Berek and Novak's Gynecology 15th Ed.

32 Infertility and Assisted Reproductive Technology

Mira Aubuchon

Richard O. Burney

Danny J. Schust

Mylene W.M. Yao

• The physician’s initial encounter with the infertile couple is extremely important because it sets the tone for subsequent evaluation and treatment. Factors from either or both partners may contribute to difficulties in conceiving; therefore, it is important to consider all possible diagnoses before pursuing invasive treatment.

• The main causes of infertility include male factor, decreased ovarian reserve, ovulatory disorders (ovulatory factor), tubal injury, blockage, or paratubal adhesions (including endometriosis with evidence of tubal or peritoneal adhesions), uterine factors, systemic conditions (including infections or chronic diseases such as autoimmune conditions or chronic renal failure), cervical and immunologic factors, and unexplained factors (including endometriosis with no evidence of tubal or peritoneal adhesions).

• Basic investigations that should be performed before starting any infertility treatment are semen analysis, confirmation of ovulation, and the documentation of tubal patency.

• Male factor is the sole cause of infertility in 20% of infertile couples and may be a contributing factor in as many as 40% of cases. Treatment of reversible endocrine or infectious causes of subfertility, such as sexually transmitted diseases and thyroid disorders, tends to be efficacious. Intrauterine insemination (IUI) is the best studied and most widely practiced of all the insemination techniques. Intracytoplasmic sperm injection (ICSI) has allowed couples with male factor infertility to achieve assisted reproductive technology (ART) pregnancy outcomes that are comparable with those of couples with non–male factor infertility using conventional in vitro fertilization (IVF) treatment.

• An association between the age of the woman and reduced fertility is well documented. The decline in fecundability begins in the early 30s and accelerates during the late 30s and early 40s.

• Disorders of ovulation account for about 20% to 40% of all cases of female infertility. These disorders are generally among the most easily diagnosed and treatable causes of infertility.

• The most common cause of oligo-ovulation and anovulation—both in the general population and among women presenting with infertility—is polycystic ovarian syndrome (PCOS).

• Tubal and peritoneal factors account for 30% to 40% of cases of female infertility. Cervical factor is estimated to be a cause of infertility in no more than 5% of infertile couples. Uterine pathologies constitute the etiologic factor in infertility in as many as 15% of couples seeking treatment and are diagnosed in as many as 50% of infertile patients. Leiomyomas have not been shown to be a direct cause of infertility.

• All methods of ART, by definition, involve interventions to retrieve oocytes. These techniques include IVF, ICSI, gamete intrafallopian transfer (GIFT), zygote intrafallopian transfer (ZIFT), cryopreserved embryo transfers, and the use of donor oocytes. Because of improved success rates associated with IVF-embryo transfer, the performance of GIFT and ZIFT has declined.

• Multiple gestation, especially higher-order multiple gestation, is a serious complication of infertility treatment and has tremendous medical, psychological, social, and financial implications and complications.

• Fortunately, recent studies have not shown an increased risk for breast, uterine, or ovarian cancer secondary to medications used for superovulation in the treatment of infertility.

• Information on the Society for Assisted Reproductive Technology (SART) and registered ART clinics are accessible on the Internet.

Infertility is defined as 1 year of unprotected intercourse without pregnancy (1). This condition may be further classified as primary infertility, in which no previous pregnancies have occurred, and secondary infertility, in which a prior pregnancy, although not necessarily a live birth, has occurred. About 90% of couples should conceive within 12 months of unprotected intercourse (2). Subfertility refers to couples who conceive after 12 months of attempted impregnation (2). Fecundability refers to the probability of pregnancy per cycle, which is considered to be at 20% in fertile couples (1). Fecundity refers to the probability of achieving a live birth in a single cycle and, by definition, has a value lower than fecundability. The diagnosis of impaired fecundity has been proposed to include couples with 36 months or more without conception or physical inability or difficulty in having a child; however, there is currently no clear consensus on any of these terms (3–5).

Epidemiology

Twenty-one percent of couples in the United States are expected to experience infertility in their lifetimes, with a current prevalence of 7.4% (6). In 2002, over 7 million US women age 22 to 44 reported using infertility services in their lifetimes (7). Once diagnosed, 13% of couples will not pursue treatment (8). The diagnosis of impaired fecundity has been rising, reaching 15% in 2002 and largely resulting from the trend toward delayed childbearing in developed countries (3). Worldwide, male factor accounts for 51.2% and tubal blockage for 25% to 35% of infertility and subfertility (conception after attempting for 1 year) (9,10). In Europe, ovulatory dysfunction accounts for 21% to 32%, male factor 19% to 57%, tubal factor 14% to 26%, unexplained 8% to 30%, endometriosis 4% to 6%, and combined male and female factors 34.4% of infertility (11–13). The odds of infertility increase with female age and in general among patients who have not graduated from college (14). The high cost of infertility treatment is a barrier for many in the United States where insurance typically does not cover these services (15). Language and other cultural barriers affect access for many minority groups (15,16). The women most likely to obtain specialized treatment are 30 years of age or older, white, married, and of relatively high socioeconomic status (7).

Initial Assessment

The physician’s initial encounter with the infertile couple is of primary importance because it sets the tone for subsequent evaluation and treatment. Ideally, both partners should be present at this first visit. It cannot be overemphasized that infertility is a problem of the couple. The presence of both partners, beginning with the initial evaluation, jointly involves them in the therapeutic process. This essential shared involvement demonstrates that the physician is receptive to the partner’s needs as well as those of the patient and offers the partner an opportunity to ask questions and voice concerns.

The physician should obtain a complete medical, surgical, and gynecologic history from the woman. Specifically, information regarding menstrual cycle regularity, pelvic pain, and previous pregnancy outcomes is important. Risk factors for infertility, such as a history of pelvic inflammatory disease (PID) or pelvic surgery, should be reviewed. A history of intrauterine exposure to diethylstilbestrol (DES) is significant. In addition, a review of systems relevant to pituitary, adrenal, and thyroid function is useful. Questions regarding galactorrhea, hirsutism, and changes in weight are particularly relevant. A directed history, including developmental defects such as undescended testes, past genital surgery, infections (including mumps orchitis), previous genital trauma, and medications should be obtained from the male partner. A history of occupational exposures that might affect the reproductive function of either partner is important, as is information about coital frequency, dyspareunia, and sexual dysfunction. Finally, information should be obtained on any family history of infertility, premature ovarian failure, congenital or developmental defects, mental retardation, and hereditary conditions relevant to preconceptional planning, such as cystic fibrosis, thalassemias, and Tay Sachs disease.

The initial interview provides the physician with the opportunity to assess the emotional impact of infertility on the couple. It presents a time for the physician to emphasize the emotional support available to the couple as they proceed with the diagnostic evaluation and suggested treatments. In some cases, referral to a trained social worker or psychologist may be beneficial.

The physical examination of the woman should be thorough, with particular attention given to height, weight, body habitus, hair distribution, thyroid gland, and pelvic examination. Referral of the male partner to a urologist for examination often is beneficial if historic information or subsequent evaluation suggests an abnormality. This initial encounter is an excellent time to outline the general causes of infertility and to discuss subsequent diagnostic and treatment plans (Figs. 32.1–32.3).

Figure 32.1 Diagnostic and treatment algorithm: infertility. HSG, hysterosalpingography. (From Yao M. Clinical management of infertility. Washington, DC: The Advisory Board: 2000, with permission.)

The basic investigations that ideally should be performed before starting any infertility treatment are semen analysis, confirmation of ovulation, and the documentation of tubal patency. Other noninfertility assessments should include rubella immunity testing (17). If a patient with severe systemic illness, such as renal failure, liver failure, or cancer, wishes to conceive, careful preconceptional assessment and counseling is advisable because the risks of fertility treatment and pregnancy can be substantial.

Causes of Infertility

The main causes of infertility include:

1. Male factor

2. Decreased ovarian reserve

3. Ovulatory factor

4. Tubal factor

5. Uterine factor

6. Pelvic factor

7. Unexplained

Factors from either or both partners may contribute to difficulties in conceiving; therefore, it is important to consider all possible diagnoses before pursuing invasive treatments. The relative prevalence of the different causes of infertility varies widely among patient populations (Table 32.1). In many cases, no specific cause is detected despite a thorough evaluation, and the couple’s infertility is categorized as unexplained.

Very few couples have absolute infertility, which can result from congenital or acquired irreversible loss of functional gametes in either partner or the absence of reproductive structures in either partner. In these specific instances, couples should be counseled regarding their options for adoption, use of donor gametes, or surrogacy.

Impact of Lifestyle on Fertility

Overweight and obese women have higher rates of ovulatory dysfunction and infertility, along with 30% lower pregnancy rates with in vitro fertilization (IVF) compared to normal-weight women (18,19). Obese men have higher rates of hypogonadotropic hypogonadism and sperm DNA damage compared to normal-weight men (20,21). Substance abuse in men is discussed in the next section. Women who smoke need twice the number of IVF cycles to conceive as nonsmokers, but the effect of alcohol on fertility is less clear (18).

Figure 32.2 Diagnostic and treatment algorithm: anovulation. FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; TSH, thyroid-stimulating hormone; T4, thyroxine; GH, growth hormone; ACTH, adrenocorticotropic hormone; BMI, body mass index; MRI, magnetic resonance imaging; GnRH, gonadotropin-releasing hormone. (From Yao M. Clinical management of infertility. Washington, DC: The Advisory Board: 2000, with permission.)

Figure 32.3 Diagnostic and treatment algorithm: ovarian disorders. FSH, follicle-stimulating hormone; LH, luteinizing hormone; CCCT, clomiphene citrate challenge test ART, assisted reproductive technology. (From Yao M. Clinical management of infertility. Washington, DC: The Advisory Board: 2000, with permission.)

Male Factor

Male factor is the only cause of infertility in about 20% of infertile couples, but it may be a contributing factor in as many as 50% of cases (9,11–13). The concept of a global decline in sperm counts is controversial (22,23). A decline in sperm density has been observed in the United States, Europe and Australia, while decreased motility and semen volume have been reported in India (22). Given that decreases in sperm parameters have been noted in fertile men, the clinical relevance for fecundability is unknown (24). However, one simulation model has suggested that if sperm concentrations decline by 21% to 47%, fecundability would decrease by 7% to 15% (25).

Table 32.1 Causes of Infertility

Relative prevalence of the etiologies of infertility (%)
Male factor	20–30
Both male and female factors	10–40
Female factor	40–55
Unexplained infertility	10–20
Approximate prevalence of the causes of infertility in the female (%)
Ovulatory dysfunction	20–40
Tubal or peritoneal factor	20–40
Miscellaneous causes	10–15

Physiology

Spermatogenesis

The male reproductive tract consists of the testis, epididymis, vas deferens, prostate, seminal vesicles, ejaculatory duct, bulbourethral glands, and urethra. Gonadotropin-responsive cells in the testes include Leydig cells (the site of androgen synthesis) and Sertoli cells, which line the seminiferous tubules (the site of spermatogenesis). The pituitary gland secretes luteinizing hormone (LH), which stimulates the synthesis and secretion of testosterone by the Leydig cells, and follicle-stimulating hormone (FSH), which acts with testosterone on the Sertoli cells to stimulate spermatogenesis (26). In humans, a new cohort of spermatogonia enter the maturation process every 16 days, and the development from spermatogonia stem cells to the mature sperm cells takes about 75 days (27). Spermatogonia undergo mitotic division to give rise to spermatocytes. These diploid spermatocytes subsequently undergo meiosis to produce haploid spermatids, which contain 23 (rather than 46) chromosomes (26). Maturation of spermatids is called spermiogenesis and involves condensation of the nucleus, formation of the flagellum, and the formation of the acrosome (a structure derived from the Golgi complex covering the tip or head of the sperm nucleus) (28). The resultant spermatozoa are released into the seminiferous tubule lumen and then enter the epididymis, where they continue to mature and become progressively more motile during the 2 to 6 days that are required to traverse this tortuous structure and reach the vas deferens (29).

Sperm Transport

During ejaculation, mature spermatozoa are released from the vas deferens along with fluid from the prostate, seminal vesicles, and bulbourethral glands. The released semen is a gelatinous mixture of spermatozoa and seminal plasma; however, this thins out 20 to 30 minutes after ejaculation. This process, called liquefaction, is the direct result of proteolytic enzymes within the prostatic fluid (30). Following ejaculation, the released spermatozoa must undergo capacitation to become competent to fertilize the oocyte. Capacitation occurs within the cervical mucus and involves removal of inhibitory mediators such as cholesterol from the sperm surface, tyrosine phosphorylation, and calcium ion influx, all of which allow the sperm to recognize additional fertilization cues during travel through the female reproductive tract. When the sperm reach the tubal isthmus they are slowly released into the ampulla, further reducing the number of sperm that reach the oocyte (31). Sperm transport from the posterior vaginal fornix to the fallopian tubes occurs within 2 minutes during the follicular phase of the menstrual cycle (32).

Fertilization

As the capacitated sperm near and pass through cumulus cells surrounding the oocyte, hydrolytic enzymes are released from the acrosome via exocytosis in a process called the acrosome reaction.Both capacitation and the acrosome reaction can be induced in vitro (28,31). Following the acrosome reaction, the sperm binds to and penetrates the zona pellucida (the extracellular coat surrounding the oocyte). This allows the sperm to fuse with the plasma membrane of the oocyte, an event that promotes changes in the oocyte and prevent entry by additional sperm (31). As the first sperm penetrates the zona pellucida, cortical granules are released (the cortical reaction) from the oocyte into the perivitelline space. This stops the oocyte’s zona pellucida from binding new sperm and inhibits penetration by previously bound sperm, further reducing the possibility of polyspermy (33).

Sperm Sensitivity to Toxins

Decreased sperm concentration and motility have been noted in areas of the United States with heavy agriculture and pesticide use, but occupational exposures have not been linked to infertility (24,34). Higher intake of food containing soy is associated with lower sperm concentrations (35). Alcoholism negatively affects all semen analysis parameters, and either smoked or chewed tobacco is associated with decreased density and motility (36–39). Marijuana inhibits motility and the acrosome reaction in vitro, and cocaine inhibits sperm motility and is associated with male infertility (40–42). Certain drugs may reduce sperm numbers or function or may cause ejaculatory dysfunction (Table 32.2). Vaginal lubricants such as Astroglide, KY Jelly, saliva, and olive oil inhibit sperm motility in vitro, while no adverse effects are seen with hydroxyethylcellulose (Pre-Seed), mineral oil, or canola oil(32).

Table 32.2 Drugs that Can Impair Male Fertility

Impaired spermatogenesis	Sulfasalazine, methotrexate, nitrofurantoin, colchicine, chemotherapy
Pituitary suppression	Testosterone injections, gonadotrophin-releasing hormone analogues
Antiandrogenic effects	Cimetidine, spironolactone
Ejaculation failure	α-blockers, antidepressants, phenothiazines
Erectile dysfunction	β-blockers, thiazide diuretics, metoclopramide
Drugs of misuse	Anabolic steroids, cannabis, heroin, cocaine
From Hirsh A. Male infertility. BMJ 2003;327:669–672, with permission.

Semen Analysis

The basic semen analysis measures semen volume, sperm concentration, sperm motility, and sperm morphology (30). Recently revised, the normal values suggested by the World Health Organization (WHO) in 2010 are listed along with the previously published guidelines in Table 32.3 (30,43). Both criteria were developed using fertile men whose semen parameters were in the lowest fifth percentile of the group studied, but values above the reference ranges do not guarantee male fertility. Furthermore, since infertile men were not used to develop the criteria, values below the cutoffs may not necessarily indicate infertility (30). However, significant deviations from the reference limits are generally classified as male factor infertility (44). Given regional differences in semen quality and between laboratories, laboratories are encouraged to develop their own reference ranges. Typically, semen is assessed manually, but computer-aided sperm analysis (CASA) may be used. Limitations of CASA include a lack of standardization among instruments, an inability to differentiate intact from nonintact sperm, possible bias from artifacts during preparation, and a paucity of studies on fertility outcomes in large popu-lations (30).

Table 32.3 Semen Analysis Terminology and Normal Values

Terminology
Normozoospermia	All semen parameters normal
Oligozoospermia	Reduced sperm numbers Mild to moderate: 5–20 million/mL Severe: <5 million/mL
Asthenozoospermia	Reduced sperm motility
Teratozoospermia	Increased abnormal forms of sperm
Oligoasthenoteratozoospermia	Sperm variables all subnormal
Azoospermia	No sperm in semen
Aspermia (anejaculation)	No ejaculate (ejaculation failure)
Leucocytospermia	Increased white cells in semen
Necrozoospermia	All sperm are nonviable or nonmotile
Normal Semen Analysis: World Health Organization
	1992 Guidelines	2010 Guidelines
Volume	2 mL	≥1.5 mL
Sperm concentration	20 million/mL	≥15 million/mL
Sperm motility	50% progressive or	≥32% progressive
	>25% rapidly progressive
Morphology (strict criteria)	>15% normal forms	≥4% normal forms
White blood cells	<1 million/mL	<1 million/mL
Immunobead or mixed antiglobulin	<10% coated with antibodies	<50%
reaction test
Terminology from Hirsh A. Male infertility. BMJ 2003;327:669–672, with permission.

Abstinence

Abstinence of a minimum of 2 to a maximum of 7 days usually is recommended prior to the semen analysis, but the optimal duration is unknown (30). The epididymis stores the equivalent of three ejaculations (29). With prolonged abstinence, sperm overflow into the urethra and are flushed out into the urine (30). A study of men attending an infertility clinic found that the total motile sperm count and normal morphology decreased after 10 days' abstinence in normozoospermic men but after 5 days' abstinence in oligozoospermic men (45). There are conflicting reports regarding the impact of shorter abstinence of 1 or 2 days on semen parameters (45,46), but one study suggested intrauterine insemination success rates might be improved by shortening abstinence times prior to specimen collection (46).

Specimen Collection

The specimen should be obtained by masturbation and collected in a clean container kept at ambient temperature (30). The patient should report any loss of the specimen, particularly the first portion of the ejaculate, which contains the highest sperm concentration. Collection may be performed either at home or in a private room near the laboratory. The sample should be taken to the laboratory within 30 minutes to 1 hour of collection to prevent dehydration and degradation. If masturbation into a container is not possible, condoms specially designed for semen analysis should be used rather than latex condoms, which are toxic to sperm. Intercourse to collect the sample is discouraged because of the risk of contamination. Even when the specimen is obtained under optimal circumstances, interpretation of the results of the semen analysis is complicated by variability within the same individual and wide differences in normal semen parameters. Semen parameters may vary widely from one man to another and among men with proven fertility. In many circumstances, several specimens are necessary to verify an abnormality(30).

Sperm Volume and pH

The lower limit of normal semen volume is 1.5 mL or more and the pH should be 7.2 or higher. These parameters are affected mainly by the balance between the acidic secretions of the prostate gland and the alkaline fluid from the seminal vesicles. Low volume along with pH less than 7 suggests obstruction of the ejaculatory ducts or absence of the vas deferens. Difficulties with collection, retrograde ejaculation, or androgen deficiency can contribute to low volume. High volumes greater than 5 mL suggest inflammation of the accessory glands (30).

Sperm Concentration

Sperm concentration or density is defined as the number of sperm per milliliter in the total ejaculate. The normal lower limit is 15 million/mL or more, recently revised from 20 million/mL or more(30,43). Only intact sperm are counted in determining sperm concentration. Fifteen percent to 20% of infertile men are azoospermic (no sperm) and 10% have a density of less than 1 million/mL (30,47).

Sperm Motility and Viability

Sperm motility is the percentage of progressively motile sperm in the ejaculate. The normal lower limit is 32% or more, recently revised from 50% or more (30,43). Viability should be at least 58%.Progressive motility refers to movement either linearly or in a large circle regardless of speed. Nonprogressive motility describes sperm that display only small movements or twitching or no movement at all (immotile). Assessments of speed of progression, either rapid or slow, have been removed from the revised guidelines because of difficulty in unbiased measurement of this parameter. A reduction in sperm motility is referred to as asthenozoospermia. Leukocytes can impair sperm motility through oxidative stress. When a large number of immotile sperm are present or when progressive motility is less than 40%, viability studies should be performed. Viable immotile sperm may have flagellar defects, while the presence of nonviable immotile sperm (necrozoospermia) suggests epididymal pathology. Viable sperm have intact plasma membranes, which will not stain (dye exclusion) but will swell in hypoosmotic solutions (hypoosmotic swelling test) (30).

Sperm Morphology

Morphology refers to anatomic malformations of the sperm. The lower limit for normal morphology is 4% or more using strict criteria, a change from previous guidelines using a more lenient assessment and a cutoff of 30% or more (30,43). Assessment of sperm morphology involves fixing and staining of a portion of the specimen. The strict Tygerberg criteria were introduced by Kruger et al. in 1986 to assess sperm morphology (30,48,49). Using this system, the entire spermatozoon—including the head, midpiece, and tail—is assessed, and even mild abnormalities in head forms are classified as abnormal. Most sperm from normal men exhibit minor abnormalities when subjected to Tygerberg standards. An abnormality of sperm morphology is known as teratozoospermia, and these sperm have poor fertilizing potential and may have abnormal DNA. A disadvantage to any morphology assessment is that reproducibility may be hampered by the subjective nature of the assessment (30).

Nonsperm Cells

These include epithelial cells, round cells, and isolated sperm heads or tails. Round cells include immature germ cells and leukocytes. Immature germ cell elevation suggests testicular damage, while leukocytes (predominantly neutrophils) are associated with inflammation. Leukocytes can be distinguished by peroxidase positive staining, and normal leukocyte concentrations should be less than 1 million/mL. However, the prognostic significance of leukocytes in the semen is controversial (30,50). When bacterial colonization is found, the most common pathogens are Chlamydia trachomatis (41.4%), Ureaplasma urealyticum (15.5%), and Mycoplasma hominis (10.3%) (51).

Antisperm Antibodies

Antisperm antibodies, particularly those found on the surface of sperm, are associated with decreased pregnancy rates. Testing may be indicated with a history of ductal obstruction, prior genital infection, testicular trauma, and prior vasectomy reversal. It may be useful with oligozoospermia in the setting of normal hormonal levels, asthenospermia with normal sperm concentration, sperm agglutination, or unexplained infertility. Antisperm antibody testing is not needed if the sperm are to be used for intracytoplasmic sperm injection (ICSI) (44,47). Using the immunobead test, washed spermatozoa are exposed and assessed for binding to labeled beads. In the mixed agglutination reaction, human red blood cells sensitized with human immunoglobulin G (IgG) are mixed with the partner’s semen. Spermatozoa that are coated with antibodies form mixed agglutinates with the red blood cells (30).

Other Sperm Tests

Although the standard semen analysis and associated tests provide a fairly reasonable picture of semen quality, they yield little information about sperm function. These specialized tests may be pursued to assess DNA integrity, fertilization potential (zona-free hamster oocyte test), and the effect of cervical mucus on sperm viability and function (postcoital test) (44,52). In general, these tests are not currently considered part of the standard assessment because their prognostic value and impact on management are limited by poor specificity, poor reproducibility, or controversies concerning the interpretation of the results (44,47,52).

Differential Diagnosis of Male Factor

If abnormalities in the semen are detected, further evaluation of the male partner by a urologist is indicated to diagnose the defect. Table 32.4 lists the differential diagnoses for male factor infertility (53). Several groups have attempted to assess the distribution of male infertility diagnoses; two such distributions are shown in Table 32.5 (54,55). The first is the result of a WHO study of 7,057 men with complete diagnoses based on the WHO standard investigation of the infertile couple (54). The figures include data from cases in which the male partner was normal and the presumed cause of the couple’s infertility was a female factor. The second distribution is the result of a study of 425 subfertile male patients (54). Although the two studies represent different populations (one is from a study of couples, the other from a urologic practice) and differ in their distribution of male infertility diagnoses, idiopathic male factor and varicocele predominate. Other anatomic and endocrine causes occur less frequently. Same sex female couples or single women without a male partner who desire pregnancy may be considered as part of this category.

