factors, endometriosis consistently adversely affected IVF outcome. Endometriosis patients were compared with patients with tubal factor infertility on all of the following parameters: peak estradiol levels, oocyte yield, fertilization rates, implantation rates, and clinical pregnancy rates. Overall, all factors were negatively affected. The overall pregnancy rates for endometriosis patients were consistently lower (odds ratio [OR] 0.56; 95% confidence interval [CI] 0.44 to 0.70). There was also a dose-response relationship to the severity of endometriosis and inverse relationship to pregnancy outcome. Pregnancy rates were better in patients with minimal to mild disease than in patients with moderate to severe disease (OR 0.46; 95% CI 0.28 to 0.74). The authors concluded that "patients with endometriosis should be referred for early, aggressive infertility treatment, including IVF, to increase the chances of conception." Whether removal of large endometriomas before WF improves the response to controlled ovarian stimulation remains controversial. Nevertheless, these patients may benefit from gonadotropinreleasing hormone (GnRH) agonist suppression before stimulation [20-22]. Because endometriomas provide a good culture media for bacteria, we, therefore, routinely use antibiotic prophylaxis at oocyte retrieval in patients who have endometriomas demonstrated during their IVF cycles.

3. Male Factor

Male factor infertility accounts for approximately 40 to 50% of couple infertility. In the past, the only available therapy for significant male factor infertility was therapeutic donor insemination. In 1992, however, the development of a technique called ICSI revolutionized the treatment of male infertility [23]. It is now possible to achieve conception with a single viable spermatid by direct, facilitated injection through the zona pellucida of the oocyte. A recent study suggested that clinically significant semen analysis cut-offs for spontaneous conception are as follows: concentration less than 13.5 million sperm/ml, motility less than 32%, and less than 9% normal morphology according to strict World Health Organization (WHO) criteria [24]. Even with conventional IVF, insemination rates are poor with motile sperm concentrations of less than 3 million per ejaculate [25]. With ICSI and, more recently, advances in sperm retrieval directly from the testes or epididymis even men with obstructive or nonobstructive azoospermia have the potential to father their own genetic offspring.

C H A P T E R 5 0 . F E M A L E I N F E R T I L I T Y AND A S S I S T E D R E P R O D U C T I V E T E C H N O L O G Y

The indications for ICSI are varied and differ slightly from center to center [26]. In general they include the following: (1) previous failed IVF fertilization, (2) sperm concentrations less than 2 to 5 x 106 sperm/ml, (3) motility less than 5%, (4) normal morphology less than 4% by Kruger's strict criteria, and (5) PGD because conventional insemination techniques may result in extra spermatozoa attached to the zona and therefore contaminate the sample for polymerase chain reaction (PCR) diagnosis. The factors that affect the success of ICSI have been shown to be relatively independent of the semen parameters and mostly dependent on factors such as techniques and experience of the embryologist performing ICSI, and also egg quality (age of the female). It is still too early to conclude on the absolute safety of the use of ICSI in IVF. Some studies have looked at sex chromosome aberrations and ICSI [32]. In our center, 15% of azoospermic males were found to have chromosomal abnormalities such as 47,XXY (Klinefelter syndrome). Also there was a higher incidence of aneuploidy in oligospermic men (3 to 6%). Y microdeletions studies of azoospermic or severely oligospermic men have also identified a higher incidence of nonkaryotypic genetic abnormalities (up to 13 %). Therefore, genetic screening of azoospermic males should be recommended before IVF/ICSI. The partners of male patients with congenital unilateral or bilateral absence of the vas deferens should be screened for cystic fibrosis as should the partners of patients with idiopathic epididymal obstruction because a large proportion of these men are carriers of the cystic fibrosis mutation. The topic of ICSI is addressed in more detail elsewhere in this book.

4. Fertility Preservation Most recently, IVF has also been used for the preservation of reproductive capacity for both men and women. Female cancer patients who face the prospect of decreased reproductive potential as a result of chemotherapy may consider embryo cryopreservation. Not only does embryo cryopreservation allow preservation of reproductive potential but it also allows couples to proceed with family planning at a time when the primary disease may be in remission or when pregnancy may not present a health risk to the mother. Recently, medications such as tamoxifen and letrozole have been investigated as protective agents for patients suffering from breast cancer who wish to undergo ovarian hyperstimulation and embryo cryopreservation before antineoplastic therapy [27]. Current research efforts are focusing on the preservation of ovarian tissue and oocytes (e.g., prereproductiveaged patients and those without a male partner). Ovarian reserve is the most important limiting factor for IVF. Oocyte donation was initially developed for patients with ovarian failure. Through oocyte donation, a woman may gestate and deliver a pregnancy that carries the genetic contribution of her male partner. Women who have gonadal dysgenesis, premature ovarian failure, or early ovarian failure benefit from this procedure. Also, couples who have experienced repeated IVF failure, either because of implantation failure or fertilization failure secondary to oocyte health, are candidates for oocyte donation. Oocyte donation may also be indicated in cases in which the mother carries a genetic risk/disorder for which PGD is not available.

555

In-vitro fertilization for the treatment of irreparable uterine abnormalities is also possible. Patients with congenital absence of the uterus (Mayer-Rokitansky Hauser syndrome) or patients who have undergone hysterectomy can undergo IVF with embryo transfer to a gestational surrogate resulting in birth of genetic offspring. Women who have medical disorders, which contraindicate pregnancy, can also conceive children through surrogacy. 5. Ovulatory Disorders Women with ovulatory disorders (hypogonadotropic hypogonadism or PCOS) typically respond well to ovulation induction. For those women who have such an exaggerated response that the risk of higher order multiples or ovarian hyperstimulation is significant, conversion from a controlled ovarian hyperstimulationIUI cycle to an IVF cycle is possible and can prevent cycle cancellation given the ability to restrict the number of embryos transferred. Success rates from these converted cycles appear comparable to cycles initially started as IVF. In addition, patients who fail to conceive despite an ovulatory response to ovulation induction agents are reasonable candidates for IVF, if other treatable causes of infertility have been excluded. We have found that patients with unexplained infertility, some of whom who have failed controlled ovarian hyperstimulationin utero insemination (COH-IUI) may benefit from IVF. As discussed earlier, the category of unexplained infertility may be considered "as yet" unexplained infertility before IVF. In-vitro fertilization may offer information on fertilization and early embryo development, which will not be available without IVF. Although a recent review of available literature by Evers [1] suggested that the benefit of IVF over controlled ovarian hyperstimulation for patients with unexplained infertility may be marginal, other studies demonstrated a higher success rate in patients with a diagnosis of unexplained infertility with IVF than with more conservative measures [28-30]. However, they all agreed that IVF may be beneficial by uncovering a cause such as fertilization failure or poor embryo development or quality. 6. Genetic Disorders Preimplantation genetic diagnosis allows the detection of significant genetic disease before embryo transfer and conception. Preimplantation genetic diagnosis is most commonly used for autosomal recessive and sex-linked disorders. Other indications for PGD include the diagnosis of aneuploidy (associated with age); assessment of patients with a history of recurrent abortions, especially if a balanced translocation has been identified in one of the parents; and the evaluation of embryos of women with repeated, unexplained IVF failure despite good embryo morphology. 7. Age Age is the strongest predictor of ovarian reserve. Reproductive success diminishes exponentially with advancing maternal age. The classic studies of the Hutterite population evaluated a society in which the average age of marriage is 22 and the only fertility barriers include lactational amenorrhea and a natural ageand parity-related decrease in coitus [31]. Donor insemination programs also demonstrate a female-dependent decrease in

556

fecundity. A recent epidemiologic study of almost 800 couples practicing natural family planning revealed that female fecundability begins to decline in the late twenties and male fertility demonstrated a lesser, although significant, decline in the late thirties. Biochemical measures of ovarian reserve are available that may predict a patient's prognosis and response to stimulation. Day 3 FSH and estradiol have been correlated with IVF success in terms of pregnancy rates, number of oocytes retrieved, and peak estradiol levels [32-34]. For younger patients, FSH and estradiol levels may more accurately reflect a patient's ovarian reserve than her age alone. However, it must be emphasized that normal FSH and estradiol levels in the older patient do not override the impact of chronologic age on outcome. The Clomid challenge test (CCCT) can also be used to prognosticate ovarian reserve. The FSH is measured on day 3 and then again on day 10 following administration of Clomid 100 mg po on days 5 to 9. Poor ovarian reserve is defined as a day 10 FSH level greater than 2 standard deviations above the mean. Navot et al [34] reported that of patients with an exaggerated response only 5.5% subsequently conceived versus 42% of patients with a normal response. Some authors have suggested that the CCCT might be more sensitive than basal FSH levels alone, but it is not clear whether basal estradiol levels were taken into account in these studies or whether basal FSH levels were assessed in more than one cycle.

