OBSTETRICS & GYNECOLOGY

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1 Clinical Expert Series Continuing medical education credit is available online at Outcomes From Assisted Reproductive Technology Bradley J. Van Voorhis, MD The use of assisted reproductive technology (ART) for treating the infertile couple is increasing in the United States. The purpose of this paper is to review the short-term outcomes after ART. Pregnancy rates after ART have shown nearly continuous improvement in the years since its inception. A number of factors affect the pregnancy rate, with the most important being a woman s age. Certain clinical diagnoses are associated with a poorer outcome from ART, including the presence of hydrosalpinges, uterine leiomyomata that distort the endometrial cavity, and decreased ovarian reserve. Multiple gestations are the major complication after ART. New laboratory techniques, including extended embryo culture, may allow the transfer of fewer embryos to maintain pregnancy rates while reducing the risk of multiple gestations. Although much of the morbidity in children born after ART is the result of multiples, recent analysis suggests that even singletons are at higher risk for perinatal morbidity, including preterm delivery and small for gestational age infants. In vitro fertilization may be associated with a slight increased risk for birth defects. The major short-term complication of ART in women is the development of ovarian hyperstimulation syndrome. This syndrome is difficult to predict, but new treatments are being developed that may limit its frequency. Because of its high pregnancy rate, couples are moving to ART more quickly in the management of their infertility. All outcomes of ART, including pregnancy rates and adverse complications, need to be compared with standard non-art therapy when deciding the appropriate course of treatment for a given couple. (Obstet Gynecol 2006;107: ) It would be difficult to name a field of medicine that has undergone more rapid and profound advancement over the past 50 years than the field of infertility treatment. The use of human gonadotropins and antiestrogens for induction of ovulation was first reported in the late 1950s, 1,2 paving the way for the first medical treatment of anovulatory infertility. Perhaps the most remarkable feat in our field, the birth of the first child from in vitro fertilization (IVF), occurred just over 25 years ago. 3 Although originally developed for women with tubal factor infertility, IVF From the Department of Obstetrics and Gynecology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa. Corresponding author: Brad Van Voorhis, MD, Department of Obstetrics and Gynecology, 200 Hawkins Drive, Iowa City, IA ; bradvan-voorhis@uiowa.edu by The American College of Obstetricians and Gynecologists. Published by Lippincott Williams & Wilkins. ISSN: /06 is now used for all causes of infertility, and use has grown to the point that nearly 1% of babies born in the United States are now conceived by IVF. Because of intense research efforts, pregnancy rates with assisted reproductive technology (ART) have shown continuous improvement. However, in recent years, outcomes other than live birth rates have become an important focus of investigation. Problems including multiple gestations, ovarian hyperstimulation, and the health of children born from these techniques have gained new prominence as researchers are now evaluating the health consequences of these procedures. The focus of this review is to evaluate the short-term outcomes from ART, including expected pregnancy rates and complications. Clinical factors that may affect the outcomes of ART will be discussed. Finally, the appropriate implementation of ART procedures within the treatment strategy of infertile couples will be examined. VOL. 107, NO. 1, JANUARY 2006 OBSTETRICS & GYNECOLOGY 183

2 OUTCOMES OF ART: PREGNANCY RATES Treatment-Independent Pregnancy Rates Although commonly referred to as infertile, many couples seeking treatments are actually subfertile and capable of conception without treatment. Often, the difference between undergoing infertility treatment or not is conceiving sooner rather than later. 4 Therefore, the background fertility rate in subfertile couples is important for counseling and for weighing the risks and benefits of a proposed treatment. The treatmentindependent pregnancy rate varies depending upon the population studied. In a primary care population of infertile patients, a treatment-independent cumulative pregnancy rate of 27.4% after 12 months was found. 5 In a population of infertile patients referred to specialists for non-art infertility evaluation and treatments, a 12.3% cumulative pregnancy rate after 12 months was reported. 6 Finally, in couples referred for IVF, a 12-month cumulative pregnancy rate of 2 6% was determined. 7 Factors that have been found to negatively affect the treatment-independent pregnancy rate include the presence of tubal disease, anovulation, male factor infertility, and endometriosis. 6 Factors associated with a higher spontaneous pregnancy rate include history of a previous pregnancy, a duration of infertility of less than 24 months, and female age less than 30 years. 5 Thus, it can be seen that the expectations for a given couple will vary depending on the severity of the infertility problem. Couples in which the woman is relatively younger or who have a shorter duration of infertility will have a significantly better chance of spontaneous conception than couples with a long history of infertility, previous infertility treatment cycles, and certain infertility diagnoses mentioned above. After a complete infertility evaluation, couples, who often believe there is no hope for spontaneous conception, should be advised regarding their prognosis for pregnancy without treatment (often in the range of 1 3% per month for unexplained infertility). Definitions and Pregnancy Rates Assisted reproductive technology includes all fertility treatments in which both eggs and sperm are handled in vitro. Assisted reproductive technology procedures typically involve stimulating the growth of multiple ovarian