Spencer S. Richlin
Aida Shanti
Ana A. Murphy
On July 25, 1978, the field of reproductive medicine and infertility changed forever with the birth of Louise Brown in England by in vitro fertilization (IVF). Her birth gave hope to many couples not able to conceive conventionally. The initial indication for IVF was tubal factor or pelvic adhesive disease as IVF bypasses the fallopian tubes by using controlled ovarian hyperstimulation (COH), egg retrieval, and insemination with ultimate embryo transfer. COH produces large numbers of mature eggs for insemination extracorporally. Intracytoplasmic sperm injection (ICSI) injects a single sperm into the cytoplasm of an egg. It has revolutionized the treatment of men with oligospermia or azoospermia in whom intratesticular sperm are present. The success rate of IVF with ICSI approaches the pregnancy rate with conventional IVF for nonmale factor etiologies. Women with premature ovarian failure, decreased ovarian reserve, or age factor infertility, can chose to undergo an IVF cycle with donor oocytes and enjoy excellent pregnancy rates.
INITIAL EVALUATION FOR IVF
The initial evaluation includes a review of the couples' medical, surgical, and infertility history and informed consent process is obtained. Topics of discussion should include:
· genetic screening, testing, and counseling
· COH and retrieval techniques and risk factors
· fresh and frozen embryo transfer procedures
· a discussion of nonselective reduction
· ICSI and its indications
· an estimate of the number of embryos to be transferred
· an estimate of the individual couple's pregnancy rate with IVF
· potential adverse events such as cancellation of cycles, gamete/fertilization problems, and pregnancy risks (multiple gestation, spontaneous abortion, and ectopic pregnancy).
The couple should have access to a referral for psychological testing, particularly in cases of donor egg.
Gamete intrafallopian transfer (GIFT) and zygote intrafallopian transfer (ZIFT) are alternatives to conventional IVF. GIFT places retrieved oocytes and sperm into the fallopian tube via laparoscopy or laparotomy where fertilization takes place, while ZIFT places zygotes created by IVF, at the stage prior to cleavage, into the fallopian tube. ZIFT and GIFT require at least one normal fallopian tube. ZIFT allows evaluation of the rate of fertilization while GIFT does not. Initially, pregnancy rates with GIFT and ZIFT were higher than conventional IVF, but this occurred at a time when IVF success rates were relatively low. For religious reasons, some couples prefer GIFT because fertilization occurs in the fallopian tube and not in the laboratory. Both GIFT and ZIFT require an additional procedure—a mini-laparotomy or laparoscopy, for placement of the gametes or zygotes. This additional procedure carries an inherent surgical risk and added cost, which makes IVF the choice of virtually all couples. Pregnancy rates with IVF have improved sufficiently so that that the added surgical risk of GIFT and ZIFT is simply not warranted. With the advent of ICSI, assisted hatching, and advanced laboratory techniques (i.e., culture media and embryo day 3 or 5 transfers), IVF success rates rival and exceed ZIFT/GIFT rates and very few of these procedures are performed.
PATIENT SELECTION FOR IVF
Although tubal factor infertility was the original indication for IVF, the indications for IVF have now been expanded to make this the final step in virtually all infertile couples. Tubal factor infertility can arise from various etiologies, including pelvic inflammatory disease, endometriosis, prior abdominopelvic surgery (including tubal ligations), ectopic pregnancy, or a ruptured appendix. This diagnosis is made by hysterosalpingogram (HSG) or laparoscopy. Although surgical therapy may be a very reasonable option in some patients, IVF may be the best option, especially for women with significant tubal damage, older women, or those with multiple factor infertility. Because of the poor prognosis for repeat tuboplasty, IVF is the treatment of choice.
The treatment of male factor infertility has been drastically advanced by IVF and ICSI. Men who have a motile sperm count below 10 × 106 with poor morphology have a significant reduction in pregnancy rates. ICSI has been used to treat severe male factor infertility, with pregnancy and fertilization rates similar to couples with normal semen parameters who are undergoing conventional IVF. Oligospermic and selected azoospermic males, who have intratesticular sperm, have high fertilization and pregnancy rates with ICSI, regardless of motility, morphology, or sperm concentrations.
Reduced fertility with advancing maternal age seems to be more common as women delay childbearing. In noncontracepting populations, fertility starts to decrease at age 30 with a severe decrease in fertility after age 40. This decline is manifested as an age-related decrease in ovarian follicles and a decline in oocyte quality. Women over 35 have a significantly decreased chance of pregnancy, compared to younger patients. Data from women of all ages, who have received an oocyte from a young donor, reveal that pregnancy rates are excellent, and independent of the recipient's age, implying that the uterus does not age. At present there are provocative tests that evaluate a patient's fecundity.
Women with diminished ovarian reserve (DOR), regardless of their age, have significantly lower pregnancy rates with any modality. They also have an increased risk of aneuploid pregnancy and spontaneous miscarriage compared to patients with normal ovarian reserve. Day 3 or basal follicle-stimulating hormone (FSH) and the clomiphene challenge test (CCT), correlate best with conception using assisted reproductive technologies (ARTs), and are also applicable to an infertility population. Typically, an abnormal day 3 FSH is ≥12.6 IU/L. These values are dependent on the particular assay used and are patient population–dependent. Infertile patients who are older than 43 years or have an elevated FSH, have a less than 5% pregnancy rate per cycle, and are encouraged to pursue donor egg or adoption.
Endometriosis affects 30% to 40% of infertile women. Endometriosis involving the ovaries, fallopian tubes, and pelvis may cause anatomic distortion and adhesions of the tubes and ovaries hampering ovum pickup. Patients with moderate to severe endometriosis (stages III and IV) are encouraged to attempt IVF, especially those with severe adhesive disease. Many of these patients have had one or more conservative surgeries and have not achieved pregnancy despite optimal debulking of their disease. Surgery prior to IVF to improve pregnancy rates is not recommended. Surgical excision of endometriomas prior to stimulation improves the stimulation and oocyte retrieval.
Many couples who fail clomiphene citrate (CC) and gonadotropin therapy eventually require IVF. Patients under 36 years of age, World Health Organization (WHO) group I and II, frequently try three to four cycles of CC or gonadotropins with intrauterine insemination (IUI). Of patients who become pregnant with CC and IUI, over 85% become pregnant within the first four cycles of treatment.
Patients with unexplained infertility are by definition ovulatory, with normal fallopian tubes and uterine cavity or stage I or II treated endometriosis, and a normal semen analyses by WHO criteria. The majority of these patients have already attempted other therapeutic strategies such as timed intercourse, CC, or gonadotropins with IUI. Women with unexplained infertility have a combined per cycle IVF pregnancy rate of approximately 20% compared to 8% and 17% for CC with IUI and gonadotropin with IUI, respectively. In 2000, IVF pregnancy rates for unexplained infertility were appreciably higher indicating this technique should be more rapidly offered.
Premature ovarian failure (POF) is defined as hypergonadotropic hypogonadism prior to the age of 40. Causes of premature oocyte loss include gonadal dysgenesis (Turner syndrome), failure of germ cell migration in fetal life, and postnatal germ cell loss (castration, autoimmune disease, infections). Iatrogenic oocyte depletion occurs with chemotherapy, radiotherapy, and ovarian surgery for benign disease. Patients with POF have excellent success when receiving donor oocytes with a delivery rate/cycle of approximately 30% and a cumulative delivery rate after four cycles approaching 86%. There is no decrease in pregnancy or delivery rates with increasing age or diagnosis of the recipient (i.e., POF, surgical castration, previous IVF failure, menopause, DOR). Thus, endometrial receptivity does not appear to be altered by recipient age or diagnosis. Using young oocyte donors optimizes success rates by having a greater chance of transferring healthy embryos.
TESTING PRIOR TO ASSISTED REPRODUCTION
Previous IVF cycles should be reviewed in detail focusing on the number and quality of embryos, the method of insemination (conventional vs. ICSI), follicular and endometrial development, estradiol levels throughout the stimulation, length of the stimulation, and the pregnancy outcome. In addition we review the method and number of embryos transferred. Frozen embryo transfer cycles are also assessed, noting the endometrial stimulation and embryo quantity and quality after thawing.
Evaluation should also consider thyroid disease (thyroid-stimulating hormone), hyperprolactinemia (prolactin), an insulin and glucose test to screen for hyperinsulinemia (if indicated), and a blood type and Rh. An infectious disease panel will consist of hepatitis B and C, syphilis, human immunodeficiency virus (HIV), and rubella titer. Initial selected genetic tests for carrier status are offered and include Tay-Sachs disease (Ashkenazi Jews), sickle cell anemia (African descent), β-thalassemia (Mediterranean/Chinese), α-thalassemia (Southeast Asians), and cystic fibrosis (Caucasians). A day 3 or basal FSH and estradiol may be useful in selected patients.