Table 32.4 Etiologic Factors in Male Infertility

Pretesticular	Testicular
Endocrine	Genetic
Hypogonadotropic hypogonadism	Klinefelter’s syndrome
Coital disorders	Y chromosome deletions
Erectile dysfunction	Immotile cilia syndrome
Psychosexual	Congenital
Endocrine, neural, or vascular	Cryptorchidism
Ejaculatory failure	Infective (orchitis)
Psychosexual	Antispermatogenic agents
After genitourinary surgery	Heat
Neural	Chemotherapy
Drug related	Drugs
Posttesticular	Irradiation
Obstructive	Vascular
Epididymal	Torsion
Congenital	Varicocele
Infective	Immunologic
Vasal	Idiopathic
Genetic: cystic fibrosis
Acquired: vasectomy
Epididymal hostility
Epididymal asthenozoospermia
Accessory gland infection
Immunologic
Idiopathic
Postvasectomy
From De Kretser DM. Male infertility. Lancet 1997;349:787–790, with permission.

Table 32.5 Frequency of Some Etiologies in Male Factor Infertility

Male Age

Men reportedly have fathered children into their 90s, but pregnancy rates are decreased with paternal older than age 40 to 45 and particularly over age 50. Increasing paternal age is associated with a higher frequency of disomic sex chromosomes and structural chromosomal abnormalities in the sperm. With respect to the offspring, paternal age confers higher rates of autosomal dominant diseases such as achondroplasia and craniosynostotic conditions and somewhat higher rates of trisomy 21 (56). In mice, offspring of older fathers have decreased survival (57). Increased paternal age is associated with recurrent pregnancy loss (58).

Treatment of Male Factor Not From Azoospermia

Medical treatment of reversible infectious or endocrine causes of male subfertility, such as sexually transmitted diseases and thyroid disorders, tends to be efficacious. Although injections of exogenous FSH have been reported to improve pregnancy rates, the benefit is less clear for clomiphene citrate, an estrogen antagonist at the hypothalamus and pituitary that promotes gonadotropin release. Exogenous testosterone is not recommended in the treatment of male subfertility because of negative feedback inhibition at the pituitary that results in decreased spermatogenesis, but the combination of testosterone and an antiestrogen may be of benefit. Antioxidant food supplements have been evaluated (59). Glutathione, carnitine, and vitamin E do not appear to affect semen parameters, but administration of zinc and folic acid were associated with improved sperm concentration and morphology (60). The diagnosis and treatment of azoospermia (no sperm on semen analysis) will be discussed separately from other forms of male factor infertility.

Varicocele Repair

A varicocele is an abnormal dilation of the veins within the spermatic cord. Varicoceles are present in 15% of normal men and 40% of men seeking infertility treatment, but semen parameters are lower among infertile men with varicoceles (61,62). The pathophysiologic effects of a varicocele appear to be mediated by an associated rise in testicular temperature, reflux of toxic metabolites from the left adrenal or renal veins, or higher reactive oxygen species (61,63). Although treatment is associated with improved semen parameters in some studies, it is not clear whether varicocele repair definitely improves fertility (61,64,65). Treatment is typically considered if the varicocele is palpable and the semen analysis is abnormal, but is not indicated if IVF would be required to treat the female partner and the existing semen analysis would be acceptable for ICSI. Treatment methods include surgical repair and percutaneous embolization. Complications of treatment include infection, varicocele persistence, recurrence, and hydrocele formation (61).

Artificial Insemination

Artificial insemination has been used mainly to treat unexplained infertility (usually combined with superovulation) and male factor infertility (including same sex female couples). All artificial insemination procedures involve the placement of whole semen or processed sperm into the female reproductive tract, which permits sperm–ovum interaction in the absence of intercourse. The placement of whole semen into the vagina as a mode of fertility treatment is now rarely performed except in cases of severe coital dysfunction. Currently, all of the common forms of artificial insemination involve processed sperm obtained from the male partner or a donor. Many techniques for artificial insemination have been described, but only intracervical and intrauterine inseminations (IUI) have been routinely employed. The success rates with intracervical insemination are lower than those with IUI, particularly when using frozen sperm (66–68).

Insemination Processing

During and after intercourse, seminal fluid usually is prevented from reaching the intrauterine cavity and intra-abdominal space by the cervical barrier. The introduction of seminal fluid past this barrier may be associated with pelvic infection and severe uterine cramping or anaphylactoid reactions, possibly mediated by seminal factors such as prostaglandins. Thus, protocols for processing whole semen include the washing of specimens to remove seminal factors and to isolate pure sperm. Additional processing may include centrifugation through density gradients, sperm migration protocols, and differential adherence procedures (66). Finally, phosphodiesterase inhibitors, such as pentoxiphylline, have been used during semen processing in an attempt to enhance sperm motility, fertilization capacity, and acrosome reactivity for IVF procedures (60,69).

Intrauterine Insemination

IUI is the best studied and most widely practiced of all the insemination techniques. It involves placement of about 0.3 to 0.5 mL of washed, processed, and concentrated sperm into the intrauterine cavity by transcervical catheterization (66). Patients should remain immobile for approximately 15 minutes following the procedure (70). Studies of the efficacy of IUI in the treatment of male factor infertility have been difficult to assess because of variations in inclusion criteria and the limitations of sperm function tests. Although it makes intuitive sense that IUI would result in higher pregnancy rates rather than timed intercourse in the case of male factor subfertility, there are conflicting reports of the benefits of IUI (66,71–74). Ideally, the total motile sperm count in the IUI specimen should be 5 million or 10 million or more (75–78). Pregnancy rates with semen meeting those thresholds have been reported to be 10.5% per cycle and 38% after 4 to 6 cycles (77,78). No benefit has been found with respect to double IUI versus single IUI during a single cycle (79).

Intracytoplasmic Sperm Injection

In general, ICSI has allowed couples with male factor infertility to achieve assisted reproductive technology (ART) pregnancy outcomes that are comparable with those of couples with non–male factor infertility using conventional IVF treatment (80). ICSI has been used since 1992 to increase the fertilization rate of oocytes retrieved during ART by direct injection of a live sperm into the oocyte, thereby theoretically bypassing limitations imposed by sperm motility and defects in capacitation, the acrosome reaction, and/or sperm binding the zona pellucida (81). This microsurgical procedure involves stripping the aspirated cumulus complex of all surrounding granulosa cells, followed by insertion of a single viable sperm into the cytoplasm (ooplasm) of the mature metaphase II egg (9,82). ICSI should be offered if the semen analysis shows less than 2 million motile sperm, less than 5% motility, or if surgically recovered sperm are used. Its use for abnormal morphology is more controversial (47,83). Immature or round spermatid nucleus injection (ROSNI) for ICSI is considered experimental at this time (84). Higher pregnancy rates have been noted with fresh compared to frozen specimens and with ejaculated compared to surgically retrieved sperm. Success rates are affected by the age of the female partner and oocyte quality (47). Non–male factor indications for ICSI include a history of fertilization failure with conventional IVF and the fertilization of oocytes before preimplantation genetic diagnosis (85). Other uses are described under “Unexplained Infertility.”

Risks of Intracytoplasmic Sperm Injection

In skilled hands, oocyte damage with ICSI is reportedly 10% (81). However, oocyte degeneration can follow even an uncomplicated ICSI procedure, with rates as high as 30% to 50%. This is most likely a function of oocyte quality and/or patient factors rather than the ICSI process itself (82). Based on data from 5-year-old children, ICSI is associated with a higher congenital anomaly risk (4.2%) when compared with conventional IVF (typically 2% to 3%) (80). There may be an increased risk of imprinting disorders and hypospadias with ICSI (47). Reports indicate a slightly higher risk of sex chromosome abnormalities (0.8% to 1% ICSI vs. 0.2% IVF) and translocations (0.36% ICSI vs. 0.07% general population). It is unclear whether these relate to the ICSI procedure or inherent gamete defects; men with abnormal semen parameters have higher rates of sperm aneuploidy. Recent studies have not found an association with impaired intellectual or motor development among ICSI children. In the setting of specific genetic abnormalities such as Y chromosome microdeletions, abnormal karyotypes, cystic fibrosis mutations, or congenital absence of the vas deferens, genetic counseling should be offered to address the possible risk of infertility or other abnormality in the offspring (80).

Azoospermia: Classification and Treatment

Azoospermia describes the absence of spermatozoa in the ejaculate and is found in 1% of all men and up to 15% to 20% of infertile men. Causes are categorized into pretesticular (nonobstructive), testicular (nonobstructive), and posttesticular (includes obstructive and nonobstructive) etiologies, but in some cases the condition is idiopathic (47,86).

Pretesticular Azoospermia

Pretesticular azoospermia is relatively rare and results from gonadotropin deficiency, which leads to loss of spermatogenesis. The physician should perform a full endocrine history that includes information on puberty and growth and check for low serum levels of LH, FSH, and testosterone (44,47,86). Prolactin levels and pituitary imaging are indicated in cases of hypogonadotropic hypogonadism (47). Hormonal treatment includes pulsatile gonadotropin-releasing hormone (GnRH), human chorionic gonadotropin, and exogenous gonadotropins (44,47,86). The best predictors of good response are postpubertal onset of gonadotropin deficiency and testicular volume greater than 8 mL (47).

Testicular Azoospermia

Gonadal failure is the hallmark of testicular azoospermia. Causes of this condition may be genetic, acquired (e.g., radiation therapy, chemotherapy, testicular torsion, varicocele, or mumps orchitis), or developmental (e.g., testicular maldescent). Testicular atrophy is often present. Because of the low chance of obtaining sperm, testicular biopsy is generally not recommended with hypergonadotropic hypogonadism (elevated levels of LH and FSH with low serum levels of testosterone) and consideration should be given to using donor sperm. Diagnostic testicular biopsies may be indicated in the setting of normal hormonal testing. If sperm are present on diagnostic testing, consideration can be given to using surgically retrieved spermatozoa for ICSI (44,47,86). If no sperm are present, consideration can be given to correcting acquired conditions such as varicocele, which may restore sperm to the ejaculate to permit ICSI or spontaneous pregnancy (87,88). Chromosomal abnormalities by peripheral karyotype testing are present in about 7% of infertile, 5% of oligospermic, and 10% to 15% in azoospermic men. Sex chromosome aneuploidies such as Klinefelter syndrome (47,XXY) encompass two-thirds of these infertility-associated chromosome abnormalities. Microdeletions in the Y chromosome have been identified in 10% to 20% of men with idiopathic azoospermia or severe oligospermia with concentration less than 5 million/mL (44,47,86) (Table 32.6). These microdeletions can be transmitted to the male offspring, who may then suffer from infertility. Therefore, screening for genetic causes is indicated in nonacquired cases of testicular azoospermia so that genetic counseling can be provided before treatment (80). The two most commonly implicated candidate gene families are the RNA-binding motif (RBM) and the “deleted in azoospermia” (DAZ) families, but microdeletions at various loci on the Y chromosome have been described (44,47,86,89–91). For example, microdeletions in Yq11.23 can occur in one or more of three regions: AZFa (proximal), AZFb (central), and AZFc (distal) (44,47,86).

Table 32.6 Genetics and Male Infertility

Posttesticular Azoospermia

Posttesticular or obstructive etiologies are associated with normal gonadotropin and testosterone levels and are present in up to 40% of azoospermic men (47,86). Ejaculatory dysfunction is associated with oligospermia or aspermia but rarely azoospermia (86). Obstructive causes include congenital absence or obstruction of the vas deferens or ejaculatory ducts, acquired obstruction of these ducts, or ductal dysfunction, including retrograde ejaculation (47,86). In the absence of congenital bilateral absence of the vas deferens (CBAVD) or hypogonadism, men with low volume ejaculate should have a postejaculatory urinalysis to check for retrograde ejaculation, which is associated with diabetes and surgery to the bladder or prostate (44,47). Sperm may be isolated from the neutralized urine of men with retrograde ejaculation and processed for insemination or for ART (30). Transrectal ultrasound may be of use to diagnose ejaculatory duct obstruction or unilateral vasal agenesis (to demonstrate contralateral atresia) but is generally not needed for CBAVD (86). Renal imaging is necessary when either unilateral or bilateral vasal absence is diagnosed as a result of the 10% to 25% incidence of renal agenesis. Most men with CBAVD will have seminal vesicle agenesis, so almost all will have low semen volume, low pH, and low fructose levels (86). Spermatogenesis can be expected to be normal in CBAVD, so generally diagnostic biopsy is not indicated (92). In some cases, testicular biopsy may be indicated to differentiate between testicular and posttesticular causes. At least two-thirds of men with CBAVD have mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR). However, because many CFTR mutations are undetectable, CBAVD patients should be assumed to have a mutation and thus testing of the female partner’s carrier status should be performed (86).

Vasectomy Reversal and Treatment of Obstructive Azoospermia

Vasectomy can be reversed effectively using microsurgical vasovasostomy or vasoepididymostomy. These techniques can be used for epidymal obstructions (93). Patency and subsequent pregnancy rates approach 100% and 80%, respectively. Pregnancy typically occurs within 24 months of reversal (94). Rates of patency and pregnancy vary inversely with the length of time from vasectomy, particularly for reversal procedure performed after 15 years or more (93,94). Although 60% of reversal patients develop antisperm antibodies, these do not appear to affect fecundability. Following surgery, periodic semen analyses can identify reobstruction, which can range from 3% to 21% depending on which segments were anastamosed. For those patients with azoospermia 6 months after reversal, the procedure is considered a failure and testicular sperm aspiration with ICSI could be considered. However, repeat vasovasostomy is associated with patency rates of 75% and pregnancy rates of 43% (94).

Surgical Sperm Recovery for Intracytoplasmic Sperm Injection

Among the many surgical methods for sperm recovery, the most widely described are microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular sperm extraction (TESE), and percutaneous testicular sperm fine-needle aspiration (TESA, also called fine-needle aspiration, or FNA). Both MESA and TESE are open surgical procedures performed with an operating microscope and general or regional anesthesia, whereas the percutaneous procedures need only local anesthesia. The optimal choice for surgical sperm recovery method has not been determined and certainly varies based on patient history. Hematoma risk appears to be low regardless of method. Testicular atrophy is a rare complication of TESE and TESA even when biopsies are obtained from multiple testicular sites (92).

With obstructive azoospermia, pregnancy rates using sperm retrieval and ICSI are 24% and 64%, respectively, and outcomes using either frozen-thawed or fresh sperm are comparable. Because MESA allows for diagnosis and possible reconstruction of ductal pathology and because it usually yields very large numbers of sperm, sperm cryopreservation and avoidance of repeat surgery may be possible (92). If repeat sperm retrievals are needed, the minimum interval between procedures is 3 to 6 months to allow for adequate healing (93).

For nonobstructive (testicular) azoospermia, epididymal aspiration is not an option. Although pregnancies have been reported following testicular sperm retrieval with nonobstructive azoospermia, the likelihood of retrieving sperm is low as are subsequent pregnancy rates (92,95). Patients with obstructive azoospermia must be counseled regarding the risk of transmitting genetic disorders to their offspring (47,86).

Donor Insemination

For men with azoospermia, couples with significant male factor infertility who do not desire ART, or women without a male partner who are seeking pregnancy, therapeutic donor insemination offers an effective option (96). In several prospective randomized or crossover trials, IUI was shown to be superior to intracervical insemination for donor insemination (68). In patients younger than 30 years of age who have no other infertility factors, delivery rates approach 90% after 12 cycles of IUI treatment with frozen sperm, so patients who do not conceive within 6 to 12 months should be assessed for female factors and encouraged to terminate treatment or proceed with alternative forms of therapy. The concomitant use of clomiphene citrate or gonadotropin (hMG) for controlled ovarian hyperstimulation (COH) did not result in higher fecundity rates in these patients (97). Psychological counseling should be offered because of the potential repercussions of using donor gametes (96).

Donor Sperm Screening

Although the use of fresh donor semen is associated with higher pregnancy rates than the use of frozen specimens, both the Centers for Disease Control and Prevention and the American Society for Reproductive Medicine recommend the use of frozen samples (96,98,99). This recommendation stems from the increasing incidence of human immunodeficiency virus (HIV) infection in the general population and the lag time between HIV infection and seroconversion. Currently, semen donors are screened for HIV infection, hepatitis B, hepatitis C, syphilis, gonorrhea, chlamydia, and cytomegalovirus infections, all of which may be transmitted through semen. All cryopreserved samples are quarantined for 6 months, and the donor is retested for HIV before clinical use of the specimen. Donors are likewise questioned about any family history of genetically transmitted disorders, including both Mendelian (e.g., hemophilia, Tay-Sachs disease, thalassemia, cystic fibrosis, congenital adrenal hyperplasia, Huntington disease) and polygenic or multifactorial conditions (e.g., mental retardation, diabetes, heart malformation, spina bifida). Those with a positive family history of these conditions are eliminated as donor candidates (96).

Figure 32.4 Live births per embryo transfer comparing use of a patient’s own versus donor oocytes. (Adapted from CDC Reproductive Health. 2009 Assisted Reproductive Technology Success Rates. National Summary and Fertility Clinic Reports. http://www.cdc.gov/nccdphp/drh/art.htm.)

Female Age and Decreased Ovarian Reserve

Decreased Fecundability

Most women will experience a decline in fecundability as they age that is physiologic rather than pathologic. This decline begins in the early 30s and accelerates during the late 30s and early 40s, reflecting declines in oocyte quantity and quality. Among populations that do not practice contraception, fertility peaks at age 20, declines somewhat at age 32, steeply declines after the age of 37, and is rare after age 45 (100). Wives of azoospermic husbands who received donor IUI had cumulative pregnancy rates over 12 cycles of 74% (age <31 years), 62% (age 31 to 35), and 54% (age >35) (101). Chronologic aging of the endometrium does not seem to play an appreciable role in reduced fertility, given the excellent pregnancy and live birth rates using donor oocytes (Fig. 32.4) (102,103). IVF success rates similarly decrease with age and will be further discussed later in the chapter (100). Reproductive aging is related to the stock of primordial follicles that are established early in fetal life and decline to near zero at meno-pause (104).

Spontaneous Pregnancy Loss

Reproductive aging is associated with abnormalities in the oocyte meiotic spindles that lead to chromosome alignment errors and increase rates of conceptus aneuploidies, particularly trisomies.This serves to increase the risk for spontaneous pregnancy loss and thereby decrease live birth rates in older women (100,105). A large study based on the Danish national registry estimated the rates of clinically recognized spontaneous pregnancy loss for various age groups to be 13.3% (12 to 19 years), 11.1% (20 to 24 years), 11.9% (25 to 29 years), 15.0% (30 to 34 years), 24.6% (35 to 39 years), 51.0% (40 to 44 years), and 93.4% (older than 45 years) (106). In addition, using sensitive hCG assays in women during their reproductive years, 22% of all pregnancies were found to be lost before they could be clinically diagnosed (107).

Ovarian Reserve

Ovarian reserve refers to the size of the nongrowing, or resting, primordial follicle population in the ovaries. This, in turn, presumably determines the number of growing follicles and the “quality” or reproductive potential of their oocytes (105). Tests of ovarian reserve are often used, but the predictive values of these tests for fertility potential are limited, particularly when normal testing is found in older women and abnormal testing is documented in younger women. The tests appear to be better suited for determining how the ovaries will respond to pharmacologic doses of exogenous gonadotropins in terms of follicle count, the number of oocytes produced, serum estradiol levels during stimulation, the duration of stimulation, and the quantity of exogenous gonadotropins required in a given cycle (108). However, unlike age, the results of ovarian reserve testing are poorly predictive of pregnancy outcomes (109–111). These tests appear to be more indicative of oocyte quantity rather than quality (110).

Serum Day 3 Follicule Stimulating Hormone

As women age, FSH physiologically rises in the early follicular phase (cycle day 3), with levels of 5.74 IU/L at age 35 to 39 and 14.34 IU/L at age 45 to 59. Higher levels are seen after unilateral oophorectomy. In women in their 40s, levels greater than 20 IU/L are predictive of menopause. Because the incidence of abnormal values is lower in younger women, testing is typically performed for women aged 35 or older (108). In subfertile women with an FSH 8 IU/L or more, spontaneous pregnancy rates decrease by 7% per unit of FSH increase, with a 40% reduction at 15 IU/L and 58% at 20 IU/L (112). FSH levels vary widely by assay, laboratory, and population (109). Because of the poor sensitivity of high basal FSH values in determining fecundability, they should not be used as the sole basis for excluding women from consideration for ART (113). Likewise, the poor specificity of low basal FSH values in determining fecundability makes them unreliable when used to reassure patients, particularly those of increased reproductive age (109).

Basal Estradiol Level

Basal day 3 FSH is often combined with estradiol (E2) testing. Estradiol levels on day 3 of the menstrual cycle reflect follicular growth rather than the number of antral follicles (114). Elevations in FSH and decreases in inhibin B (see below) that accompany aging results in advanced follicular growth at the end of the preceding luteal phase. In response, early follicular E2 levels are typically higher in older women and in women with advanced reproductive aging (104).

Clomiphene Citrate Challenge Test

Clomiphene citrate is thought to have antiestrogenic effects on the hypothalamic–pituitary axis, resulting in a decrease in E2-mediated suppression of FSH production by the pituitary. The clomiphene citratechallenge test (CCCT) involves the measurement of serum FSH and estradiol on day 3 of the menstrual cycle, and again on day 10 after administration of clomiphene citrate (100 mg orally each day) from days 5 to 9. The CCCT has been reported to be more sensitive than basal FSH alone in identifying poor response to exogenous gonadotropins, but others report that the predictive values of the tests do not differ substantially (109,110).

Serum Inhibin B

Serum inhibin B is secreted by ovarian granulosa cells starting at the preantral follicle stage and therefore reflects the size of the growing follicular cohort (111). Reduced inhibin B levels are seen with aging even in normal fertile women (114). Inhibin B alone has poor predictive value for ovarian response but improves the predictive value when added to the CCCT (109,110). Unlike basal testing, levels of inhibin B measured on the 5th day of ovarian stimulation were predictive of live birth following IVF/ICSI (115).

Serum Antimüllerian Hormone

Antimüllerian hormone (AMH) is produced by the granulosa cells of preantral and small antral follicles (111,114). The serum level of AMH in women with normal cycles declines with age and becomes undetectable after menopause (114,116). AMH appears to be a good predictor of both excessive (>3.5 ng/mL) and poor (<1 ng/mL) IVF stimulation response and is strongly correlated to the antral follicle count (111,116,117). Unlike other serum markers, AMH can be measured at any time in the menstrual cycle (116).

Antral Follicle Count

Using transvaginal ultrasound in the early follicular phase, all ovarian follicles 2 to 10 mm are counted and the total for both ovaries is called the basal antral follicle count (AFC). The AFC correlates well with chronologic age in normal fertile women and appears to reflect what remains of the primordial follicular pool (104). Decreases in AFC with age are gradual rather than sudden (118). A total AFC less than 4 is predictive of poor response and higher cancellation rates with IVF (119,120).

Treatment of Diminished Ovarian Reserve

Treatments of diminished ovarian reserve include autologous IVF, use of donor oocytes or embryos, and adoption. Pretreatment of women with diminished ovarian reserve for 4 to 5 months with dehydroepiandrosterone(DHEA; 25 mg three times daily) has been described to improve oocyte yield and pregnancy rates with IVF (121–123).