follicles, surgically removing eggs from a woman s ovaries, and then combining them with sperm in the laboratory. With gamete intrafallopian transfer (GIFT), a laparoscopy is performed, and sperm and unfertilized eggs are placed into the fallopian tube immediately after egg retrieval. With all other ART procedures, eggs are fertilized in vitro, either by culturing eggs with sperm or by injecting a single sperm into the egg by a process known as intracytoplasmic sperm injection (ICSI). Zygote intrafallopian transfer (ZIFT) is the laparoscopic transfer of fertilized eggs (zygotes) to the fallopian tube. The large majority of ART cycles are IVF cycles, performed by culturing embryos for variable numbers of days before transferring the embryos through the cervix to the uterus. Assisted reproductive technology includes the use of donor eggs and cryopreserved embryos. Although previously very intuitive to clinicians treating infertile couples, the fact that IVF is more effective than awaiting spontaneous conception is now evidence-based. 8 In a Canadian prospective multicenter trial, couples were randomized to immediate IVF or 3 months without treatment. Study entrance criterion ensured that only couples with a relatively good prognosis (female age 40 years, open fallopian tubes, motile sperm in the ejaculate) were included. Of 71 couples randomized to expectant management, only 1 couple conceived a twin gestation in the 3 months of waiting. In contrast, of the 68 couples randomized to IVF, 29 couples conceived an ongoing pregnancy resulting in a live birth. Thus, this trial demonstrated a 21-fold increase in the live birth rate after one IVF cycle, compared with 3 months of nontreatment. In 1992, Congress passed the Fertility Clinical Success Rate and Certification Act requiring all clinics performing ART in the United States to annually report success rates to the Centers for Disease Control and Prevention (CDC). Data accuracy is verified by medical directors at individual clinics and validated by committee members performing site visits. This reporting system serves as a rich source of outcomes from ART in the United States. Although the outcome data are not corrected for all possible confounding variables, because of the large size and comprehensive nature of the report, important insights into outcomes of ART can be gleaned from the composite statistics. The most recent data available on the CDC Web site ( index.htm) is from the year 2002, the delay being necessary to collect the most important outcome of ART, the live birth rate. In that year, 391 clinics out of a total of 428 ART clinics reported data on 115,392 cycles. Clinics that did not report either were not functioning in 2002 or simply failed to report as required. By analyzing annual reports, several trends can be seen in the practice of ART in the United States. 184 Van Voorhis Outcomes From ART OBSTETRICS & GYNECOLOGY

3 Most importantly, pregnancy rates have improved over time (Fig. 1). Pregnancy rates from IVF have steadily increased in the United States virtually every single year since these rates have been compiled, with a current (2002 data) live birth rate of 28.3% per cycle. 9 The live birth rate per retrieval was 32.6%, and the live birth rate per transfer was 34.8%. Rates are higher in the latter categories because of the smaller denominator as couples with canceled cycles or failed fertilization and no embryo transfer are eliminated. Pregnancy rates with IVF now equal or exceed those of both GIFT and ZIFT. As a result, GIFT and ZIFT, procedures which once represented about 25% of all ART cycles, currently represent less than 1% of ART cycles. This is a major advancement in the treatment of infertile women because the cost and morbidity of a laparoscopic procedure is avoided. Pregnancy rates are also improving for the transfer of cryopreserved embryos (Fig. 1). Cryopreserved embryos result in a lower pregnancy rate than fresh embryo transfers (live birth rate of 24.8% versus 34.8% per transfer). However, frozen embryo transfer cycles are very cost-effective because ovarian stimulation and retrieval are not necessary before embryo transfer. 10 In addition, when couples are finished with fertility treatments, excess cryopreserved embryos can be donated to other infertile couples, a practice which is also quite cost-effective for the recipient couple. 11 Another major advancement in ART is ICSI, which was developed for the treatment of severe male factor infertility sperm. 12 These men, previously at very high risk for failed fertilization using routine insemination, are now selected to have ICSI, resulting in similar fertilization rates as for men without sperm abnormalities. It has since been discovered that ICSI can be used with ejaculated sperm, sperm from the epididymis, and even sperm recovered from testicular tissue. Intracytoplasmic sperm injection is being used with increasing frequency in ART cycles. In 2002, 53% of ART cycles used ICSI for fertilization of eggs. Furthermore, pregnancy rates with ICSI are now very similar to (although slightly below) rates achieved with IVF and standard insemination (CDC Web site). Intracytoplasmic sperm injection is being used more routinely in some clinics in an attempt to reduce the rate of failed fertilization of oocytes. This complication of IVF is rare, difficult to predict based on seminal fluid parameters, and very disappointing to couples after the effort of ovarian stimulation and egg retrieval. A recent meta-analysis of studies evaluating the use of ICSI in nonmale factor infertility cases concluded that there was no advantage to using ICSI rather than IVF and standard insemination. 13 In fact, the fertilization rates were slightly higher with standard insemination in nonmale factor cases. Because ICSI is more expensive, most centers are restricting the use of ICSI for the indication of male factor infertility. Fig. 1. Trends in live birth rates per embryo transfer. Results are shown for in vitro fertilization cycle using fresh and frozen embryos in both nondonor cycles and donor egg cycles. ART, assisted reproductive technology. Modified from Centers for Disease Control and Prevention, American Society for Reproductive Medicine, Society for Assisted Reproductive Technology Assisted Reproductive Technology Success Rates: National Summary and Fertility Clinic Reports. Atlanta (GA): Centers for Disease Control and Prevention; Available at index.htm. Retrieved November 2, Van Voorhis. Outcomes From ART. Obstet Gynecol VOL. 107, NO. 1, JANUARY 2006 Van Voorhis Outcomes From ART 185