Ovarian Reserve Testing
Testing ovarian reserve can be useful for all IVF patients regardless of age. These tests are inexpensive, noninvasive, and highly predictive of ultimate outcome with ARTs. Most IVF programs routinely check all patients 35 years or older or those at risk, including patients with multiple surgeries, stage III or IV endometriosis, severe adhesive disease, and cigarette smokers. A day 3 basal FSH is obtained during a natural cycle. Based on our laboratory assay, a day 3 FSH greater than 12.6 mIU/mL predicts a less than 5% chance of achieving a pregnancy per IVF cycle, regardless of age. In order to counsel patients effectively, each institution and laboratory must establish their own critical cutoff point, above which, a pregnancy becomes highly unlikely. In a large study, both FSH and estradiol were shown to be excellent predictors of IVF performance. As a “single predictor,” basal FSH was better than age at predicting a successful pregnancy. Regardless of age or fertility history, women with diminished ovarian reserve as manifested by an elevated day 3 FSH, ultimately conceived with a high rate of first trimester pregnancy loss. The live-birth rate in patients with DOR was less than 1%.
Significant intercycle variability can be found among basal FSH values, especially in patients with widely fluctuating FSH levels. This variability reflects the physiologic inability of the follicle cohort to suppress FSH as a result of declining follicular health. FSH levels rise as granulosa cell production of inhibin-B decreases. Inhibin exerts negative control on the pituitary. Studies have shown that patients with intermittently elevated basal FSH levels stimulate poorly, need more medication for stimulation, have fewer aspirated follicles, and lower peak estradiol levels compared to patients with minimal intercycle variability. Once a patient has an isolated elevated day 3 FSH, even among normal cycles, her chance for IVF pregnancy is poor.
Due to the intercycle variability of day 3 FSH values, a normal value has limitations. The CCT uses the day 3 FSH value, and a provoked day 10 response to 100 mg of clomiphene citrate, administered cycle days 5 through 9. The CCT specifically evaluates the day 10 FSH. An abnormal day 10 FSH carries the same poor prognosis as an abnormal day 3 FSH. A poor CCT response predicts a decreased pregnancy rate for an infertile population as well as for IVF patients. Unexplained infertility comprises 52% of the patients with an abnormal CCT.
A CCT should be performed on all patients over the age of 37, or in women who have a borderline day 3 FSH. Patients may be tested earlier if they have had ovarian surgery, are diagnosed with stage III or IV endometriosis, or have severe adhesive disease. The results of these tests help direct patient counseling. An elevated day 3 basal FSH value, or an abnormal CCT, predicts a less than 5% chance of pregnancy with ART.
Evaluation of the Uterus and Fallopian Tubes
An HSG, sonohysterogram, or office hysteroscopy should be done before starting IVF. An HSG aids in the diagnosis of hydrosalpinges or an intrauterine filling defect, both of which decrease IVF pregnancy rates. Since patients can develop uterine or tubal pathology in intervening years, a repeat study should be considered if the previous uterine evaluation is more than 1 year old. A complete pelvic ultrasound, a trial embryo transfer, vaginal cultures, and a Pap smear are done on all patients. During the ultrasound, the adnexae are evaluated for ovarian cysts, endometriomas, and hydrosalpinges, while any leiomyomas or polyps are noted in the uterus. Sonohysterography is almost as accurate as hysteroscopy, and more sensitive and specific than transvaginal ultrasound or HSG in diagnosing intrauterine pathology. Additionally, sonohysterography is less invasive, relatively inexpensive, and does not require the use of contrast dyes.
Hydrosalpinges and IVF
Tubal disease is one of the main indications for IVF with hydrosalpinges accounting for 10% to 30% of tubal factor infertility. Compared to other types of tubal pathology, women with hydrosalpinges (including surrogate carriers) have reduced IVF success. Hydrosalpinges decrease IVF implantation, pregnancy, and delivery rates while increasing early pregnancy loss. This has been confirmed by large meta-analyses as well as numerous retrospective studies.
Only two randomized, prospective studies have evaluated whether prophylactic salpingectomy of hydrosalpinges prior to IVF improves pregnancy rates. Pregnancy rates after a single IVF cycle are approximately 37% in women after salpingectomies and 24% when the hydrosalpinges were not removed. Subgroup evaluation of these patients revealed that women with bilateral hydrosalpinges, visible on ultrasound, had a significant increase in implantation and delivery rates after salpingectomy compared to the nonintervention group. Ultimate delivery rates more than double in patients with ultrasound visible hydrosalpinges after salpingectomy.
Many mechanisms have been proposed to explain the adverse effects hydrosalpinges have on IVF success rates. Fluid that accumulates in the hydrosalpinx can move retrograde into the uterine cavity. Endometrial receptivity marker molecules have been shown to be decreased in patients with hydrosalpinges, while inflammatory cytokines within the fluid may act as inhibitors. Salpingectomy is recommended when the fallopian tubes have visible fluid within the tube on sonogram. Polyps, müllerian anomalies, intrauterine synechiae, and leiomyoma are all associated with reproductive loss or failure. In women undergoing IVF, a midfollicular office sonohysterogram (intrauterine saline infusion) can further delineate the nature of the lesion. After the lesion is identified, a diagnostic or operative hysteroscopy should be performed to correct the abnormality.
Uterine leiomyomas may contribute to infertility and miscarriage but the data are limited by the uncontrolled retrospective nature of the reports. Most IVF studies distinguish between submucosal (leiomyomas that distort the uterine cavity), intramural (leiomyomas confined to the myometrium with no cavity distortion), and subserosal leiomyomas (leiomyomas protruding from the serosal surface). Subserosal leiomyomas, which are not adjacent to the endometrium, do not decrease pregnancy rates or increase miscarriages rates. Submucosal leiomyomas, which protrude into the uterine cavity, have been shown to decrease IVF pregnancy and implantation rates when compared to nonprotruding intramural and subserosal myomas. Prior to starting an IVF cycle, removal of submucosal distorting myomas is strongly recommended. Depending on location, leiomyomas are removed abdominally or hysteroscopically.
The literature is controversial regarding the removal of intramural leiomyomas prior to an IVF cycle, especially if they do not distort the endometrial cavity. In retrospective studies in patients under 40 years of age with intramural myomas, there is a significant decrease in implantation rate. There is only a trend toward decreased live-birth rates in women with intramural myomas, independent of the size of the leiomyoma volume.
Debate remains as to whether endometriomas should be removed prior to IVF. The few retrospective studies performed have not shown an increase in pregnancy rates with removal of endometriomas. However, some studies have shown a decrease in peak estradiol, fertilization, and implantation rates with advanced stages of endometriosis, including endometriomas. Most experienced clinicians remove endometriomas prior to IVF because there appears to be a significant improvement in the recruitment of follicles. However, debulking endometriosis is not warranted before IVF except in cases of endometriomas or pelvic pain. Women with stage III or IV endometriosis have a decreased pregnancy rate and their embryos are often poor in quality with thickened zona pellucida.
EVALUATION OF MALE FACTOR INFERTILITY
Male factor infertility will affect 50% of infertile couples in some fashion. In the vast majority of cases, this diagnosis is known prior to IVF. During the initial IVF consultation, the male partner is encouraged to be present so we can review his medical history and any prior semen analyses. Men suspected of male factor infertility are typically evaluated with regard to the following:
· infertility history
· sexual history
· childhood and developmental history
· medical history
· surgical history
· prior infections
· gonadotoxins
· family history
· a review of systems.
Multiple semen analyses should be evaluated in addition to any appropriate hormonal and genetic screening.
Semen Analysis and Sperm Preparation
Semen samples should be collected after an abstinence interval of 2 to 3 days. WHO criteria are a rough guideline for evaluating semen samples. These criteria include assessment of volume, sperm concentration, motility, morphology, pH, and total sperm count. Sperm morphology has become a useful indicator of successful fertilization with IVF. Kruger coined the term “strict criteria,” which includes only morphologically normal sperm. In studies using strict morphologic criteria, men with greater than 14% normal forms had normal fertilization rates in vitro. Patients with 4% to 14% normal forms had intermediate fertilization rates, while men with less than 4% normal forms had fertilization rates of 7% to 8%. ICSI should be recommended for any factor or combination of factors that are abnormal, including count, motility and score, as well as sperm morphology. In men with poor sperm parameters, ICSI produces fertilization and pregnancy rates similar to conventional IVF.
Semen samples need to be washed and processed prior to their use in IVF. Washing techniques isolate motile capacitated sperm for insemination. Sperm isolation may be performed on ejaculated semen, sperm retrieved from the epididymis or testes, or cryopreserved samples. Several isolation techniques are currently used. The “swim-up” method is used for samples with normal concentration and motility. For low motility and decreased sperm counts, the semen sample is layered over discontinuous Percoll gradients (45% and 90%) and centrifuged. The resulting pellet separates normal spermatozoa from lymphocytes, epithelial cells, abnormal sperm, cell debris, and bacteria.