C. Basic Evaluations Before In-vitro Fertilization The initial evaluation for IVF should include a basic infertility workup, as previously outlined, and a thorough history including a review of previous pregnancies, pregnancy outcomes, and fertility treatments including controlled ovarian hyperstimulation-IUI and IVF. Physical examination should assess abnormalities of the thyroid, galactorrhea, and the pelvic examination. The pelvic examination generally includes cultures for Chlamydia, Neisseria gonorrhoeae, and other organisms as indicated. Cervical cytology should be up-to-date. A sounding (trial transfer) of the endometrial cavity for depth, position, and accessibility should also be performed, once negative cultures have been obtained. An ultrasound assessment of the uterus, endometrial cavity, and adnexa should also be performed to rule out any abnormalities such as ovarian cysts. If previous HSG films are available, these should be reviewed for the presence of intracavitary defects and hydrosalpinges. Depending on the history, surgical correction of hydrosalpinges may be indicated. The male partner should also be evaluated. The evaluation of the male is addressed in further detail elsewhere in this book. Previously fathered pregnancies; history of congenital defects or infections, prior surgery including varicoceles, vasectomy, and vasovasostomy; previous semen analyses and/or tests for anti-sperm antibodies, social habits including the use of tobacco, alcohol, prescription medications, and nonprescription substances; exposure to chemicals, toxins, radiation, or extremes of temperature (e.g., saunas, hot tubs); and sexual function should be questioned on history. A semen analysis should be obtained before IVF. Semen cultures may be performed as indicated.

SECTION 6 - REPRODUCTIVE BIOLOGY

D. Protocols for Ovarian Stimulation for In-vitro Fertilization The first IVF baby (Louise Brown, England) resulted from harvest of a single oocyte in a spontaneous, nonstimulated menstrual cycle [12]. The second IVF baby was born after a similar cycle in Australia. The third baby, the first North American baby, was born in the United States. Human menopausal gonadotropin was used to achieve multiple follicular development and therefore improved the number of oocytes retrieved and the number of embryos formed. Ovarian hyperstimulation protocols have since been used to maximize the efficiency of the IVF process. Natural cycle IVF has been limited by relatively poorer success rates. Natural cycle IVF involves the retrieval of a single oocyte, which effectively eliminates the ability to select and/or cryopreserve embryos and limits the pregnancy rates to the implantation rate of a single conceptus. In addition, ancillary reproductive technology procedures such as PGD and ICSI optimally require the harvest of multiple mature oocytes. Moreover, patients undergoing natural cycle IVF may ovulate spontaneously before oocytes retrieval. As described later, the introduction of GnRH antagonists may, however, avoid cancellations resulting from premature LH surges and improve success rates following natural cycle IVF. Nevertheless, natural cycle IVF remains an option for patients who respond poorly to ovarian stimulation (i.e., produce only one or two follicles) or, more importantly who have medical reasons to avoid supraphysiologic estradiol levels. The use of ovulation induction medications such as CC and, more significantly, gonadotropins (hMG and both urinary and recombinant FSH) have allowed the recruitment of multiple oocytes per IVF cycle, improving the odds for fertilization, increasing the number of embryos available for selection and transfer, and improving pregnancy rates. Clomiphene citrate is seldom used alone for IVF but can be used in combination with gonadotropins.

1. GnRH Agonists/Antagonists The development of the GnRH agonists has greatly enhanced the efficiency of IVF. Before the routine use of GnRH agonists, premature luteinization accounted for cancellation of up to 20% of IVF cycles [35]. Currently fewer than 2% of cycles are canceled because of premature LH surges [36]. For over a decade, GnRH agonists have been used in most ART stimulation protocols. Native GnRH is a decapeptide produced in the arcuate nucleus of the hypothalamus and first characterized in 1967. However, GnRH agonists (e.g., leuprolide acetate) and more recently GnRH antagonists did not become available until much later. The GnRH agonists are produced by substitution of the amino acids at positions 6 and 10. Native GnRH has a halflife of 2 to 4 minutes. The substituted agonist has half-lives of up to 3 hours. Agonists result in pituitary desensitization through prolonged receptor occupancy. However, the initial effect of GnRH agonists is a flare through the increased release of stored gonadotropins. This effect is especially noticeable if given during the early follicular phase. After the initial flare, suppressive effect of the GnRH agonist, which is desired for

CHAPTER 5 0 . FEMALE I N F E R T I L I T Y AND A S S I S T E D R E P R O D U C T I V E T E C H N O L O G Y

IVF to prevent early luteinization, is observed as early as after 1 week of administration. GnRH agonists do not block pituitary gonadotropin production completely, and small LH pulses may still be observed. GnRH agonists can be used in long, short, or ultra-short protocols, as described later, to maximize the benefit of their differential effects dependent on dose and duration of use. Development of well-tolerated, effective GnRH antagonists occurred more recently. The second-generation compounds exhibit side effects of histamine release and potentially anaphylaxis. Third-generation compounds have now become available for clinical use in the United States. Side effects appear to be mainly limited to those of estrogen withdrawal and localized, short-duration erythema at the injection site. GnRH antagonists have altered amino acids at positions 6, 10, 1, 2, 3, and 8. They inhibit pituitary gonadotropin output through competitive inhibition of the pituitary GnRH receptors but, unlike the agonists, are not accompanied by the flare effect (i.e., the suppression is immediate). The half-life is 6 to 30 hours for the nondepot form. The half-lives of the available antagonists suggest that minimal GnRH antagonist activity should be available in the peri-implantation window [37]. The debate regarding the importance of LH for ovarian stimulation is ongoing. The GnRH agonist does not completely abolish endogenous LH production, whereas the antagonist does. At many centers, antagonist cycles are supplemented with exogenous LH (e.g., hMG) at the time of initiation of the antagonist, if recombinant FSH is being used as the sole initial agent. Without adjuvant LH, estradiol levels may drop, although clinical significance of this phenomenon is not clear [38]. No adverse effects on children born from GnRH antagonist cycles have been reported [39]. In-vitro fertilization medication protocols are optimally selected according to a patient's age, ovarian reserve, body mass index, and history of prior response to ovarian stimulation. For patients who are likely to have a good response, one of the objectives is to avoid overstimulation, including full-blown ovarian hyperstimulation syndrome (OHSS), which is potentially lifethreatening. One commonly used protocol is the long GnRH agonist protocol. Here, the agonist is initiated 1 week following ovulation (e.g., documentation of the LH surge). Once adequate suppression has been documented by withdrawal bleeding and appropriately suppressed estradiol, exogenous gonadotropins are initiated at a dose and in a combination tailored to the individual patient. A typical starting dose is three to four ampules (225 to 300 IU) of FSH and/or hMG per day. Either a fixed, step-up or a step-down (which we recommend) protocol may be used (i.e., the gonadotropins can be started at a low dose and adjusted according to estradiol levels) or a protocol with a higher starting dose can be employed with the dosage decreased according to the response. Once the lead follicles attain a mean diameter of approximately 16 to 18 mm, hCG is administered usually at a dose of 5000 to 10,000 IU. This allows maturation of the oocytes (resumption of meiosis) for retrieval 34 to 36 hours later. Patients are advised not to try to conceive in the peri-ovulatory window of the cycle, when they are to start the agonist. The latter may rescue the corpus luteum and therefore rescue a very early pregnancy. Although it is prudent to avoid administering

557

GnRH agonist during a known pregnancy, GnRH agonists taken inadvertently have not demonstrated any adverse impact on the very early pregnancy.