Ovulatory Factor

Ovulatory factor accounts for 30% to 40% of all cases of female infertility. Initial diagnoses among women with ovulatory factor infertility may include anovulation (complete absence of ovulation) or oligo-ovulation (infrequent ovulation). Menstrual history may be suggestive if oligomenorrhea, amenorrhea, polymenorrhea, or dysfunctional uterine bleeding is present (124). Menstrual dysfunction is present in 18% to 20% of the general population (125). Figure 32.5 shows the fluctuations of E2, progesterone, FSH, and LH in a normal, 28-day ovulatory cycle. The normal length of the menstrual cycle in reproductive-age women varies from 21 to 35 days, with a mean of 27 to 29 days (126). Most of the variability in cycle length occurs during the follicular phase (127), but the luteal phase, often considered to be fixed at 14 days, can range from 7 to 19 days (128). Even women with regular monthly menses may have anovulation, although the presence of moliminal symptoms such as premenstrual breast swelling, bloating, and mood changes are much more suggestive of ovulatory cycles (125).

Figure 32.5 Relative hormonal fluctuations in a normal, ovulatory, 28-day menstrual cycle.

Methods to Document Ovulation

The “Fertile Window”

The fertile window is the 6 day interval ending on the day of ovulation, but not after ovulation. Sperm can survive for up to 6 days in well-estrogenized cervical mucus, but the egg may be fertilizable for less than a day. Daily intercourse during this window may increase the probability of conception (32,127). The average woman is fertile between days 10 and 17 of the menstrual cycle, but many women can conceive outside of this range (128). Therefore, if timed intercourse is too cumbersome, intercourse two to three times weekly throughout the menstrual cycle will likely result in at least some of those occasions falling within the fertile window (129). Duration of abstinence prior to the fertile window has not been established, although one author suggests 5 days (127).

Basal Body Temperature

This inexpensive method involves daily recording of oral or rectal temperature using a basal body temperature (BBT) thermometer before the patient arises, eats, or drinks. The secretion of progesterone following ovulation causes a temperature increase of about 0.5 to 1°F over the baseline temperature of 97°F to 98.8°F that is typically recorded during the follicular phase of the menstrual cycle. Ovulation is assumed after 3 consecutive days of raised temperatures. Charting of daily BBTs produces a characteristic biphasic pattern in women with ovulatory cycles (130). Limitations to BBT include its inability to prospectively predict ovulation and its frequent false-negative results (131). Smoking and irregular sleep patterns can interfere with accurate BBT testing (130).

Cervical Mucus

During the fertile window, cervical secretions at the vaginal introitus are slippery and clear, while secretions at other times of the menstrual cycle are dry and sticky (32,130). The volume of cervical mucus peaks 2 to 3 days prior to ovulation, thus identifying higher day-specific probabilities of conception (32).

Luteinizing Hormone Monitoring

At a mean time of 2 hours following the peak of the serum LH surge, urinary LH can be detected. Commercially available kits for documenting the LH surge are generally accurate, quick, convenient, and relatively inexpensive enzyme-linked immunosorbent assays (ELISA) that use 35 to 50 mIU/mL as their threshold for detection (132,133). Once the LH surge is detected, ovulation may occur within the next 48 hours (32,132,133). The positive-predictive and negative-predictive values for these kits have been described to be 92% and 95%, respectively (132,134). Because the duration of the surge may be less than 12 hours, twice daily testing may increase detection rates (133). However, the 2 days of highest probability of conception are the day of and the day prior to the LH surge, so this may actually lead to abstaining from intercourse during a potentially fertile time (127). False-positive rates occur in 7% of cycles, which may reflect urinary clearance of unsustained premature LH surges (32,131,135). The tests cannot be used in patients with irregular cycles (127).

Midluteal Serum Progesterone

When used to document ovulation, serum progesterone measurement should coincide with peak progesterone secretion in the midluteal phase (typically on days 21 to 23 of an ideal 28-day cycle or 7 days following the LH surge). The lower limit of progesterone levels in the luteal phase varies among laboratories, but a level above 3 ng/mL (10 nmol/L) typically confirms ovulation. However, interpretation of isolated luteal-phase measurements of serum progesterone is complicated by the frequent pulses that characterize the secretion of this hormone. Although ovulatory levels are often considerably higher than 3 ng/mL, low midluteal serum levels of progesterone are not necessarily diagnostic of anovulation (124).

Ultrasound Monitoring

Ovulation is characterized both by a decrease in the size of a monitored ovarian follicle and by the appearance of fluid in the cul-de-sac using transvaginal ultrasound (124). Follicles reach a preovulatory diameter of 17 to 19 mm in spontaneous cycles or 19 to 25 mm for clomiphene-induced cycles (136,137). A combination of LH testing and ultrasound can be used, with LH kit testing starting when the ultrasound-measured follicle size reaches 14 mm (131). Ten percent of the cycles of normally fertile women may have a luteinized unruptured follicle, whereby progesterone is released and the luteal phase progresses normally without visible signs of follicle rupture when daily ultrasounds are performed from cycle days 10 to 20. This incidence is increased to 25% in women with unexplained infertility (138). Because of the inconvenience and expense of serial measurements, ultrasound monitoring should be reserved for patients who fail less expensive methods for detecting ovulation or for certain types of ovulation induction (124).

Follow-Up Tests

In women with absent or infrequent ovulation, serum FSH, prolactin, and thyroid-stimulating hormone (TSH) testing should be performed (124).

Polycystic Ovarian Syndrome

The most common cause of oligo-ovulation and anovulation—both in the general population and among women presenting with infertility—is polycystic ovarian syndrome (PCOS) (139). The diagnosis of PCOS is determined by exclusion of other medical conditions such as pregnancy, hypothalamic–pituitary disorders, or other causes of hyperandrogenism (e.g., androgen-secreting tumors or nonclassical congenital adrenal hyperplasia) and the presence of two of the following conditions (140):

• Oligo-ovulation or anovulation (manifested as oligomenorrhea or amenorrhea)

• Hyperandrogenemia (elevated levels of circulating androgens) or hyperandrogenism (clinical manifestations of androgen excess)

• Polycystic ovaries detected by ultrasonography

Documentation of elevated serum LH:FSH ratios and hyperinsulinemia are not required for either diagnosis or treatment of PCOS (139,140). Patients with PCOS should be counseled and screened regarding potential metabolic disease and obstetric complications prior to fertility treatment (141).

Ovulation Induction in Women with Polycystic Ovarian Syndrome

Despite the use of similar medications, the indications and goals of ovulation induction should be distinguished from those of superovulation. The goal of ovulation induction refers to the therapeutic restoration of the release of one egg per cycle in a woman who either has not been ovulating regularly or has not been ovulating at all. In contrast, the explicit goal of superovulation is to cause more than one egg to be ovulated, thereby increasing the probability of conception in women with unexplained infertility (136).

Weight Loss

Obesity in PCOS patients is associated with poor infertility treatment outcomes (139,142–144), although the impact on pregnancy loss rates is less clear (141,142,145). Given that even a 5% weight loss may improve pregnancy rates, weight loss should be encouraged in all overweight and obese infertility patients (139). Generally, lifestyle modification is the first-line therapy, followed by pharmacologic treatment and weight-loss surgery (139). Lifestyle recommendations include a decrease in daily caloric consumption by 500 kcal and regular physical exercise, although the optimal regimen for the latter is unknown (139,146). Weight loss interventions should be undertaken prior to attempting conception in patients with excess body weight (139).

Clomiphene Citrate (Clomid, Serophene) Pharmacology

Clomiphene citrate is a weak synthetic estrogen that mimics the activity of an estrogen antagonist when given at typical pharmacologic doses for the induction of ovulation. It is cleared through the liver and excreted into the stool, with 85% clearance in 6 days. A functional hypothalamic–pituitary–ovarian axis is usually required for appropriate clomiphene citrate action. More specifically, clomiphene citrate is thought to bind and block estrogen receptors in the hypothalamus for prolonged periods, thereby decreasing the normal ovarian–hypothalamic estrogen feedback loop. This blockade increases GnRH pulsatility, leading to increased pituitary secretion of gonadotropins, which promote ovarian follicular development (136).

Clomiphene Citrate Outcomes

Clomiphene is considered a first-line treatment for anovulatory infertility (147). Over the course of 6 months, clomiphene is associated with 49% ovulation, 23.9% pregnancy, and 22.5% live birth rates in women with anovulatory infertility (142). Clomiphene effectiveness is decreased by obesity, increased age, and hyperandrogenic states (142,148,149). Side effects of clomiphene citrate include vasomotor flushes, mood swings, breast tenderness, pelvic discomfort, and nausea. In a minority of individuals, the antiestrogenic effects of clomiphene citrate at the level of the endometrium or the cervix may have adverse effects on fertility. In the presence of visual abnormalities, clomiphene citrate should be discontinued promptly. Multiple gestation rates with clomiphene citrate are approximately 8%, most of which are twins (136). Treatment should be limited to 6 ovulatory cycles or 12 total cycles (141,150,151).

Clomiphene Citrate Dosing

The drug is supplied in 50 mg tablets; the usual starting dose is 50 mg per day, but patients who are very sensitive may respond to 12.5 to 25 mg per day. Therapy is typically begun within the first 5 days after the onset of a spontaneous or progesterone-induced menses and is continued for 5 days (i.e., treatment on days 2 to 6, 3 to 7, or 5 to 9 of the menstrual cycle) (136). It has been reported to be effective when started the day following progesterone withdrawal therapy, without waiting for menses (152). If ovulation does not occur at the initial dosage of clomiphene citrate, the dosage is increased in each subsequent cycle by 50 mg per day. Seventy-four percent of women will ovulate with 100 mg per day, the maximum dose approved by the U.S. Food and Drug Administration (153). However, some patients need higher doses, which have been safely given up to 250 mg per day (136). A novel stair-step method has been proposed, which increases the dose within a single cycle without intervening menses if there was no follicular response documented by ultrasound 4 to 5 days after the last pill (154). However, this particular protocol raises concern for unknown endometrial and embryonic effects (155).

Ovulation Monitoring during Clomiphene Citrate Therapy

If preovulation monitoring is not performed, patients should be instructed to have intercourse every 2 to 3 days following the last day of therapy and check a serum progesterone weekly for 5 weeks before inducing a withdrawal bleed or increasing the clomiphene citrate dose (142). Although no clear advantage has been demonstrated for any ovulation monitoring technique, regular contact should be maintained with patients to review response to therapy. The urinary LH surge may be detected 5 to 12 days after treatment is completed. When clomiphene is given cycle days 5 to 9, the surge typically occurs on cycle day 16 or 17 and can be confirmed by midluteal serum progesterone testing 7 days later. With ultrasound monitoring, treatment should be withheld if large cysts are seen on baseline testing. Following clomiphene, follicles typically reach a preovulatory diameter of 19 to 25 mm by ultrasound, but may be as large as 30 mm (136,137). A combination of LH testing and ultrasound can be used, with LH kits starting when the largest ultrasound-measured follicle reaches 14 mm in diameter (131).

Human Chorionic Gonadotropin

If a dominant follicle develops, but there is no spontaneous LH surge, human chorionic gonadotropin (hCG) can be used to induce final follicular maturation, with ovulation occurring approximately 40 hours following administration (136,156). Although administration of hCG at midcycle does not appear to improve conception chances in most infertility patients using clomiphene citrate, it may be useful for patients with known ovulatory dysfunction(157–159). The medication may be derived from urine (5,000 to 10,000 IU intramuscularly) or manufactured with recombinant technology (250 μg subcutaneously, equivalent to 5,000 to 6,000 IU urinary) (156).

Timing of Intrauterine Insemination

When IUI is added to the therapeutic protocol, insemination typically is performed 24 hours following the LH surge (157). However, given data that ovulation can occur much later than this, it is not surprising that no differences in pregnancy rates were found when IUI was performed between 24 and 60 hours following the LH surge (160). Although timing of IUI is typically performed 36 hours following hCG administration to coincide with follicle rupture when hCG is added to trigger ovulation, no significant differences in pregnancy or live birth rates are seen whether IUI is performed at 24 or 36 hours following the hCG trigger (161).

Table 32.7 Clinical Findings that Suggest Insulin Resistance and Hyperinsulinemia

Physical findings associated with insulin resistance

Body mass index >27 kg/m2

Waist-to-hip ratio >0.85

Waist >100 cm

Acanthosis nigricans

Numerous achrochordons (skin tags)

From Barbieri RL. Induction of ovulation in infertile women with hyperandrogenism and insulin resistance. Am J Obstet Gynecol 2000;183:1412–1418, with permission.

Insulin Sensitizers

Insulin resistance is thought to play a central role in the pathogenesis of PCOS (Table 32.7). Metformin is an oral biguanide that is approved for the treatment of non–insulin-dependent diabetes and has been used in PCOS to increase the frequency of spontaneous ovulation. Metformin acts by several mechanisms, including inhibition of gluconeogenesis in the liver and increasing the uptake of glucose in the periphery (162). Although the literature is conflicting, larger studies have suggested that the live birth rate with metformin alone (7.2%) is lower than that achieved with clomiphene, and the combination does not confer additional benefit over clomiphene alone (142,163). Obese patients are less likely to respond to metformin (164). It remains unclear whether metformin decreases miscarriage rates in PCOS, although it appears to be safe when used during pregnancy (142,162). Risks of metformininclude gastrointestinal upset and rare lactic acidosis, so it should be avoided in settings of hepatic and renal dysfunction and prior to surgery or use of contrast radiologic dye. Reported effective doses include 500 mg three times daily, 850 mg twice daily, or 1,000 mg twice daily (142,162). The medication is best tolerated when started at the lowest dose and increased gradually. Extended release formulations are associated with less gastrointestinal upset (142). Because regular ovulation may be delayed for 3 to 6 months or may not occur with metformin alone, patients may need medications to induce withdrawal bleeding to mitigate the risk of continued anovulation and endometrial hyperplasia (141). Thiazolidinediones, which include rosiglitazone and pioglitazone, have been used for ovulation induction in PCOS patients (165–168). They have less associated gastrointestinal upset than metformin. All carry the risk of liver toxicity and, in the case of rosiglitazone, cardiovascular harm, and they are typically avoided during pregnancy (141).

Dexamethasone

Adjunctive oral dexamethasone may improve ovulation rates in patients resistant to clomiphene alone (147). These improvements have been reported even in the absence of adrenal hyperandrogenemia, so the mechanism of action remains unclear (169,170). Dose regimens have included 0.5 mg for 5 days starting on the first day of clomiphene, 0.5 mg for 6 weeks prior to starting clomiphene, and 2 mg for 10 days starting on the first day of clomiphene (169–172).

Oral Contraceptive Pretreatment

Administration of oral contraceptives for 2 months prior to beginning a cycle of clomiphene may improve ovulation and pregnancy rates in clomiphene-resistant patients, perhaps by improving a preexisting hyperandrogenic environment (147,173).

Tamoxifen

Tamoxifen is an oral antiestrogen similar in structure to clomiphene that is commonly used as an adjuvant therapy for breast cancer but has been used off-label to induce ovulation (174). Ovulation and pregnancy rates are similar with tamoxifen and clomiphene (147,174). Doses start at 20 mg per day for 5 days (similar in timing to clomiphene), and can be increased to 40 or 60 mg per day in subsequent cycles. The mechanism of action for tamoxifen in inducing ovulation appears to be similar to that of clomiphene, although tamoxifen exerts less negative endometrial effects (175).

Aromatase Inhibitors

These drugs include letrozole and anastrazole. Through their inhibition of aromatase-mediated conversion of androgens to estrogens and subsequent decreases in circulating estradiol, aromatase inhibitors have been approved for treatment of breast cancer. The off-label use of letrozole for ovulation induction in clomiphene-resistant patients was first reported in 2001 (176). Typical doses are 2.5 to 5 mg of letrozole or 1 mg of anastrazole daily for 5 days. Timing of administration is similar to that of clomiphene, although longer durations of 10 days have been reported (177,178). Letrozole appears to have less negative effects on endometrial development when compared to clomiphene (176). Although concerns have been raised regarding possible associations between fetal congenital anomalies and the use of aromatase inhibitors, no such increase was observed when letrozole was compared to clomiphene (179). Patients should be counseled regarding the absence of prospective, sufficiently powered studies to assess safety of off-label use of aromatase inhibitors for ovulation induction (141).

Gonadotropin Therapy

Anovulatory PCOS patients who fail to ovulate or conceive with oral agents should be considered for ovulation induction with exogenous gonadotropin injections. Evening medication administration allows for morning monitoring and midday decision making. Monitoring involves serum estradiol levels and transvaginal ultrasound measurements of follicle development. Typical protocols monitor at baseline, 4 to 5 days after treatment initiation, then every 1 to 3 days until follicular maturation (expected follicle growth is 1 to 2 mm daily after achieving 10 mm diameter) (180). Given the goal of promoting growth of a single mature follicle, low initial gonadotropin doses of 37.5 to 75 IU per day are generally recommended, with increases in doses by 50% of the previous dose after 7 days if no follicle greater than 10 mm is observed (141,180). Typical treatment duration is 7 to 12 days, but some patients require longer medications regimens for adequate stimulation. The maximum required gonadotropin dose seldom exceeds 225 units per day. Ovulation triggering with hCG is recommended for gonadotropin cycles and is used when one or two follicles are 16 to 18 mm in diameter and the E2 level per dominant follicle is 150 to 300 pg/mL. Ovulation can be expected 24 to 48 hours after the hCG trigger. GnRH agonists such as leuprolide (500 μg subcutaneously) can be used to trigger ovulation, but they require progesterone supplementation following administration. Intercourse should be recommended within 24 to 48 hours of ovulation triggering or IUI 24 to 36 hours after triggering (180). Testing for pregnancy should be performed 15 to 16 days after ovulation triggering and the cycle reviewed if pregnancy testing is negative. Gonadotropin dosage in future cycles should be altered if the prior response was inadequate or excessive.

Gonadotropin Preparations

Several gonadotropin preparations are available. Human menopausal gonadotropin (HMG) is derived from human urine and includes approximately equivalent FSH and LH (derived from hCG) activity of 75 IU each. Current formulations of HMG and FSH can be administered either subcutaneously or intramuscularly. FSH-only preparations may be derived either from urine or via recombinant methods and are packaged either as lyophilized powder or premixed-liquid cartridges/pens. With the exception of highly purified urinary FSH, which has an FSH activity of 82.5 IU per ampule, all other products contain 75 IU of gonadotropin when supplied in ampules. All preparations of FSH are highly purified, with minimal to no batch-to-batch variation and a high level of safety regardless of the derived source. Despite being differentially marketed as follitropin-α and -β, these recombinant FSH preparations still contain combinations of 1-α and 1-β glycoprotein chain. Rather, they differ in their posttranslational modifications and processes for purification. Recombinant LH is available in syringes delivering 75 IU (156). For PCOS patients, FSH alone (either recombinant or urinary) appears to be sufficient in the gonadotropin preparation, although LH is not harmful (180) (Table 32.8). Contraindications to gonadotropin therapy are listed in Table 32.9.

Table 32.8 Different Types of Available Gonadotropin Preparations

Table 32.9 Contraindications to Gonadotropins for the Treatment of Infertility in Women

1. Primary ovarian failure with elevated follicle-stimulating hormone levels

2. Uncontrolled thyroid and adrenal dysfunction

3. An organic intracranial lesion such as a pituitary tumor

4. Undiagnosed abnormal uterine bleeding

5. Ovarian cysts or enlargement not caused by polycystic ovary syndrome

6. Prior hypersensitivity to the particular gonadotropin

7. Sex hormone–dependent tumors of the reproductive tract and accessory organs

8. Pregnancy

From Physicians desk reference. Micromedex (R) Healthcare Series Vol. 107. Thompson PRD and Micromedex Inc., 1974–2004, with permission.

Gonadotropin Outcomes

Cumulative live birth rates are similar when gonadotropins are compared to clomiphene for ovulation induction when the goal is monofollicular ovulation and a maximum of six ovulatory cycles is similarly recommended (141). When compared to other anovulatory patients, PCOS patients using gonadotropins are at higher risk for multiple gestations (36%), ovarian hyperstimulation syndrome (4.6%), and cycle cancellation (10%) because of their high numbers of baseline antral follicles (141,180, 181). Cancellation should be strongly considered in patients who reach E2 levels 1,000 to 2,500 pg/mL, have three or more follicles 16 mm or larger, or two or more follicles 16 mm or larger plus two or more follicles 14 mm or larger (141). Sequential use of gonadotropins and either clomiphene or aromatase inhibitors has been associated with lower gonadotropin requirements and lower cancellation rates and treatment duration without compromising pregnancy rates. The addition of aromatase inhibitors is associated with a lower number of dominant follicles and lower maximum E2 levels (182–184).

Surgical Treatment

For clomiphene-resistant patients, surgical ovarian drilling has been performed as an alternative to the outdated ovarian wedge resection in an effort to decrease ovarian androgen-producing tissue and to promote ovulation without the risk of multiple pregnancy seen with gonadotropin administration (141,185). Drilling 3 to 15 puncture sites per ovary is typically performed via laparoscopy using electrocautery and diathermy or laser, although transvaginal ultrasound-guided and vaginal hydro-laparoscopy procedures have been reported (186–195). Successful drilling has been performed with the harmonic scalpel (190). Within 12 months after ovarian drilling, cumulative ovulation, clinical pregnancy, and live birth rates are 52%, 26% to 48%, and 13% to 32%, respectively. These outcomes are similar to those using gonadotropins, but ovarian drilling carries a lower multiple gestation rate. Outcomes are not significantly different when diathermy or laser are used for drilling, but they are compromised with patient age greater than 35 years or basal FSH greater than 10 mIU/mL (185,187). The risks of ovarian drilling include surgical complications, adhesions, recurrence of anovulation, and a theoretical risk of ovarian failure (185,196).

Ovulation Induction for Other Anovulatory Disorders

Hyperprolactinemia

Hyperprolactinemia can be associated with oligomenorrhea or amenorrhea and should be evaluated with pituitary magnetic resonance imaging (MRI) to exclude macroadenoma or other intracranial pathology (197). Dopamine agonists are first-line agents in otherwise asymptomatic oligoovulatiory or anovulatory patients to restore ovulation (197). Bromocriptine normalizes prolactin levels and induces ovulation in 80% to 90% of patients. It is taken two to three times daily, and most patients will respond to a total dose less than 7.5 mg daily. Side effects can be bothersome and include nausea, vomiting, postural hypotension, and headache. Cabergoline has similarly high efficacy and the advantage of a 0.25 mg twice-weekly dosing schedule and fewer side effects (198).

Hypogonadotropic Hypogonadism

Anovulation in the presence of low serum LH, FSH, and estradiol levels defines hypogonadotropic hypogonadism and reflects dysfunction within the hypothalamic–pituitary axis. Causes of hypogonadotropic hypogonadism, including craniopharyngiomas, pituitary adenomas, arteriovenous malformations, or other central space-occupying lesions, should be excluded using MRI. Stress, extreme weight loss, anorexia, excessive exercise, and low body mass index are all associated with functional hypothalamic suppression, so good nutrition and optimal body weight should be encouraged to restore ovulation (197,199). Leptin is a hormone produced by peripheral adipocytes that reflects energy stores and is deficient in women with diet or exericise-induced amenorrhea (200). Exogenous leptin has been reported to restore ovulation in these women (200,201). Other conditions of hypothalamic dysfunction, such as congenital hypothalamic failure (Kallmann syndrome), can be treated using pulsatile GnRH therapy or gonadotropins. In these patients, both FSH and LH should be administered(197,202). Pulsatile GnRH agonist therapy (25 ng/kg every 60 to 90 minutes) simulates normal physiology and offers some advantages over gonadotropin injections, including fewer multiple gestations and less ovarian hyperstimulation syndrome (OHSS) while maintaining excellent pregnancy rates (202).