Many tests have been proposed to evaluate sperm function, including the immunobead test, the interspecies sperm penetration assay, a hemizona assay, the hypoosmotic swelling test, and the mannose binding assay. These are not part of a semen analysis and purportedly predict the ability of sperm to fertilize an oocyte. These tests have poor inter- and intralaboratory reproducibility and the information gained from these studies rarely changes management, which is ultimately to proceed to IVF either with or without ICSI. Even without these studies, the nonfertilization rate is less than 1%. If the semen analysis is normal, a fresh sample for IVF insemination will be produced the day of oocyte retrieval.
Azoospermic or severely oligospermic men will be further evaluated. If necessary, a sperm retrieval procedure may be required. One or two of these sperm samples will be cryopreserved prior to starting an IVF/ICSI cycle. At the time of oocyte retrieval, a frozen sample will be thawed for ICSI.
Genetic and Hormonal Evaluation of the Infertile Male
Azoospermic and severely oligospermic males require genetic and hormonal evaluation. Men with nonobstructive azoospermia or severe oligospermia have a Y chromosome microdeletion assay performed. Thirteen percent of men with nonobstructive azoospermia will have a deletion in the gene cluster termed DAZ(deleted in azoospermia), which resides in the AZFc region, within the DAZ gene cluster. Men with this Y chromosome deletion produce male offspring that are severely infertile or sterile. Prior to ICSI, we test for DAZ, and provide genetic counseling if needed. Nonobstructive azoospermic men often have karyotype abnormalities that cause reproductive failure. Ten percent of these men have a sex chromosome abnormality, with the most common being Klinefelter syndrome (47 XXY). Men who have obstructive azoospermia, with congenital bilateral absence of the vas deferens (CBAVD), require testing for mutations in the cystic fibrosis gene. Ninety percent of patients with CBAVD will have gene aberrations. Since these genes are transmitted in an autosomal recessive fashion, genetic counseling is advised for these men and their families. Evaluation of oligospermic and azoospermic men requires serum measurements of luteinizing hormone (LH), FSH, prolactin, and testosterone. These often aid in the diagnoses of testicular resistance or failure, and hypogonadotropic hypogonadism.
PREPARATION FOR IVF
A trial or “mock” embryo transfer should be performed with the same catheter used during the actual embryo transfer. This enables the physician to measure the uterine length and note the curvature of the cervix and uterus. The goal of this precycle trial transfer is to facilitate a nontraumatic embryo transfer which has been shown to increase pregnancy and implantation rates. If there is cervical stenosis precluding easy access to the uterine cavity, it will be detected before undergoing an IVF cycle. Severe cases of cervical stenosis may require dilation of the cervix with hysteroscopic guidance prior to starting the IVF cycle.
IVF PROTOCOLS
Many protocols have been described for COH designed to increase the number of mature follicles for retrieval. All protocols aim to maximize the number of mature oocytes retrieved per stimulation cycle. The information needed to make a protocol decision is based on:
· medical and surgical history
· results from laboratory tests (i.e., ovarian reserve testing)
· extensive review of any previous assisted reproduction cycles (stimulation response, embryo quantity and quality, fertilization rate, embryo transfer).
In general, patients fit into one of three groups—normal, high, and poor responders.
COH Medications and Strategies
Gonadotropin releasing hormone-agonists (GnRH-a) have revolutionized the use of gonadotropin-based stimulation protocols. Pregnancy rates per IVF cycle have been shown in meta-analyses to significantly improve with gonadotropins because of the increased number of oocytes retrieved. GnRH-a, such as leuprolide acetate, substitute amino acids at position 6 and 10 on the GnRH molecule. This decreases the degradation rate and increases the binding affinity of the agonist to the GnRH receptor. GnRH-a initially produce a surge or flare that increases circulating LH and FSH levels. The flare lasts 7 to 10 days, followed by down-regulation and desensitization, resulting in a hypogonadal state and eventual withdrawal bleeding. This suppression of endogenous gonadotropins prevents the spontaneous LH surge prior to oocyte retrieval. The flare protocols begin GnRH-a the day before or the first day of gonadotropin stimulation. Before the advent of GnRH-a, detection of the LH surge was necessary as 15% of IVF cycles were cancelled due to premature LH surges. Protocols using GnRH-a either capitalize on the down-regulation (long protocol) feature of GnRH-a or the flare (short protocol), which adds endogenous gonadotropins to those administered therapeutically.
At the present time, the majority of IVF cycles use GnRH-a in either a “long” or “short” protocol. GnRH antagonists have been introduced for COH. Third-generation antagonists (ganirelix and cetrorelix) are more complex than agonists, having substitutions on the GnRH molecule at positions 1, 2, 3, 6, and 10. They bind competitively to the GnRH receptor, decreasing gonadotropin secretion within 4 to 8 hours in a dose-dependent manner. GnRH antagonists prevent the risk of a premature LH surge by using either a “single-dose” or “multiple-dose” protocol. Both regimens initiate stimulation with gonadotropins on day 2 of a spontaneous menstrual cycle. The “single-dose” protocol uses 3 mg of cetrorelix based on follicle size and LH level, while the “multiple-dose” protocol uses 0.25 mg of antagonist, starting on stimulation day 6 and then daily up to and including the day of human chorionic gonadotropin (hCG) administration.
Large, prospective, randomized clinical trials comparing agonist to antagonist protocols have demonstrated no significant differences in fertilization, implantation, and pregnancy rates between the two analogues. With GnRH-a there can be difficulty timing cycles since the antagonist protocol depends on the initiation of a natural cycle. GnRH-a protocol requires 2 to 4 weeks for complete pituitary desensitization before starting stimulation, requires longer treatment, and increases total number of gonadotropin ampules needed to achieve follicular maturation. When compared to antagonists, long-acting agonists appear to have an increased risk of ovarian hyperstimulation syndrome (OHSS) and produce estrogen withdrawal effects. While the ideal stimulation protocol has yet to be developed, GnRH agonist and antagonist protocols will undoubtedly continue to be universally used.
There are two major classes of gonadotropins, urinary–derived human menopausal gonadotropins (hMG) which contain both LH and FSH, and recombinant gonadotropins which contain pure FSH. The goal of any gonadotropin therapy is to maximize follicular recruitment for oocyte retrieval. The urinary hMG typically has 75 IU of FSH and 75 IU of LH per ampule. Since FSH is the most important gonadotropin for folliculogenesis, newer preparations of hMG have decreased substantially or removed LH by purification techniques. This can result in a highly purified gonadotropin product with only negligible amounts of LH present (<0.1 IU LH/ampule). The newer recombinant gonadotropins are genetically engineered by inserting the human gene for FSH in cultured immortal mammalian cells. Mammalian cells are needed as bacteria are unable to glycosylate FSH and glycosylation is required for FSH to be biologically active. Recombinant gonadotropin products (r-FSH) are devoid of LH and are highly pure, have specific activity, maintain excellent batch-to-batch consistency, and are tolerated well upon injection. It remains uncertain whether the recombinant FSH products (r-FSH) or hMG (u-FSH and u-LH) preparations have any advantages in clinical pregnancy rates with IVF but if a difference exists, it is modest.
Metformin, an insulin-sensitizing agent, has been successfully used to induce ovulation in a small number of women with polycystic ovary syndrome (PCOS) and is now being investigated in women with PCOS undergoing IVF. Early reports suggest increased fertilization and pregnancy rates when using metformin in patients with PCOS who are resistant to clomiphene citrate. Metformin should be started at least 1 month prior to the IVF cycle with a slow increase to a therapeutic metformin dose to minimize the gastrointestinal side effects. Hopefully, this approach will result in fewer cases of OHSS due to a decreased number of small follicles and an increased number of larger follicles (>16 mm).
Those with a high response rate to gonadotropin stimulation are typically young and thin, and often have the diagnosis of PCOS. They are at risk for producing elevated estradiol levels (>5,000 pg/mL) with few mature preovulatory follicles. This results in cycle cancellation (withholding hCG), to avoid the development of OHSS. Poor responders recruit fewer oocytes than normal responders, and have decreased pregnancy rates. Patient characteristics that predict a poor response include women over 40 years of age, a prior cancelled standard long protocol IVF cycle, severe endometriosis (stage III or IV), diminished ovarian reserve, and cigarette smoking. Normal responders are often patients with unexplained infertility, mild endometriosis, tubal disease, or male factor infertility.