2. High Responders Several stimulation approaches have been used in an effort to improve outcome in the anticipated high responder (young, PCOS, history of high response) who is at increased risk for OHSS. The objective is to suppress ovarian response to allow only 5 to 15 oocytes to develop and to maintain an estradiol level less than 3000 pg/ml at the time of hCG administration. Dual suppression with both an oral contraceptive pill overlapping with a GnRH agonist has been shown to attenuate the response to stimulation and to reduce the risk of excess numbers of oocytes and excessive estradiol concentrations. Lower starting doses of gonadotropins are administered in these patients as well (e.g., 150 IU/day).

3. Poor Responders The poor responder poses a far greater challenge. The true test of ovarian reserve is the response to ovarian hyperstimulation. The approaches to stimulation of the poor responder are generally 3-fold: (1) to maximize exogenous gonadotropin stimulation by increasing the number of ampules of gonadotropins prescribed, (2) to prevent ovarian suppression by avoiding the use of luteal GnRH agonist, and (3) to maximize the simultaneous endogenous pituitary gonadotropin complement by using protocols that produce an early follicular, pituitary flare effect in addition to maximal exogenous injectable gonadotropins. E. The In-vitro Fertilization Procedure

1. Oocyte Retrieval Oocyte retrieval is generally performed 34 to 36 hours post hCG administration. Either urinary hCG or recombinant hCG may be used. The interval between hCG and retrieval allows resumption of meiosis I. The first human successful IVF attempt occurred in 1944 [40]. The eggs were retrieved during laparotomy, between cycle days 10 and 12, and then incubated in human serum for 27 hours. Hopkins et al first introduced gynecologic laparoscopy in 1954 [12]. In 1968, Steptoe [41] published an article about ovulation and laparoscopy. Steptoe and Edwards [42] then collaborated to develop a laparoscopic method for timed oocyte retrieval following superovulation and hCG administration. Transvaginal ultrasound has since become the standard route of oocyte harvest and requires minimal anesthesia and recovery time. Another advantage of ultrasound-guided retrieval is that inadequate ovarian access is an extremely rare phenomenon, even in patients with extensive previous surgical history. The patient is prepped and draped in the dorsal lithotomy position under intravenous sedation. Antiseptics may be toxic to oocytes and are therefore prohibited in some centers. A transvaginal ultrasound probe with a high frequency transducer (5 to 7 MHz) and needle guide is used to identify the follicles and align the follicles in their largest diameter. The follicles are then aspirated under negative pressure (100 to 120 mm Hg) with flushing of the tubing following each withdrawal of the needle, to maximize

558

SECTION 6 - REPRODUCTIVE

oocyte recovery. "Empty follicle syndrome" is a clinical scenario in which, despite the presence of ovarian follicles, no oocytes are retrieved at harvest; although this could rarely be due to technical difficulties, most often it occurs when the patient fails to appropriately administer the hCG. We therefore measure serum LH and/or hCG levels the day before retrieval to ensure adequate hCG exposure. The debate regarding the need for prophylactic antibiotics is ongoing. We typically advocate a 4-day course of oral tetracycline started immediately following retrieval. We routinely use pre-retrieval intravenous antibiotic prophylaxis (e.g., cefoxitin) for high-risk patients (i.e., with a history of pelvic inflammatory disease and/or endometriomas) because it has been shown that endometriomas are a risk for abscess formation postretrieval. Postoperative complications are reported to occur in 0.3 to 3% of cases. The most common complication postretrieval is pelvic infection. Bleeding is also a risk and may result from injury to uterine, vaginal, infundibulopelvic, or iliac vessels as well as from the ovary itself whose vascularity is increased during stimulation. Injury to abdominal viscera is extremely rare but possible. In addition, anesthesia carries some inherent risks.

2. In-vitro Insemination and Fertilization As described previously, harvested oocytes exhibit various stages of maturity and, therefore, require varying intervals of preincubation before insemination. For mature oocytes (metaphase II), the oocytes are incubated briefly and insemination is performed at approximately 4 hours (range 2 to 8) following retrieval. A semen sample should be obtained by masturbation just before or after retrieval. It is usually collected in a sterile plastic jar or a Silastic condom. The sample is allowed to liquefy at room temperature before preparation. Two methods are commonly used for sperm preparation: the swim-up method or the gradient centrifugation method. The objective is to isolate a highly motile fraction of sperm for insemination. The highly motile fraction is then incubated in a high-protein supplemented media for 30 minutes to 4 hours to initiate capacitation. Generally each oocyte is incubated with between 50,000 to 200,000 motile sperm for a period of 12 to 18 hours at 37~ 5% CO 2 in air, and 98% relative humidity. The acrosome reaction, which is necessary for the spermatozoa to penetrate the zona pellucida, is initiated by contact between the zona pellucida and the sperm. Exocytosis of cortical granules from the ooplasm (cortical reaction) causes the zona pellucida to become relatively refractory to polyspermy. Occasionally incubation with greater than 200,000 sperm per oocyte is undertaken in male factor cases to improve fertilization rates. This practice can result in a higher incidence of polyspermy. Sperm penetration of the oocyte induces oocyte activation and initiates the second meiotic division, which then divides the chromatids between the oocyte and second polar body. Oocytes are evaluated for fertilization at 18 hours post insemination. The presence of two pronuclei, one each from the oocyte and spermatozoa, and two polar bodies in the perivitelline space indicates normal fertilization. Polyploidy occurs in 5 to 10% of IVF embryos with 1 to 2% in mature oocytes and up to 30% in immature oocytes. In addition

BIOLOGY

to polyspermy, polyploidy may result from digyny, with origin of the extra chromosomal complement from the oocyte, which may occur because meiotic spindle errors or failure to extrude a polar body. These events are more common in aging oocytes or immature or postmature oocytes. Intracytoplasmic sperm injection may result in a polyploid embryo because of digyny (retention of the second polar body). The process of fertilization takes approximately 24 hours and is completed with the initiation of the first mitotic cleavage.

3. Embryo Transfer Embryo transfer is most commonly performed after 72 hours (day 3 postretrieval). "Blastocyst transfer" is generally performed at 120 hours (day 5 postretrieval). Blastocyst transfer is detailed later; the principal advantage of blastocyst transfer is the replacement of fewer embryos (generally one to two), given their apparent higher implantation potential. Transfer of fewer day 3 embryos reduces the incidence of higher order multiple gestations. Pre-embryos transferred on day 3 have generally cleaved to six to eight cells. Techniques for grading of the quality of embryos vary from center to center. The objective of embryo transfer is to maximize the chance for pregnancy while limiting the number of multiple gestations. Both of these outcomes are directly correlated with the number of pre-embryos transferred [43]. The optimal number of embryos to transfer is individualized, based on the individual's expected implantation rate per embryo. Maternal age and embryo quality are important factors determining the implantation potential for each embryo [44]. Some centers calculate the number of embryos for transfer on the basis of a cumulative embryo score. The cumulative embryo score is derived from morphologic analysis of the embryo and the number of blastomeres. Maternal age is also an important predictor for implantation potential. Fewer embryos are generally transferred to younger patients, and some authors advocate the replacement of only a single embryo, in these cases; this cannot be universally applied to all patients given different anticipated implantation and pregnancy rates [45]. Schoolcrafl etal [46] recently published a summary of the literature regarding variables that can affect the success of embryo transfer. Although much of the literature is based on retrospective observational data, this paper attempted to address such issues as bedrest post-transfer, physician factor, catheter type, loading of the catheter, placement of the catheter tip, trial transfer, uterine contractions, effect of blood or mucus on or in the catheter, and perceived difficulty of transfer. Frank blood on the catheter may be an indicator of endometrial trauma. Ultrasonography at the time of transfer has been suggested to improve implantation rates, but randomized controlled trials are lacking [47]. It is likely that ultrasound guidance is most useful for difficult transfers (e.g., as with a tortuous cervical canal). The transfer is generally performed with the patient in the dorsal lithotomy position. The cervix is cleansed with transfer medium and a mock transfer may be performed to ensure accessibility of the cavity and the correct bend, if necessary, of the catheter. We perform most transfers with a soft Wallace