Hypothyroidism

The prevalence of hypothyroidism among mid-reproductive aged women is 2% to 4% and is mostly a result of autoimmune factors. Menstrual abnormalities, including those from anovulation, are present in 23% to 68% of overtly hypothyroid women and can be corrected with levothyroxine replacement. Subclinical hypothyroidism and the presence of antithyroid antibodies (even if euthyroid) are associated with increased rates of infertility and spontaneous pregnancy loss, although there exists a lack of consensus as to appropriate upper normal limits of TSH and selection bias limits interpretation. In any case, because even very mild or subclinical hypothyroidism can have adverse effects on fetal brain development and subsequent intelligence quotient, it is prudent to screen and treat women with thyroid hormone abnormalities before commencing infertility treatment (203).

Tubal Factor

Tubal factor accounts for 25% to 35% of infertility. Noninfectious causes for tubal factor include tubal endometriosis, salpingitis isthmica nodosa, tubal polyps, tubal spasm, and intratubal mucous debris (10). The incidence of tubal infertility has been reported to be 8%, 19.5%, and 40% after one, two, and three episodes of PID, respectively (204). Live birth rates are negatively affected by the severity of a single episode of PID (205). C. trachomatis and Neisseria gonorrhoeae are common pathogens associated with PID and infertility. M. hominis and U. urealyticum have been implicated in PID, but their contribution to infertility is less clear (17). Many patients with documented tubal damage have no history of PID and are presumed to have had subclinical chlamydial infections (206,207).

Hysterosalpingography

Hysterosalpingography (HSG) is performed after menses but prior to ovulation between cycle days 7 and 12 to avoid potential pregnancy and take advantage of the thinner proliferative phase endometrium. The patient is typically premedicated 30 to 60 minutes prior to the procedure with ibuprofen or related medication (208). Lidocaine injected intracervically may provide further pain relief (209). With the patient in the dorsal lithotomy position, either a metal cannula or a balloon catheter is inserted through the cervix and past the internal cervical os. Contrast dye is then injected under fluoroscopy to visualize the uterine cavity, fallopian tube architecture, and tubal patency (208,210). Certain disease processes, such as salpingitis isthmica nodosa, have a characteristic appearance on HSG, with typical cornual or isthmic honeycombing resulting from contrast-filled diverticular projections (211). Compared to laparoscopy, HSG has a sensitivity and specificity of 86.5% and 79.8% for bilateral tubal patency and 90% and 97% for bilateral tubal occlusion, respectively. The specificity of HSG remains high but the sensitivity drops for unilateral tubal patency (212).

Hysterosalpingography Risks

Although use of oil-based contrast has been associated with higher pregnancy rates following HSG when compared to water-based contrast (213,214), water-based contrast is generally preferred to avoid oil embolism or granuloma formation. Furthermore, patients should be screened for and pretreated with glucocorticoids if iodine allergy is found (210). The risk of PID after HSG is 0.3% to 3.1% overall but is greater than 10% in the setting of hydrosalpinges (215,216). Therefore, HSG should be avoided in the setting of known hydrosalpinges and/or current or suspected PID (208,216). The role of routine antibiotic prophylaxis for HSG is controversial, but in high-risk patients doxycycline could be considered (217). Recommended dosing is 100 mg twice daily, beginning the day before HSG and continuing for 3 to 5 days. If prophylaxis is not used and hydrosalpinges are noted on examination, postprocedure doxycycline treatment is recommended. Other rare complications of HSG include vascular intravasation, cervical laceration, uterine perforation, hemorrhage, vasovagal reactions, severe pain, and allergic response to the contrast dye (208).

Chlamydia Serology

Chlamydia antibody testing appears to have comparable sensitivity and specificity to HSG but does not localize pathology and the utility of testing is controversial (211). It has been proposed that positive serologies in the setting of normal HSG should still prompt laparoscopy to rule out peritubal adhesions (207).

Laparoscopy

Laparoscopy is considered the gold standard for diagnosing tubal and peritoneal disease. It allows visualization of all pelvic organs and permits detection and potential concurrent treatment of intramural and subserosal uterine fibroids, peritubal and periovarian adhesions, and endometriosis. Abnormal findings on HSG can be validated by direct visualization on laparoscopy using chromopertubation, which involves the transcervical installation of a dye such as indigo carmine to directly visualize tubal patency and fimbrial architecture (212). However, even laparoscopy has been reported to have a false-positive rate of 11% for proximal tubal occlusion when resected tubal segments are examined pathologically (218).

Other Diagnostic Modalities

Falloposcopy is used in conjunction with hysteroscopy and allows direct fiberoptic visualization of tubal ostia and intratubal architecture for identification of tubal ostial spasm, abnormal tubal mucosal patterns, and even intraluminal debris causing tubal obstruction (218,219). At present, instrumentation availability and technical complications, including tubal perforation, limit its routine use (219). Alternatively, sonohysterography offers a much less invasive method of diagnosing fallopian tubal obstruction. The use of contrast media during sonohysterography is preferred to improve accuracy in documenting fallopian tubal patency, but these contrast agents are not available in the United States. As a substitute, the use of agitated saline (air-saline) during sonohysteroscopy provides good negative predictive value when compared to HSG or laparoscopy (220).

Treatment of Tubal Factor Infertility

As success rates for ART continue to improve, the indications for surgical approaches in the treatment of tubal infertility have become increasingly limited (221). Still, surgery can be effective in several situations and may be the optimal approach in some patients.

Proximal Tubal Occlusion

Proximal tubal catheterization and cannulation performed either via HSG or hysteroscopy can restore tubal patency in up to 85% of obstructions, although the reocclusion rate approaches 30%. The best candidates for proximal tubal catheterization or cannulation have muscle spasm, stromal edema, amorphous debris, mucosal agglutination, or viscous secretions, while nonresponders include those with luminal fibrosis, failed tubal reanastamosis, fibroids, congenital atresia, or tuberculosis. Occlusion from salpingitis isthmica nodosa, endometriosis, synechiae, salpingitis, and cornual polyps only occasionally will respond to catheterization or cannulation (10). Catheterization involves passage of a soft catheter into the tubal ostia, while cannulation passes a guidewire thru the ostia and injects contrast media or colored dye. Tubal perforation, typically minor, occurs in 1.9% to 11% of cases (10,211,218). Catheterization under fluoroscopy during HSG is referred to as selective salpingography (222). Visualization of patency with the hysteroscopic approach can be accomplished using laparoscopy or ultrasound (10,218). Ongoing pregnancy rates following proximal tubal catheterization or cannulation are 12% to 44% regardless of hysteroscopic or HSG approach. If occlusion persists or recurs, IVF is usually recommended. Microsurgical tubocornual anastomosis is an option with small studies reporting pregnancy rates of up to 68% (10). This procedure typically is performed via laparotomy and involves excision of the tubal isthmus, followed by reimplantation of the residual tube into a new opening made through the uterine cornua (211).

Distal Tubal Occlusion (Excluding Sterilization or Hydrosalpinx)

Distal tubal disease and occlusion are causal in 85% of all tubal infertility and can be secondary to a variety of inflammatory conditions including infection, endometriosis, or prior abdominal or pelvic surgery (211,223). Patients younger than 35 years of age with mild distal tubal disease, normal tubal mucosa, and absent or minimal pelvic adhesions are the best candidates for corrective microsurgery (223). In vitro fertilization should be considered for older patients or those with diminished ovarian reserve, combined proximal and distal tubal disease, severe pelvic adhesions, tubal damage that is not amenable to reconstruction, or additional infertility factors (223,224). Fimbrioplasty involves lysis of fimbrial adhesions or dilation of fimbrial phimosis, whereas salpingostomy (also known as salpingoneostomy) involves the creation of a new tubal opening in an occluded fallopian tube (223). In well-selected patients, pregnancy rates are reported to be 32% to 42.2%, 54.6% to 60%, 30% to 34.6%, and 55.9% for adhesiolysis, fimbrioplasty, salpingostomy, and nonsterilization-related anastamosis, respectively (211,223). As a group, these procedures are associated with a 7.9% rate of subsequent ectopic pregnancy (223).

Sterilization Reversal

Twenty percent of women express regret following sterilization, and 1% to 5% of those will request reversal, often after a change in marital status. The technique for sterilization reversal involves microsurgical dissection of the occluded ends of the fallopian tube followed by a layered reapposition of the proximal and distal tubal segments. Surgical approaches include minilaparotomy, laparoscopy, and robotic-assisted laparoscopy (224,225). Pregnancy rates following microsurgical tubal reanastamosis for sterilization reversal are 55% to 81%, with most pregnancies occurring within 18 months of surgery (224). Ectopic pregnancy rates following the procedure are generally less than 10% but may approach 18% (223,226). The main predictors of success are age younger than 35 years, isthmic-isthmic or ampulo-ampullar anastamosis, final anastamosed tubal length greater than 4 cm, and less-destructive sterilization methods such as use of rings or clips (224,226). Unlike vasectomy reversal, the length of time between fallopian tubal sterilization and reversal does not seem to affect outcome. IVF should be considered in lieu of sterilization reversal for older patients or those with diminished ovarian reserve, severe pelvic adhesions, additional infertility factors, or prior unsuccessful reanastamosis (223,224).

Hydrosalpinx

Distal occlusion may lead to fluid buildup in the fallopian tube, causing a hydrosalpinx. Hydrosalpinx fluid impedes embryo development and implantation (211). A meta-analysis of 14 studies and 1,004 patients with hydrosalpinges concluded that IVF pregnancy rates were significantly lower in the presence of hydrosalpinges (227). Salpingectomy for hydrosalpinx prior to IVF significantly improves both pregnancy and live birth rates when compared to IVF performed with the fallopian tubes in situ, although laparoscopic tubal occlusion appears to be a reasonable alternative (228–230). There are significantly less outcome data on the use of transvaginal needle drainage and salpingostomy for treatment of hydrosalpinges prior to IVF (211).

Uterine Factors

Pathologies within the uterine cavity are the cause of infertility in as many as 15% of couples seeking treatment and are diagnosed in greater than 50% of infertile patients (231). Therefore, the evaluation of the couple with infertility should consistently include an assessment of the endometrial cavity. Uterine cavity abnormalities include endometrial polyps, endometrial hyperplasia, submucous myomas, intrauterine synechiae, and congenital uterine anomalies (232).

Diagnostic Imaging for Uterine Pathology

Hysteroscopy

Hysteroscopy is considered the gold standard for uterine cavity evaluation because it allows for direct visualization. The procedure involves insertion of an endoscope through the cervical canal into the uterine cavity and instillation of distension media to allow for visualization (231–233). Diagnostic hysteroscopy may be performed in the office using a small-diameter hysteroscope and saline distension, often without need for anesthesia(232). To optimize visualization of the endometrial cavity and avoid performing the procedure during early pregnancy, hysteroscopy is typically scheduled during the early- to midfollicular phase of the cycle. Disadvantages to the procedure include poor visualization when uterine bleeding is present and the inability to evaluate structures outside the uterine cavity, including those in the myometrium and adnexa. Office hysteroscopy is reported to have a 72% sensitivity for cavity abnormalities when compared to operative hysteroscopy using general anesthesia (231).

Hysterosalpingogram

Insofar as it allows assessment of both tubal and intrauterine pathology, HSG is a reasonable initial imaging technique to use in the basic infertility evaluation. Hysterosalpingogram shows the general configuration of the uterine cavity and indicates endometrial lesions as filling defects or irregularities of the intrauterine wall. Excessive contrast may lead to false-negative findings, which may account for the 50% sensitivity of HSG compared to hysteroscopy for endometrial polyps. Inability to discriminate air bubbles, mucous, and debris from true intracavitary pathology may account for HSG’s high false-positive rate when compared with hysteroscopy (232,233). Other drawbacks include patient discomfort, use of iodinated contrast, and radiation exposure (232).

Transvaginal Ultrasound

Compared to hysteroscopy, transvaginal ultrasound has a 75% positive predictive value and 96.5% negative predictive value for intracavitary polyps but a 0% positive predictive value for intrauterine adhesions. Similar to HSG, it has a sensitivity of 44% for uterine malformations (233). However, this may be improved significantly with the use of three-dimensional technology.

Sonohysterography

Saline infusion sonography (SIS), synonymous with sonohysterography, involves the transcervical instillation of saline, often via a balloon catheter, during transvaginal ultrasound to distend the uterine cavity and delineate the endometrium. As with office hysteroscopy and HSG, SIS is performed during the follicular phase of the cycle and anesthesia typically is not required. Endometrial polyps appear as hyperechogenic pedunculated lesions, submucous fibroids have mixed echogenicity, and adhesions contain densely echogenic and cystic areas (234). Compared to hysteroscopy, SIS has 100% sensitivity, specificity, and positive and negative predictive values for uterine polyps (233). Because of a smaller volume of distension media used, SIS is generally better tolerated than HSG or hysteroscopy (231,234). Another advantage of SIS is the ability to evaluate the myometrium and the adnexa for fibroids or adenomyosis. When combined with three-dimensional technology, SIS is particularly good at assessing the overall uterine contour and delineating congenital anomalies such as septate uteri (234). Standard SIS has somewhat lower sensitivity of 77.8% in detecting uterine congenital anomalies when compared to hysteroscopy, but this is higher than both two-dimensional transvaginal ultrasound and HSG. Hysterosalpingogram and SIS perform similarly for intrauterine adhesions, each having approximately 50% positive predictive value and greater than 90% negative predictive value (233).

Magnetic Resonance Imaging

Although transvaginal ultrasound, HSG, SIS, and hystero-scopy may suggest congenital uterine anomalies, pelvic MRI is considered the gold standard for imaging and is particularly useful for diagnosing rudimentary uterine horns (235). Pelvic MRI has the best sensitivity and specificity for intramural and submucous myomas compared to pathologic examination and is especially useful for detecting large or multiple fibroids (236,237). MRI has been suggested as a tool to differentiate fibroids from adenomyosis, but routine substitution for ultrasound is not recommended (237,238).

Congenital Anomalies of the Uterus

Congenital uterine anomalies occur in 3% to 4% of women. This increases to 5% to 10% in women with early pregnancy loss and up to 25% in those with second and third trimester pregnancy losses (235). During female embryonic development, the paired paramesonephric or müllerian ducts elongate toward each other and fuse in the midline. This is followed by resorption of the intervening septum to form the upper vagina, cervix, uterus, and fallopian tubes by week 20 of gestation. Failure of any of these steps leads to absent uterine development or development of unicornuate, bicornuate, arcuate, didelphic, or septate uteri. Because of the proximity of the paramesonephric ducts to the urinary system, renal anomalies often coexist with müllerian anomalies. Appropriate urologic imaging should be performed whenever a müllerian anomaly is diagnosed (235,239). Uterine anomalies are more closely associated with pregnancy wastage and poor obstetric outcomes than with infertility, as the prevalence of congenital uterine defects is generally similar among fertile and infertile women. The exception to this is müllerian agenesis. Patients with müllerian agenisis can have genetically related children only through the use of IVF and a gestational carrier. The arcuate uterus is the mildest congenital uterine anomaly and typically live birth rates are comparable to those in women with normal uteri (235). Surgical uterine repair to improve obstetric outcomes is controversial for most anomalies. However, rudimentary uterine horns require removal on diagnosis, and hysteroscopic metroplasty of the septate uteri significantly reduces the rates of pregnancy loss, but not infertility (235,240). The number of reproductive-age patients presenting with in utero exposure to DES is declining rapidly and will continue to decline in the future, because the substance was banned in 1971. Women whose mothers were exposed to DES have higher rates of uterine malformations (e.g., T-shaped uterus) and associated obstetric complications (235).

Acquired Abnormalities of the Uterus

Leiomyomas

Leiomyomas, also called myomas or fibroids, are benign monoclonal uterine myometrial tumors that affect 25% to 45% of reproductive-age women, particularly African Americans (241). The mechanisms by which fibroids cause infertility are unknown, but may involve altered uterine contractility, impaired gamete transport, or endometrial dysfunction (242). Among women with infertility and uterine leiomyomas, pregnancy rates are primarily affected by leiomyoma location (236,242). Subserosal fibroids do not appear to affect fertility or obstetric outcomes, while intramural (regardless of cavity distortion) and submucosal myomas are associated with lower implantation and live birth rates (236,242,243). It remains unclear whether the size of intramural fibroids determines pregnancy rates and obstetric outcomes, because lower or unchanged pregnancy rates have been reported among patients with fibroids of varying sizes including >2cm, >4cm, or 4–8 cm when compared to patients without fibroids (242).

Myomectomy

In women desiring fertility who require treatment for fibroids, myomectomy is the preferred approach, and uterine artery embolization is relatively contraindicated (242). Removal of cavity-distorting intramural and submucous myomas is generally recommended prior to proceeding with infertility treatment. The utility of surgical removal of non-cavity-distorting intramural fibroids is presently unknown (236,242, 243). Myomectomy can be performed hysteroscopically, via laparotomy, laparoscopically (alone or with robotic assistance), or vaginally (237,241,244–246). Hysteroscopic removal is generally preferred for small submucous fibroids without intramural involvement, while use of the other methods generally depends on patient preference, operator skill, or the presence of other pelvic pathology (237,241,244,245). The utility of pretreatment with GnRH agonists prior to surgery is debatable. GnRH agonists may shrink larger fibroids (5 to 6 cm) enough to allow hysteroscopic resection and may decrease the risk of intraoperative blood loss and postoperative anemia. Fluid overload and uterine perforation are the most common complications of hysteroscopic myomectomy, while bleeding and adjacent organ injury are more often associated with alternative approaches (237,241). Fibroids that are located low on the uterus and posteriorly are less amenable to laparoscopic resection. Transmyometrial approaches raise concern for uterine rupture during pregnancy, although this risk appears to be very low (241).

Endometrial Polyps

The incidence of asymptomatic endometrial polyps among women with infertility has been reported to range from 6% to 8%, but may be as high as 32% (247–249). Risk factors for polyp development include obesity, unopposed estrogen exposure, and polycystic ovary syndrome. The mechanisms by which endometrial polyps may impair fertility are incompletely described but may relate to disordered endometrial receptivity (250). One report localized 32% of endometrial polyps in infertile women to the posterior uterine wall, indicated that 40.3% of patients had multiple polyps, and stated a 6.9% hyperplasia rate (251). Polypectomy is generally performed via curettage, blind avulsion, or hysteroscopic removal (249). Although the efficacy of polypectomy prior to infertility treatment has not been clearly established, a prospective randomized trial showed a 2.1-fold higher rate of pregnancy among women who underwent the procedure prior to IUI (249,252). Higher pregnancy rates have been noted for polyps removed from the uterotubal junction when compared to those removed from other locations (251). Smaller nonrandomized studies provide conflicting data on the negative fertility effects of polyps less than 1.5 to 2 cm (251,253,254).

Intrauterine Synechiae or Asherman’s Syndrome

Severe trauma to the basalis layer of the endometrium with subsequent tissue bridge formation leads to intrauterine synechiae or Asherman’s syndrome. Symptoms of severe disease include amenorrhea, menstrual irregularities, spontaneous abortion, and recurrent pregnancy loss. The causes of intrauterine adhesions are often iatrogenic, with patients typically reporting intraoperative or postoperative complications of uterine evacuations for incomplete pregnancy loss, pregnancy termination, or postpartum hemorrhage. Myomectomy, hysterotomy, diagnostic curettage, cesarean section, tuberculosis, caustic abortifacients, and uterine packing are less-common causes in Western countries (255). In developing countries, Asherman’s syndrome resulting from genital tuberculosis is quite common (256). Hysteroscopic resection of synechiae is the preferred treatment to restore fertility in women with Asherman’s syndrome, and success rates are generally very high. Patients with genital tuberculosis have a very poor prognosis. Postoperative prevention of adhesion reformation disease may involve estrogentherapy alone for 1 month or in combination with intraoperative placement of an intrauterine device (such as a small Malecot catheter or pediatric Foley catheter) for 1 to 2 weeks. There is no standard regimen for estrogen therapy, but oral conjugated estrogens 2.5 mg daily overlapping with progestin or estradiol valerate 2 mg injections daily have been suggested (255,257).

Luteal-Phase Defect and Progesterone Supplementation

Mechanisms

The luteal phase is normally characterized by progesterone secretion by the corpus luteum and appropriate endometrial secretory transformation that allow for embryonic implantation in the endometrium and support of early pregnancy for the first 7 to 8 weeks of gestation (258,259). Luteal phase defect (LPD) is a failure to develop a fully mature secretory endometrium during the implantation window and is thought to account for 4% of infertility (260,261). Proposed mechanisms for LPD include inadequate production of progesterone following ovulation, improper GnRH pulsatility causing insufficient gonadotropin production during the LH surge, and inadequate endometrial responsivity to progesterone (260–262). ART or gonadotropin ovulation induction medications may induce iatrogenic LPD via disruption of granulosa cells from follicular aspiration and suppression of endogenous LH secretion through a combination of supraphysiologic estradiol levels and GnRH agonist or antagonist therapy (259,262).

Diagnosis

Diagnostic criteria for LPD have been varyingly defined, but have included a low mid-luteal phase serum progesterone levels of less than 5 to 10 ng/mL, a delay of 2 days or more in endometrial histology when compared to chronologic cycle day in two or more cycles, a BBT rise lasting less than 11 days, and a shortened luteal phase of less than 14 days (259–261). Unfortunately, the characteristic pulsatile secretion of progesterone during the luteal phase of the menstrual cycle combines with wide temporal variations (even within a 60- to 90-minute time span) to make interpretation of midlueal progesterone levels difficult (258). Similar rates of shortened luteal phase are found in fertile and infertile women, and there is significant variability of luteal phase length from cycle to cycle in an individual woman (261). Finally, there is significant interobserver variability in pathologic interpretation of endometrial biopsies from infertile women, and out-of-phase biopsy results poorly discriminate between fertile and infertile women (263,264).

Treatment

Progesterone therapy is considered standard practice during ART cycles, but is more controversial during non-ART fertility treatments (258,259,262). When used, progesterone supplementation can be administered via oral, vaginal, or intramuscular routes. Intramuscular progesterone is dosed at 25 to 50 mg daily. Most products are delivered in oil, and caution should be used to ascertain the presence of sesame or peanut allergies. Oral micronized progesterone is associated with erratic absorption and decreased bioavailability, so it is typically administered via an off-label delivery route—vaginally 200 to 600 mg daily, and often in divided doses. The side effects of this delivery route, however, can include vaginal discharge and irritation. Other vaginal preparations include a once-daily gel and a 100 mg insert that is given two to three times daily. There remains no consensus regarding the superiority of vaginal versus intramuscular administration. Progesterone therapy typically begins 3 to 4 days following the hCG trigger or LH surge and, if pregnancy occurs, continues for at least 8 to 9 weeks of gestation (258,259,262). Although some progestins can stimulate the androgen receptor, there is no evidence of teratogenicity with the progesterone supplementation described herein (258).

Pelvic Factor

Endometriosis

Endometriosis affects 6% to 10% of all women during their reproductive years but is present in 25% to 50% of infertile women (212,265). It is characterized by the presence of endometrial tissue growing outside the uterine cavity and is found primarily on the peritoneum, ovaries, and rectovaginal septum (265). Fecundability rates in affected patients are estimated at 2% to 10% per month (266). Possible mechanisms for infertility among women with endometriosis include anatomic distortion from adhesions or fibrosis and the known presence of inflammatory mediators that exert toxic effects on gametes, embryos, tubal fimbria, and eutopic endometrium (265,266). Laparoscopy for direct visualization remains the mainstay in the diagnosis of endometriosis. The disease is staged laparoscopically according to the Revised American Society for Reproductive Medicine’s classification, with stages III and IV (moderate to severe) including ovarian endometriomas, dense tubal or ovarian adhesions, and/or cul-de-sac obliteration (265). However, laparoscopy can miss deep disease that may better be detected by ultrasound (i.e., endometriomas) and rectovaginal examination (267). It is unclear whether the presence of endometriosis negatively affects IVF outcomes, although some reports indicate a worse prognosis for more severe disease (stages III and IV) (221,268).