Normal and high responders use leuprolide (1.0 mg subcutaneous daily), given in the midluteal phase (7 days after a positive LH surge) of the menstrual cycle preceding their stimulation (Fig. 39.1). Patients who are on a contraceptive pill, for cycle control and gonadotropin suppression, simply overlap the last 7 days of their pill with leuprolide. The leuprolide dose is decreased to 0.5 mg per day on the day gonadotropin stimulation is continued until the day of hCG administration. An estradiol level and baseline transvaginal ultrasound are obtained. Gonadotropin stimulation is initiated when:
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FIG. 39.1. Example of a long gonadotropin-releasing hormone (GnRH) protocol. The dosage of follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) will vary depending on the patient characteristics. hCG, human chorionic gonadotropin. |
· the uterine lining is less than 6 mm
· no functional ovarian cysts are larger than 1 cm
· women are adequately suppressed with an estradiol level less than 20 pg/mL and an FSH less than 12.6 pg/mL.
Women with functional ovarian cysts larger than 1 cm should continue to be suppressed until the cysts are resolved before initiating gonadotropin stimulation. Those with an endometrial thickness greater than 6 mm should continue leuprolide for another week, and have a repeat scan. In the majority of cases the lining will then be sufficiently thin, allowing the start of gonadotropin stimulation.
To maximize follicular recruitment in normal and high responders, gonadotropin stimulation is started (day 2–3) at a dose between 3 to 6 ampules per day. The starting dosage of gonadotropin is based on age, prior stimulation cycles, and the patient's diagnosis. Women who are poor responders, with a history of pelvic adhesive disease or endometriosis stage III or IV will start at a higher dose (up to 8 ampules/d), while patients with PCOS start at a much smaller dose. The daily dose of gonadotropins is fixed until day 5 of stimulation.
After the first 5 days of stimulation, the daily dose of gonadotropin is adjusted based on the patient's ultrasound and estradiol level. When at least two lead follicles reach 18 mm, or one follicle becomes 19 mm, ovulation is triggered with 10,000 IU of hCG and the leuprolide is discontinued. In addition to follicular size, the decision to trigger ovulation depends upon the rate of follicular growth and estradiol rise. Oocytes are retrieved 35 hours later.
On occasion, high responders will hyperstimulate, producing multiple follicles with estradiol levels greater than 3,000 pg/mL. These patients are at risk for OHSS. Cancellation of the cycle prior to hCG stimulation almost always thwarts the development of OHSS. Another approach to avoid OHSS is to retrieve all of the patient's follicles after hCG, and cryopreserve the embryos for future frozen embryo transfers.
“Coasting” refers to withholding gonadotropin administration for 1 to 3 days while continuing leuprolide until the estradiol falls below 3,000 pgmL so as to be able to proceed with retrieval. Follicles at the time of coasting are typically 16 mm, and are able to continue developing without gonadotropin stimulation. If the estradiol level declines and the number of small follicles declines, hCG may be given and the cycle proceeds to retrieval. Very acceptable pregnancy rates with very few cancelled cycles for fear of OHSS can be achieved when employing “coasting” or cryopreserving all embryos for later transfer.
While antagonist protocols are becoming more commonplace in IVF centers, the efficacy of antagonist stimulation protocols only will be realized when supported by prospective randomized studies. Presently there are two antagonist protocols, a “single-dose” and a “multiple- dose” regimen, both of which are evolving as clinicians become more experienced. When using the “multiple-dose” protocol, antagonist is given when follicles reach 12 to 14 mm. From that day onward, the gonadotropin dose is adjusted depending upon ovarian response. At this point, one ampule of r-FSH is substituted for one ampule of hMG. The antagonist is given (0.25 mg of ganirelix/d) until the triggering hCG injection. The natural cycle length of the patient is taken into consideration when deciding when to trigger ovulation.
Several protocols have been proposed for the poor responder. Simply increasing the dose of gonadotropins may increase numbers of follicles and oocytes, but pregnancy rates remain low. An alternative protocol and strategy is the flare-up or co-flare. The “co-flare” regimen, which is a short protocol, uses the initial increase of gonadotropins from leuprolide to begin follicular stimulation. Leuprolide is initiated on cycle day 2 after an estradiol and FSH are drawn. On day 3, eight ampules of gonadotropins are administered. Target follicle size is 16 to 17 mm, with a progesterone level below 1.0.
Ovulation is usually triggered on day 8 or later as a shorter interval is associated with immature oocytes. Poor responders often have estradiol levels that plateau associated with postmature oocytes. Most patients receive 10,000 IU hCG at 16 mm. Poor responders are most often placed on a “co-flare” protocol rather than a long, down-regulation protocol.
OOCYTE RETRIEVAL
Oocyte retrieval is performed 35 hours after hCG injection. Retrieval is performed in an operating room or procedure room using conscious sedation. The perineum and vagina are prepped with warm sterile saline prior to retrieval. Oocytes are retrieved by vacuum aspiration of follicles under transvaginal ultrasound guidance. A sharp 17-gauge needle is used, allowing precise penetration of the ovary. The ovary is stabilized with the vaginal probe prior to needle penetration. To reduce patient discomfort, follicles are usually aspirated through a single entry into each ovary (Fig. 39.2). Aspirates are collected into 15-mL heated collection tubes. The patient is discharged once she is tolerating liquids and has recovered sufficiently from the conscious sedation.
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FIG. 39.2. Transvaginal ultrasound probe with needle-guided aspiration. Oocytes are removed through the needle and tubing using negative vacuum pressure. |
In the laboratory, follicular aspirates are examined under a dissecting microscope and blood and cumulusgranulosa cells are removed. Oocytes are placed in culture media, and graded for maturity and quality. The optimum stage of oocyte maturity is metaphase II, with one polar body extruded. At this stage, conventional insemination or ICSI can be performed (Fig. 39.3). Oocytes are placed into culture media in microdroplets under oil. With conventional insemination, oocytes are inseminated 40 hours after hCG injection with 50,000 normal motile sperm per mL, with greater than 4% normal forms by strict Kruger criteria. One hour later, oocytes are removed from the insemination droplets and placed into clean, sperm-free microdroplets under oil. Fertilization is checked 18 hours later. Normal fertilized embryos are moved to individual microculture droplets and remain there until embryo transfer. Embryo transfer occurs 72 hours after insemination.
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FIG. 39.3. Oocyte in metaphase II about to undergo intracytoplasmic sperm injection (ICSI). Ooplasm is clear with the first polar body at 12 o'clock. During ICSI, the introducing pipette (right) places a single sperm (at the tip of the pipette) into the cytoplasm of the egg. The holding pipette (left) holds the egg in place with gentle suction. |
LUTEAL PROGESTERONE SUPPORT AFTER OOCYTE RETRIEVAL
The use of exogenous progesterone, during the luteal phase of an IVF cycle, to increase pregnancy rates is supported by large meta-analyses. Progesterone prepares the endometrial lining for implantation and pregnancy maintenance. The timing of progesterone supplementation seems to be critical and is started the evening of the day of retrieval. There has been much debate in the literature with regard to the route of administration of progesterone for IVF luteal support.
Proponents of intramuscular administration argue that levels are measured more accurately in the serum, helping clinicians modify progesterone administration throughout the luteal phase and early pregnancy. Physicians who prefer intravaginal progesterone point to its local effects on the uterus and endometrial lining.
Most programs prefer progesterone in oil (given intramuscularly), starting the night of oocyte retrieval and continuing until the patient has her first pregnancy test. If the peak progesterone level is greater than 40 µg/L, the supplementation is continued until the patient is 8 weeks pregnant and then a change to vaginal suppositories, 100 mg twice daily, is made until 12 weeks. If the progesterone level is less than 20 µg/L, 100 mg vaginal progesterone suppository twice a day is added. Levels will be rechecked in 1 week and are titrated accordingly.
ICSI AND SPERM RETRIEVAL TECHNIQUES FOR SEVERE MALE FACTOR
ICSI has given men with severe male factor infertility the ability to produce offspring. Prior to microinsemination techniques, severe male factor infertility was essentially untreatable. ICSI delivers a single sperm into the cytoplasm of an egg through the zona pellucida and egg membrane (see Fig. 39.3). Fertilization rates with ICSI using viable motile spermatozoa are very similar to rates achieved with conventional IVF with standard insemination.