C H A P T E R 5 0 . FEMALE I N F E R T I L I T Y AND A S S I S T E D R E P R O D U C T I V E T E C H N O L O G Y

catheter. Soft catheters have been shown to be associated with less local trauma. The patient is transferred to a holding area where she remains supine for 30 minutes or more. The interval of rest following transfer does not appear to be clinically important. Zygote intrafallopian tube transfer (ZIFT) and gamete intrafallopian tube transfer (GIFT) entail the laparoscopic transfer of the zygotes or oocytes/sperm, respectively. At one time these techniques were advocated as being more successful than IVF for patients with normal fallopian tubes, but advances in the IVF laboratory have all but relegated these procedures to the archives.

4. Blastocyst Transfer The first human IVF pregnancy was achieved after blastocyst transfer [1]. Since then most transfers have been performed with day 2 or 3 pre-embryos because of difficulties in successfully maintaining pre-embryos in culture to the blastocyst stage. Recently, newer sequential media have led to renewed interest in blastocyst transfer. Compaction of the pre-embryo usually occurs at the 8 to 16 cell stage. Before the 8-cell stage, assessment of the pre-embryo is preliminary because the embryonic genotype has not yet been activated. Therefore, it is difficult to specifically predict pregnancy success rates from a day 3 pre-embryo assessment. Advantages to blastocyst transfer include excellent implantation rates in good prognosis patients, an extended window of opportunity for ancillary procedures such as PGD, and a decrease in the number of embryos transferred because of better implantation rates. Patients who have a good response to stimulation and at least four good quality pre-embryos on day 3 may be good candidates for blastocyst culture; it should be noted that generally fewer than 50% of in-vitro fertilized oocytes will attain the blastocyst stage even in sequential media. The debate is still ongoing as to whether patients with poor prognosis might benefit from blastocyst culture. For pre-embryos with a poorer prognosis, earlier placement in the uterus may be advised. Single blastocyst transfer should be advocated in women who have contraindications of carrying twins or more, as in the case of some congenital uterine anomaly. Donor egg recipients are excellent candidates for blastocyst transfer as well in light of the young age of the embryos.

5. Luteal Phase Support Centers vary in their approach to management of the luteal phase after embryo transfer. The prevalent practice is to provide luteal phase support in the form of either supplemental hCG or progesterone until sonographic documentation of viable pregnancy and placental progesterone production (approximately 7 to 8 weeks' gestation) [48]. Ovarian hyperstimulation results in supraphysiologic estradiol levels. Although we assume that multiple corpora lutea are developed post-hCG, aspiration of follicles may debulk some of the granulosa-theca cells destined to produce progesterone. Therefore, to ensure counterbalance of supraphysiologic estradiol levels with adequate progesterone, we supplement progesterone. Luteal phase support may be accomplished with additional hCG injections or daily progesterone supplementation. However,

559

the risk of OHSS is increased because exogenous hCG restimulates the ovaries. Progesterone supplementation may be administered once daily in an intramuscular dose of 25 to 50 mg or it can also be given as oral, or more commonly vaginal, micronized progesterone, (e.g., 100 to 200 mg tid). At our center, we recommend intramuscular progesterone. The initial dose is given the day postretrieval. Studies to date have suggested superior efficacy of intramuscular progesterone [49]. Patients who experience local reactions to intramuscular progesterone in oil may be switched to vaginal progesterone. Progesterone supplementation is maintained until the placental progesterone production is established and adequate. This usually occurs between 6 and 7 weeks' gestatational age and is clinically documented by the presence of a fetal heart. Care must be taken that progesterone supplements are in fact progesterone (often micronized) and not progestin. Progestin is a synthetic, androgenic compound, which may potentially be teratogenic. Progesterone supplements should not be continued beyond 9 to 10 weeks' gestational age because of theoretical androgenic effects on external female genital differentiation. The exception to this rule is in patients who are recipients of donor embryo in a programmed cycle who may not produce adequate endogenous progesterone.

F. Ancillary Techniques and Micromanipulation 1. Embryo Co-culture Systems Standard culture media for human pre-embryos are typically formulated to mimic human tubal fluid. In a natural cycle the oocyte is usually fertilized in the distal third of the fallopian tube. Ham's F-10 or HTF are media commonly used in U.S. IVF programs. Maternal serum or protein substitutes are often used to supplement the media. Optimal conditions for preembryonic culture remain elusive. Embryo co-culture is an attempt to improve cleavage and implantation rates by co-incubation of pre-embryos with another in-vitro cell system that more closely mimics the in-vivo system. Indications for embryonic co-culture include history of poor quality embryo or implantation failure despite good embryo quality. The original attempts at coculture involved incubation of embryos with culture media that included tubal cells, usually harvested from bovine or primate monkey Species. For obvious reasons, access to human tubal cells would be extremely limited. Because of the theoretical concerns about infection, we developed a system at our center whereby autologous endometrial cells collected at biopsy before IVF are used in the culture media. A good mix of glands and stroma is preferred. Recent clinical trials suggest that co-culture may be of particular benefit to patients with poor IVF prognosis, particularly those who have failed multiple previous cycles [50-52]. Current Food and Drug Administration (FDA) regulations have significantly limited the application of nonautologous co-culture systems given concern for transmission of potential infectious agents.

2. Assisted Hatching Observations of improved implantation rates in pre-embryos that had undergone partial zona drilling led to the concept of assisted hatching (AHA). Assisted hatching involves the thinning

560

or partial disruption of the zona pellucida just before embryo transfer. The objective of this procedure may be 2-fold. It may improve implantation rates for patients with a thick zona pellucida and allow removal of fragments from the perivitelline space. Such fragments have been correlated with embryo implantation failure (although they may be increased with chromosomal abnormalities, this correlation is not perfect). AHA may be performed early or late (two-cell embryo to a blastocyst), but it is generally performed on day 3 embryos. The size of the zona gap should not be too small so as to interfere with escape of the embryo from the zona pellucida. Complications of AHA include embryo loss at the time of transfer if the hole is too large. Also, if the hole is too small, the blastocyst may be trapped. The incidence of monozygotic twinning appears to be increased by AHA [53]. In the early to mid-1990s AHA gained popularity especially for the following indications: advanced maternal age, poor reproductive history with IVF, poor ovarian reserve, poor embryo morphology, increased cytoplasmic fragmentation, and thick/abnormal zona pellucida. However, some more recently small randomized controlled trials have challenged this concept and now AHA at our institution is not performed routinely but only on indicated patients.