Endometriosis Infertility Managment

Hormonal suppression of endometriosis typically has a minimal benefit for endometriosis-related infertility (265). In minimal to mild disease, laparoscopic ablation appears to significantly improve pregnancy rates when compared to diagnostic laparoscopy alone, although there remains some dissent (267,269). One major randomized trial reported 31% versus 17% pregnancy rates over 3 years with a subsequent meta-analysis supporting these findings (265,266,270,271). Although authors have estimated that eight laparoscopies involving treatment of mild or minimal endometriosis would need to be performed for each pregnancy gained, that number is likely to be much higher given that not everyone who undergoes laparoscopy will have endometriosis (267,270). The benefit of surgical management of endometriosis is even less clear for moderate to severe disease, although removal of endometriomas may be indicated prior to IVF when they would interfere with oocyte retrieval (265–267). Endometrioma resection during IVF or ICSI treatment is associated with decreased ovarian function in up to 13% of cases (272,273). Furthermore, 40% of endometriomas recur postoperatively, and conflicting reports exist that show increases and decreases in pregnancy and live birth rates after surgery (267,272,273). Therefore, IVF is considered a reasonable first-line therapy for endometriosis-associated infertility because of the short time to pregnancy and avoidance of surgery (221).

Adhesions

Adhesions may result from sharp, mechanical, or thermal injury, infection, radiation, ischemia, dessication, abrasion, or foreign body reaction. Adhesiolysis improves pregnancy rates by 12% at 1 year and by 29% at 2 years in infertile women with adnexal adhesions. Use of adhesion barriers reduces adhesion formation following laparoscopy and laparotomy, but there is no evidence to date for improvement in pregnancy rates (274,275).

Unexplained Infertility

Thirty percent of couples are diagnosed with unexplained infertility, in which the basic infertility evaluation reveals normal semen parameters, evidence of ovulation, patent fallopian tubes, and no other obvious cause of infertility. Patients with unexplained infertility may be reassured that even after 12 months of failed attempts, 20% will conceive in the following 12 months and over 50% in the following 36 months. This suggests that in couples with the good prognostic factors of female age less than 30, less than 24 months of infertility, and a previous pregnancy in the same partnership, unexplained infertility may merely reflect the lower extreme of normal fertility. It is likely that current technology is limited in terms of diagnosing all causes for infertility, and the utility of evaluations other than basic testing in an infertile couple has yet to be proven (2,150,276).

Proposed Mechanisms for Unexplained Infertility

Luteinized Unruptured Follicle Syndrome

This condition involves luteinization of a follicle that has failed to rupture and release its oocyte, leading to a normal menstrual cycle but infertility. It is thought to occur in up to 25% of patients with unexplained infertility, more than twice the incidence in fertile women (138). The diagnosis may justify the use of IVF whereby follicles are aspirated and oocyte are retrieved and fertilized in vitro.

Immunologic Factors

Although serum antiphospholipid antibodies and antithyroid antibodies are more prevalent among patients with unexplained infertility than among fertile women, the presence of antiphospholipid antibodies has not been found to adversely affect IVF outcomes, so screening is discouraged (277,278). The association between the presence of antithyroid antibodies and infertility is inconsistent, so screening is not recommended (279–282). Unexplained infertility has been associated with antisperm antibodies, but the extent to which these antibodies affect fertility treatment outcomes and whether IUI, ICSI, or glucocorticoids should be used remains unclear (280,282,283). A similar lack of consensus exists concerning the assessment of peripheral natural killer cell number and/or activity in infertility patients (276,282).

Decreased Endometrial Perfusion

Using ultrasound-based endometrial Doppler studies, women with unexplained infertility have been shown to exhibit abnormal endometrial perfusion when compared to fertile women, but, at present, there is no direct link to fertility treatment outcomes and no recommendations to act on these findings (284).

Infection

C. trachomatis and related clinical and subclinical infections have been discussed in the “Tubal Factor” section. To date, no consistent associations have been reported between chlamydial species, M. hominis, and unexplained infertility in men or women (207,285,286). U. urealyticum and M. genitalium, however, may be more of a concern (287). Prophylactic doxycycline (100 mg twice daily for 4 weeks) given to infertile couples improved pregnancy rates only for those couples in which the male partner was able to clear a ureaplasma infection (288). Antimicrobial prophylaxis is often given for ART, but it is not clear the extent to which clearance of these organisms improves pregnancy rates (287,289).

Undiagnosed Pelvic Pathology

Following a negative infertility workup, laparoscopy has been proposed to evaluate for peritubal adhesions and endometriosis. However, there is a lack of consensus as to the frequency of these abnormalities in women with unexplained infertility, and many practitioners will forgo laparoscopy in lieu of a few cycles of less invasive interventions in such patients (290–292).

Occult Male or Oocyte Factors

Occult male factor despite normal semen analysis and oocyte factors, such as premature zona hardening, mitochondrial dysfunction, and/or aberrant spindle formation, has been suggested as a mechanism for unexplained infertility (293).

Treatment of Unexplained Infertility

It is reasonable to discuss no intervention or expectant management with younger patients presenting with unexplained infertility. Some patients will want to proceed with diagnostic (and potentially therapeutic) laparoscopy. Typical interventions proceed stepwise with superovulation (first with clomiphene or letrozole for three to four cycles, then gonadotropins for three to four cycles) combined with intrauterine insemination, followed by ART (294). ART options for unexplained infertility include conventional IVF, split IVF/ICSI, and full ICSI, even though no male factor has been identified. Risks with clomiphene, letrozole, and non-ART gonadotropins, as well as gonadotropin preparations, have been discussed above.

Baseline Ovarian Cysts

Prior to beginning therapy, a baseline ultrasound may be performed on cycle day 2 or 3 during menses (or following GnRH agonist suppression) to confirm an optimally thin (<4 mm) endometrium and quiescent ovaries. With clomiphene citrate cycles, the presence of ovarian cysts on a baseline scan was associated with decreased rates of ovulation but not of pregnancy, although cyst size was not predictive of response (295). Ovarian cysts on baseline evaluation have been associated with decreased pregnancy rates in ovulation induction cycles employing gonadotropins (296). In IVF patients, functional (estrogen-producing) cysts are seen in 9.3% of women following GnRH agonist suppression (297). Although nonfunctional ovarian cysts up to 5.3 cm did not affect IVF outcomes, functional ovarian cysts (mean diameter 2 cm and baseline estradiol 180 pg/mL) have been associated with increased gonadotropin requirements (dosing and duration), higher cancellation rates, fewer retrieved oocytes, poorer embryo quality, and lower pregnancy rates per cycle (9.6% vs. 29.7% in cyst-free cycles) (297,298). Ovarian cysts greater than 10 mm that are seen on the baseline scan tend to resolve spontaneously within 1 to 2 months, and oral contraceptive administration does not appear to hasten their resolution (299). However, oral contraceptive pretreatment prior to GnRH agonist cycles is associated with decreased risk of cyst formation (300). Functional cyst aspiration prior to gonadotropin stimulation, which is commonly performed to expedite initiation of treatment, did not improve IVF outcomes in one large study (297).

Superovulation (Controlled Ovarian Hyperstimulation)

Unlike ovulation induction, in which the goal is to stimulate the release of a single oocyte in a women who was not previously ovulating, the explicit goal of superovulation (for non-ART or ART purposes) is to cause more than one egg to be ovulated, thereby increasing the probability of conception in women with unexplained infertility (136). In most superovulation protocols, a baseline scan is performed on day 2 of the menstrual cycle to assess antral follicle count and the presence or absence of ovarian cysts. Baseline estrogen and progesterone levels are typically obtained on this day (301). Starting daily doses in superovulation cycles are higher for both clomiphene(100 mg for 5 days) and gonadotropins (150 to 300 IU daily) than those used for ovulation induction (111,301,302). Superovulation with clomiphene is otherwise conducted as described above for ovulation induction. When gonadotropins are used for normal responders with unexplained infertility, FSH doses of 225 IU and 300 IU lead to similar outcomes with IVF (111). The maximal gonadotropin dosage is typically 450 IU per day because higher dosages do not increase ovarian response (301). In most superovulation protocols, gonadotropins are started on day 2 or 3 of menses (301–303). The starting dose of gonadotropins is maintained each day until cycle day 6 or 7 (stimulation day 4 or 5), when the serum estradiol level and transvaginal ultrasound are first measured to document ovarian response (111,302,303). Gonadotropin dosage is increased by 50 to 100 IU per day every 2 to 4 days until a response is evident (304). Estradiol levels typically double every 48 hours, with follicle growth of 1 to 2 mm daily after a 10 mm diameter is reached (305). Triggering of ovulation typically occurs when at least two follicles have reached an average diameter 17 to 18 mm and the endometrial thickness is 8 mm or more (111,302,303,306–308). For non-ART cycles, cancellation should be strongly considered for E2 levels 1,000 to 2,500 pg/mL, for three or more follicles 16 mm or more, or for two or more follicles 16 mm or more plus two or more follicles 14 mm or more (141). Ultrasound monitoring of IVF cycles without estradiol levels can be considered (309).

Treatment Outcomes

It should be noted that pregnancy rates in unexplained infertility patients vary widely among studies, even when the same treatments are used, perhaps as a result of heterogeneity in treatment protocols and patient age. Interpretation of data on the use of clomiphene in patients with unexplained infertility is particularly difficult because the studies are heterogeneous and their results conflicting (150,151). One meta-analysis of 1,159 participants with unexplained infertility involving seven randomized controlled trials of clomiphene citrate with and without IUI indicated no improvement in pregnancy or live birth rates when compared to no treatment or placebo(151). Still, oral therapies continue to be used, and clomiphene and letrozole have recently been shown to have comparable efficacy when combined with IUI for treating unexplained infertility, with pregnancy rates approximately 18% per cycle (310). Combined therapy with gonadotropins and IUI is more likely to result in pregnancy (9% per cycle) than either superovulation (4%) or IUI (5%) alone (302). Laparoscopy to treat minimal or mild endometriosis is associated with live birth rates similar to those in patients with unexplained infertility who receive COH/IUI (70% over four cycles) (311). In women over the age of 40, non-ART therapies are much less effective and IVF should be offered as an initial or early treatment option (312). Pregnancy rates per cycle for unexplained infertility in women who are less than 40 years old have been grouped by treatment modality and reported as follows: (i) clomiphene/IUI 7.8%, (ii) gonadotropin/IUI 9.8%, and (iii) IVF 30.7% (313). The use of ICSI or splitting sibling oocytes between IVF and ICSI has been proposed as an approach to unexplained infertility patients to diagnose and treat underlying occult male or oocyte factors (293,314). Although ICSI does improve fertilization rates and reduce fertilization failure, no differences have been noted in pregnancy or live birth rates when comparing IVF to ICSI for unexplained infertility (314,315).

Cost-Effectiveness

According to computerized decision-tree modeling, laparoscopy followed by expected management for unexplained infertility may be cost-effective when compared to no intervention, non-ART treatment, or IVF (294). Given that 15 gonadotropin/IUI cycles are needed to produce one additional pregnancy when compared to intracervical insemination alone, IVF seems a reasonable option (150). In Massachusetts, where insurance companies are required to cover infertility treatment, the cost-effectiveness of IVF in the treatment of unexplained infertility was determined. Couples were randomized to accelerated treatment with three cycles of clomiphene citrate/IUI followed by up to six cycles of IVF if no pregnancy occurred (n = 256) versus conventional treatment with clomiphene/IUI for three cycles, then injectable gonadotropins/IUI for three cycles, then IVF for six cycles (n = 247). Median time to pregnancy was 8 months in the accelerated versus 11 months in the conventional treatment groups. The authors used an average cost per cycle with clomiphene/IUI of $500, with gonadotropin/IUI of $2,500, and assuming IVF costs were less than $17,749 per cycle. They reported that the accelerated treatment protocol resulted in a savings of $2,624 per couple and lowered the per delivery charges by $9,856 compared to conventional treatment regimens. The study concluded that couples with unexplained infertility who have not achieved pregnancy after three cycles of clomiphene/IUI should proceed directly to IVF (313).

Assisted Reproductive Technologies

Process of Assisted Reproductive Technologies

ART include IVF, ICSI, gamete intrafallopian transfer (GIFT), zygote intrafallopian transfer (ZIFT), cryopreserved embryo transfers, and the use of donor oocytes. Because of improved success rates associated with IVF-embryo transfer, the performance of GIFT and ZIFT has declined in the United States, so this review will focus primarily on IVF and ICSI (316). Both processes involve:

• Prevention of a premature LH surge

• Follicle growth

• Pretreatment

• Adjunctive medications

• Oocyte maturation/ovulation triggering

• Oocyte retrieval

• Luteal support

• Fertilization by IVF or ICSI

• In vitro embryo culture

• Transfer of fresh embryos

• Cryopreservation of surplus embryos

• First trimester pregnancy monitoring

Prevention of Premature Luteinizing Hormone Surge

Luteinizing Hormone surge and Premature Luteinization

Without GnRH agonist or antagonist suppression, LH surges occur in IVF cycles resulting from high estradiol levels in the early follicular phase. This, in turn, results in a lower oocyte yield and reduced pregnancy rates (317). Spontaneous ovulation prior to oocyte retrieval is reported to occur in 16% of nonsuppressed IVF cycles (318). The premature LH rise typically occurs after 5 to 7 days of stimulation (317). In contrast, premature luteinization is a somewhat misleading term that refers to a rise in serum progesterone (cutoffs vary from 0.8 to 2 ng/mL) observed on the day of hCG administration and occurs in the setting of low LH levels secondary to gonadotropin-suppressive medications (319). The incidence of premature luteinization has been estimated at 6% to 7% when the progesterone cutoff level is set at greater than 1.5 ng/mL, but it may be as high as 35%. Mechanisms underlying premature luteinization may include incomplete pituitary desensitization, increased granulosa cell receptor sensitivity to LH secondary to aggressive COH or innately poor responders, and/or the presence of multiple follicles each producing a normal amount of progesterone consistent with the late follicular phase (319,320). Although the literature on this subject remains inconsistent, the negative effects of premature luteinization have been attributed to endometrial advancement rather than oocyte or embryonic dysfunction (319–321). Methods proposed to address premature luteinization have included earlier triggering with hCG, less aggressive stimulation protocols, cryopreservation of embryos (freeze-all), and administration of the antiprogestin mifepristone at the time of hCG (320,322).

GnRH Agonists

Native GnRH is rapidly degraded in the circulation. Commercial preparations of GnRH agonists consist of decapeptides similar to GnRH but for modification at two amino acid residues, which increase both the half-life and the receptor binding affinities (323). Over the course of 10 to 14 days, agonists initially bind to and upregulate pituitary GnRH receptor activity, leading to a flare response of increased gonadotropin secretion. This is followed by receptor desensitization (depleted gonadotropin pools along with rapid uncoupling of the GnRH receptor from its regulatory protein and loss of signal transduction) that suppresses circulating levels of pituitary gonadotropins and, with high doses and prolonged use, eventually decreases GnRH receptor numbers (301,317,323–325). Therefore, prolonged use of GnRH agonists induces a menopause-like state characterized by low estradiol levels and accompanied by common side effects such as hot flashes and moodiness. The flare effect may cause ovarian cyst formation as described in the “Unexplained Infertility” section of this chapter (301). GnRH agonists are commercially available for either depot or daily use and can be administered intranasally (buserelin and nafarelin) or by intramuscular or subcutaneous injection (leuprolide, triptorelin, or buserelin). Intranasal preparations have lower absorption rates when compared to injectable agonists and are associated with milder suppression (326). Typical starting daily doses of leuprolide are 1 mg, 0.5 mg, or 25 μg (microdose) (324).

Gonadotropin-Releasing Hormone Agonist Protocols

Some of the more commonly used ART medications and protocols are summarized in Fig. 32.6 and Table 32.8. In the long protocol, a GnRH agonist is started in the luteal phase (day 21) of the previous cycle. This diminishes the GnRH agonists flare effect and suppresses endogenous FSH and dominant follicle selection to promote synchronous follicular growth (317). After 10 to 14 days of GnRH agonist administration, a pelvic ultrasound and estradiol level are used to confirm suppression and gonadotropin stimulation begins. The GnRH agonist is continued (the dose may be halved or unchanged) throughout the cycle until the hCG trigger (317,324). Mean serum progesterone levels on the day of hCG administration using the long protocol are reportedly 0.84 ng/mL (320). The long protocol provides for better oocyte yields and pregnancy rates in normal responders when compared with shorter protocols that use later administration or early cessation of agonists (317). Shorter protocols using lower GnRH agonist doses have been advocated for poor responders in whom excessive suppression may be undesirable, perhaps from direct negative effects of the agonist on the ovary (324). However, long protocols, particularly those using single-dose depot rather than daily GnRH agonist formulations, are associated with higher gonadotropin dose and duration, which themselves have been associated with lower pregnancy rates (327–329). Alternatively, GnRH microdose flare protocols have been developed that may improve oocyte yield in poor responders. Microdose flare regimens involve pretreatment with 14 to 21 days of combination oral contraceptives. Four days following the cessation of the oral contraceptive pills, a microdose (leuprolide 25 μg) of agonist is added in the early follicular phase to take advantage of the agonist flare effect. Gonadotropin stimulation is initiated 1 to 2 days later while continuing the agonist (324).

Figure 32.6 In vitro fertilization protocols using gonadotropin-releasing hormone (GnRH) agonist or antagonist with gonadotropins in controlled ovarian hyperstimulation. hCG, human chorionic gonadotropin.

Gonadotropin-Releasing Hormone Antagonists

GnRH antagonists (cetrorelix and ganerelix) were developed by modifying the GnRH decapeptide at six positions. They compete with endogenous GnRH for binding to pituitary GnRH receptors. Because they have no agonistic activity, GnRH antagonists lead to almost immediate suppression of FSH and LH and do not require the additional time for pituitary down-regulation that characterizes the GnRH agonists. With prolonged use, GnRH antagonists down-regulate GnRH receptors (323). At this time, the only delivery method available for GnRH antagonist is subcutaneous injection, although orally active agents are in development. GnRH antagonists can be given as 0.25 mg daily doses or as a single 3 mg dose with no difference in outcome (325). The time between daily injections should not exceed 30 hours (301). With single dose regimens, avoidance of multiple injections is attractive, but additional small doses starting 4 days after initial dose are required in 10% of cycles (325). Use of GnRH antagonists in ART protocols is typically begun on day 4 to 7 of stimulation. This timing balances the risk for premature LH surges with the need for initiation of endogenous FSH-mediated follicular recruitment and endogenous estradiol production prior to administration (301,317,325).

Gonadotropin-Releasing Hormone Antagonists Fixed versus Flexible Protocols

Several GnRH antagonist protocols have been developed for use in ART cycles. Fixed protocols involve starting the antagonist on day 4, 5, 6, or 7 of stimulation regardless of follicular response(301,308,317,325). Flexible protocols were developed to reduce gonadotropin stimulation dose and duration (317). In flexible protocols, varying thresholds have been described for antagonist initiation. These include addition of the antagonist when the leading follicle has reached 12 to 16 mm in average diameter or when the estradiol level has risen above 600 pg/mL (303,307,317,324,325). When flexible protocols are used, pregnancy rates are similar when antagonist is initiated on day 4 or 5 of stimulation but drop significantly when antagonist is initiated after day 6. This suggests that rapid follicular growth may be more important in preventing LH surges and improving pregnancy rates than the day the antagonist is initiated (303). Fixed initiation of a GnRH antagonist on day 6 of stimulation has been associated with higher pregnancy rates when compared to flexible protocols in one meta-analysis involving four randomized trials; however, the true superiority of one approach over the other remains to be determined (303,324,325). Diminished estradiol levels can be expected following GnRH antagonist administration, but this does not appear to affect follicular growth (308). The addition of exogenous FSH or LH does not appear to be necessary (301,324,325).

Gonadotropin-Releasing Hormone Agonists Compared with Antagonists

Because there is no pituitary desensitization period required when GnRH antagonists are used in ART, cycles can begin more quickly than those that employ GnRH agonists (301). Antagonists are associated with lower duration of stimulation, lower stimulation dose, and reduced rates of ovarian hyperstimulation syndrome (330). Antagonists are often used in the absence of pretreatment with oral contraceptives. Initiation of antagonist cycles therefore relies on the start of spontaneous menses, often making scheduling easier for GnRH agonist cycles (331). Study and protocol heterogeneity make it difficult to detect consistent differences in IVF pregnancy outcomes after GnRH agonist and antagonist cycles (330,332). A Cochrane review of 27 randomized controlled trials indicated that while the number of good quality embryos produced for transfer are similar, clinical pregnancy rates are higher by 4.7% in agonist compared to antagonist cycles. They concluded that for every 21 couples, one additional pregnancy would be gained by using a GnRH agonist protocol. The number of oocytes retrieved and the live birth rates favored agonist usage (330).

Follicular Growth

Follicular Recruitment

Although the inciting stimulus is unclear, cohorts of small antral follicles (<2 mm) become responsive to FSH stimulation only after 45 days of initial growth. They are then considered recruitable and begin to slowly enlarge in the late luteal phase of the cycle preceding ovulation. Once the antral follicle grows beyond 10 mm, FSH induces the appearance of LH receptors on the granulosa cells. At that point, somewhat contrary to the 2-cell-2-gonadotropin model, LH can exert FSH-like actions on the granulosa cell, including stimulation of aromatase. With complete absence of endogenous LH, such as that seen with hypothalamic hypogonadism, exogenous FSH produces fewer preovulatory follicles, inadequate estradiol, lower ovulation rates, and thinner endometrium when compared to ovaries stimulated with exogenous HMG (FSH and LH). Therefore, in the setting of normal hypothalamic function, it seems likely that even with suppressive therapy, enough endogenous LH is present to synergize with exogenous FSH to provide adequate stimulation for recruitment (326).

Follicular Waves

Seventy-five percent of unstimulated human menstrual cycles have two waves of follicular development: one major wave in which a dominant follicle is selected to the detriment of other follicles and one minor wave during which all follicles undergo atresia. When progesterone is available to block LH, the first wave is typically minor and occurs one day after ovulation in the previous cycle. The subsequent major wave occurs around the time of menses, with the dominant follicle being selected by its ability to reach a diameter of 10 mm by cycle day 6 or 7, which coincides with an estradiol rise that inhibits FSH. One-quarter of cycles will exhibit an additional wave that occurs 2 days prior to menses and may be minor or major. If the dominant follicle regresses or is removed, a new follicular wave begins 2 days later. In light of this, future efforts toward greater synchrony during stimulation might include ablation of the early dominant follicle using aspiration or administration of exogenous estrogen and progesterone during the follicular phase (333).