Sperm retrieval techniques have given hope to men with obstructive and nonobstructive azoospermia. The etiology of obstructive azoospermia includes men with a previous vasectomy, ejaculatory duct obstruction, and congenital bilateral absence of the vas deferens. Sperm production is normal in these men, though sperm parameters may decrease with chronic obstruction. Sperm are extracted using both percutaneous sperm retrieval and open microsurgical techniques. In percutaneous epididymal sperm aspiration (PESA), sperm are aspirated through a butterfly needle that is placed into the caudal portion of the epididymis. Adequate numbers of sperm are often retrieved allowing for cryopreservation and future ICSI cycles. With the advent of microsurgical epididymal sperm aspiration (MESA), sperm are retrieved in higher numbers than with PESA, allowing for cryopreservation of large numbers of sperm. In our practice, PESA is attempted first since it is a less invasive procedure that often produces enough sperm for ICSI. If sperm collection fails, MESA is the second option. Men with nonobstructive azoospermia have an impairment of normal spermatogenesis. Their pregnancy and fertilization rates with ICSI are lower than for men with obstructive azoospermia. These men normally do not have sperm present in their epididymis for retrieval. Thus, testicular sperm retrieval is performed with either testicular fine- needle aspiration of the testes (TESA) or open testicular biopsy. Recovered sperm can either be used for ICSI or cryopreserved and thawed on the day of retrieval. Nonobstructive azoospermic patients need to be counseled that sperm retrieval techniques may fail to recover sperm. In the event that sperm is not retrieved, couples may opt for donor sperm.
ICSI uses a single washed sperm placed into a viscous solution of 10% polyvinyl pyrrolidine which impedes sperm movement. The spermatozoa flagellum is crushed, which causes immobilization and increases permeability of the sperm membrane, enhancing nuclear decondensation and pregnancy rates. A morphologically normal sperm is aspirated tail-first into an injecting pipette. The sperm is injected through the zona pellucida and into the ooplasm with the polar body at 12 o'clock (see Fig. 39.3). Injected sperm are placed into culture microdroplets under oil. Eighteen hours later, oocytes are examined for fertilization comparable to standard insemination in vitro. Normal fertilized eggs are moved to individual culture microdroplets under oil and transferred 72 hours from insemination.
ASSISTED HATCHING PRIOR TO EMBRYO TRANSFER
The majority of morphologically “normal” embryos do not implant. Other embryos may have decreased cell numbers, cleave slowly, or have impaired blastocyst hatching. The embryo “hatches” from the zona pellucida before implantation. Assisted hatching (AH) has been suggested to enhance the ability of viable embryos to implant after transfer in IVF cycles. AH is the process by which the zona pellucida is artificially breached or opened (e.g., drilling) (Fig. 39.4). This disruption of the zona pellucida theoretically facilitates the hatching of embryos by opening a hole and allowing blastomeres to more easily be extruded. Studies suggest that thick and hardened zona may prevent or reduce the efficiency of hatching of otherwise normally developing embryos and, hence, improve the rate of implantation. A thickened or hardened zona has been postulated to result from gonadotropin stimulation, the laboratory environment, culture techniques, age (>38), or in women with an elevated day 3 FSH. Most commonly, zona drilling is performed with acid Tyrode solution approximately 72 hours after oocyte retrieval, on the day of embryo transfer. This “drilling” creates a defect in the zona pellucida approximately 30 µm in diameter (see Fig. 39.4). Indications for AH include:
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FIG. 39.4. Assisted hatching. The zona pellucida is opened with acid Tyrode solution. To the left is the holding pipette. The pipette on the right delivers acid Tyrode next to the zona pellucida. |
· age greater than 38
· elevated day 3 FSH
· a prior failed IVF cycle with suspected implantation failure
· increased zona thickness on microscopy
· excess oocyte fragmentation.
AH for the selected indications may increase implantation and pregnancy rates but randomized trials are needed in order to determine the best candidates for AH.
EMBRYO TRANSFER
Embryo transfer melds the efforts of the clinician and laboratory. The success of implantation hinges in part on the success of the embryo transfer. The controversy remains as to the optimal timing of embryo transfer. Embryos can be transferred up to 6 days after fertilization. However, most embryos are transferred at 3 days, at the 8 to 10 cell stage (Fig. 39.5). Proponents of later embryo transfer argue that implantation and pregnancy rates are higher at the blastocyst stage as the embryo is further developed and the embryos that progress in vitro to this stage are healthier and more likely to implant. This has the theoretic advantage of decreasing the need to transfer more than one or two embryos, and thereby reduce the multiple pregnancy rate.
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FIG. 39.5. Grade I embryo ready for transfer. This excellent embryo has 10 to 12 even cells and less than 15% fragmentation. |
Embryos can be transferred with a variety of soft catheters. A soft-tipped catheter conforms to the contour of the cervix and endometrial cavity and reduces tissue trauma. The catheter is attached to a 1-mL syringe containing approximately 30 µL of transfer medium in a continuous column with the embryos placed toward the tip. Patients should have a full bladder prior to transfer which helps straighten out the angle between the uterus and cervix in women with an anteverted uterus. The cervix and vagina are prepped and draped and care is taken not to introduce blood, mucus, or bacteria with the catheter tip as these contaminants produce a poor environment for implantation. Using abdominal ultrasound guidance, embryos are deposited between 1 and 1.5 cm from the fundus without actually contacting it. Touching the fundus during transfer may cause uterine contractions thereby increasing the intrauterine pressure and expelling the embryos through the cervix, which reduces the pregnancy rate. Ultrasound guidance facilitates embryo transfer and confirms that the embryos are placed correctly high in the uterine fundus. There appears to be a significant increase in implantation and pregnancy rates using ultrasound-guided embryo transfer when compared to touching the fundus prior to transfer to determine the catheter tip location in the uterine fundus. After transfer, the catheter is checked for any retained embryos that were not expelled.
The best quality day 3 embryos are selected for fresh embryo transfer. The morning of transfer, embryos are graded and selected for transfer in order of their grade. They are graded as I, II, or III depending on the number and evenness of the dividing blastomeres, the percentage of extracellular fragmentation, and their cleavage rates. Ideally, grade I embryos (see Fig. 39.5) are selected first for transfer. These are “excellent” embryos with 8 even cells and less than 15% fragmentation, while grade II embryos have 6 to 8 even or slightly uneven cells with up to 25% fragmentation. Grade III embryos have less than 6 uneven cells and excessive fragmentation and are typically not transferred fresh or are cryopreserved because their obvious degeneration renders them virtually unable to develop further. If the patient has a significant number of excellent embryos or a uterine anomaly she may be offered day 5 embryo transfer with one or two embryos.
Patients 35 years and under are routinely transferred with two embryos; three embryos are transferred in women over age 35.
OOCYTE DONATION
Oocyte donation consists of oocyte retrieval with subsequent embryo transfer to a third party (the infertile patient or surrogate carrier). Oocyte donors can be either known or anonymous. Oocyte donation is used for women with:
· POF
· a history of surgical castration
· diminished ovarian reserve
· a risk of transmitting a heritable disease to an offspring
· older menopausal patients.
Donor-recipient IVF cycles typically have the highest pregnancy rates among all ARTs and the number of donor-recipient cycles is increasing. In 1999, the ASRMSART (American Society for Reproductive Medicine/Society for Assisted Reproductive Technology) registry reported that 10% (9,066) of all ART cycles used donor oocytes.
The success of donor recipient cycles is based on the tenet that the capacity to conceive is not based on uterine aging but on the quality of the oocyte. Younger donors have higher implantation and pregnancy rates when compared to older donors, with a decreased incidence of preclinical and clinical pregnancy loss. There does not appear to be a decrease in the per cycle or cumulative pregnancy rates as endometrial receptivity is maintained and not altered by advancing maternal age. In addition, there was no association between cumulative pregnancy rates and the etiology of the infertility of the recipient.
Patients with Turner syndrome are a special class of patient using donor oocytes. They are infertile due to gonadal dysgenesis, with natural pregnancies occurring in approximately 2% of these women, usually in patients with mosaicism (XX,XO). Small studies comparing oocyte donation in patients with Turner syndrome to controls report conflicting pregnancy and implantation rates. There may be an inherent endometrial receptivity problem in Turner syndrome with lower implantation and pregnancy rates. Women with Turner syndrome should be carefully screened as cardiovascular abnormalities and arterial hypertension are common, with coarctation of the aorta seen in 10% of patients, and bicuspid aortic valve noted in about one-third of patients without coarctations. Even fatal aortic dissection has been reported in pregnancies established by oocyte donation in women with Turner syndrome.
Donor Oocyte Recipient Endometrial Preparation
Embryos are transferred into the recipient's uterus after exogenous endometrial preparation using sequential estrogen and progesterone and gonadotropin down-regulation if they are still cycling. This hormonal manipulation primes the endometrium for embryo transfer by mimicking the natural menstrual cycle. Leuprolide acetate (0.1 µg) is started on cycle day 21 for gonadotropin down-regulation. Bleeding will typically begin after adequate gonadotropin suppression and the decline in the luteal support. After a normal baseline ultrasound and low endogenous estradiol level is created, estrogen therapy is initiated, based on the date of anticipated embryo transfer. Transdermal estradiol patches (0.1 mg) are used in increasing doses that mimic the progressively increasing estradiol level in the follicular phase. By day 11, most women are using four patches.