3. Assisted Fertilization (Intracytoplasmic Sperm Injection) During the last decade and a half rapid advances have been seen in micromanipulation technology. Assisted fertilization and PGD are two commonly applied micromanipulation procedures. Intracytoplasmic sperm injection may be addressed in more detail elsewhere in this book. Before the advent of assisted fertilization, couples who suffered from severe male factor infertility experienced very limited success with IVF. The first human births from ICSI were reported in 1992 [23]. Intracytoplasmic sperm injection involves the injection of a single, viable sperm into a single oocyte. The development of ICSI has revolutionized the treatment of male factor infertility. Previously, semen concentrations of less than 5 x 106 sperm/ml were associated with poor IVF outcome. With ICSI and, more recently, advances in sperm retrieval directly from the testes or epididymis (TESE, MESA) even men with obstructive or nonobstructive azoospermia have the potential to fertilize oocytes and father their own genetic offspring. Much attention has recently been focused on the outcome of ICSI pregnancies. Several initial reports did not observe untoward neonatal effects from ICSI, and a recent update of a study done at our center again confirmed these findings [54]. This study also suggested a lower incidence (0.17%) of sex chromosomal abnormalities than the previous Lancet article [55]. Palermo etal did not observe a higher incidence of other chromosomal abnormalities, spontaneous abortions, or congenital anomalies when compared with IVF or general population statistics for women of similar ages. Bowen et al [56] evaluated medical and developmental outcome of children born from ICSI at 21 years of age. They compared children born from ICSI, IVF, and natural conception. Although there were no significant health problems and the mean Bayley MDI for development was within the normal range for most children, the ICSI subset did appear to have a significantly lower score than the IVF and natural conception group. However, fathers of ICSI children in this

SECTION 6 - REPRODUCTIVE BIOLOGY

study were found to be significantly different from the other groups in that the former were more likely to hold "unskilled" occupations. Nevertheless, when the subgroup analysis was done a significant difference remained. Another recent study suggested that the incidence of major birth defects is increased after ISCI [57]. Nevertheless, this study did not differentiate major from minor birth defects and was suspect to multiple biases characteristic of observational data such as surveillance bias and confounding and lack of an appropriate control group. Obviously, more controlled studies are needed to evaluate the offspring of ICSI treatment.

4. Cryopreservation Successful cryopreservation of embryos has been a revolution in ART. The first pregnancy from a cryopreserved conceptus was reported in 1983. Before embryo cryopreservation, patients and their physicians often faced a dilemma regarding the optimal number of oocytes to inseminate to maximize the likelihood of pregnancy but minimize potential embryo wastage. Advances in the success of cryopreservation have drastically improved the efficiency and safety of IVF. Approximately 60 to 70% of pre-embryos survive the thaw process. Once the conceptus has survived the thaw process, the success rate of a frozen cycle transfer almost approaches two thirds of that of a fresh cycle. Cryopreserved embryos from IVF cycles in which the fresh embryos resulted in pregnancy are also more likely to result in pregnancy. Success of frozen cycle transfers increases the overall pregnancy rate per retrieval [58]. In some instances, patients may complete their family through a single IVF cycle that results in frozen embryos that subsequently are transferred. Patients who are identified as having significant risk for ovarian hyperstimulation may benefit from cryopreservation of all pre-embryos and transfer in a later frozen cycle [59]. Conceptuses may be frozen safely for at least 7 years. Interestingly, those frozen for more than 12 months appear to have better success than those frozen for less. This fact may reflect that patients who have a successful pregnancy from the fresh transfer return later for the frozen transfers [60]. Fertility preservation for patients with cancer or other significant medical illnesses often depends on the success of embryo cryopreservation. Such patients may undergo ovarian hyperstimulation and IVF with cryopreservation of resultant embryos for a later date when their active disease is in remission or their prognosis is known. Frozen pre-embryos may be transferred into the endometrial cavity in a natural cycle for patients with regular ovulation and menses. For patients without regular cycles, programmed use of exogenous estrogen and progesterone can be performed to prepare the endometrial lining. The transfer is timed so that the endometrium is synchronous with the embryo development stage as per the number of days postinsemination. No differences in success between natural or programmed cycles are apparent [61].

5. Preimplantation Genetic Diagnosis Preimplantation genetic diagnosis has become a relatively recent indication for IVF. Preimplantation genetic diagnosis

C H A P T E R 5 0 . F E M A L E I N F E R T I L I T Y AND A S S I S T E D R E P R O D U C T I V E T E C H N O L O G Y

allows diagnosis at three levels: sex chromosome abnormalities/ aneuploidy, structural chromosomal abnormalities, and single gene defects. The first reported cases of PGD were undertaken for sex determination of embryos to prevent transmission of X-linked genetic disorders. These initial cases were reported in 1989. Subsequently, PGD was used to prevent single gene disorders such as cystic fibrosis. The two most common single gene disorders diagnosed by PGD are cystic fibrosis and sickle cell disease [62]. Recently the indications for PGD have been expanded to include the diagnosis of embryo aneuploidy in women of advanced maternal age, previous IVF failures, and history of previously affected embryos or offspring. Diagnosis of structural chromosomal abnormalities in couples having balanced translocations is also possible with PGD, particularly in the treatment of recurrent miscarriage. Recently, whole genome amplification with comparative genomic hybridization has been used to derive the entire karyotype for PGD, but the results are still very preliminary and the procedure is lengthy [63]. There are many potential advantages to PGD. Preimplantation genetic diagnosis can limit the occurrence of known lethal or severely disabling inherited genetic diseases and potentially even remove these diseases from a familial lineage completely. Preimplantation genetic diagnosis can be used to identify some specific causes of recurrent implantation or pregnancy failure. This ability may serve two purposes: (1) it may help resolve issues for such couples who have been unable to succeed with reproductive technology and allow them closure so that they may decide to proceed with alternatives such as oocyte donation or adoption and (2) once we are able to identify a particular disorder, we may be able to improve the implantation rates and ongoing pregnancy rates by transferring only those embryos that are genetically normal. This would help reduce the emotional burden of recurrent miscarriages for affected couples. Another advantage of PGD is the ability to avoid later pregnancy termination in patients who have a high risk of genetically abnormal conceptuses. Many couples may consider PGD a preferable alternative to prenatal diagnosis and pregnancy termination. PGD to avoid a particular disease (e.g., Fanconi's anemia), while producing an offspring who may be human leukocyte antigen (HLA) compatible with an affected sibling is also possible and has been previously achieved [64]. There are several potential limitations for PGD. Preimplantation genetic diagnosis opens many ethical controversies with regards to selection for traits, which do not specifically make a difference in the viability of offspring. Also, with the availability of HLA typing, the risk exists of couples reproducing for the express purpose of providing a marrow donor or rescue sibling for an affected child. There are several technical limitations to PGD. The technology has been mostly limited to centers, which have experts in both molecular genetics and reproductive technology, although collaboration among centers has recently become popular. The relatively small number of PGD cycles completed so far has limited the availability of outcomes studies. This has several implications. Prenatal diagnosis is still recommended to confirm the accuracy of PGD, which is possible for errors through several mechanisms. For single gene defects, PCR is used to amplify the genetic signal. Contamination from several sources is possible including the maternal cumulus cells, copies of paternal DNA available from extra sperm attached to the zona

561

pellucida (ICSI is therefore used), and any other contaminants. Also, the DNA may be obtained at different stages: from the polar body, from the cleavage stage (six to eight cell) embryo or from the blastocyst trophoectoderm. The sources may not accurately reflect the DNA constitution of the remainder of the embryonic cells (e.g., mosaicism may exist). For single gene diagnosis, allelic dropout poses a serious threat of misdiagnosis. A limited number of liveborn infants have been available to ensure that there are no long-term consequences of these procedures and not all outcomes have been reported [65-67]. The diagnoses are also limited by incomplete identification of all possible mutations. For example, for a single gene defect such as cystic fibrosis the most common allelic mutation is the delta 508; however, several other mutations may lead to compound heterozygotes who also may be affected. The ability to identify these compound heterozygotes depends on the availability of the appropriate probes and the fact that certain mutations have not yet been identified. Also, when one is searching for numerical abnormalities, aneuploidy, only six to seven chromosomes can be evaluated at one time (e.g., 13, 16, 18, 21, X, Y). Therefore, only those numerical chromosomal abnormalities related to the tested chromosomes can be identified. The two most common materials used in PGD are the first polar body and more commonly the blastomere. First polar body biopsy is often used for structural chromosomal analysis for anomalies of maternal origin, because fertilization has not yet occurred. Blastomere biopsy may be safely performed at the six- to eight-cell stage, before which it will be detrimental to the embryo by taking out one cell. Theoretically, biopsy of one blastomere should represent all other embryonic cells at this stage, although embryonic chromosomal mosaicism as mentioned previously can interfere with the diagnosis. Structural chromosomal analysis is generally achieved with florescent in situ hybridization (FISH), whereas PCR is required for single gene diagnosis.