Controlled Ovarian Hyperstimulation

The goal of gonadotropin stimulation for ART is the synchronous growth of dominant follicles in quantities greater than that associated with non-ART therapy. FSH is the key hormone in this regard (111). Gonadotropin preparations are described in the “Ovulatory Factor” section, while dosing and the relevance of baseline ovarian cysts are described in the “Unexplained Infertility” section. ART pregnancy outcomes are not affected by the source of FSH, the delivery system, or the route of administration (156). In one recent report, women who were predicted to be normal responders using 225 to 300 IU of FSH required 11 days of stimulation, had 11 to 13 follicles 15 mm or larger on the day of hCG trigger, had a peak E2 level of approximately 2,100 pg/mL, and had 10 to 11 oocytes retrieved, of which 82% to 83% were mature (111). Most investigators suggest that the optimal number of retrieved oocytes in a given cycle is between 5 and 15, and the optimal level of estradiol at the time of hCG administration is between 70 and 140 pg/mL per oocyte or follicle (306,329). It should be noted that the utility of estradiol monitoring during ART is controversial, and that ultrasound monitoring alone may be adequate to maximize pregnancy and live birth rates (309). Cycle cancellation in normal responders occurs in up to 6% of the cycles because of inadequate response and 1.5% of cycles for excessive response(111). Cycle cancellation increases with older age and decreased ovarian reserve, but is reportedly decreased by 2% for every additional 100 IU gonadotropins used (328). There appears to be no benefit of exceeding a total daily gonadotropin dose of 450 IU in any patient (301).

Follicle-Stimulating Hormone versus Human Menopausal Gonadotropin

Higher androgen and lower progesterone levels on the day of hCG trigger are observed when HMG is used for stimulation rather than FSH alone, indicating a more favorable endocrine profile with HMG (334). Concern has been raised that exogenous LH might be needed to address the sudden decrease in endogenous LH that is associated with GnRH antagonist use and the possible excess endogenous LH suppression in long GnRH agonist protocols (326). However, there is no consensus as to whether the addition of LH to exogenous FSH improves pregnancy and live birth outcomes in women with normal hypothalamic function (156,335,336).

Less Aggressive Stimulation

Although it reduces cancellation rates, each additional 100 IU of gonadotropins is correspondingly associated with a 2% lower rate of clinical pregnancy and live birth (328). This seems to support the findings of premature luteinization and lower pregnancy rates when more aggressive FSH stimulation protocols are used (320). When comparing normal responders using a flexible antagonist protocol and a fixed FSH dose of 150 IU (mild) to those using conventional FSH doses, implantation rates were maximized when a median of 5 oocytes were retrieved in the mild stimulation group, while 10 oocytes were necessary in the conventional dose group. Outcomes were compromised when more than eight oocytes were obtained in the mild group, and only the high dose group experienced poorer outcomes with low oocyte yield. Pregnancy rates seem to level off or decrease when stimulation is pushed beyond a certain threshold, but this threshold is unknown (329).

Pretreatment

Combined oral contraceptives (OCs) are commonly taken for 14 to 28 days prior to GnRH analogues to ease cycle scheduling, synchronize follicular development, further prevent LH surges, reduce the incidence of ovarian cysts, and reduce cancellation rates resulting from hyperstimulation (331,337,338). Patients can begin OCs anytime between days 1 and 5 of menses (337). For antagonist cycles, COH begins 2 to 5 days after stopping OCs (irrespective of menses) (331). During long GnRH agonist protocols, the agonist overlaps the final 5 days of OC use, followed by initiation of COH on the second or third day of withdrawal bleeding (300,338). As previously discussed, microdose flare protocols involve pretreatment with 14 to 21 days of OC, followed 4 days later by a microdose of agonist. COH typically starts 1 to 2 days later (324). The progestins norethindrone acetate 10 mg orally daily, medroxyprogesterone acetate 10 mg orally daily, or a single intramuscular dose of progesterone (not specified) can be used in place of an OC in ART cycles, but recommendations on the duration of treatment and the timing of initiation vary widely: a duration of 5 to 20 days and initiation anytime between days 1 and 19 of menses have been reported. A dose of 4 mg of daily micronized 17β estradiol or estradiol valerate has been used in lieu of OCs in ART cycles, with initiation between cycle days 15 and 21 and a duration of 10 to 15 days. Although OC pretreatment for antagonist cycles has been shown to increase both the duration of stimulation and the amount of medication used, its effect on pregnancy rates is controversial (331,337). OC pretreatment during GnRH agonist cycles is associated with higher pregnancy rates than those in cycles without pretreatment (300). Progestin and estradiolpretreatment do not affect live birth rates in either agonist or antagonist cycles (337).

Adjunctive Medications

Prenatal vitamins should be given to all infertility patients beginning at least 1 month prior to initiation of infertility treatment. Although aspirin is commonly used during IVF regimens, recent studies have failed to demonstrate beneficial changes in pregnancy rates (339,340). Antimicrobial prophylaxis using doxycycline or azithromycin for both partners is often given during ART cycles, particularly those involving assisted hatching procedures, although these medications have not been clearly associated with improved pregnancy rates (287,289,341). Glucocorticoids given to women during the peri-implantation period may improve pregnancy rates in women with autoimmune disease, those undergoing assisted hatching or frozen/thaw embryo transfers, and in women of advanced maternal age (341). Metformin may limit ovarian hyperstimulation (OHSS) in PCOS patients, but shows no benefit for pregnancy or live birth rates (342).

Oocyte Maturation/Ovulation Triggering

Physiology of Oocyte Maturation

Prior to maturation, oocytes are arrested in the prophase stage of meiosis I, also known as the germinal vesicle (343,344). Meiosis I oocytes must reach at least the early antral follicle stage to respond to FSH and be competent to resume meiosis. In vivo, LH receptors on the follicle are induced by FSH during later stages of follicular development (345). Therefore, only fully grown oocytes respond to the LH surge in vivo to begin the cytoplasmic and nuclear maturation that are required for developmental progression toward the metaphase stage of meiosis II. At this point, the developmentally competent oocyte will extrude the first polar body, the oocyte-cumulus complex will detach from the ovarian wall, ovulation will occur, and fertilization is possible (343–346).

Oocyte Maturation during Assisted Reproductive Technology Cycles

Because spontaneous LH surges occur inconsistently during non-ART gonadotropin cycles and are suppressed in ART cycles, hCG has been used to trigger ovulation. In combination with its long half-life, homology between hCG and LH (identical α subunits) allows for cross-reactivity with the LH receptor and induction of final ococyte maturation and ovulation (347). hCG is derived from urine (5,000 to 10,000 IU intramuscularly) or through recombinant technology (250 μg subcutaneously, equivalent to 5,000 to 6,000 IU of intramuscular urinary product) (156). The half-life of hCG is 2.32 days, compared to 1 to 5 hours for LH (347). Ovulation is typically triggered when at least two follicles are 17 to 18 mm or larger in average diameter (but <24 mm) and the endometrial thickness is 8 mm or more (111,302,303,306–308,348). Similar clinical outcomes have been noted when 5,000 IU or 10,000 IU of hCG (349) and urinary or recombinant preparations are used for triggering (348,350). If there is concern for ovarian hyperstimulation syndrome (OHSS, see below), GnRH agonists can be substituted for hCG to trigger ovulation in antagonist protocols or recombinant LH can be substituted for hCG in agonist protocols; however, both protocols are associated with decreased pregnancy rates in nondonor ART cycles (347,351).

Oocyte Retrieval

Oocyte retrieval is performed via transvaginal ultrasound-guided needle puncture into each follicle followed by aspiration of follicular fluid. Either general anesthesia or intravenous conscious sedation may be used (352). Prophylactic antibiotics such as ceftriaxone are recommended at the time of retrieval (353). The vaginal preparation can be performed either with sterile saline alone or with povidone iodinefollowed by vigorous saline flushing (354,355). The highest oocyte yield is obtained when oocyte retrieval is performed 36 to 37 hours after the hCG injection. Earlier retrieval (35 hours) is associated with a much lower oocyte yield, and later retrieval risks ovulation; spontaneous follicular rupture appears to occur at a mean of 38.3 hours following hCG administration (348).

Luteal Support

The rationale and regimens for luteal phase support with progesterone are discussed in the “Uterine Factors” section. The timing for luteal progesterone support varies, but lower pregnancy rates have been noted with initiation prior to oocyte retrieval or later than 5 days following retrieval (356,357). Luteal phase estradiol supplementation is not necessary (358,359).

Fertilization by In Vitro Fertilization or Intracytoplasmic Sperm Injection

Following semen collection and sperm processing (described in the “Male Factor” section), sperm are incubated in media for 3 to 4 hours to promote sperm capacitation and the acrosome reaction. Before fertilization, retrieved oocytes are cultured in media. Conventional IVF involves insemination concentrations of 100,000 to 800,000 motile sperm/mL per oocyte with each oocyte in a small droplet of media under oil (9,360,361). For every three cycles done for severe male factor, the use of ICSI prevents one case of fertilization failure when compared to conventional IVF (9). The indications for and the procedure and risks of ICSI are discussed under “Male Factor.”

In Vitro Embryo Culture

Embryo Development

Initial embryo development is typically assessed 15 to 20 hours after insemination or ICSI, when fertilization is characterized by the presence of two pronuclei and the extrusion of the second polar body (9,361,362). Embryos are examined again for cleavage after 24 to 30 hours of culture (9). The first embryo cleavage occurs approximately 21 hours after fertilization, and subsequent divisions occur every 12 to 15 hours up to the eight-cell stage on the 3rd day of embryo development (363). Compaction to form the 16-cell morula occurs on the 4th day of embryo development, and differentiation of the inner cell mass and trophectoderm to form a blastocyst (containing a fluid-filled area called a blastocele) is completed by the 5th or 6th day (364,365).

Culture Environment

Sequential media systems are preferred during embryo culture to adjust for each stage of embryo development. Prior to compaction, the embryo is under genetic control of the oocyte, it uses a pyruvate-based metabolism, it requires at least a few amino acids, and it prefers a relatively oxygenated environment (though much lower than atmospheric oxygen) similar to that found in the fallopian tube. Following compaction, amino acid needs increase (stable dipeptide glutamine instead of glutamine will avoid toxic ammonium buildup), the embryonic genome is activated, and metabolism requires both glucose and a very low oxygen environment similar as that found in the uterus (363,366). Supplementation of culture media with hyaluron and albumin is beneficial in postcompaction media preparations (363).

Extended Culture to Blastocyst

Although precompaction human embryos can survive when placed in the uterus, the uterine cavity is a nonphysiological location for them, and there is greater uterine pulsatility during this period that may cause the embryos to be expelled. Therefore, the blastocyst stage represents a more physiologic time for embryo transfer. Since nearly 60% of morphologically normal cleavage embryos but only 30% of blastocysts are chromosomally abnormal, extended culture allows for better selection of embryos with improved quality (364,367).

Blastocyst versus Cleavage Transfer Outcomes

Comparisons involving equal numbers of transferred embryos demonstrate that blastocyst transfer is associated with lower implantation failure, a higher pregnancy rate, and a 7% higher live birth rate than cleavage stage transfer. This is of particular interest in programs that offer elective single embryo transfer (364,367,368). Given that blastocyst formation rates range from only 28% to 60%, disadvantages of extended culture include the possibility that no embryos will survive to transfer (8.9% vs. 2.8% for cleavage transfer) and a reduced opportunity for embryo cryopreservation. Monozygotic twinning rates may be higher with blastocyst culture, although this has not been a consistent finding (364,369).

Criteria for Extended Culture

There are no established guidelines or criteria that determine when to utilize extended culture. Varying suggestions include maternal age 42 or younger with five or more two pronuclear stage (2PN) embryos on postretrieval day 1; maternal age of 40 or younger and three or more good-quality day 3 embryos having 4 to 10 cells with less than 15% fragmentation; maternal age of 41 to 42 or younger and four or more good-quality day 3 embryos having 4 to 10 cells with less than 15% fragmentation; and age less than 37 with four or more morphologically good embryos on day 3, or four or more embryos with 6 cells and less than 10% fragmentation (362,364,370).

Embryo Transfer

Embryo Morphology

Embryo morphology guides the choice of embryo for transfer. Pronuclear embryos are assessed by their distribution and number of nucleoli, the position of the second polar body relative to the first, and cleavage rates (abnormal rates are too fast, too slow, or arrested) (365). Preferred cleavage stage embryos have a normal developmental pattern characterized by early cleavage on day 1, four cells on day 2, and eight cells on day 3. Embryo fragmentation should be 10% or less, the blastomere size should be regular, and there should be no multinucleation (362). The Gardner and Schoolcraft system for scoring blastocysts uses a scale from 1 (worst) to 6 (best), with grades 1 to 3 indicating growth of the blastocele until it completely fills the embryo. Grade 4 blastocysts are expanded with a larger blastocele volume and a thinning zona pellucida. The trophectoderm in a grade 5 blastocyst is starting to hatch though the zona, and the grade 6 blastocyst has completely escaped or hatched from the zona. The inner cell mass is graded A to C based on tightness and cellularity (A is best), and the trophectoderm is assessed from A to C based on cohesiveness and cellularity (A is best) (361).

Number of Embryos to Transfer

High-order multiple pregnancy (three or more fetuses) increases complications for mothers and fetuses, so guidelines have been developed to minimize this adverse outcome (371). Single embryo transfer should be considered for patients younger than age 35, particularly those undergoing their first ART cycle who have a large quantity of good quality embryos or patients who have conceived in a prior cycle. Otherwise, transfer should be limited to two embryos in women under 35 years of age. For older women, the maximum number of transferred cleavage-stage embryos should be three in women aged 35 to 37, four in women aged 38 to 40, and five in women older than 40 years of age. Because of their high implantation potential, no more than three blastocysts should be transferred to any woman regardless of her age. Limits on the number of embryos transferred when the embryos were created from donor oocytes should be based on the age of the donor, rather than the recipient (371).

Transfer Procedure

The goal of transcervical embryo transfer is to atraumatically deliver the embryos to an optimal intrauterine location for implantation. Implantation is more likely after an easy transfer using a soft catheter and when fundal contact is avoided (353). Trial transfer, although not required, allows advance preparation such as cervical dilation or placement of a traction stitch, although uterine position and depth can be different at the time of the actual procedure. When performed at the time of embryo transfer, trial transfer should not go past the internal os. Trial transfer can be combined with an afterloading technique in which the outer sheath of the transfer catheter is left in place and the transfer catheter is threaded through the trial transfer sheath and into the uterus, although there is no advantage when compared to routine transfer (353,372). Soft catheters such as those made by the Cook or Wallace companies are preferred to rigid catheters to minimize prostaglandin release after cervical and/or endometrial trauma (353). The utility of cervical mucus removal prior to embryo transfer in improving embryo delivery remains controversial (353,372). Although intrauterine infections decrease pregnancy rates, the efficacy of antibiotic administration at the time of transfer is not clear. During a conventional embryo transfer, embryos are suspended in 20 μL of media at the tip of a syringe with air on either side of the fluid. This creates an air–fluid interface easily seen with ultrasound. Abdominal ultrasound visualization during embryo transfer is useful to ensure deposit of the embryos 1.5 to 2 cm from the uterine fundus. Once the embryos are deposited, the inner and outer sheath should be removed as a unit to avoid suction within the device (353). No changes in patient position are necessary (372). Following transfer, the catheter is checked for retained embryos. If present, retained embryos should be transferred because there is no detriment in pregnancy rates (353).

Figure 32.7 Regimens of ovarian stimulation and hormone replacement used to synchronize the development of ovarian follicles in the oocyte donor and the endometrial cycle in the recipient. hCG, human chorionic gonadotropin; OCP, oral contraceptive pill; GnRH-a, gonadotropin releasing hormone antagonist; TVA, ultrasound-guided transvaginal aspiration of oocytes. (Adapted from Chang PL, Sauer MY. Assisted reproductive techniques. Stenchever MA, ed.; Atlas of clinical gynecology, Mishell DR, ed, Reproductive endocrinology. Vol. 3. Philadelphia, PA: Current Sciences Group, 1998, with permission.)

Cryopreservation of Embryos

Embryo cryopreservation at the pronuclear, cleavage, and blastocyst stages has allowed for multiple transfer cycles from a single oocyte retrieval. Because transfer of cryopreserved embryos is less expensive than a second fresh cycle, overall fertility treatment costs can be optimized. Embryo cryopreservation can be considered as a means to prevent ovarian hyperstimulation syndrome. Techniques for embryo cryopreservation include slow freezing and rapid freezing or vitrification. Slow freezing protocols use lower concentrations of cryoprotectants but are more time-consuming when compared to vitrification, which uses high-concentration cryoprotectants for rapid cooling and is less expensive. Embryo thawing is accomplished by brief exposure to air and warm water followed by rehydration (373). Although pregnancy rates for freeze/thaw transfer (FET) cycles using the two cryopreservation methods are similar, vitrification is associated with higher postthaw embryo survival (93% vs. 76% with slow freezing) (374). Infant outcomes are reassuring for slow freezing but are more limited for the newer technique of vitrification (375). Overall, use of frozen embryos results in lower pregnancy rates when compared to fresh transfer cycles, but this may be a result of embryo selection (the best embryos are typically used for fresh transfer and lesser quality embryos are frozen) (373,376). Frozen transfer outcomes are heavily dependent on the characteristics of the fresh cycle that generated the frozen embryos; excellent pregnancy rates are noted when the fresh cycle resulted in conception or when all the embryos were frozen from the fresh cycle (376).

Endometrial Preparation for Frozen Embryo Transfer

When FET is combined with a recipient’s natural cycle, no exogenous treatment is given, and transfer is timed to spontaneous ovulation. In medicated FET cycles, estradiol supplementation begins in the early follicular phase and is continued for 13 to 15 days (373). Multiple estradiol preparations have been described for use in FET cycles, but none has been proven superior (377). Transvaginal ultrasound is used to assess endometrial thickness during estrogentherapy, and estrogen administration continues until an optimal thickness of greater than 8 mm is reached (373). Progesterone supplementation begins 48 to 72 hours prior to transfer when cleavage-stage embryos are used and 6 to 7 days prior to transfer when blastocysts will be thawed (373,377). Again, several progesterone preparations have been described for use in FET cycles, but none has been proven superior (discussed in the “Uterine Factors” section). GnRH agonists are commonly used during medicated cycles to prevent premature LH surges that might adversely affect endometrial maturation (377) (Fig. 32.7).

First-Trimester Pregnancy Monitoring

The production of hCG by the blastocyst can be detected as early as 7 days posttransfer, and serum quantitative hCG levels may be obtained 11 to 14 days following embryo transfer (373,378). A serum threshold of 200 mIU/mL of hCG measured 12 days after transfer is 92% and 80% predictive of ongoing pregnancies for day 3 and day 5 embryos, respectively, with levels normally rising approximately 40% per day. If normally rising hCG levels are detected, transvaginal ultrasound is planned at 6 to 7 weeks of gestation to determine the location of the pregnancy, the number of gestational sacs, and pregnancy viability (378).

Table 32.10 In Vitro Fertilization Success Rates

Assisted Reproductive Techonology Success Rates

Since 1992, all clinics performing ART in the United States have been required to submit annual success rates to the Centers for Disease Control and Prevention (379). Success rates of IVF vary from program to program. The most comprehensive assessment of the efficacy of ART programs in North America comes from the database of the Society for Assisted Reproductive Technology (SART). Information on SART and registered ART clinics are accessible by the public online. Because ICSI and IVF rates are similar, statistical analyses combine them for the annual SART reports. IVF success rates are largely dependent on maternal age, and data from recent studies, including those from SART, are summarized in Table 32.10 (380). SART statistics should not be used to compare IVF clinics since success rates are largely dependent on patient demographics, including the cause of infertility, which will be different for each individual clinic. Lifestyle factors affecting IVF success are discussed under “Causes of Infertility.” Since SART does not publish success rates for non-ART cycles, it can often be helpful for physicians to assist patients in comparing the success rates for clomiphene, gonadotropins, and IVF, although rates vary widely between studies (Table 32.11).

A new approach to predicting live birth probabilities focuses on the use of machine learning or the mining of clinic-specific IVF outcomes data to provide a personalized per-cycle prognosis that pertains to the clinical scenario of each patient (381). Briefly, a live birth prediction model was developed by boosted tree analysis of baseline clinical data, uterine response to a patient’s first IVF treatment, and embryo developmental parameters, with no preselection of prognostic factors. The model predicted live birth outcomes in a subsequent IVF treatment cycle. Validation by an independent dataset and comparison with a control model that is based on chronological age alone showed that the boosted tree model was more than 1,000 times better in fitting new data and improved discrimination (i.e., the ability to discern among patients with different prognoses) by receiver–operator curve analysis. Approximately 60% of patients were found to have significantly different predicted live birth probabilities when compared to the use of age categories. Further testing across different clinics will be required to determine whether this approach may be generally valid and applicable. Nevertheless, the nonredundant and unique contribution of the patient’s age to predicting outcomes was found to be limited if data pertaining to the response to gonadotropins and embryo development in the first IVF treatment were available. These findings may move prognostic counseling away from an age-centric paradigm and toward a more objective extraction of predictive value from simultaneous analysis of a wide array clinical factors.

Table 32.11 Fertility Treatment Success Rates

Cessation of Therapy

Patients must be accurately informed of estimated success rates and reasonable expectations for all therapeutic interventions. Patients with a very poor prognosis have a 2% to 5% chance of achieving a live birth with fertility therapy, and those with a futile prognosis have a 1% or less chance. Refusing or limiting therapy may be justified if the risk of intervention outweighs the potential benefits (382).

Third Party Reproduction

When gametes and the ability to gestate a pregnancy are compromised through circumstances or disease, other reproductive options can be considered. These include use of donor sperm (discussed in “Male Factor”), donor oocytes, donor embryos, a gestational carrier, or a combination of these approaches. In contrast to donor gametes (sperm or oocytes), the decision to donate embryos is typically made after the embryos have been generated and there is a known surplus. A gestational carrier receives and gestates birth embryos created from the intended mother’s oocytes. Patients who choose to utilize a gestational carrier may have irreparable uterine factor infertility or suffer from medical conditions that contraindicate pregnancy. In true surrogacy, the birth mother is the genetic mother but not the intended mother. Legal and psychosocial counseling are suggested for all parties embarking on any form of third party reproduction.

Donor Oocyte

Patients with ovarian failure, poor oocyte quality, poor ovarian response to stimulation, or failed fertilization or implantation after multiple ART cycles may be candidates to receive donated oocytes. A female same-sex couple may choose to have one partner undergo IVF and place the resulting oocytes fertilized with donor sperm into the other partner (383). At present, donor oocytes must be used to create embryos during the retrieval cycle, because oocyte freezing is considered experimental (384). With carefully selected donors, live birth rates per cycle of donor oocyte IVF are 50% to 60% regardless of the recipient’s age (Table 32.10). However, recipients must be aware that advanced recipient age is associated with higher risk for preeclampsia, diabetes, and cesarean section (385–387). Oocyte donors must endure all the interventions and risks of the ART process described herein except for embryo transfer and luteal support. Because of the intensity of therapy and the potential infectious disease and genetic risks for donor, recipient, and the resulting offspring, oocyte donors must be screened for infectious and heritable disorders similar to those performed for sperm donors (see “Male Factor”) and undergo meticulous informed consent and a comprehensive psychosocial evaluation. Oocyte donors may be anonymous or known to the recipient (96). Oocyte recipients undergo endometrial preparation (as described in the “Process of Assisted Reproductive Technologies” section), but GnRH analogues are not required if there is no endogenous ovarian function. Recipients may initiate progesterone one day prior, on the same day, or one day after the donor’s oocyte retrieval, but a randomized trial found lower pregnancy rates when progesterone was initiated prior to the retrieval (377). Other topics, such as methods for donor recruitment and financial compensation for the donor, are much more challenging issues (388).

Complications of Assisted Reproductive Technology

Cycle Cancellation

Cycle cancellation in normal responders occurs in up to 6% of cycles because of inadequate stimulation response and in 1.5% of cycles because of excessive response (111). In 0.2% to 7% of retrievals, no ooctyes will be obtained. Two proposed explanations include human error during the administration of hCG and early oocyte atresia despite normal follicular response (346).