Most IVF programs initiate aspirin (81 mg) at the start of stimulation in the hope of improving uterine and ovarian blood flow, implantation, and pregnancy rates. In an optimum fresh embryo transfer cycle, Lupron is maintained while transdermal estradiol is adjusted according to endometrial thickness and plasma estradiol levels. The recipient will receive between 2 and 3 weeks of estrogen for endometrial preparation. Once the endometrial lining is ≥8 mm (14 days) the recipient is ready for the pharmacologic conversion to luteal phase in coordination with the status of the oocyte donor. Both intramuscular (50 mg of progesterone in oil) and vaginal progesterone (100 mg b.i.d.) is started along with Medrol and tetracycline. The majority of fresh embryos are transferred on day 3 which translates into a day 18 transfer for cryopreserved embryos. After embryo transfer, the estradiol, the aspirin, and progesterone in oil are continued until the first pregnancy test, at which time the dose of progesterone is adjusted upward.
Oocyte donors must be screened prior to donation as outlined in the ASRM guidelines for gamete and embryo donation. Donors should be between the ages of 21 to 34 years. Screening for a personal genetic history as well as a sexual history is complemented by testing for infectious and genetic diseases. Psychological assessment by a qualified mental health professional for the donor and her partner is required. Minimal genetic laboratory screening is typically required.
Once prospective donors complete this initial evaluation, they are matched with potential recipients based on both physical and personal characteristics. Prior to starting a donor–recipient cycle, recipients have a precycle evaluation that includes an evaluation of the uterine cavity by sonohysterography or office hysteroscopy. If an intracavitary lesion or a hydrosalpinx is noted, surgical removal is conducted prior to embryo transfer.
Oocyte donation is not without risks to the donor. Donors are often concerned about the risks of ovulation-inducing drugs and ovarian cancer, although the aggregate of studies does not support any association. With COH, the risk of OHSS is always present, although it is decreased in donor oocyte cycles since conception does not ensue. Oocyte retrieval places the donor at risk for anesthetic complications, pelvic infection, and intraperitoneal hemorrhage. These complications are real, though exceedingly rare. There are also potential psychological risks that donors undertake. With appropriate pretreatment screening, we can hopefully minimize donors who may have feelings of ambivalence or regret after donation.
Recipients over 45 years of age are at an increased risk for obstetric complications including gestational diabetes mellitus, hypertensive disorders, cesarean delivery, intrauterine growth restriction, abruptio placentae, and preterm labor. These patients are referred to maternal–fetal medicine specialists for consultation prior to and after conception with IVF. Since there is an increasing risk of adverse pregnancy outcome with advanced maternal age, many centers do not perform donation for women over 50 years of age.
CRYOPRESERVATION
After COH and fresh embryo transfer, 60% of stimulated IVF cycles will produce excess viable embryos which are available for cryopreservation. Cryopreserved or frozen embryos can be thawed and transferred back into the uterus, during a subsequent frozen embryo transfer cycle. This allows for higher overall pregnancy rates per attempted IVF cycle. The indications for embryo cryopreservation include:
· storing excess embryos for future use after a fresh embryo transfer
· decreasing the risk of OHSS in a fresh embryo transfer cycle at very high risk of OHSS
· uterine conditions that are unfavorable for fresh embryo transfer after retrieval (e.g., uterine bleeding, polyps, leiomyomas, severe cervical stenosis, or a thin endometrial lining).
Uterine polyps, leiomyomas, and cervical stenosis are usually identified by sonohysterography, ultrasound, or at trial transfer prior to oocyte retrieval and corrected prior to IVF.
Cryopreservation techniques attempt to minimize cell damage to embryos during the freezing and thawing process with the aid of cryoprotectants. Embryos are frozen at a slow rate with the cryoprotectant. A gradient is induced that allows intracellular water to leave the cell. The embryo is dehydrated to avoid the formation of cytotoxic intracellular ice crystals. Once they are frozen, the embryos are loaded into cryostraws and stored in liquid nitrogen at -196°C. When embryos are needed for transfer, they are thawed rapidly to avoid formation of intracellular ice crystals. Typically, cryopreservation results in an 80% survival rate after thawing frozen embryos.
Patients should be extensively counseled prior to oocyte retrieval with regard to cryopreserving excess embryos. Informed consent is obtained as outlined in the ASRM committee opinion on elements to be considered in obtaining informed consent for ART.
Semen is cryopreserved in men who are not able to produce a sample on the day of oocyte retrieval (due to performance anxiety), who are oligospermic, or azoospermic (post-MESA, PESA, or TESA). Even with the use of cryoprotectants, freezing and thawing sperm samples can decrease motility by 50%. This is usually not a problem as original ejaculate samples contain large numbers of sperm. In men with azoospermia, epididymal or testicular samples are usually cryopreserved until the day of oocyte retrieval. On the day of oocyte retrieval, the semen sample(s) are thawed, the cryoprotectant is removed, and ICSI is performed.
FROZEN EMBRYO TRANSFER
The morning of “frozen embryo transfer,” embryos are thawed and surviving embryos are again graded. Pregnancy rates for fresh embryo transfer and frozen embryo transfer are very similar when similar grade embryos are transferred. This is due to selection criteria for embryo freezing. As discussed previously, day 3 embryos are graded as excellent (grade I), average (grade II), and poor (grade III). After the highest graded embryos are transferred fresh, the remaining grade I and II embryos are cryopreserved, while grade III embryos are not usually transferred or cryopreserved. The number of frozen embryos that are transferred is based on the same criteria as for fresh embryo transfer. Prior to frozen embryo transfer, the recipient will receive between 2 and 3 weeks of estrogen for endometrial preparation. Once the endometrial lining is ≥8 mm (14 days), intramuscular and vaginal progesterone is started for endometrial support.
RISKS OF IVF
Couples who present for ARTs are usually healthy with no significant medical history aside from their infertility. In the treatment of their infertility, they are asked to take significant risks. The two major complications of ARTs are OHSS and multiple gestation. There has been a proposed association between ovulation-induction drugs and gynecologic cancers, although this is not supported by well-controlled retrospective trials. Prior to initiating an IVF cycle, the risks and benefits of ARTs should be discussed, and formal written consent is obtained. In addition, patients should be counseled on fetal reduction of higher-order multiple pregnancies to decrease perinatal morbidity of infants and mother.
OHSS
OHSS is a potentially life-threatening complication of gonadotropin-induced COH. Although the signs and symptoms of impending OHSS usually become evident during stimulation, this syndrome does not become fully manifested until after hCG administration and oocyte retrieval. Patients typically resolve the OHSS within 10 to 14 days after onset of their initial symptoms. The syndrome is prolonged if a triggering dose of hCG is followed by a supplemental dose of hCG or if the patient becomes pregnant and produces her own hCG.
Patients who develop OHSS complain of abdominal bloating and pain due to ovarian enlargement. The pathophysiology of OHSS includes increased capillary permeability with “third spacing” of protein-rich fluid. This can result in hemoconcentration and ascites with dramatic fluid accumulations in the abdomen, pleura, or pericardial spaces. Patients can develop nausea, vomiting, diarrhea, and a decrease in appetite if OHSS progresses. As significant amounts of “third spacing” ensue, we often see shortness of breath and decreased urine output. The precise pathophysiology of OHSS is still not completely understood, though vascular endothelial growth factor (VEGF) also known as “vascular permeability factor” has been implicated. It has been shown that follicular fluid from patients with OHSS produce VEGF in increased quantities which, in a dose-dependant manner, increase vascular permeability.
OHSS can be staged based on clinical signs and symptoms, ultrasound features, and laboratory findings and is used to help predict which women require hospitalization. Patients with mild OHSS present with abdominal discomfort, distention and pain, with ovaries enlarged up to 12 cm. Patients may have nausea, vomiting, or diarrhea. Moderate OHSS includes all the features of mild OHSS, but ultrasonographic evidence of ascites as well. Severe OHSS complicates less than 2% of stimulations. It includes features of mild and moderate OHSS as well as clinical evidence of ascites or hydrothorax and difficulty breathing. In addition, severe OHSS patients are at risk for hemoconcentration, coagulation and electrolyte abnormalities, diminished renal perfusion and function, and even adult respiratory distress syndrome.
Risk factors for developing OHSS include young age, low body mass index, and elevated estradiol levels with an increased number of stimulated follicles during COH. In addition, women with a previous pregnancy complicated by a history of OHSS are at great risk of recurrence. If early manifestations of OHSS appear (i.e., abdominal pain, rapidly increasing estradiol levels [>3,000 pg/mL], and extensive follicular recruitment), preventive treatment strategies are employed. On rare occasions, when follicular development and estradiol levels are excessively high early in the stimulation protocol, all stimulation medications are stopped and the cycle cancelled. More often, gonadotropin dosages are adjusted so that estradiol levels increase more slowly, or “freeze all” the embryos rather than transfer them as fresh embryos. Alternatively, using a “coast” by withdrawing gonadotropins and withholding hCG until the estradiol level decreases often allows retrieval and transfer of fresh embryos.