4 PROGNOSTIC FACTORS FOR PREGNANCY AFTER ART Demographic Factors A woman s age is the most important factor affecting the chances for live birth after ART (Fig. 2). This point needs to be emphasized with older patients who may have inflated perceptions about the chances for pregnancy. Misperceptions may arise from media reports of celebrity pregnancies that fail to distinguish between donor and nondonor oocyte sources. From 2002 CDC data, live birth rates per cycle range from just over 40% in women aged 27 years, down to 6% at age 43, and only 2% in women who are over 43 years of age. A large increase in miscarriage rate with aging (reaching nearly 45% at age 43) contributes to the low live birth rate after IVF in relatively older women. Primary care physicians should consider prompt referral of older women ( 36 years of age) with infertility. Data from donor oocyte cycles demonstrate that the reduced fertility associated with aging is linked primarily to aging of the ovary and oocyte rather than aging of the uterus and endometrium. Donor oocyte cycles result in high pregnancy rates among recipient women that are independent of the age of the recipient (Fig. 2). Based on 2002 CDC data, parous women have consistently higher pregnancy rates with ART than nulliparous women. Women who have been pregnant, but miscarried, have the same pregnancy rate as age-matched women who have never been pregnant. Among women aged 40 or younger, those who have had no previous IVF cycles have a slightly higher pregnancy rate than those women who have had 1 or more previous IVF cycles that have not resulted in pregnancy. Infertility Diagnosis Based on CDC data, several infertility diagnoses appear to be associated with a worsened prognosis for pregnancy after ART. Compared with the average live birth rate of 28.3%, a distinctly lower live birth rate of 13.9% is seen with the diagnosis of reduced ovarian reserve. Women with multiple diagnoses had a live birth rate of 23.4%, and couples with both male and female diagnoses had a live birth rate of 26.4%. Uterine factor infertility, defined as a structural or functional disorder of the uterus, was associated with a reduced pregnancy rate of 22.9%. In contrast, very little difference in pregnancy rates is seen when comparing couples with the diagnosis of tubal factor infertility, ovulation dysfunction, endometriosis, male factor infertility, or unexplained infertility. Couples in all of these diagnostic groups had similar live birth rates per cycle of 30 35%. It is important to remember that, with these national statistics, the extent to which the infertility workup was completed is not known and definitions may vary between clinics. In addition these pregnancy rates per diagnostic group are not corrected for other potentially important confounding variables, including the age of the woman. The effect of endometriosis on ART outcomes is difficult to determine from national data due to selec- Fig. 2. The effect of female age on assisted reproductive technology (ART) pregnancy rate in couples using a woman s own eggs or using donor eggs. Modified from Centers for Disease Control and Prevention, American Society for Reproductive Medicine, Society for Assisted Reproductive Technology Assisted Reproductive Technology Success Rates: National Summary and Fertility Clinic Reports. Atlanta (GA): Centers for Disease Control and Prevention; Available at reproductivehealth/art/index.htm. Retrieved November 2, Van Voorhis. Outcomes From ART. Obstet Gynecol Van Voorhis Outcomes From ART OBSTETRICS & GYNECOLOGY

5 tion bias because not all cases have been ascertained in the diagnostic evaluation of infertile couples. A meta-analysis of studies evaluating endometriosis and IVF outcomes has concluded that patients with endometriosis have a significantly lower pregnancy rate (odds ratio 0.56, 95% confidence interval ) than women with tubal factor infertility. Women with more severe disease had lower pregnancy rates than women with mild disease. 14 It should be noted that women with endometriosis still had reasonably high pregnancy rates with ART, and this finding does not imply that treatment of endometriosis will result in higher pregnancy rates with ART. Hydrosalpinges and Pregnancy After ART In vitro fertilization was initially developed as a treatment of tubal factor infertility, although it is now used as treatment for virtually all causes of infertility. Ironically, several retrospective series noted that women with tubal disease appeared to have poorer results from IVF than women with other types of infertility. In 1994, the first study found a worsened prognosis for women with a hydrosalpinx. 15 Since then, numerous studies have confirmed that women with hydrosalpinges have approximately a 50% reduction in both clinical pregnancy and delivery rates compared with age-matched women without hydrosalpinges but being treated by ART. 16 Some have also noted an association between the presence of a hydrosalpinx and an increased miscarriage rate after ART. Hydrosalpinges vary in the amount of fluid they contain and how they are diagnosed. Large hydrosalpinges that are easily visible by ultrasonography may differ in prognostic significance from hydrosalpinges not present by ultrasonography but only demonstrated after filling of the tubes with fluid either at hysterosalpingography (HSG) or laparoscopy. Several retrospective studies have found that ultrasonography-visible hydrosalpinges carry the worst prognosis, indicating that the size of the hydrosalpinx and volume of fluid contained are important factors. 