Oocyte Retrieval

The risks of oocyte retrieval include bleeding requiring transfusion, injury to adjacent structures requiring laparotomy, formation of a pelvic abscess leading to loss of reproductive function despite prophylaxis, and risks related to anesthesia (389).

Multiple Gestation

As the majority of ART cycles involve the transfer of more than one embryo, multiple gestation occurs at higher rates than the 3% rate for spontaneous conception. This has social, medical, emotional, and financial ramifications (379,380). Although the majority of complications of multiple gestations occur with high-order (three or more) multiples, twins have increased risks for low birth weight, preterm birth, and neurologic deficits when compared to singletons (380,390). Despite this, 20% of infertile patients view multiple pregnancy as a desired outcome. Because most patients lack insurance coverage for IVF, patients may push their physicians to take greater risks when deciding on the number of embryos to transfer (379). The multiple pregnancy risk is higher in patients under the age of 35 who are undergoing IVF because their embryos are typically of better quality and implantation rates are higher, but multiple pregnancy can occur at any reproductive age (371). From 1998 to 2007, the overall twinning rate in ART cycles remained stable at 29% to 32%, but the rate of high-order multiples decreased from 6% to 2%. In 2007, the live birth rates for twins and triplets or more for women under the age of 35 who underwent ART in the United States were 33.2% and 3.5%, respectively. Women aged 43 or 44 had twinning live birth rates of 10.6% and triplet or more live birth rates of 0.8% (380). Most multiple pregnancies arising from ART are dizygotic, but monozygotic twinning occurs in 3.2% of IVF cycles (compared to a background rate of 0.4%) (379). Concerns have been raised that monozygotic twinning might increase after blastocyst culture, but this finding has not been consistent (364,369). With increasing adherence to guidelines suggesting limitations on the number of embryos to transfer in a given ART cycle (discussed in “Process of Assisted Reproductive Technologies”) and improved implantation rates that allow single embryo transfer, the multiple pregnancy rates associated with ART should continue to decrease.

Selective Reduction

Ten percent of multifetal pregnancies spontaneously lose at least one gestational sac during the first trimester. This loss rate increases to 21% in women older than 35 years of age. The spontaneous reduction rate to twins or singletons is much higher for triplets (14%) than for quadruplets (3.5%) (391,392). Selective pregnancy termination or multifetal reduction may be an option for some patients in whom spontaneous reduction does not occur by 11 to 13 weeks. This involves first karyotyping each fetus through transabdominal chorionic villous sampling to preferentially reduce those that are abnormal, then injecting potassium chloride into the heart of the targeted fetus. Although selective reduction carries a 3% to 7% risk of losing the entire pregnancy when performed prior to 19 weeks, this is still lower than the 15% chance of spontaneously losing an entire triplet pregnancy (390,393). Selective fetal reduction is typically considered for triplet or higher pregnancies, but even reduction from twins to singletons may have benefit (390).

Ectopic and Heterotopic Pregnancy

Up to 3.4% of ART pregnancies are ectopic (implantation outside the uterus) and require treatment with either surgery or methotrexate. The absence of an intrauterine pregnancy on transvaginal ultrasound evaluation in conjunction with a maternal serum hCG level above a threshold of 1,500 mIU/mL suggests the diagnosis (394,395). Heterotopic pregnancy involves concurrent intrauterine and ectopic pregnancy, usually within the fallopian tube, but ovarian implantations have been reported (395,396). The incidence of heterotopic pregnancy, which is normally rare, is particularly high (1%) after IVF treatment. Multiple gestation, smoking, previous tubal surgery, and prior PID are potential risk factors in addition to ART. As with standard ectopic pregnancies, pain and bleeding are the most common presenting findings with heterotopic pregnancies. Heterotopic pregnancies are most often diagnosed in the first 5 to 8 weeks of gestation using laparoscopy or laparotomy. Only 26% of heterotopic cases can be diagnosed with transvaginal ultrasound, possibly as a result of difficulties in sonographic interpretation in the presence of concomitant ovarian hyperstimulation. After treatment of a heterotopic gestation with laparoscopy, laparotomy, or ultrasound-guided injection of potassium chloride into the extrauterine pregnancy, the overall delivery rate for the intrauterine pregnancy is nearly 70% (395).

Ovarian Hyperstimulation Syndrome

OHSS is a medical complication that is both completely iatrogenic and unique to stimulatory infertility treatment (397). Its symptoms are the result of ovarian enlargement and fragility, extravascular fluid accumulation, and intravascular volume depletion. Proposed mechanisms for the characteristic fluid shifts that accompany OHSS include increased protein-rich fluid secretion from the stimulated ovaries, increased renin and prorenin within follicular fluid, and increased capillary permeability mediated by angiotensin. Vascular endothelial growth factor (VEGF), whose expression in granulosa cells and serum is augmented by hCG, and a variety of other inflammatory cytokines have been implicated in the pathogenesis of this disease (398). Two distinct patterns of OHSS onset have been described. Early OHSS occurs 3 to 7 days following the hCG trigger and is associated with the administration of exogenous hCG. Late onset disease occurs 12 to 17 days after the hCG trigger; it is the result of endogenous hCG secretion from the pregnancy and tends to be more severe with multiple gestation. Pregnancy outcomes are inconsistently affected by the presence of OHSS, with higher rates of biochemical losses but similar rates of clinical losses when compared to patients without OHSS (399).

Severity Classification

Mild OHSS (grades 1 and 2) is associated with high serum estradiol levels and ovarian enlargement to less than 5 cm and has minimal clinical significance. Grade 3 moderate OHSS is accompanied by mild abdominal distention and has minimal clinical significance (397). Grade 3 OHSS occurs in one-third of all COH cycles (398). Grade 4 moderate OHSS is accompanied by gastrointestinal upset and ovarian enlargement to 5 to 12 cm (397). The presence of fluid shifts from intra- to extravascular spaces are the hallmark of severe disease, and grade 5 OHSS includes tense ascites or hydrothorax. Grade 6 OHSS is accompanied by hemoconcentration, coagulation abnormalities, respiratory failure, and renal dysfunction (397). Other markers of severe OHSS include hyponatremia, hyperkalemia, elevated liver function tests, and an elevated white blood cell count (397,398). Concerning symptoms for severe disease include rapid weight gain (≥2 lb per day), increased measurable abdominal girth, hypotension, tachypnea, tachycardia, oliguria, and severe abdominal pain. The latter symptom is suggestive of ovarian cyst rupture or hemorrhage (398).

Risk Factors

Mild OHSS occurs in 13.5% of clomiphene cycles, but more severe disease is rare. With gonadotropin injections, the incidence of moderate disease is 3% to 6% and severe OHSS occurs in 0.1% to 2% of patients. Polycystic ovary syndrome, polycystic ovarian morphology with attendant elevated antimüllerian hormone levels (>3.36 ng/mL), and previous episodes of OHSS are major risk factors for the disease; the impact of young age and lean body mass is controversial (351,398,400). Estradiol levels greater than 800 pg/mL on day 9 of stimulation have been associated with a 55.8% risk for the development of OHSS (severity not specified). Estradiol concentrations of greater than 3,500 pg/mL and greater than 6,000 pg/mL at the time of hCG trigger were associated with severe OHSS in 1.5% and 38% of patients, respectively. During antagonist cycles, the presence of 13 or more follicles at 11 mm or greater average diameter was predictive of the development of OHSS. More than 20 preovulatory follicles were associated with a 15% incidence of severe OHSS. When 20 to 29 oocytes were collected at retrieval, 1.4% of patients developed severe OHSS; this raised to 22.7% with 30 or more oocytes (400).

Management

If outpatient management is appropriate, the patient should be instructed to limit her activity, to weigh herself daily, and to monitor her fluid intake (at least 1 L per day of mostly electrolyte-balanced fluid) and output. Daily follow-up by telephone or visit is important and the patient should be reassessed if she notes worsening of the symptoms or if her weight gain increases to more than 2 lb per day. Indications for hospitalization include an inability to tolerate oral hydration, hemodynamic instability, respiratory compromise, tense ascites, hemoconcentration, leukocytosis, hyponatremia, hyperkalemia, abnormal renal or liver function, and decreased oxygen saturation. Fluid intake and urine output need to be carefully measured, and admission to an intensive care setting can be considered if the patient has hyperkalemia, renal failure, respiratory failure, or thromboembolic disease. Although intravenous fluids may worsen ascities, they are essential to correct hypovolemia, hypotension, electrolyte abnormalities, and oliguria. Albumin 25% can be dosed 50 to 100 mg intravenously every 4 to 12 hours if further intravascular volume expansion is needed. Diuretics can be considered to improve weight gain and oliguria only after hypovolemia has been corrected. Thromboembolic prophylaxis should be given. Single or repeated transvaginal or transabdominal ultrasound-guided paracentesis may relieve pain, hydrothorax, or persistent oliguria. Rapid large-volume fluid removal can be considered, as compensatory fluid shifts are unlikely to occur in this typically young healthy population as long as the patient is carefully monitored (398).

Prevention

No method will prevent OHSS completely, but steps can be taken to decrease risk. Careful gonadotropin stimulation for monofollicular development is discussed under “Ovulatory Factor.” For ART, stimulation protocols for high-risk patients include lower initial COH doses of 150 IU and GnRH antagonists for LH surge prevention, which reduce the total dosage and duration of gonadotropin stimulation (401,402). Reintroduction of GnRH antagonists following retrieval may be of benefit (351).

Decreasing the dose of hCG to reduce the incidence of OHSS is controversial (401,402). Exogenous recombinant LH is available for COH, but the dose required to trigger ovulation has not been established (402). The very short half-life of endogenous LH may in turn reduce the incidence and/or severity of OHSS (351). GnRH agonists can be used instead of hCG during antagonist cycles to induce an endogenous LH surge. As GnRH agonist triggers are associated with lower pregnancy rates, these may be a better option for oocyte donors or for patients who are not planning a fresh embryo transfer (351,402).

Coasting may be considered when estradiol levels are less than 4,500 pg/mL and/or there are 15 to 30 mature follicles present. During coasting, gonadotropin stimulation is withheld and estradiol levels are checked daily (351). An initial rise of estradiol is typically observed within the first 48 hours of the coast, but the levels should subsequently plateau or decrease (402). The patient may then be triggered when serum estradiol levels fall to less than 3,500 pg/mL. If GnRH agonists are used for initial LH surge suppression, switching to an antagonist during the coast has been associated with improved outcomes (351).

The cycle should be cancelled and the trigger withheld if there are greater than 30 mature follicles, the coast duration is more than 4 days, or if estradiol levels rise to more than 6,500 pg/mL during coasting (351,402).

The adjunctive use of metformin is associated with decreased OHSS rates in PCOS patients (342). Cabergoline, a dopamine agonist that inhibits VEGF production, decreased OHSS rates when given at 0.5 mg daily for 7 or 8 days following retrieval. Long-term use of cabergoline may be associated with valvular heart disease (351,401,402). Albumin given at oocyte retrieval does not appear to decrease subsequent rates of OHSS (401).

Cryopreservation of all embryos without transfer will prevent late onset OHSS (402), but pregnancy outcomes vary for frozen/thaw transfers and early onset OHSS may still occur (401,402). More information on embryo crypreservation can be found in the “Process of Assisted Reproductive Technologies” section.

In vitro oocyte maturation completely obviates the need to stimulate the ovaries with gonadotropins. During in vitro oocyte maturation cycles, immature follicles are aspirated following hCG administration, and the retrieved oocytes are grown in vitro until mature. Mature oocytes are then fertilized by insemination or ICSI. Randomized trials are still needed to further evaluate this technique (403).

Risk of Cancer after Fertility Therapy

Infertility by itself is a predisposing factor for ovarian cancer and breast cancer (404,405). Although treatments that promote incessant ovulation and elevated estrogen levels offer biologic plausibility for further increased cancer risk, data regarding the impact of infertility therapy on neoplasias have been inconsistent and conflicting (406). A retrospective cohort study of women in the United States found no association between prior use of clomiphene or gonadotropins and subsequent development of ovarian or breast cancer, although the authors suggested that very high doses or long duration of administration may warrant closer attention (404,405). A large British cohort of women with ovulatory disorders who were given ovulation-stimulation drugs did not find evidence to suggest causation for cancer of the breast, ovary, colon, skin, or thyroid. However, there was a dose–response relationship between the development of uterine cancer and prior use of clomiphene, particularly with a lifetime exposure of 2,250 mg or more (406).

Stress

Stress, as manifested by anxiety or depression, is thought to be increased among women experiencing infertility (407). Stress is the most common reason for patients, even those with insurance coverage, to terminate fertility treatment (408). However, it is not clear whether stress worsens infertility, nor is there clear evidence that psychological treatment improves fertility (407). If psychological disorders are present, treatment should be offered regardless of fertility status.

Preimplantation Genetic Diagnosis

The primary indication for preimplantation genetic diagnosis (PGD) is to improve the chances of having healthy infants in families at high risk for a specific genetic disease (409). Following embryo biopsy, genetic testing can be performed on a blastomere (cell from day 3 embryo), polar body, or on blastocyst trophectoderm prior to transferring the embryo. Aneuploidy in embryos most commonly affects chromosomes X, Y, 13, 14, 15, 16, 18, 21, and 22. Fluorescence in situ hybridization (FISH) is a technique that assesses aneuploidy, translocation, other structural chromosomal defects and sex chromosome content (410). FISH is technically limited by the number of distinct chromosomes that can be evaluated, which results in an inherently high false-negative rate. Newer methods are being developed that assess the entire genome using comparative genomic hybridization or DNA microarrays (411). One-quarter of the cases of PGD are performed for single gene disorders, most commonly myotonic dystrophy, Huntington disease, cystic fibrosis, fragile X syndrome, spinal muscular atrophy, tuberous sclerosis, Marfan syndrome, thalassemia, and sickle cell anemia. Because polymerase chain reaction (PCR) is required for single gene disorder diagnosis, ICSI is performed during ART to avoid contamination from sperm bound to the zona pellucida. PGD can be used for HLA antigen tissue matching in an effort to produce a child whose cord blood or stem cells could potentially help an existing affected child. Disadvantages to PGD include decreased postbiopsy embryo survival, requirements for extended culture with an associated possibility that no embryos will be available for transfer or cryopreservation, false-positive and false-negative testing results, and controversies regarding disposition of nontransferred embryos (409,410). PGD is not synonymous with preimplantation genetic screening (PGS), which is performed in couples without known chromosomal anomaly, mutation, or other genetic abnormality.Although it seems intuitive that replacing only euploid embryos should improve pregnancy and live birth rates in patients with advanced age, recurrent pregnancy loss, or implantation failure, study outcomes have not been consistent (409,410,412–415).

Preservation of Fertility in Cancer Patients

Improved cancer treatments such as chemotherapy, surgery, and radiotherapy have greatly enhanced survival, such that many cancer survivors contemplate parenthood. Unfortunately, those life-saving treatments can diminish fertility potential in both men and women. Cancer itself does not usually affect oocytes, but certain chemotherapeutic drugs or radiation damage may adversely affect ovarian reserve and uterine function, particularly in older women (416). Ovarian transposition prior to radiation therapy seems to be effective in preserving ovarian function (417). It is unclear whether ovarian suppression with GnRH analogues or oral contraceptives before or during chemotherapy or radiation is beneficial. If time allows prior to commencement of cancer treatment, women can undergo IVF with embryo cryopreservation (oocyte and ovarian tissue preservation are not routine). In women with cervical cancer, radical trachelectomy allows for retention of the uterus, those with uterine cancer may respond to progestin therapy in lieu of hysterectomy, and some ovarian tumors are amenable to unilateral oophorectomy if future fertility is desired. In men, cancer directly affects gametogenesis, and cancer treatments cause more fertility damage when given at younger ages. Semen and sperm cryopreservation prior to cancer treatment are often recommended for fertility preservation in men (416).

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157. Vlahos NF, Coker L, Lawler C, et al. Women with ovulatory dysfunction undergoing ovarian stimulation with clomiphene citrate for intrauterine insemination may benefit from administration of human chorionic gonadotropin. Fertil Steril 2005;83:1510–1516.

158. Kosmas IP, Tatsioni A, Fatemi HM, et al. Human chorionic gonadotropin administration vs. luteinizing monitoring for intrauterine insemination timing, after administration of clomiphene citrate: a meta-analysis. Fertil Steril 2007;87:607–612.

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162. Practice Committee of American Society for Reproductive Medicine. Use of insulin-sensitizing agents in the treatment of polycystic ovary syndrome. Fertil Steril 2008;90:S69–S73.

163. Tang T, Lord JM, Norman RJ, et al. Insulin-sensitising drugs (metformin, rosiglitazone, pioglitazone, D-chiro-inositol) for women with polycystic ovary syndrome, oligo amenorrhoea and subfertility. Cochrane Database Syst Rev 2010;1:CD003053.

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176. Mitwally MF, Casper RF. Use of an aromatase inhibitor for induction of ovulation in patients with an inadequate response to clomiphene citrate. Fertil Steril 2001;75:305–309.

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180. Practice Committee of American Society for Reproductive Medicine. Use of exogenous gonadotropins in anovulatory women: a technical bulletin. Fertil Steril 2008;90:S7–S12.

181. Ganesh A, Goswami SK, Chattopadhyay R, et al. Comparison of letrozole with continuous gonadotropins and clomiphene-gonadotropin combination for ovulation induction in 1387 PCOS women after clomiphene citrate failure: a randomized prospective clinical trial. J Assist Reprod Genet 2009;26:19–24.

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183. Sipe CS, Davis WA, Maifeld M, et al. A prospective randomized trial comparing anastrozole and clomiphene citrate in an ovulation induction protocol using gonadotropins. Fertil Steril 2006;86:1676–1681.

184. Mitwally MF, Casper RF. Aromatase inhibition reduces the dose of gonadotropin required for controlled ovarian hyperstimulation. J Soc Gynecol Investig 2004;11:406–415.

185. Farquhar C, Lilford RJ, Marjoribanks J, et al. Laparoscopic “drilling” by diathermy or laser for ovulation induction in anovulatory polycystic ovary syndrome. Cochrane Database Syst Rev 2007;3:CD001122.

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195. Gordts S, Gordts S, Puttemans P, et al. Transvaginal hydrolaparoscopy in the treatment of polycystic ovary syndrome. Fertil Steril 2009;91:2520–2526.

196. Mercorio F, Mercorio A, Di Spiezio Sardo A, et al. Evaluation of ovarian adhesion formation after laparoscopic ovarian drilling by second-look minilaparoscopy. Fertil Steril 2008;89:1229–1233.

197. Practice Committee of American Society for Reproductive Medicine. Current evaluation of amenorrhea. Fertil Steril 2008;90:S219–S225.

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207. Guven MA, Dilek U, Pata O, et al. Prevalance of Chlamydia trochomatis, Ureaplasma urealyticum and Mycoplasma hominis infections in the unexplained infertile women. Arch Gynecol Obstet 2007;276:219–223.

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210. Baramki TA. Hysterosalpingography. Fertil Steril 2005;83:1595–1606.

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227. Camus E, Poncelet C, Goffinet F, et al. Pregnancy rates after in-vitro fertilization in cases of tubal infertility with and without hydrosalpinx: a meta-analysis of published comparative studies. Hum Reprod 1999;14:1243–1249.

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235. Rackow BW, Arici A. Reproductive performance of women with mullerian anomalies. Curr Opin Obstet Gynecol 2007;19:229–237.

236. Pritts EA, Parker WH, Olive DL. Fibroids and infertility: an updated systematic review of the evidence. Fertil Steril 2009;91:1215–1223.

237. Di Spiezio Sardo A, Mazzon I, Bramante S, et al. Hysteroscopic myomectomy: a comprehensive review of surgical techniques. Hum Reprod Update 2008;14:101–119.

238. Moghadam R, Lathi RB, Shahmohamady B, et al. Predictive value of magnetic resonance imaging in differentiating between leiomyoma and adenomyosis. JSLS 2006;10:216–219.

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245. Nezhat C, Lavie O, Hsu S, et al. Robotic-assisted laparoscopic myomectomy compared with standard laparoscopic myomectomy—a retrospective matched control study. Fertil Steril 2009;91:556–559.

246. Plotti G, Plotti F, Di Giovanni A, et al. Feasibility and safety of vaginal myomectomy: a prospective pilot study. J Minim Invasive Gynecol 2008;15:166–171.

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248. Karayalcin R, Ozcan S, Moraloglu O, et al. Results of 2500 office-based diagnostic hysteroscopies before IVF. Reprod Biomed Online 2010;20:689–693.

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250. Onalan R, Onalan G, Tonguc E, et al. Body mass index is an independent risk factor for the development of endometrial polyps in patients undergoing in vitro fertilization. Fertil Steril 2009;91:1056–1060.

251. Yanaihara A, Yorimitsu T, Motoyama H, et al. Location of endometrial polyp and pregnancy rate in infertility patients. Fertil Steril 2008;90:180–182.

252. Perez-Medina T, Bajo-Arenas J, Salazar F, et al. Endometrial polyps and their implication in the pregnancy rates of patients undergoing intrauterine insemination: a prospective, randomized study. Hum Reprod 2005;20:1632–1635.

253. Isikoglu M, Berkkanoglu M, Senturk Z, et al. Endometrial polyps smaller than 1.5 cm do not affect ICSI outcome. Reprod Biomed Online 2006;12:199–204.

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255. Robinson JK, Colimon LM, Isaacson KB. Postoperative adhesiolysis therapy for intrauterine adhesions (Asherman’s syndrome). Fertil Steril 2008;90:409–414.

256. Sharma JB, Roy KK, Pushparaj M, et al. Genital tuberculosis: an important cause of Asherman’s syndrome in India. Arch Gynecol Obstet 2008;277:37–41.

257. Roy KK, Baruah J, Sharma JB, et al. Reproductive outcome following hysteroscopic adhesiolysis in patients with infertility due to Asherman’s syndrome. Arch Gynecol Obstet 2010;281:355–361.

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260. Jones HW Jr. Luteal-phase defect: the role of Georgeanna Seegar Jones. Fertil Steril 2008;90:e5–e7.

261. Bukulmez O, Arici A. Luteal phase defect: myth or reality. Obstet Gynecol Clin North Am 2004;31:727–744.

262. Hubayter ZR, Muasher SJ. Luteal supplementation in in vitro fertilization: more questions than answers. Fertil Steril 2008;89:749–758.

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273. Esinler I, Bozdag G, Aybar F, et al. Outcome of in vitro fertilization/intracytoplasmic sperm injection after laparoscopic cystectomy for endometriomas. Fertil Steril 2006;85:1730–1735.

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281. Kilic S, Tasdemir N, Yilmaz N, et al. The effect of anti-thyroid antibodies on endometrial volume, embryo grade and IVF outcome. Gynecol Endocrinol 2008;24:649–655.

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283. Omu AE, al-Qattan F, Ismail AA, et al. Relationship between unexplained infertility and human leukocyte antigens and expression of circulating autogeneic and allogeneic antisperm antibodies. Clin Exp Obstet Gynecol 1999;26:199–202.

284. Edi-Osagie EC, Seif MW, Aplin JD, et al. Characterizing the endometrium in unexplained and tubal factor infertility: a multiparametric investigation. Fertil Steril 2004;82:1379–1389.

285. Gorini G, Milano F, Olliaro P, et al. Chlamydia trachomatis infection in primary unexplained infertility. Eur J Epidemiol 1990;6:335–338.

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287. Grzesko J, Elias M, Maczynska B, et al. Occurrence of Mycoplasma genitalium in fertile and infertile women. Fertil Steril 2009;91:2376–2380.

288. Toth A, Lesser ML, Brooks C, et al. Subsequent pregnancies among 161 couples treated for T-mycoplasma genital-tract infection. N Engl J Med 1983;308:505–507.