Once OHSS develops, a complete physical exam and ultrasound are performed including blood tests for sodium, potassium, creatinine, and hematocrit. On physical exam, patients often have abdominal distension and tenderness. Decreased breath sounds may be heard at the bases with pleural effusions. Ultrasound is performed to note the presence of ascites and ovarian size. Patients with OHSS can develop hyponatremia, hemoconcentration, hyperkalemia and even decreased renal perfusion. These patients are monitored as outpatients daily until the condition improves significantly. Patients with severe OHSS are admitted to the hospital for inpatient management using intravenous albumin. Patients are instructed to weigh and measure their abdominal girth daily, drink eight glasses of a high sodium drink, and maintain a high protein diet. In addition, they monitor their urine output, and are instructed to report any changes. An increase in abdominal pain or girth, nausea, vomiting, or decreased appetite necessitates a prompt phone call to the health care provider and an office visit.
Fertility Drugs and Cancer
The potential association between ovulation induction drugs and ovarian cancer remains controversial. The data suggesting an association have been based on meta-analyses, but more recent, well-done case-control studies reassuringly have not found any association when evaluating treatment of infertile women with fertility medications compared with infertile women not treated with ovulation-induction drugs. Patients should be informed about the available studies on this issue. It is appropriate to minimize the number of ovulation-induction cycles, both with clomiphene citrate and gonadotropins, as long as there is any concern surrounding ovulation-induction agents and ovarian cancer.
Multiple Pregnancy
Multiple pregnancies occur with a higher frequency in pregnancies resulting from ARTs than spontaneous conception. Since there is a low implantation rate per embryo (10%–25%) with IVF, more than one embryo is often placed into the uterus. The goal is to minimize the number of multiple pregnancies while maintaining good pregnancy rates. Multifetal gestations confer significant morbidity and mortality to both the mother and fetuses because of a high rate of prematurity and in utero mortality. Maternal risks include preterm labor, placental abruption and previa, cesarean section, postpartum hemorrhage, gestational diabetes, and preeclampsia. According to the U.S. Department of Health/Centers for Disease Control and Prevention's 1999 National Summary and Fertility Clinic Reports, 37% of all ART births using fresh nondonor eggs were multiple births (84% twins and 16% triplets and higher), while less than 3% of births in the general population are multiples.
Patients under 34 years of age with a history of a full-term pregnancy and no history of female infertility have the highest implantation rates with IVF. This includes women with previous tubal ligations, normal ovarian reserve testing, donor oocyte recipients, and couples with male factor infertility. In contrast, women older than 34 years of age, with no pregnancies, and extended histories of infertility, have lower implantation rates. Quite often these patients have a history of endometriosis, poor ovarian reserve, and previous failed IVF cyclesembryo transfers.
During the initial IVF consultation, the IVF success rates and the patient's infertility history are reviewed. Based on this information, the number of embryos we recommend transferring and the number of embryos a couple is willing to accept is defined. The goal is that each patient will have two excellent quality embryos replaced, adjusting (up or down) the number of embryos according to embryo quality and each patient's infertility history. Multifetal pregnancy reduction should be discussed as an option for couples with high-order multiple pregnancy. The aim is to reduce preterm deliveries by decreasing the number of fetuses a woman carries. Besides being an invasive procedure, pregnancy reduction, either transabdominal or transvaginal, risks the loss of the entire pregnancy. In experienced centers, pregnancy loss after transabdominal fetal reduction occurs in 6% to 8% of these pregnancies. In addition, the data suggest that pregnancies reduced to twins proceed as if the fetuses were naturally conceived as twins. There is a psychological toll on couples who are making decisions regarding multifetal reduction, especially in women who have experienced difficulty becoming pregnant. If fetal reduction is not acceptable to a patient under 40, a maximum of three embryos is transferred.
High-order multiple pregnancies (triplets and higher) can no longer be viewed as an acceptable risk of IVF and transferring two embryos will decrease the risk. Proponents of two-embryo transfers argue that pregnancy rates in large series are equivalent when compared to three-embryo transfers. Multiple pregnancy rates in patients 30 to 35 years of age are significantly increased from approximately 29% when two embryos were transferred to 40% with the transfer of three embryos. Interestingly, the twin birth rate with transfers of two and three embryos is 26% and 29%, respectively. Thus, a decrease in embryos transferred from three to two decreases the incidence of high-order pregnancies, but does not reduce the twinning rate.
Preimplantation Genetic Diagnosis
Preimplantation genetic diagnosis (PGD) enables couples who are carriers of genetic diseases to test embryos in vitro for genetic abnormalities prior to embryo transfer and select the embryos free of the genetic error for transfer. Traditionally, carrier couples have used chorionic villus sampling and amniocentesis, during the first and second trimesters respectively, to determine if they have a genetically abnormal fetus. The major disadvantage of this type of prenatal diagnosis is the need for second trimester pregnancy termination when genetically abnormal fetuses are detected. PGD provides couples the option to select genetically normal embryos for embryo transfer prior to transfer. PGD involves performing a blastomere biopsy and then genetic testing to determine the genetic complement of the embryo. Blastomeres can be removed from oocytes and embryos at three different stages, polar body analysis, 6- to 10-cell cleavage stage biopsy, and blastocyst stage biopsy. Cleavage stage biopsy is the most widely used technique. In order to biopsy a blastomere or polar body a hole is made in the zona pellucida using micromanipulation instruments with either acid Tyrode or laser. Since these embryos are transferred back into the uterus on day 4 or 5, a genetic diagnosis is determined within 48 hours. First and second polar body biopsies evaluate only maternal chromosomes. Crossover recombination events between homologous chromosomes can create improper diagnoses of heterozygous genetic defects. Presently, polar body biopsy is not performed by most centers offering PGD.
Cleavage stage biopsies are performed on one or two blastomeres of 6- to 10-cell stage embryos 3 days after insemination. These cells are used for diagnosis with polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH). PCR amplifies fragments of DNA for specific single gene defects. DNA amplification requires careful monitoring to avoid the problems of accidental contamination and allele dropout (ADO) leading to an erroneous genetic diagnosis. ADO or preferential amplification occurs when one of two alleles amplifies preferentially over the other. In a heterozygous cell, the normal allele may be amplified over the abnormal allele and the embryo would be diagnosed as “normal,” and then transferred. PGD has been applied to many monogenic diseases including autosomal recessive, dominant, and X-linked diseases. Examples include cystic fibrosis, Tay–Sachs, spinal muscular dystrophy, Huntington disease, Marfan syndrome, and fragile X syndrome.
Defining the number and structure of metaphase chromosomes is carried out by FISH, not PCR. FISH is used to examine chromosomes in embryos for aneuploidy in patients with male factor infertility and women of advanced maternal age, as well as to detect chromosomal translocations. Common aneuploidies occurring and screened for with FISH include defects in chromosomes 13, 16, 18, 21, 22,X, and Y. FISH uses fluorochrome-labeled probes that hybridize to complementary DNA sequences. The fluorescence produced by the probes allows a color and number notation of these probes which aids in diagnosis. PGD is successful in about 80% to 90% of biopsied embryos, with 50% of these embryos being unaffected and suitable for transfer. Amniocentesis and chorionic villus sampling continues to be recommended because of possible errors in cell sampling or technical difficulties with PCR or FISH. Despite this concern, the risk of carrying a genetically abnormal fetus is limited to the risk of a testing error which is far less than the spontaneous risk of the genetic disease.
SUCCESS RATES
The Fertility Clinic Success and Certification Act of 1992 required all clinics performing ARTs in the United States to annually report their success rates to the Centers for Disease Control and Prevention (CDC). Through SART, the CDC obtains clinic data. The CDC compiles these data and publishes a yearly report which is also available on the CDC website. The first year this clinical data became available for review was 1995. There is a 3-year lag between the time clinics report to the CDC and the time that these reports are available to the public which reflects the time for the last delivery in a calendar year and the subsequent time needed to compile and publish the data.
Each clinic reports on their “pregnancy success rates” based on the type of cycle performed (fresh embryos from nondonor eggs, frozen embryos from nondonor eggs, and donor eggs) as well as the age of the women (<35, 35–37, 38–40, and >40.). The number of ART cycles in the United States has steadily increased as well as the success rates. In 1995, 45,906 fresh nondonor IVF cycles were performed as compared to 65,751 in 1999 (Table 39.1). For patients using their own oocytes, pregnancy rates decrease with increasing age. It is difficult to directly compare the success rates between individual ART programs as they each treat different populations of infertile couples, they may focus on specific infertility factors rather than others (e.g., male factor, preimplantation genetic diagnosis, etc.), and different judgments are often made as to the number of embryos to transfer, IVF protocols, and success rates.