15,17,18 The mechanism of the impaired pregnancy rates with hydrosalpinges and IVF is unknown. Theories have focused on the toxic effect of this fluid on embryo development, the effect of the fluid on endometrial receptivity and implantation, and the simple mechanical wash out of embryos by hydrosalpinx fluid. Each of these theories is supported by in vitro data, but the true cause of reduced pregnancy rates is unknown. Retrospective data suggested that women who had a hydrosalpinx removed by salpingectomy had an improved pregnancy rate after ART treatment. As a result, a randomized, multicenter, controlled trial was performed in several Scandinavian countries. 19 A total of 204 patients with uni- or bilateral hydrosalpinges were randomized to either laparoscopic salpingectomy or no intervention before an IVF cycle. Because of slow recruitment, the trial was stopped before the targeted sample size was reached. In total, the women treated by salpingectomy before IVF had a 30% delivery rate compared with an 18% delivery rate in women with no treatment, a difference that did not reach statistical significance. However, statistically improved delivery rates were noted in the subset of women who had ultrasonography-visible hydrosalpinges, particularly if they were bilateral. A recent meta-analysis combining the Scandinavian study with 2 other prospective randomized trials concluded that surgical treatment for hydrosalpinges significantly increased the odds of a live birth after IVF. 20 In addition, a later analysis of the cumulative results experienced by the women enrolled in the Scandinavian trial found a significantly higher pregnancy rate with ART after salpingectomy. 21 Again, the improvement was most notable in patients with hydrosalpinges visible by ultrasonography. Salpingectomy is not without some morbidity. In addition to the usual morbidities associated with operative laparoscopy, concern has been raised that dissection of the fallopian tube from the ovary may compromise blood flow to the ovary and thus impair ovarian responsiveness in the subsequent IVF cycle. 16 Therefore, experienced laparoscopists should perform this surgery, and if extensive dissection is necessary to completely remove the tube, then consideration should be given to simply proximally obstructing the fallopian tube. Given the proposed mechanisms of impaired IVF pregnancy rates, theoretically, this should be successful and is supported by retrospective data showing equivalent outcomes after proximal obstruction compared with salpingectomy. 22 Suggested alternatives to salpingectomy have included aspiration of the hydrosalpinx at the time of oocyte retrieval or prolonged treatment with antibiotics before ART. 17,23,24 Retrospective series have reached different conclusions regarding the effectiveness of these practices, and they require further study. Consideration can always be given to performing operative salpingostomy, which would have the advantage of possibly permitting pregnancies without ART. However, with large hydrosalpinges, the pregnancy rates following tubal surgery are disappointingly low and the risk of ectopic pregnancy is quite VOL. 107, NO. 1, JANUARY 2006 Van Voorhis Outcomes From ART 187

6 high. In addition, hydrosalpinges often reform after distal salpingostomy. The data now seem to clearly suggest that the presence of a hydrosalpinx, particularly when visible with ultrasonography, is associated with worsened outcome from IVF. Furthermore, evidence is mounting that salpingectomy is associated with improved outcomes in these patients with their IVF cycle. In our clinical practice, we perform ultrasound examinations of all women who are suspected of having tubal disease, either by clinical factors or based on hysterosalpingogram. If a hydrosalpinx is present on ultrasonogram, we always determine whether or not ART is an option for the couple before performing a laparoscopic procedure. If ART is a possibility, it has been our practice to perform laparoscopic salpingectomy for its beneficial effects on subsequent ART cycles. Only if ART is not possible for the couple, either for economic or ethical reasons, will salpingostomy and lysis of adhesions be performed in an attempt to improve the fertility of the woman. Leiomyomata and ART Outcomes The effect of uterine leiomyomata on outcomes with ART likely depends on the size and location of the tumor. Current expert opinion is that uterine leiomyomata that are intracavitary or distort the endometrium have an adverse effect on live birth rates after ART. However, this opinion is based on only 2 retrospective series 25,26 that reported a 50 70% reduction in pregnancy rates in women with leiomyomata in this location. Most studies do not include women with intracavitary leiomyomata because they have typically been removed empirically before any ART attempts. The effect of intramural leiomyomata with no apparent distortion of the endometrial cavity is more controversial, with some studies reporting impaired pregnancy 27,28 or embryo implantation rates, 29 whereas others find no effect. 30 The differences in these studies may be related to how the location of the leiomyomata and how cavity distortion was determined. The techniques of hysteroscopy and saline infusion sonography may more sensitively detect leiomyomata impinging on the endometrial cavity than a hysterosalpingogram or standard transvaginal ultrasonography. 30 Based on the available evidence, if intramural or subserosal leiomyomata that do not impinge on the endometrial cavity have an effect on pregnancy rates after ART, the effect is likely to be small. Certainly there is no evidence that myomectomy improves ART outcomes. We currently recommend removal of leiomyomata that impinge on the endometrial cavity before ART although evidence from a randomized trial is lacking. Clinical judgment that takes into consideration patient symptomatology and reproductive history is necessary in deciding whether or not to perform surgery for intramural leiomyomata not impinging on the endometrium. 30 Smoking and ART Outcomes Cigarette smoking is associated with reduced ovarian function because it contributes to an earlier age of menopause. 31 Meta-analyses of studies evaluating the effects of smoking on IVF outcomes have concluded that smoking reduces the pregnancy rate by approximately 50%. 