289. Moore DE, Soules MR, Klein NA, et al. Bacteria in the transfer catheter tip influence the live-birth rate after in vitro fertilization. Fertil Steril 2000;74:1118–1124.

290. Lavy Y, Lev-Sagie A, Holtzer H, et al. Should laparoscopy be a mandatory component of the infertility evaluation in infertile women with normal hysterosalpingogram or suspected unilateral distal tubal pathology? Eur J Obstet Gynecol Reprod Biol 2004;114:64–68.

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294. Moayeri SE, Lee HC, Lathi RB, et al. Laparoscopy in women with unexplained infertility: a cost-effectiveness analysis. Fertil Steril 2009;92:471–480.

295. Csokmay JM, Frattarelli JL. Basal ovarian cysts and clomiphene citrate ovulation induction cycles. Obstet Gynecol 2006;107:1292–1296.

296. Akin JW, Shepard MK. The effects of baseline ovarian cysts on cycle fecundity in controlled ovarian hyperstimulation. Fertil Steril 1993;59:453–455.

297. Qublan HS, Amarin Z, Tahat YA, et al. Ovarian cyst formation following GnRH agonist administration in IVF cycles: incidence and impact. Hum Reprod 2006;21:640–644.

298. Penzias AS, Jones EE, Seifer DB, et al. Baseline ovarian cysts do not affect clinical response to controlled ovarian hyperstimulation for in vitro fertilization. Fertil Steril 1992;57:1017–1021.

299. Altinkaya SO, Talas BB, Gungor T, et al. Treatment of clomiphene citrate-related ovarian cysts in a prospective randomized study. A single center experience. J Obstet Gynaecol Res 2009;35:940–945.

300. Biljan MM, Mahutte NG, Dean N, et al. Effects of pretreatment with an oral contraceptive on the time required to achieve pituitary suppression with gonadotropin-releasing hormone analogues and on subsequent implantation and pregnancy rates. Fertil Steril 1998;70:1063–1069.

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302. Guzick DS, Carson SA, Coutifaris C, et al. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. National Cooperative Reproductive Medicine Network. N Engl J Med 1999;340:177–183.

303. Lainas T, Zorzovilis J, Petsas G, et al. In a flexible antagonist protocol, earlier, criteria-based initiation of GnRH antagonist is associated with increased pregnancy rates in IVF. Hum Reprod 2005;20:2426–2433.

304. Kolibianakis EM, Albano C, Camus M, et al. Initiation of gonadotropin-releasing hormone antagonist on day 1 as compared to day 6 of stimulation: effect on hormonal levels and follicular development in in vitro fertilization cycles. J Clin Endocrinol Metab 2003;88:5632–5637.

305. Baerwald AR, Walker RA, Pierson RA. Growth rates of ovarian follicles during natural menstrual cycles, oral contraception cycles, and ovarian stimulation cycles. Fertil Steril 2009;91:440–449.

306. Lass A. Monitoring of in vitro fertilization-embryo transfer cycles by ultrasound versus by ultrasound and hormonal levels: a prospective, multicenter, randomized study. Fertil Steril 2003;80:80–85.

307. Pinto F, Oliveira C, Cardoso MF, et al. Impact of GnRH ovarian stimulation protocols on intracytoplasmic sperm injection outcomes. Reprod Biol Endocrinol 2009;7:5.

308. de Jong D, Macklon NS, Eijkemans MJ, et al. Dynamics of the development of multiple follicles during ovarian stimulation for in vitro fertilization using recombinant follicle-stimulating hormone (Puregon) and various doses of the gonadotropin-releasing hormone antagonist ganirelix (Orgalutran/Antagon). Fertil Steril 2001;75:688–693.

309. Kwan I, Bhattacharya S, McNeil A, et al. Monitoring of stimulated cycles in assisted reproduction (IVF and ICSI). Cochrane Database Syst Rev 2008;2:CD005289.

310. Badawy A, Elnashar A, Totongy M. Clomiphene citrate or aromatase inhibitors for superovulation in women with unexplained infertility undergoing intrauterine insemination: a prospective randomized trial. Fertil Steril 2009;92:1355–1359.

311. Werbrouck E, Spiessens C, Meuleman C, et al. No difference in cycle pregnancy rate and in cumulative live-birth rate between women with surgically treated minimal to mild endometriosis and women with unexplained infertility after controlled ovarian hyperstimulation and intrauterine insemination. Fertil Steril 2006;86:566–571.

312. Tsafrir A, Simon A, Margalioth EJ, et al. What should be the first-line treatment for unexplained infertility in women over 40 years of age—ovulation induction and IUI, or IVF? Reprod Biomed Online 2009;19(Suppl 4):4334.

313. Reindollar RH, Regan MM, Neumann PJ, et al. A randomized clinical trial to evaluate optimal treatment for unexplained infertility: the fast track and standard treatment (FASTT) trial. Fertil Steril 2010;94:888–899.

314. Foong SC, Fleetham JA, O'Keane JA, et al. A prospective randomized trial of conventional in vitro fertilization versus intracytoplasmic sperm injection in unexplained infertility. J Assist Reprod Genet 2006;23:137–140.

315. Bhattacharya S, Hamilton MP, Shaaban M, et al. Conventional in-vitro fertilisation versus intracytoplasmic sperm injection for the treatment of non-male-factor infertility: a randomised controlled trial. Lancet 2001;357:2075–2079.

316. Toner JP. Progress we can be proud of: U.S. trends in assisted reproduction over the first 20 years. Fertil Steril 2002;78:943–950.

317. Huirne JA, Homburg R, Lambalk CB. Are GnRH antagonists comparable to agonists for use in IVF? Hum Reprod 2007;22:2805–2813.

318. Kadoch IJ, Al-Khaduri M, Phillips SJ, et al. Spontaneous ovulation rate before oocyte retrieval in modified natural cycle IVF with and without indomethacin. Reprod Biomed Online 2008;16:245–249.

319. Elnashar AM. Progesterone rise on the day of HCG administration (premature luteinization) in IVF: an overdue update. J Assist Reprod Genet 2010;27:149–155.

320. Bosch E, Labarta E, Crespo J, et al. Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles. Hum Reprod 2010;25:2092–3100.

321. Sonmezer M, Pelin Cil A, Atabekoglu C, et al. Does premature luteinization or early surge of LH impair cycle outcome? Report of two successful outcomes. J Assist Reprod Genet 2009;26:159–163.

322. Escudero EL, Boerrigter PJ, Bennink HJ, et al. Mifepristone is an effective oral alternative for the prevention of premature luteinizing hormone surges and/or premature luteinization in women undergoing controlled ovarian hyperstimulation for in vitro fertilization. J Clin Endocrinol Metab2005;90:2081–2088.

323. Ortmann O, Weiss JM, Diedrich K. Gonadotrophin-releasing hormone (GnRH) and GnRH agonists: mechanisms of action. Reprod Biomed Online 2002;5(Suppl 1):1–7.

324. Reh A, Krey L, Noyes N. Are gonadotropin-releasing hormone agonists losing popularity? Current trends at a large fertility center. Fertil Steril 2010;93:101–108.

325. Tarlatzis BC, Fauser BC, Kolibianakis EM, et al. GnRH antagonists in ovarian stimulation for IVF. Hum Reprod Update 2006;12:333–340.

326. Filicori M, Cognigni GE, Samara A, et al. The use of LH activity to drive folliculogenesis: exploring uncharted territories in ovulation induction. Hum Reprod Update 2002;8:543–557.

327. Albuquerque LE, Saconato H, Maciel MC. Depot versus daily administration of gonadotrophin releasing hormone agonist protocols for pituitary desensitization in assisted reproduction cycles. Cochrane Database Syst Rev 2005;1:CD002808.

328. Pal L, Jindal S, Witt BR, et al. Less is more: increased gonadotropin use for ovarian stimulation adversely influences clinical pregnancy and live birth after in vitro fertilization. Fertil Steril 2008;89:1694–1701.

329. Verberg MF, Eijkemans MJ, Macklon NS, et al. The clinical significance of the retrieval of a low number of oocytes following mild ovarian stimulation for IVF: a meta-analysis. Hum Reprod Update 2009;15:5–12.

330. Al-Inany HG, Abou-Setta AM, Aboulghar M. Gonadotrophin-releasing hormone antagonists for assisted conception: a Cochrane review. Reprod Biomed Online 2007;14:640–649.

331. Griesinger G, Venetis CA, Marx T, et al. Oral contraceptive pill pretreatment in ovarian stimulation with GnRH antagonists for IVF: a systematic review and meta-analysis. Fertil Steril 2008;90:1055–1063.

332. Lainas TG, Sfontouris IA, Zorzovilis IZ, et al. Flexible GnRH antagonist protocol versus GnRH agonist long protocol in patients with polycystic ovary syndrome treated for IVF: a prospective randomised controlled trial (RCT). Hum Reprod 2010;25:683–689.

333. Bianchi P, Serafini P, da Rocha AM, et al. Follicular waves in the human ovary: a new physiological paradigm for novel ovarian stimulation protocols. Reprod Sci 2010;17:1067–1076.

334. Smitz J, Andersen AN, Devroey P, et al. Endocrine profile in serum and follicular fluid differs after ovarian stimulation with HP-hMG or recombinant FSH in IVF patients. Hum Reprod 2007;22:676–687.

335. Daya S. Follicle-stimulating hormone and human menopausal gonadotropin for ovarian stimulation in assisted reproduction cycles. Cochrane Database Syst Rev 2009;1.

336. Coomarasamy A, Afnan M, Cheema D, et al. Urinary hMG versus recombinant FSH for controlled ovarian hyperstimulation following an agonist long down-regulation protocol in IVF or ICSI treatment: a systematic review and meta-analysis. Hum Reprod 2008;23:310–315.

337. Smulders B, van Oirschot SM, Farquhar C, et al. Oral contraceptive pill, progestogen or estrogen pre-treatment for ovarian stimulation protocols for women undergoing assisted reproductive techniques. Cochrane Database Syst Rev 2010;1:CD006109.

338. Damario MA, Barmat L, Liu HC, et al. Dual suppression with oral contraceptives and gonadotrophin releasing-hormone agonists improves in-vitro fertilization outcome in high responder patients. Hum Reprod 1997;12:2359–2365.

339. Dirckx K, Cabri P, Merien A, et al. Does low-dose aspirin improve pregnancy rate in IVF/ICSI? A randomized double-blind placebo controlled trial. Hum Reprod 2009;24:856–860.

340. Lambers MJ, Hoozemans DA, Schats R, et al. Low-dose aspirin in non-tubal IVF patients with previous failed conception: a prospective randomized double-blind placebo-controlled trial. Fertil Steril 2009;92:923–929.

341. Boomsma CM, Macklon NS. Does glucocorticoid therapy in the peri-implantation period have an impact on IVF outcomes? Curr Opin Obstet Gynecol 2008;20:249–256.

342. Tso LO, Costello MF, Albuquerque LE, et al. Metformin treatment before and during IVF or ICSI in women with polycystic ovary syndrome. Cochrane Database Syst Rev 2009;2:CD006105.

343. Zhang M, Ouyang H, Xia G. The signal pathway of gonadotrophins-induced mammalian oocyte meiotic resumption. Mol Hum Reprod 2009;15:399–409.

344. Marteil G, Richard-Parpaillon L, Kubiak JZ. Role of oocyte quality in meiotic maturation and embryonic development. Reprod Biol 2009;9:203–224.

345. Mehlmann LM. Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation. Reproduction 2005;130:791–799.

346. Stevenson TL, Lashen H. Empty follicle syndrome: the reality of a controversial syndrome, a systematic review. Fertil Steril 2008;90:691–698.

347. European Recombinant LH Study Group. Human recombinant luteinizing hormone is as effective as, but safer than, urinary human chorionic gonadotropin in inducing final follicular maturation and ovulation in in vitro fertilization procedures: results of a multicenter double-blind study. J Clin Endocrinol Metab 2001;86:2607–2618.

348. Ludwig M, Doody KJ, Doody KM. Use of recombinant human chorionic gonadotropin in ovulation induction. Fertil Steril 2003;79:1051–1059.

349. Tsoumpou I, Muglu J, Gelbaya TA, et al. Symposium: update on prediction and management of OHSS. Optimal dose of HCG for final oocyte maturation in IVF cycles: absence of evidence? Reprod Biomed Online 2009;19:52–58.

350. Al-Inany HG, Aboulghar M, Mansour R, et al. Recombinant versus urinary human chorionic gonadotrophin for ovulation induction in assisted conception. Cochrane Database Syst Rev 2005;2:CD003719.

351. Garcia-Velasco JA. How to avoid ovarian hyperstimulation syndrome: a new indication for dopamine agonists. Reprod Biomed Online 2009;18(Suppl 2):71–75.

352. Vlahos NF, Giannakikou I, Vlachos A, et al. Analgesia and anesthesia for assisted reproductive technologies. Int J Gynaecol Obstet 2009;105:201–205.

353. Mains L, Van Voorhis BJ. Optimizing the technique of embryo transfer. Fertil Steril 2010;94:785–790.

354. Tsai YC, Lin MY, Chen SH, et al. Vaginal disinfection with povidone iodine immediately before oocyte retrieval is effective in preventing pelvic abscess formation without compromising the outcome of IVF-ET. J Assist Reprod Genet 2005;22:173–175.

355. van Os HC, Roozenburg BJ, Janssen-Caspers HA, et al. Vaginal disinfection with povidon iodine and the outcome of in-vitro fertilization. Hum Reprod 1992;7:349–350.

356. Sohn SH, Penzias AS, Emmi AM, et al. Administration of progesterone before oocyte retrieval negatively affects the implantation rate. Fertil Steril 1999;71:11–14.

357. Williams SC, Oehninger S, Gibbons WE, et al. Delaying the initiation of progesterone supplementation results in decreased pregnancy rates after in vitro fertilization: a randomized, prospective study. Fertil Steril 2001;76:1140–1143.

358. Engmann L, DiLuigi A, Schmidt D, et al. The effect of luteal phase vaginal estradiol supplementation on the success of in vitro fertilization treatment: a prospective randomized study. Fertil Steril 2008;89:554–561.

359. Serna J, Cholquevilque JL, Cela V, et al. Estradiol supplementation during the luteal phase of IVF-ICSI patients: a randomized, controlled trial. Fertil Steril 2008;90:2190–2195.

360. Tournaye H. Management of male infertility by assisted reproductive technologies. Baillieres Best Pract Res Clin Endocrinol Metab 2000;14:423–435.

361. Gardner DK, Lane M, Stevens J, et al. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73:1155–1158.

362. Papanikolaou EG, D'Haeseleer E, Verheyen G, et al. Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study. Hum Reprod2005;20:3198–3203.

363. Lane M, Gardner DK. Embryo culture medium: which is the best? Best Pract Res Clin Obstet Gynaecol 2007;21:83–100.

364. Blake DA, Farquhar CM, Johnson N, et al. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Syst Rev 2007;4:CD002118.

365. Ambartsumyan G, Clark AT. Aneuploidy and early human embryo development. Hum Mol Genet 2008;17:R10–R15.

366. Bavister B. Oxygen concentration and preimplantation development. Reprod Biomed Online 2004;9:484–486.

367. Papanikolaou EG, Kolibianakis EM, Tournaye H, et al. Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis. Hum Reprod 2008;23:91–99.

368. Papanikolaou EG, Camus M, Kolibianakis EM, et al. In vitro fertilization with single blastocyst-stage versus single cleavage-stage embryos. N Engl J Med 2006;354:1139–1146.

369. Papanikolaou EG, Fatemi H, Venetis C, et al. Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer. Fertil Steril 2010;93:592–597.

370. Reh A, Fino E, Krey L, et al. Optimizing embryo selection with day 5 transfer. Fertil Steril 2010;93:609–615.

371. Practice Committee of the American Society for Reproductive Medicine; Practice Committee of the Society for Assisted Reproductive Technology. Guidelines on number of embryos transferred. Fertil Steril 2009;92:1518–1519.

372. Derks RS, Farquhar C, Mol BW, et al. Techniques for preparation prior to embryo transfer. Cochrane Database Syst Rev 2009;4:CD007682.

373. El-Toukhy T, Coomarasamy A, Khairy M, et al. The relationship between endometrial thickness and outcome of medicated frozen embryo replacement cycles. Fertil Steril 2008;89:832–839.

374. Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Curr Opin Obstet Gynecol 2009;21:270–274.

375. Wennerholm UB, Soderstrom-Anttila V, Bergh C, et al. Children born after cryopreservation of embryos or oocytes: a systematic review of outcome data. Hum Reprod 2009;24:2158–2172.

376. Urman B, Balaban B, Yakin K. Impact of fresh-cycle variables on the implantation potential of cryopreserved-thawed human embryos. Fertil Steril 2007;87:310–315.

377. Glujovsky D, Pesce R, Fiszbajn G, et al. Endometrial preparation for women undergoing embryo transfer with frozen embryos or embryos derived from donor oocytes. Cochrane Database Syst Rev 2010;1:CD006359:

378. Kumbak B, Oral E, Karlikaya G, et al. Serum oestradiol and beta-HCG measurements after day 3 or 5 embryo transfers in interpreting pregnancy outcome. Reprod Biomed Online 2006;13:459–464.

379. Van Voorhis BJ. Outcomes from assisted reproductive technology. Obstet Gynecol 2006;107:183–200.

380. Centers for Disease Control and Prevention. 2007 Assisted reproductive technology success rates. Available online at: http://www.cdc.gov/art/ART2007/PDF/COMPLETE_2007_ART.pdf

381. Banerjee P, Choi B, Shahine LK, et al. Deep phenotyping to predict live birth outcomes in in vitro fertilization. Proc Natl Acad Sci U S A 2010;107:13570–13575.

382. Ethics Committee of the American Society for Reproductive Medicine. Fertility treatment when the prognosis is very poor or futile. Fertil Steril 2009;92:1194–1197.

383. Marina S, Marina D, Marina F, et al. Sharing motherhood: biological lesbian co-mothers, a new IVF indication. Hum Reprod 2010;25:938–941.

384. Practice Committee of American Society for Reproductive Medicine; Practice Committee of Society for Assisted Reproductive Technology. Ovarian tissue and oocyte cryopreservation. Fertil Steril 2008;90:S241–S246.

385. Krieg SA, Henne MB, Westphal LM. Obstetric outcomes in donor oocyte pregnancies compared with advanced maternal age in in vitro fertilization pregnancies. Fertil Steril 2008;90:65–70.

386. Paulson RJ, Boostanfar R, Saadat P, et al. Pregnancy in the sixth decade of life: obstetric outcomes in women of advanced reproductive age. JAMA 2002;288:2320–2323.

387. Antinori S, Gholami GH, Versaci C, et al. Obstetric and prenatal outcome in menopausal women: a 12-year clinical study. Reprod Biomed Online 2003;6:257–261.

388. Ethics Committee of the American Society for Reproductive Medicine. Financial compensation of oocyte donors. Fertil Steril 2007;88:305–309.

389. Sharpe K, Karovitch AJ, Claman P, et al. Transvaginal oocyte retrieval for in vitro fertilization complicated by ovarian abscess during pregnancy. Fertil Steril 2006;86:219.e11–e13.

390. Evans MI, Kaufman MI, Urban AJ, et al. Fetal reduction from twins to a singleton: a reasonable consideration? Obstet Gynecol 2004;104:102–109.

391. Ulug U, Jozwiak EA, Mesut A, et al. Survival rates during the first trimester of multiple gestations achieved by ICSI: a report of 1448 consecutive multiples. Hum Reprod 2004;19:360–364.

392. Leondires MP, Ernst SD, Miller BT, et al. Triplets: outcomes of expectant management versus multifetal reduction for 127 pregnancies. Am J Obstet Gynecol 2000;183:454–459.

393. Brambati B, Tului L, Camurri L, et al. First-trimester fetal reduction to a singleton infant or twins: outcome in relation to the final number and karyotyping before reduction by transabdominal chorionic villus sampling. Am J Obstet Gynecol 2004;191:2035–2040.

394. Fernandez H, Gervaise A. Ectopic pregnancies after infertility treatment: modern diagnosis and therapeutic strategy. Hum Reprod Update 2004;10:503–513.

395. Barrenetxea G, Barinaga-Rementeria L, Lopez de Larruzea A, et al. Heterotopic pregnancy: two cases and a comparative review. Fertil Steril 2007;87:417.e9–e15.

396. Kamath MS, Aleyamma TK, Muthukumar K, et al. A rare case report: ovarian heterotopic pregnancy after in vitro fertilization. Fertil Steril 2010;94:1910.e9–e11.

397. Golan A, Weissman A. Symposium: update on prediction and management of OHSS. A modern classification of OHSS. Reprod Biomed Online 2009;19:28–32.

398. Practice Committee of American Society for Reproductive Medicine. Ovarian hyperstimulation syndrome. Fertil Steril 2008;90:S188–S193.

399. Papanikolaou EG, Tournaye H, Verpoest W, et al. Early and late ovarian hyperstimulation syndrome: early pregnancy outcome and profile. Hum Reprod 2005;20:636–641.

400. Delvigne A. Symposium: update on prediction and management of OHSS. Epidemiology of OHSS. Reprod Biomed Online 2009;19:8–13.

401. Humaidan P, Quartarolo J, Papanikolaou EG. Preventing ovarian hyperstimulation syndrome: guidance for the clinician. Fertil Steril 2010;94:389–400.

402. Aboulghar M. Symposium: update on prediction and management of OHSS. Prevention of OHSS. Reprod Biomed Online 2009;19:33–42.

403. Siristatidis CS, Maheshwari A, Bhattacharya S. In vitro maturation in subfertile women with polycystic ovarian syndrome undergoing assisted reproduction. Cochrane Database Syst Rev 2009;1:CD006606.

404. Brinton LA, Scoccia B, Moghissi KS, et al. Breast cancer risk associated with ovulation-stimulating drugs. Hum Reprod 2004;19:2005–2013.

405. Brinton LA, Lamb EJ, Moghissi KS, et al. Ovarian cancer risk after the use of ovulation-stimulating drugs. Obstet Gynecol 2004;103:1194–1203.

406. Silva Idos S, Wark PA, McCormack VA, et al. Ovulation-stimulation drugs and cancer risks: a long-term follow-up of a British cohort. Br J Cancer 2009;100:1824–1831.

407. Van den Broeck U, D'Hooghe T, Enzlin P, et al. Predictors of psychological distress in patients starting IVF treatment: infertility-specific versus general psychological characteristics. Hum Reprod 2010;25:1471–1480.

408. Domar AD, Smith K, Conboy L, et al. A prospective investigation into the reasons why insured United States patients drop out of in vitro fertilization treatment. Fertil Steril 2010;94:1457–1459.

409. Practice Committee of Society for Assisted Reproductive Technology; Practice Committee of American Society for Reproductive Medicine. Preimplantation genetic testing: a Practice Committee opinion. Fertil Steril 2008;90:S136–S143.

410. Simpson JL. Preimplantation genetic diagnosis at 20 years. Prenat Diagn 2010;30:682–695.

411. Munne S, Howles CM, Wells D. The role of preimplantation genetic diagnosis in diagnosing embryo aneuploidy. Curr Opin Obstet Gynecol 2009;21:442–449.

412. Schoolcraft WB, Katz-Jaffe MG, Stevens J, et al. Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial. Fertil Steril 2009;92:157–162.

413. Hardarson T, Hanson C, Lundin K, et al. Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial. Hum Reprod 2008;23:2806–2812.

414. Mersereau JE, Plunkett BA, Cedars MI. Preimplantation genetic screening in older women: a cost-effectiveness analysis. Fertil Steril 2008;90:592–598.

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