6. Oocyte Donation Oocyte donation has been proven to be a successful option for women who cannot conceive with their oocytes because of advanced age, diminished ovarian reserve, or genetic disease. Oocyte donation allows the female partner to carry and deliver a pregnancy with her husband' s genetic contribution. The success of oocyte donation is mainly limited by the age of the donor, who should optimally be younger than 35 years old. Although endometrial receptivity may diminish somewhat with age, the contribution of this uterine factor appears minimal in comparison to oocyte quality. For this reason, the optimal number of embryos to be transferred to the recipient is also principally determined by the donor's age, rather than that of the recipient age [68,69]. Oocyte donors may be either known or unknown to the recipient. Known donors are often biologically related donors (e.g., sister or cousin donors). Because most donors and particularly anonymous donors are young, their oocytes and resultant embryos offer excellent pregnancy rates. The risks of the ART procedure for the donor are confined to the risks associated with stimulation and retrieval because the donor does not carry the pregnancy. The primary risk of donation for the recipient is

562

the transmission of infection. Despite the fact that the donors are screened at multiple intervals for infectious disease, the fact that fresh embryos are used, because of lower implantation rates with frozen embryos, poses a small theoretical risk of transmission of diseases such as human immunodeficiency virus (HIV), although such transmission has not been documented with oocyte donation. Although this risk appears to be mainly theoretical, the alternative of cryopreserving and quarantining embryos resulting from donor oocytes should be discussed with recipients as an option. A key component of successful oocyte donation is synchronization of the recipient' s menstrual cycle with the donor's cycle. In a recipient with intact ovarian function, this can be achieved through GnRH agonist downregulation followed by hormonal support with exogenous estrogen and progesterone. A mock cycle is undertaken before actual donor oocytes IVF to ensure that the recipient responds appropriately to hormonal support and that her endometrial lining develops adequately. In the event of pregnancy via donor oocytes IVF, estrogen and progesterone support are continued throughout the first trimester. G. In-vitro Fertilization Outcome

In-vitro fertilization outcomes have improved throughout the years. In 1998, 58,937 cycles were started in the United States, and the overall delivery rate per retrieval was 29.1%. Although advancing maternal age has a negative effect on prognosis, analysis of outcomes over the past 3 years shows a steady improvement in outcomes for each of the following maternal age groups: younger than 35 years, 35 to 37 years, 38 to 40 years, and older than 40 years. Factors that have been shown to affect outcome include maternal age, clinic size, the use of fresh versus cryopreserved embryos, and oocyte donation [70]. The direct risks of IVF to the mother include the risks associated with the procedure (bleeding, infection, and injury to surrounding structures) as outlined previously and the risk of OHSS. Ovarian hyperstimulation syndrome can be mild, moderate, or severe. The trigger for OHSS is hCG. In the absence of pregnancy or if hCG administration is withheld in light of risk for OHSS, the syndrome, if it occurs, usually adopts a very mild and self-limiting course. The incidence of moderate to severe hyperstimulation has been reported to be 2 to 4%. Rarely, severe OHSS can occur. It is a condition in which enlarged ovaries, encumbered by multiple follicles, exude fluid in the third space, and ascites, pleural effusions, pericardial effusions, electrolyte imbalances, hypovolemia, oliguria, and venous thromboembolism can be observed. Therefore, it potentially may be life-threatening. Although OHSS may potentially occur in any patient, younger patients, patients with PCOS, patients with very high estradiol levels, or patients with excessive number of follicles may be at greater risk. Ovarian hyperstimulation syndrome virtually does not occur in the absence of hCG. Therefore, in the very high risk patient, a decision to withhold hCG may be prudent if estradiol levels are high during stimulation. It is also extremely important in such an individual to avoid a natural LH surge and or conception by maintaining leuprolide or GnRH-antagonist injections. Patients who appear to be at high risk after oocyte retrieval may delay embryo transfer from day 3 to 5. If sufficient resolution of symptoms has not occurred by then, consideration

S E C T I O N 6 - R E P R O D U C T I V E BIOLOGY

may be given to embryo cryopreservation and embryo transfer at a later date. Management of mild to moderate OHSS requires careful monitoring of symptoms like abdominal pain, and shortness of breath, weight, abdominal girth, fluid intake and urine output, ovarian size, and ascites. Serum for hematocrit, platelets, electrolytes, and coagulation profiles should be monitored. Because of the enlarged ovarian size, ovarian torsion is a risk and patients must be advised of inactivity and warning signs. Severe OHSS can be life-threatening. Such patients should be admitted on bedrest with frequent monitoring of vital signs and the following: daily weights, abdominal girth, fluid intake and output, oxygen saturation, and other appropriate blood tests. Abdominal and pelvic examination should be strictly avoided because enlarged ovaries may be very fragile and prone to rupture and hemorrhage. Attention should be paid particularly to renal function, pulmonary edema, and thromboembolic risk. Thromboprophylaxis should be considered, particularly if hemoconcentrated. Diuretics should generally be avoided because these patients are intravascularly volume depleted. However, some authors have recommended the use of plasma expanders (e.g., albumin, pentaspan) to draw the fluid out of the third space, followed by a touch of diuretic chasers. Therapeutic, ultrasound-guided paracentesis may be required to relieve abdominal distension, discomfort, and intra-abdominal pressure in severe situations. In general, however, primary therapy is conservative and mainly supportive. A major concern regarding reproductive technology has been an increased incidence of multiple births [76-78]. Both pregnancy rates and multiple birth rates are directly correlated to number of embryos transferred. In 1998, IVF gestations had the following constitution: 61.8% singletons, 31.7% twins, 6.2% triplets, and 0.4% higher order multiple deliveries. Morbidity and mortality are significantly increased in pregnancies complicated by multiple gestations. Approaches to decrease the number of multiple births may also decrease the number of successes per fresh IVF cycle (e.g., by decreasing the number of embryos transferred). However, with laboratory conditions being more optimized, the trend to replace fewer day 3 pre-embryos in any age category is an appropriate one. Transfer of one or two blastocysts should prevent high-order multiples. As discussed earlier, the incidence of ectopic pregnancies according to some studies is increased after IVF compared with the general population (~1%). The most recent report from SART cited an incidence of 2.1%. We recently reviewed the incidence of laparoscopically/ultragraphically confirmed ectopic pregnancy and found the incidence after fresh embryo transfer at our center to be only 0.898%. However, the incidence is higher in the specific population of patients with tubal infertility. Little practical information is available regarding the overall incidence of heterotopic pregnancies from IVF. Most of the available data is in the form of case reports. We have recently reviewed our statistics and found an incidence of 0.18% heterotopic [ 14,71 ]. Interestingly, tubal disease was a risk factor in all of the heterotopic pregnancies, and the intrauterine pregnancies were all delivered successfully near term following surgical intervention for the ectopic pregnancy. Early monitoring ultrasound surveillance is important to diagnose heterotopic pregnancies. Recent data evaluating the outcomes of children born from IVF has become available. One study specifically assessed growth

CHAPTER

50.

FEMALE

INFERTILITY

AND ASSISTED

and physical and d e v e l o p m e n t a l health of children born from cryopreserved e m b r y o s c o m p a r e d with fresh I V F cycles and also c o m p a r e d these to spontaneous conceptions. There were no significant differences with respect to chronic illness, congenital anomalies, c h r o m o s o m a l anomalies, and neurologic or d e v e l o p m e n t a l health a m o n g all groups [72]. Three other recent cohort studies have recently suggested that outcomes of I V F children, in some cases specifically ICSI children, m a y be different from spontaneous conception with regards to neurologic development, congenital anomalies, and low birth weight [57,73,74]. As with most observational data, however, these studies suffer from multiple biases including surveillance bias, confounding, reporter bias and misclassification, and lack of an adequate control group. One consistent finding, however, is that singleton births from I V F do appear to have lower gestational ages and birth weights c o m p a r e d with spontaneous conceptions [74]. This might be related to the differences in obstetrical practice patterns on I V F versus naturally conceived patients. Available data concerning the link between ovarian cancer and ovulation induction agents have recently proven to be reassuring. Several meta-analyses have shown that the risk appears to be more related to infertility rather than the use of fertility drugs [75]. In fact, a recent meta-analysis by one of the authors suggested that w o m e n w h o suffer from infertility m a y be protected f r o m ovarian cancer if they conceive with therapy [76]. Preliminary data from a large, prospective cohort study conducted with the National Institutes of Health are also reassuring.