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TABLE 39.1. 1999 CDC/SART assisted reproductive technology success rates |
SUMMARY POINTS
· IVF was initially developed to treat tubal factor infertility but now represents the final therapy for virtually all infertile couples, regardless of the etiology.
· There is a significant maternal age-related decline in IVF pregnancy and delivery rate which should be taken into account when discussing infertility therapies.
· Day 3 FSH ± estradiol and clomiphene challenge test is a more accurate predictor of IVF success than simply maternal age.
· IVF with ICSI has allowed procreation in men with severe oligospermia and in azoospermic men after retrieval of sperm from the epididymis and testes.
· High-order multiple gestation significantly increases maternal and fetal morbidity and mortality. Careful consideration should be given to transferring the fewest number of embryos possible.
· Donor oocyte IVF is very successful for women with ovarian failure, limited oocyte reserve, advanced maternal age, and genetic disorders.
· Preimplantation genetic diagnosis is being increasingly performed using blastomere biopsy and genetic screening to avoid transferring embryos with aneuploidy and autosomal recessive or autosomal dominant gene mutations.
SUGGESTED READINGS
Patient Selection for IVF
Guzick DS, Sullivan MW, Adamson GD, et al. Efficacy of treatment for unexplained infertility. Fertil Steril 1998;70:207–213.
Nestler JE, Jakubowicz DJ, Evans WS, et al. Effects of metformin on spontaneous and clomiphene-induced ovulation in the polycystic ovarian syndrome. N Engl J Med 1998;338:1876–1880.
Paulson RJ, Hatch IE, Lobo RA, et al. Cumulative conception and live birth rates after oocyte donation: implications regarding endometrial receptivity. Hum Reprod 1997;12:835–839.
Testing Prior to Assisted Reproduction
American Society for Reproductive Medicine. Guidelines for gamete and embryo donation. ASRM Practice Committee Report, 1997.
Ovarian Reserve Testing
Levi AJ, Raynault MF, Bergh PA, et al. Reproductive outcome in patients with diminished ovarian reserve. Fertil Steril 2001;76:666.
Navot D, Rosenwaks Z, Margalioth EJ. Prognostic assessment of female fecundity. Lancet 1987;2:645–647.
Toner JP, Philput CB, Jones GS, et al. Basal follicle stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril1991;55:784–791.
Evaluation of the Uterus and Fallopian Tubes
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.
Eldar-Geva T, Meagher S, Healy DL, et al. Effect of intramural, subserosal, and submucosal uterine fibroids on the outcome of assisted reproductive technology treatment. Fertil Steril 1998;70:687–691.
Hayden HA, Li TC, Cooke ID. The septate uterus: a review of management and reproductive outcome. Fertil Steril 2000;73:1–14.
Hurst BS, Tucker KE, Awoniyi CA, et al. Hydrosalpinx treated with extended doxycycline does not compromise the success of in vitro fertilization. Fertil Steril 2001;75:1017–1019.
Lass A, Williams G, Abusheikha N, et al. The effect of endometrial polyps on outcome of in vitro fertilization (IVF) cycles. J Assist Reprod Genet1999;16:410–415.
Pal L, Shifren JL, Isaacson KB, et al. Impact of varying stages of endometriosis on the outcome of in vitro fertilization-embryo transfer. J Assist Reprod Genet 1998;15:27–31.
Strandell A, Lindhard A, Waldenstrom U, et al. Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum Reprod 1999;14:2762–2769.
Surrey ES, Lietz AK, Schoolcraft WB. Impact of intramural leiomyomata in patients with a normal endometrial cavity on in vitro fertilization-embryo transfer cycle outcome. Fertil Steril 2001;75:405–410.
Evaluation of the Male Factor
Daya S. Overview analysis of outcomes with intracytoplasmic sperm injection. J SOGC 1996;18:645.
Kim ED, Lipshultz LI. Evaluation and imaging of the infertile male. Infertil Reprod Med Clin North Am 1999;10:377–409.
Semen Analysis and Sperm Preparation
Kruger TF, Acosta AA, Simmons KF, et al. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1998;49:112.
Genetic and Hormonal Evaluation of the Infertile Male
Jaffe T, Oates RD. Genetic abnormalities and reproductive failure. Urol Clin North Am 1996;21:389.
Reijo R, Lee TY, Salo P, et al. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 1995;10:383.
Preparation for IVF
Mansour R, Aboulghar M, Serour G. Dummy embryo transfer: a technique that minimizes the problems of embryo transfer and improves the pregnancy rate in human in vitro fertilization. Fertil Steril 1990;54:678–681.
COH Medications and Strategies
Agrawal R, Holmes J, Jacobs HS. Follicle-stimulating hormone or human menopausal gonadotropin for ovarian stimulation in in vitro fertilization cycles: a meta analysis. Fertil Steril 2000;73:338–343.
Benadiva CA, Davis O, Kligman I, et al. Withholding gonadotropin administration is an effective alternative for the prevention of ovarian hyperstimulation syndrome. Fertil Steril 1997;67:724–727.
Hughes EG, Fedorkow DM, Daya S, et al. The routine use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril 1992;58:888–896.
Olivennes F, Belaisch-Allart J, Emperaire JC, et al. Prospective, randomized, controlled study of in vitro fertilization-embryo transfer with a single dose of a leuteinizing hormone-releasing hormone (LH-RH) antagonist (cetrorelix) or a depot formula of an LH-RH agonist (triptorelin). Fertil Steril 2000;73:314–320.
Stadtmauer LA, Toma SK, Riehl RM, et al. Metformin treatment of patients with polycystic ovary syndrome undergoing in vitro fertilization improves outcomes and is associated with modulation of the insulin-like growth factors. Fertil Steril 2001;75:505–509.
Luteal Progesterone Support after Oocyte Retrieval
Soliman S, Daya S, Collins J, et al. The role of luteal phase support in infertility treatment: a meta-analysis of randomized trials. Fertil Steril1994;61:1068–1076.
ICSI and Sperm Retrieval Techniques for Severe Male Factor
Schlegel PN. Sperm retrieval techniques for assisted reproduction. Infertil Reprod Med Clin North Am 1999;10:539–553.
Assisted Zona Hatching Prior to Embryo T'ransfer
Cohen J, Alikani M, Trowbridge J, et al. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992;7:685–691.
Embryo Transfer
Schoolcraft WB, Surrey ES, Gardner DK. Embryo transfer: techniques and variables affecting success. Fertil Steril 2001;76:863–871.
Oocyte Donation
Navot D, Drews MR, Bergh PA, et al. Age-related decline in female fertility is not due to diminished capacity of the uterus to sustain embryo implantation. Fertil Steril 1994;61:97–101.
Donor Oocyte Endometrial Preparation
American Society for Reproductive Medicine. Guidelines for gamete and embryo donation. Fertil Steril 2002[Suppl 5];77:1S–18S.
Cryopreservation
American Society for Reproductive Medicine. Elements to be considered in obtaining informed consent for ART. Committee Opinion, June 1997
Elder E, Dale B. Cryopreservation. In: In vitro fertilization, second ed. United Kingdom: Cambridge University Press, 2000:192–224.
Ovarian Hyperstimulation Syndrome (OHSS)
Levin ER, Rosen GF, Cassidenti DL, et al. Role of vascular endothelial cell growth factor in ovarian hyperstimulation syndrome. J Clin Invest1998;102:1978–1985.
Fertility Drugs and Cancer
Mosgaard BJ, Lidegaard O, Kjaer SK et al. Infertility, fertility drugs, and invasive ovarian cancer: a case-control study. Fertil Steril 1997;67:1005–1012.
Potashnik G, Lerner-Geva L, Genkin L, et al. Fertility drugs and the risk of breast and ovarian cancers: results of a long-term follow-up study. Fertil Steril1999;71:853–859.
Multiple Pregnancy
Berkowitz R, Lynch L, Stone J, et al. The current status of multifetal pregnancy reduction. Am J Obstet Gynecol 1996;174:1265–1272.
Templeton A, Morris JK. Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. N Engl J Med 1998;339:573–577.
Preimplantation Genetic Diagnosis
Kanavakis E, Traeger-Synodinos J. Preimplantation genetic diagnosis in clinical practice. J Med Genet 2002;39:6–11.
Success Rates
US Department of Health and Human Services, Centers for Disease Control and Prevention. 1999 assisted reproductive technology success rates. National Summary and Fertility Clinic Reports, Dec 2000.