31 The mechanism of this effect is not clear, but constituents of cigarette smoke can be detected in follicular fluid and thus could affect the health of oocytes or embryos. We found that cigarette smoking was associated with a prolonged and dosedependent reduction in numbers of oocytes and embryos obtained in an ART cycle. 32 Smoking also appears to have a more transient adverse effect on fertility because current, but not past smokers, had a markedly reduced pregnancy rate after ART in our study. For many reasons, women undergoing ART cycles should be encouraged to stop smoking for as long as possible before a cycle although the time frame for the reversal of the adverse effect of smoking on ART outcomes is not clear. Decreased Ovarian Reserve and ART Outcomes In general, women have a decline in ovarian function and fertility with aging. However, age alone is not always a reliable predictor of ovarian function, so investigators have searched for means of testing ovarian reserve. Decreased ovarian reserve is defined by poor ovarian follicular response to gonadotropin stimulation during ART, probably secondary to a reduced number and quality of ovarian follicles available for stimulation. This results in a much higher rate of cycle cancellation, fewer eggs retrieved, fewer embryos, and a lower pregnancy rate with ART. 33 A number of hormones, including follicle-stimulating hormone (FSH), inhibin, and müllerian inhibiting substance, have been assessed for their ability to predict decreased ovarian reserve. The most widely used test for estimating ovarian reserve is a basal FSH value. 33,34 Follicle-stimulating hormone values have been shown to be elevated early in the menstrual cycle of women with reduced ovarian reserve. The basal FSH is typically drawn on cycle day 2, 3, or 4, and values above 15 miu/ml (in many laboratories) suggest a decreased ovarian reserve and a significantly reduced probability of pregnancy after ART. If 188 Van Voorhis Outcomes From ART OBSTETRICS & GYNECOLOGY

7 the FSH is above 20 miu/ml, the chances for pregnancy are virtually zero. Another test for ovarian reserve is the clomiphene citrate challenge test. 35 This test requires a basal FSH value on cycle day 3, followed by the administration of 100 mg of clomiphene citrate on cycle days 5 9. A second FSH value is then obtained on cycle day 10. If either FSH value is elevated, reduced ovarian reserve is diagnosed. This test is thought to be more sensitive because it has a provocative component that may unmask reduced ovarian reserve not detected by the basal FSH screening alone. A recent meta-analysis of studies using either the basal FSH measurement or the clomiphene citrate challenge test concluded that both tests were similar in their ability to predict a clinical pregnancy after ART and therefore recommended that a basal FSH be preferred over the clomiphene citrate challenge test because it is simpler and less expensive. 36 With either test, the sensitivity was very low, but specificity was very high for clinical pregnancy after ART. Thus, a normal result is not useful because of its poor sensitivity; the woman may still have reduced ovarian reserve or may not conceive after ART for some other reason. It has been suggested that ovarian reserve testing reflects oocyte numbers but not quality, and thus older women (with reduced egg quality) still have a low chance for pregnancy with ART even after the finding of a normal FSH value. 37 An abnormal result for FSH is highly predictive of a poor outcome from ART. 36 Some ART centers use an FSH cutoff for admitting patients into their program for ART cycles. It should be remembered that an elevated FSH value does not necessarily mean that a woman will be unable to conceive a pregnancy naturally. There are many examples of patients with elevated FSH values that were subsequently able to conceive without treatment. The ovarian reserve tests are better used as a predictor of who is not likely to benefit from ART. There are some problems with using the serum FSH levels for ovarian reserve testing. The first is that different studies use different cutoffs for a normal value. There is considerable difference among laboratories measuring FSH because of the use of different assays and reference standards for the assay. 38 As a result, physicians must be aware of the assay and references used in their laboratory to find relevant values for their clinical situation. The poor sensitivity of a basal FSH can be improved by also measuring the estradiol (E2) level, with values above 80 pg/ml being abnormal. 39 The improved sensitivity is seen because some normal basal FSH values will be falsely normal because, in some patients, E2 is abnormally elevated in the early follicular phase and is actively suppressing FSH. Sensitivity of the test can also be improved by repeating basal FSH values in several cycles, with any one elevated value signaling reduced ovarian reserve. 40,41 Reduced ovarian reserve can also be diagnosed through ultrasound observation of the ovaries. We first reported that the volume of the ovaries was a predictor of ovarian reserve because women with small ovarian volumes had an increased rate of cycle cancellation, achieved a lower E2 level after stimulation, had fewer oocytes retrieved, and had a lower pregnancy rate. 42,43 Others have confirmed these initial observations. More recently, investigators have found that low numbers of ovarian antral follicles ( 10 total follicles with a diameter between 2 and 10 mm) indicates reduced ovarian reserve and diminished chance for pregnancy after ART. 44,45 Laboratory Techniques and ART Outcomes New laboratory techniques have been introduced for the purpose of improving pregnancy rates from ART. One innovation is prolonged culture of embryos before transfer to the uterus. 