III. Conclusions The desire to procreate and have o n e ' s o w n genetically descended offspring is an extremely visceral desire. U n d e r s t a n d i n g of reproductive p h y s i o l o g y is essential to the treatment of such patients. Assisted reproduction has e v o l v e d i m m e n s e l y during the past decade. The development of G n R H antagonists, sequential m e d i a for blastocyst culture, assisted fertilization (especially ICSI), and advances in cryopreservation have contributed to the increased success rates of IVF. In addition, ancillary techniques such as P G D allow clinicians not only to "cure" infertility but also to prevent certain potentially devastating and tragic genetic diseases. O o c y t e donation allows couples w h o cannot conceive with their o w n oocytes to gestate and deliver a pregnancy. Multiple births from I V F are a significant issue. One problem is that in a capitalistic society where medicine is a private and competitive industry, success rates are reported by pregnancy rate per transfer procedure or retrieval. A second problem is that patients, often anxious for success and perhaps because of misinformation or lack of patient education, feel that multiple birth is a more palatable option than a negative pregnancy test. With advances in the successes of blastocyst transfer, embryo cryopreservation, physician and patient education about the complications of multiple births, and hopefully a more responsible reporting system that accounts for 'all pregnancies achieved from a single IVF cycle (either by fresh or frozen embryo transfer), we can prevent high-order multiple pregnancy. In fact, in some countries the governments regulate the number of pre-embryos to be transferred to one or two with the remainder of concepti frozen for later cycles. Assisted reproduction combines both art and science. Both meticulous clinical and laboratory m a n a g e m e n t are required to

REPRODUCTIVE

TECHNOLOGY

563

m a x i m i z e the potential of the existing technology. Future directions such as cytoplasmic transfer, nuclear cloning, and oocyte/ ovarian tissue freezing are on the horizon. W e expect that future d e v e l o p m e n t s in this field will be as exciting as its history.

IV. Suggestions for Further Investigations 9 Long term cohort follow-up studies are required to measure the impact of assisted reproductive technology on both parents and children. 9 N o v e l techniques such as oocyte m a n u f a c t u r i n g may, in the future, alleviate the b u r d e n of age-related infertility. 9 Preimplanation genetic diagnosis m a y be a significant part of future prenatal diagnosis. References

1. Evers JL. (2002). Female subfertility. Lancet. 360:151-159. 2. Westrom I. (1980). Incidence, prevalence, and trends of acute pelvic inflammatory disease and its consequences in industrialized countries. Am J Obstet Gynecol. 138:880. 3. Pisarka M, Carson S, Buster J. (1998). Ectopic pregnancy. Lancet. 351:1115. 4. Mol B, Dijkman B, Wertheim Petal. (1997). The accuracy of serum chlamydial antibodies in the diagnosis of tubal pathology: A meta-analysis. Fertil Steril. 67:1031-1037. 5. Petersen H, Xia Z, Hughes Jet al. (1997). The risk of ectopic pregnancy after tubal sterilization. N Engl J Med. 336:762. 6. Marcoux S, Maheux R, Berube S et al. (1997). Laparoscopic surgery in infertile women with minimal to mild endometriosis. N Engl J Med. 337:217-222. 7. Soules M, Spadoni L. (1982). Oil versus aqueous media for hysterosalpingography: A continuing debate based on many opinions and few facts. Fertil Steril. 38(1): 1-11. 8. Flemming R, Hopkinson ZE, Wallace Met al. (2002). Ovarian function and metabolic factors in women with oligomenorrhea treated with metformin in a randomized double blind placebo-controlled trial. J Clin Endocrinol Metab. 87:569-574. 9. Dunson DB, Colombo B, Baird DD. (2002). Changes with age in the level and duration of fertility in the menstrual cycle. Hum Reprod. 17:1399-1403. 10. Pincus G, Enzmann E. (1934). Can mammalian eggs undergo normal development in vitro? Proc Natl Acad Sci U S A. 20:121. 11. Thibault C, Dauzier L, Wintenberger S. (1954). Etude cytologique de la fecondafion in vitro de l'oeuf de la lapine. C R Soc Biol Paris. 148:789. 12. Perone N. (1994). In vitro fertilization and embryo transfer--a historical perspective. J Reprod Med. 39:695-700. 13. Estes WJ. (1924). Ovarianimplantationfor sterility. Surg Gynecol Obstet. 38:394. 14. Kashyap S, Chung P, Kligman I, Rosenwaks Z. (2002). 7 year descriptive summary of ectopic pregnancies occurring after fresh and frozen IVF cycles. Fertil Steril. 78:$137. 15. Diaz I, Navarro J, Blasco L etal. (2000). Impact of stage III-IV endometriosis on recipients of sibling oocytes: matched case-control study. Fertil Steril. 74:31-34. 16. Toya M, Saito H, Saito T et al. (2000). Moderate and severe endometriosis is associated with alterations in the cell cycle of granulose cells in patients undergoing in vitro fertilization and embryo transfer. Fertil Steril. 73:344-350. 17. Oliviennes F, Feldberg D, Liu H etal. (1995). Endometriosis: A stage by stage analysis: The role of in vitro fertilization. Fertil Steril. 64:392-398. 18. Societyfor Assisted ReproductiveTechnology (SART) and American Societyfor Reproductive Medicine. (2002). Assisted reproductive technology in the United States: 1998 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril. 77:18-31. 19. Barnhart K, Dunsmoor-Su R, Coutifaris C. (2002). Effect of endometriosis on in vitro fertilization. Fertil Steril. 77:1148-1155. 20. Marcus S, Edwards R. (1994). High rates of pregnancy after long-term down-regulation of women with severe endometriosis. Am J Obstet Gynecol. 171:812-817. 21. Nakamura K, Oosawa M, Kondou Iet al. (1992). Menotropin stimulation after prolonged gonadotropin releasing hormone agonist pretreatment for in vitro fertilization in patients with endometriosis. JAssisted Reprod Genet. 9:113-117.