46 Embryo transfer was traditionally performed 2 days after oocyte retrieval when embryos are at the 2- to 4-cell stage. The day 2 transfer of embryos may have some advantages because earlier placement in the uterus limits the amount of time spent in the in vitro environment. On the other hand, more prolonged culture (often for 3 5 days) allows embryologists to observe embryos for growth and morphology and select presumably healthier embryos for transfer to the uterus. Recent changes in culturing techniques have allowed for the culture of embryos for 5 6 days when they have developed to the blastocyst stage. (Fig. 3) The disadvantage of prolonged culture is that average rates of blastocyst formation have ranged from 28% to nearly 50% in various series. 47 Thus, more embryos need to be cultured to produce a suitable number of blastocysts for transfer, and some women may not have a good-quality blastocyst to transfer. Many programs restrict blastocyst embryo transfer to patients with a good prognosis, as determined by ovarian response to ovulation induction or a high number of embryos obtained in a given cycle. A systematic review of trials comparing day 2 with day 3 embryo transfer found no significantly different live birth rates. 48 Likewise, an analysis of trials comparing day 2 and day 3 embryo transfers with blastocyst transfers found no significantly different live birth rates. 45 However, these conclusions are VOL. 107, NO. 1, JANUARY 2006 Van Voorhis Outcomes From ART 189

8 media used in various trials, and this may make a large difference in ultimate implantation and pregnancy rates. Further studies are needed to determine the optimal strategy for embryo culture and transfer. Blastocysts appear to have a higher implantation rate than embryos transferred at cleavage stages (day 2 or day 3), allowing for transfer of fewer embryos to achieve the same pregnancy rate. Although the pregnancy rate may not differ, the major advantage of prolonged culture may ultimately be a reduction in multiple gestations after ART. Fig. 3. Human embryos cultured in vitro. A) Embryos after 2 days of culture at the 4-cell stage of development. B) An 8-cell embryo on culture day 3. C) Blastocyst embryos on culture day 5. All photographs were taken at the same magnification. Van Voorhis. Outcomes From ART. Obstet Gynecol based on a small number of prospective randomized trials. Because blastocyst transfer is a relatively new practice, there are great variations in the culture ADVERSE OUTCOMES FROM ART Multiple Gestations In the United States, rates of multiple births are increasing at a rapid rate (Fig. 4). The greatest increases in multiple birth rates have been in women of advanced maternal age and in white women. 49 Two potentially linked causes are a societal trend toward delayed childbearing and increasing use of ART. Older women are at increased risk for multiple gestations in naturally conceived pregnancies. They are also at increased risk of being infertile and of using ART and donor eggs, placing them at higher risk of multiple gestations. Multiple births are now the biggest challenge facing infertility specialists in the United States. In the year 2000, of the 35,000 infants that were born after ART procedures, 44% were twins and 9% were triplets or higher-order multiple gestations. 50 With ART, twinning rates are 22 times higher than what is seen in the general population, and triplets and higher order multiples are 50 times the natural rate of 0.18%. 50 Nationally, it has been estimated that more than 40% of the triplet and higher-order births in 1997 were the result of ART and another 40% due to use of ovulation-inducing drugs. 51 Multiple gestations conceived naturally or through ART are associated with significant morbidity. Although multifetal births account for only 3% of all live births nationally, they account for 17% of all preterm births ( 37 weeks of gestation), 23% of early preterm births ( 32 weeks of gestation), and 26% of very low birth weight infants ( 1,500 g). 52,53 There is evidence of adverse long-term health consequences for infants of multiple gestations born after ART. Although most commonly associated with higher-order multiple gestations, this is true for twins as well. Children born after IVF have been found to have an increased risk of cerebral palsy 54 and a higher hospitalization rate, mainly due to the high twinning rate associated with IVF. A recent Danish national 190 Van Voorhis Outcomes From ART OBSTETRICS & GYNECOLOGY

9 Fig. 4. High-order (triplets and greater) multiple-birth ratios among infants of all races (shaded bars), white infants (solid line), and black infants (broken line) in the United States from 1980 to Modified from Russell RB, Petrini JR, Damus K, Mattison DR, Schwarz RH. The changing epidemiology of multiple births in the United States. Obstet Gynecol 2003;101:129 35, with permission from Lippincott Williams & Wilkins. Van Voorhis. Outcomes From ART. Obstet Gynecol study found that, compared with IVF singletons, more IVF twins were admitted to a neonatal intensive care unit (NICU), had a surgical intervention, and had special needs and poor speech development. More mothers of IVF twins rated their infants general health as being poor than did IVF singleton mothers. Furthermore, analysis showed that parents of twins experienced more marital stress than did parents of singletons. 55 Multiple gestations also increase the maternal risks of hypertension, postpartum bleeding, premature labor with prolonged bedrest, and cesarean deliveries. Although rare, maternal mortality is increased in multiple gestations. Multiple gestations are costly to the health care system. The estimated charges associated with a singleton delivery are approximately $10,000, whereas with a twin delivery the charges are closer to $40, ,57 Excess hospital costs for multiple gestations born from IVF cycles have been estimated at $640 million in the United States in the year There are several reasons for the increased multiple birth rate after ART. Historically, low embryo implantation rates led clinicians to place multiple embryos in the uterus in an effort to improve the efficacy of a given IVF procedure. Although the large preponderance of twins after IVF are dizygotic as a result of transferring multiple embryos, monozygotic twinning has also been shown to be increased. We found the incidence of monochorionicity of 3.2% after IVF, compared with a background rate of 0.4% in the general population. 58 Others have confirmed the increased rate of monozygotic twinning after ART, with particular risk factors being manipulation of the zona pellucida with the use of assisted hatching or extended culture and blastocyst embryo transfer. In addition to the desire for high pregnancy rates among IVF clinicians, patient input may be partially responsible for the high multiple gestation rate after ART. We have found that over 20% of infertile patients surveyed actually desired multiples (predominantly twins) over a singleton gestation as an outcome from treatment. Our study found that younger women who were nulliparous, had a lower family income, and had a longer duration of infertility were more likely to desire a twin gestation over a singleton. Importantly, a lack of knowledge of the health consequences and risks of twins gestations was also associated with the desire for multiple gestations. Perhaps by educating our patients about the risks, the desire for multiple gestations might be lessened. 59 Physicians performing ART are aware of the problem, and improvements are being made. Advances in embryo culture techniques, as well as embryo selection (the practice of culturing multiple embryos and then selecting the most morphologically favorable embryos for transfer), have lead to improvements in embryo implantation rates. In response to VOL. 107, NO. 1, JANUARY 2006 Van Voorhis Outcomes From ART 191

10 these improvements and to changes in the Society for Assisted Reproductive Technology (SART) guidelines for numbers of embryos to transfer, IVF clinicians have reduced the number of embryos transferred over time. This has likely contributed to a recent reduction in triplet and higher-multiple gestation rate in the United States after ART. 60 Increased use of fetal reduction procedures could also contribute to the reduction. However, as yet, twinning rates remain high and have not dropped. One obvious solution to the problem of multiple gestations, and particularly twins, after ART is to transfer a single embryo in an IVF cycle. Although it is intuitive that putting in more embryos will result in a higher live birth rate, this is not necessarily the case according to CDC data. In women who are under 35 years of age and who have excess cryopreserved embryos, pregnancy rates are not greatly affected by numbers of embryos transferred, with a pregnancy rate of 47.4% after single-embryo transfer compared with 51.8% if 2 embryos were transferred. Transfer of additional embryos did not result in a higher pregnancy rate but simply increased the high-order multiple gestation rate. Therefore, among patients with a good prognosis (characterized by young age and having extra embryos for cryopreservation), singleembryo transfer results in a high pregnancy rate and virtually eliminates the risk of twins. A recent large multicenter randomized prospective trial to test this practice was conducted in several Scandinavian countries. 61 This trial compared the strategy of transferring 2 fresh embryos with the strategy of transferring a single fresh embryo (followed by the transfer of a single cryopreserved frozen and thawed embryo if needed). Entrance criteria required that women be under the age of 36 and undergoing their first or second IVF cycle. In addition, they had to have at least 2 embryos of good morphologic quality for this study. Approximately one third of patients presenting to these clinics had these favorable prognostic factors and qualified for the trial. Transferring 2 embryos resulted in a higher pregnancy rate than transfer of a single embryo (43.4% versus 29.6%). However, after the women who received a single-embryo transfer and did not conceive subsequently had a single cryopreserved embryo replaced, the cumulative pregnancy rate was 38.8%, a number similar to the double-embryo transfer group. This study did not demonstrate equivalence of the 2 treatment protocols but suggested that any reduction in live birth rate by transferring one embryo was unlikely to exceed 11.6%. Importantly, twinning was markedly reduced in the single-embryo transfer arm. These findings emphasize the importance of a high-quality embryo cryopreservation program as a means of allowing fewer embryos to be transferred, with the promise that future frozen cycles may be successful. They further suggest that singleembryo transfer should be strongly considered in couples with a good prognosis for pregnancy after IVF. In this study, a large majority of the embryos were transferred after 2 days in culture, at which time the average embryo is typically at a 4-cell stage. Some have advocated that culturing embryos for 5 days to the blastocyst stage may allow for selection of embryos that are more likely to implant, thus improving the pregnancy rate after single-embryo transfer. One pilot study has already demonstrated a very high pregnancy rate after single-blastocyst embryo transfer. 62 Unfortunately, in many states, infertility is not covered by insurance companies, resulting in out-ofpocket payments for ART services. This economic limitation to multiple treatment cycles may lead patients to take more risks in a given ART cycle in the hope that, by placing more embryos, the pregnancy rate will be improved. Indeed, a recent study found an association between state-mandated insurance coverage for IVF services and lower average numbers of embryos transferred per IVF cycle. This, in turn, resulted in a decrease in the percentage of pregnancies, with 3 or more fetuses in these states. 63 Singleembryo transfer is more likely to be embraced by couples that have insurance coverage for infertility. This strategy may be attractive to insurers because the practice of single-embryo transfer has been shown to be cost-effective compared with double-embryo transfer due to the much reduced twinning rate with single-embryo transfer. 64,65 Perinatal Outcomes for Singletons Conceived After ART Recently, data has accumulated suggesting that even singleton pregnancies conceived by IVF are at higher risk for adverse outcomes. A meta-analysis of studies evaluating perinatal outcomes for singletons conceived after IVF included 15 cohort studies from around the world. 66 All studies included in this analysis compared IVF-conceived singletons with spontaneously conceived singletons and controlled for maternal age and parity because these are 2 factors that are known to affect perinatal outcome. Singletons conceived by IVF were at approximately a 2-fold increase risk for perinatal mortality, low birth weight, very low birth weight, preterm delivery, and small for gestational age infants (Table 1). Increased risks for gestational diabetes, placenta previa, preeclampsia, 192 Van Voorhis Outcomes From ART OBSTETRICS & GYNECOLOGY