564

22. Dicker D, Goldman J, Levy T etal. (1996). The impact of long-term gonadotropin-releasing hormone analogue treatment in preclinical abortions in patients with severe endometriosis undergoing in vitro fertilization-embryo transfer. Fertil Steril. 65:791-795. 23. Palermo G, Joris H, Devroey P, Van Steirteghem AC. (1992). Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 340:17-18. 24. Guzick D, Overstreet J, Factor-Litvak P e t al. (2001). Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med. 345:1388-1393. 25. Van Uem J, Acosta A, Swanson R etal. (1985). Male factor evaluation in in vitro fertilization: Norfolk experience. Fertil Steril. 44:375. 26. Schlegel P, Girardi S. (1997). In vitro fertilization for male factor infertility. J Clin Endocrinol Metab. 82:709-716. 27. Oktay K, Buyuk E, Davis O etal. (2003). Fertility preservation in breast cancer patients: IVF and embryo cryopreservation after ovarian stimulation with tamoxifen. Hum Reprod. 18:90--95. 28. Aboulghar M, Mansour R, Serour G etal. (2001). Controlled ovarian hyperstimulation and intrauterine insemination for treatment of unexplained infertility should be limited to a maximum of three trials. Fertil Steril. 75:88-91. 29. Ruiz A, Remohi J, Minguez Y e t al. (1997). The role of in vitro fertilization and intracytoplasmic sperm injection in couples with unexplained infertility after failed intrauterine insemination. Fertil Steril. 68:171-173. 30. Crosignani P, Waiters D, Soliani AHR. (1991). The ESHRE multicentre trial on the treatment of unexplained infertility: A preliminary report. European Society of Human Reproduction and Embryology. Hum Reprod. 6:953-958. 31. Tietze C. (1957). Reproductive span and rate of reproduction among Hutterite women. Fertil Steril. 8:89. 32. Damario MA, Davis O, Rosenwaks Z. (1999). The role of maternal age in the assisted reproductive technologies. Reprod Med Rev. 7:141-160. 33. Liccardi F, Liu H, Rosenwaks Z. (1995). Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril. 64:991-994. 34. Navot D, Rosenwaks Z, Margalioth E. (1987). Prognostic assessment of female fecundity. Lancet. 2(8560):645-647. 35. Edwards R, Lobo R, Bouchard P, 29. Janssens RM LC, Vermeiden JP, et al. Dose-finding study of triptolrelin acetate for prevention of a premature LH surge in IVF: A prospective, randomized, double-blind, placebo-controlled study. Human Reproduction 2000; 15: 2333-40. 36. Diedrich K, Ludwig M, Felberbaum R. (2001). The role of gonadotropinreleasing hormone antagonists in in vitro fertilization. Semin Reprod Med. 19(3):213-220. 37. Ludwig M FR, Albano C, Oliviennes F et al. Cetrorelix levels in plasma and follicular fluid. Gynecol Endocrinol. 1000:030. 38. Levy D, Navarro J, Schattman G e t al. (2000). The role of LH in ovarian stimulation. Hum Reprod. 15:2258-2265. 39. Ludwig M, Reithmuller-Winzen H, Felberbaum R etal. (2001). Health of 227 children born after controlled ovarian stimulation for in-vitro fertilization using the luteinizing hormone-releasing hormone antagonist cetrorelix. Fertil Steril. 75:18-22. 40. Rock J, Menkin M. (1944). In vitro fertilization and cleavage of human ovarian eggs. Science. 100. 41. Steptoe P. (1968). Laparoscopy and ovulation. Lancet. 292(7574):913. 42. Steptoe P, Edwards R. (1970). Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotropins. Lancet. 295(7649):683-89. 43. Muasher S, Wilkes C, Carcia J e t al. (1984). Benefits and risks of multiple transfer with in vitro fertilization. Lancet. 323(8376):570. 44. Schulman A, Ben-Nun I, Ghetler Y etal. (1993). Relationship between embryo morphology and implantation rate after in vitro fertilization treatment in conception cycles. Fertil Steril. 60:123. 45. Dhont M. (2001). Single embryo transfer. Semin Reprod Med. 19:251-258. 46. Schoolcraft W, Surrey E, Gardner D. (2001). Embryo transfer: Techniques and variables affecting success. Fertil Steril. 67:863-870. 47. Coroleu B, Carreras O, Veiga A et al. (2000). Embryo transfer under ultrasound guidance improves pregnancy rate after in vitro fertilization. Hum Reprod. 15:616-620. 48. Soliman S, Daya S, Collins J, Hughes E. (1994). The role of luteal phase support in infertility treatment: A meta-analysis of randomized controlled trials. Fertil Steril. 61:1068. 49. Damario M, Goudas V, Session D etal. (1999). Crinone 8% vaginal progesterone gel results in lower embryonic implantation efficiency after in vitro fertilizationembryo transfer. Fertil Steril. 72:830-836.

SECTION

6 -

REPRODUCTIVE

BIOLOGY

50. Simon C, Mercader A, Garcia-Velasco J etal. (1999). Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. J Clin Endocrinol Metab. 84:2638-2346. 51. Wiemer K, Cohen J, Tucker M, Godke R. (1998). The application of co-culture in assisted reproduction: 10 years of experience with human embryos. Hum Reprod. 13:226-238. 52. Barmat L, Liu H, Spandorfer Set al. (1998). Human preembryo development on autologous endometrial coculture versus conventional medium. Fertil Steril. 70:1109-1113. 53. Schieve L, Meikle S, Peterson H et al. (2000). Does assisted hatching pose a risk for monozygotic twinning in pregnancies conceived through in vitro fertilization? Fertil Steril. 74:288-294. 54. Palermo G, Neri Q, Rafaelli R et al. (2000). Evolution of Pregnancies and Follow-up of Newborns Delivered after Intracytoplasmic Sperm Injection. In Manual on Assisted Reproduction, 2nd Edition (Rabe TDK, Strowitzki T eds.), Springer Publishers. 55. IN't Veld P, Brandenberg H, Verhoff A etal. (1995). Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet. 336:776. 56. Bowen J, Gibson F, Leslie G, Saunders D. (1998). Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet. 351:1529-1534. 57. Hansen M, Kurinczuk J, Bower C, Webb S. (2002). The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med. 346:725-730. 58. Damario M, Hammitt D, Session D, Dumesic D. (2000). Embryo cryopreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from a single oocyte retrieval. Fertil Steril. 73:757-773. 59. Ferraretti A, Gianoroli L, Magli C et al. (1999). Elective cryopreservation of all pronucleate embryos in women at risk of ovarian hyperstimulation syndrome: Efficiency and safety. Hum Reprod. 14:1457-1460. 60. Lin Y, Cassidenti D, Chacon R etal. (1995). Successful implantation of frozen sibling embryos is influenced by the outcome of the cycle from which they were derived. Fertil Steril. 63:262-267. 61. Oehninger S, Mayer J, Muasher S. (2000). Impact of different clinical variables on pregnancy outcome following embryo cryopreservation. Mol Cellular Endocrinol. 169:73-77. 62. Xu K, Shi Z, Veeck L etal. (1999). First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell anemia. JAMA. 281:1701-1706. 63. Elias S. (2001). Preimplantation genetic diagnosis by comparative genomic hybridization. N Engl J Med. 345:1569-1571. 64. Verlinsky Y, Rechitsky S, Schoolcraft W etal. (2001). Preimplantation diagnosis for Fanconi anemia combined with HLA matching. JAMA. 285:3143-3144. 65. Committee. (2000). EPCS. ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: Data collection II. Hum Reprod. 15:2673-2683. 66. Strom C, Levin R, Strom S et al. (2000). Neonatal outcome of preimplantation genetic diagnosis by polar body removal: The first 109 infants. Pediatrics. 106:650--653. 67. Bonduelle M, Van Asche E, Sermon K etal. (1999). Neonatal outcome following preimplantation genetic diagnosis (PGD) as an alternative to prenatal diagnosis. Eur J Hum Genet. 7:38. 68. Shulman A, Frenkel Y, Dor J et al. (1999). The best donor. Hum Reprod. 10:2493-2496. 69. Faber B, Mercan R, Hamacher Petal. (1997). The impact of an egg donor's age and her prior fertility on recipient pregnancy outcome. Fertil Steril. 68:370--372. 70. Templeton A, Morris J, Parslow W. (1996). Factors that affect outcome of in-vitro fertilization treatment. Lancet. 348:1402-1406. 71. Kashyap S, Kligman I, Chung P, Rosenwaks Z. (2002). Heterotopic pregnancies after IVF: A case-control study. Fertil Steril. 78:$248-$249. 72. Wennerholm U, Albertsson-Wilkand K, Bergh C etal. (1998). Postnatal growth and health of children born after cryopreservation as embryos. Lancet. 351:1085-1090. 73. Stromberg B, Dahlquist G, Ericson A et al. (2002). Neurological sequelae in children born after in-vitro fertilization: Population based study. Lancet. 359:461-465. 74. Schieve L, Meikle S, Ferre C et al. (2002). Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med. 346:731-737. 75. Ness RB, Cramer DW, Goodman MT et al. (2002). Infertility, fertility drugs, and ovarian cancer: A pooled analysis of case-control studies. Am J Epidemiol. 155:217-224. 76. Kashyap S, Moher D, Fung MFK, Rosenwak Z. (2004). Assisted Reproductive Technology and the incidence of ovarian cancer--a meta-analysis. Obset Gynecol. (In press).