11 Table 1. Perinatal Outcomes in Singletons After In Vitro Fertilization and Natural Conception: Results of a Meta-Analysis of Clinical Trials Approximate Absolute Risk (%) Outcome IVF Spontaneous Odds Ratio (95% CI) Perinatal mortality ( ) Preterm delivery ( ) Birth weight 2,500 g ( ) Birth weight 1,500 g ( ) Small for gestational age ( ) Adapted from Jackson RA, Gibson KA, Wu YW, Croughan MS. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004;103;551-63, with permission from Lippincott Williams & Wilkins. IVF, in vitro fertilization; CI, confidence interval. and stillbirth were also found among IVF-conceived singletons although these were secondary outcomes for this analysis. The odds ratios for these complications ranged from 1.55 for preeclampsia to 2.87 to placenta previa. The cause for these adverse perinatal outcomes among IVF-conceived singletons is unknown. One possibility is that some aspect of the IVF treatment (eg, ovarian stimulation or embryo culture) may increase the risk for subsequent adverse pregnancy outcomes. Alternatively, there may be an underlying disorder in the infertile couple that contributes both to the infertility and the adverse perinatal outcomes. It has been noted that IVF-conceived singletons are at a significantly higher risk of having induction of labor and both emergent and elective cesarean deliveries. 66 Thus, some of the adverse outcomes, including low birth weight, very low birth weight, and preterm delivery, may be attributable, in part, to iatrogenic intervention. Regardless of the cause, current evidence suggests that pregnancies conceived after IVF are at higher risk for adverse outcomes, and thus, patients should be advised of this risk before undergoing infertility treatment. Birth Defects After ART Several studies have been conducted to determine whether or not ART is associated with an increased risk of birth defects. 67 The results from these studies are mixed, although a majority of published studies report a slight increase in the rate of birth defects after IVF. In the largest study reported from the United States, we compared children conceived by IVF at the University of Iowa and a matched cohort of naturally conceived children. 68 Birth defects were prospectively diagnosed and recorded in a statewide birth defects registry. We found that 6.2% of IVF-conceived children had major birth defects, compared with a rate of 4.4% in naturally conceived children. There was a statistically significant difference in major birth defect rate after IVF, with an adjusted odds ratio for a major birth defect of 1.30 (95% confidence interval ). Specific birth defects that were increased in the IVF population included cardiovascular and musculoskeletal defects as well as certain known birth defect syndromes. Our study supports the findings of some, but not all, previous studies evaluating birth defects after IVF as compared with either matched cohort or national registry rates after correction for important variables. The studies listed in Table 2 are the largest studies of birth defect rates that control for maternal age and plurality This is important because birth defects are increased in multiple-gestation pregnancies. In addition, all of these studies detected and reported birth defects in a standardized fashion. However, even these studies cannot be directly compared because birth defects were detected in children for varying lengths of time after birth, and different classification systems for birth defects were used. Our interpretation of current evidence is that IVF may be associated with an increase in birth defects, but the effect is small. Findings to date are not likely to dissuade many couples from pursuing infertility treatments. Recent attention has been directed toward epigenetic errors that might be inherent in the infertile couple or induced as an adverse effect of ART itself. Differential DNA methylation leading to expression of only 1 of 2 parental alleles is a mechanism of gene regulation known as genomic imprinting. Defects in imprinting may cause either over- or underexpression of certain genes, leading to birth defects or cancer. Several syndromes caused by imprinting defects, including Beckwith-Wiedemann syndrome and Angelman syndrome, have been reported to be more prevalent in children born after IVF as reviewed in Niemitz and Feinberg. 75 Some have proposed that VOL. 107, NO. 1, JANUARY 2006 Van Voorhis Outcomes From ART 193

12 Table 2. Association of Birth Defects and In Vitro Fertilization Study Country Years Infants Studied IVF Defects n/n (%) Controls Defects n/n (%) Adjusted OR Specific Defects Increased Matched cohort studies Dhont 69 Belgium Singleton 84/3,048 (2.8) 62/3,048 (2) Nonsignificant None Twin 86/2,482 (3.5) 73/2,482 (2.9) Nonsignificant None Westergaard 70 Denmark All 107/2,245 (4.8) 103/2,245 (4.6) Not stated None Hansen 71 Australia Singleton 50/527 (9.5) 164/3,906 (4.2) 2.2 ( ) All 75/837 (9.0) 168/4,000 (4.2) 2.0 ( ) Cardiovascular, musculoskeletal, chromosomal, urogenital Koivurova 72 Finland All 20/309 (6.6) 24/569 (4.4) Not stated Cardiovascular Olson 68 United States Singleton 38/645 (5.9) 171/4,590 (3.7) 1.44 ( ) Cardiovascular, musculoskeletal, syndromal All 90/1,462 (6.2) 369/8,422 (4.4) 1.3 ( ) Population-based studies Ericson 73 Sweden All 516/9,111 (5.6) Not stated 0.89 ( ) Omphalocele, neural tube defects, anal atresia Anthony 74 Netherlands All 137/4,224 (3.2) 8,526/314,605 (2.7) 1.03 ( ) Cardiovascular, musculoskeletal IVF, in vitro fertilization; OR, odds ratio. 194 Van Voorhis Outcomes From ART OBSTETRICS & GYNECOLOGY