The Ovarian Factor in Assisted Reproductive Technology

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The Ovarian Factor in Assisted Reproductive Technology LAUREL STADTMAUER, ESTELLA JONES, AND ROGER GOSDEN

evident in the fourth and fifth decades of women’s lives. Studies based on IVF, intracytoplasmic sperm injection (ICSI), and other ARTs indicate there is a reduced potential for pregnancy among older women. National data collected by SART, along with other studies, reveal that clinic-specific and national pregnancy success rates for IVF decline markedly with age [2, 5, 6]. The age-related decline in fertility is also seen in patients receiving less aggressive treatments, such as human menopausal gonadotropins (hMG) and IUI [7]. Multiple pregnancy risk in ART cycles also declines with age, which is another indication of reduced fecundity [2]. Fertility starts to decrease at about age 30, with a precipitous decline after 37 to 38 years of age [8]. There is a maternal age-related effect on oocyte quality leading to a decline in the embryo implantation rate and a decline in pregnancy rates. In addition, a decreased ovarian reserve is observed by counting the number of follicles in ovaries from pathology specimens on autopsy, and the steady decline in follicle numbers until 37 to 38 years of age accelerates in the years before menopause [9]. The origin of the age-related decline in egg and embryo quality with advanced maternal age seen in IVF patients is unknown. However, we know that it is not just a decrease in the ability to fertilize oocytes. In addition, it is unlikely that the decline is causally linked to reduced numbers of eggs at retrieval or embryos at transfer because these parameters do not drop in proportion to the pregnancy rates. Instead, it appears to be caused by a decrease in oocyte quality related to nuclear and cytoplasmic abnormalities in oocytes of women with advanced reproductive age. A number of unanswered questions remain. Are abnormalities in nuclear and cytoplasmic components predetermined during oogenesis in fetal life? Do they reflect the impact of adverse environmental factors present at that time? Are ovarian follicles that finally develop gonadotropin responsiveness after years of inactivity unable to interact effectively with the other intraovarian factors required to ensure proper egg maturation? Are oocytes exposed to environmental toxins leading to increased cellular damage? The physiology and cell biology of oocyte for-

INTRODUCTION It has now been more than 20 years since the first successful in vitro fertilization (IVF) conception [1] and since the first conception in the United States at the Jones Institute. Assisted reproductive technology (ART) has become increasingly prevalent each year, with more than 360 ART programs available in 1999 and more than 88,000 cycles in the United States alone [2]. In addition, success rates have increased, although outcomes remain age-dependent. The 1999 database generated by the Society for Assisted Reproductive Technology (SART) showed an IVF delivery rate per cycle of 32.4% for women younger than 35 years of age, 26.4% for women aged 35 to -57 years, 18.5% for women aged 38 to 40 years, and only 8.1% for women older than 40 years. In oocyte donor programs, rates were higher with a delivery rate per transfer of 41%, which is consistent with early research that showed higher success rates for older patients using oocytes from young donors [2, 3]. Therefore the most important factor for the success of ART is oocyte quality, which is affected by ovarian age, whereas the age of the endometrium is less important [4]. The biggest challenge continues to be treating women of advanced reproductive age who wish to use their own eggs. This chapter addresses the role of the ovary in ART, the organ with the largest impact on ART success. We discuss the decline in fertility with age and the relationship between increased aneuploidy and decreased oocyte quality and ovarian reserve. We also discuss ovarian stimulation, specifically the approach to low responders, hyperresponders, and the role of luteinizing hormone (LH) in ovarian stimulation.

DECLINE IN FEMALE FERTILITY WITH AGE Overview The decline in female fertility associated with increasing chronological age has been established and is especially The Ovary

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mation and maturation will continue to be an exciting area of basic research because the findings could have immediate clinical significance. The reduction in follicle numbers with advancing age is reflected by rising levels of serum follicle-stimulating hormone (FSH) measured during the early follicular phase [10]. The number of mature follicles and oocytes that can be obtained in response to stimulation using gonadotropins is an index of total ovarian follicular reserve and correlates inversely with FSH levels. Numerous studies have shown that the rates of implantation, pregnancy, and miscarriage are not only related to age but also to FSH levels [11–16]. Moreover, young and reproductively aged women who respond poorly to stimulation share the inherent problem of ovarian aging. Biological aging of the ovaries manifests before high levels of FSH are observed, as a decline of oocyte quality is evident even while FSH levels are still within the normal range of young women [11], and weak responses to gonadotropins can occur at any age. However, young poor responders have higher pregnancy rates than older patients, although the pregnancy rates are lower than age-matched normal responders [17]. Hence, the counseling of patients must involve discussions on the impact on pregnancy rates of their prior history of ovarian response, day 3 FSH levels, and age. This subject is discussed in more detail in Chapter 28, Ovarian Function in Assisted Reproduction.

Age-Related Aneuploidy Many types of genetic abnormalities have been reported in human oocytes. The most common egg abnormalities result from an error in chromatid segregation during the first meiotic division. The frequency of chromosomally abnormal offspring resulting from errors during female meiosis, such as trisomy, is directly correlated with maternal age. In women older than 40 years, it is estimated that 50% or more of all ovulated oocytes are chromosomally abnormal [18, 19]. In the human female, oocytes enter meiosis during the fetal period and remain suspended in prophase of the first meiotic division at the diplotene stage until ovulation. Possibly, this delay in meiosis contributes to the large increase in aneuploidy, which is not paralleled in spermatogenesis. There is evidence suggesting that the age effect for trisomy is caused by events that occur prenatally at the time of entry into meiosis I [20] or postnatally at the time of reentry into meiosis I [21–23]. The ability to resume and complete the first meiotic division before ovulation is linked to the process of oocyte growth. In the neonate, inactive oocytes are surrounded by a single layer of somatic cells. During the process of folliculogenesis, these primordial follicles undergo significant growth and development to produce a preovulatory follicle containing a mature oocyte.

Recent studies in the mouse have demonstrated that the ability of the oocyte to resume and complete the first meiotic division and the capacity to undergo fertilization and cleavage are acquired by the oocyte in a stepwise fashion during the late stages of folliculogenesis [24]. The importance of the final stages of follicular maturation in the meiotic process suggests that somatic factors may play a role in subsequent meiotic events. Meiotic studies of oocytes from mice with a mutation that causes defects in folliculogenesis demonstrate strikingly similar meiotic defects to those observed in human oocytes from reproductively aged donors [24]. This may not be the best model however for the human, because the mouse does not go through such dramatic ovarian senescence as the human. The control of the female meiotic process may lack a cell cycle checkpoint control mechanism, as is found in mice, to monitor the alignment of chromosomes at metaphase [25]. A lack of checkpoint control may explain the high error frequency in female meiosis and the age-related increased meiotic nondisjunction. The disappointing success rate for IVF involving women older than 40 years indicates an age-related reduction in the developmental competence of human oocytes, including fertilization, embryo cleavage, and implantation. The agerelated reduction of pregnancy and implantation rates is associated with the increase in meiotic chromosome nondisjunction and decline in oocyte quality observed in oocytes and embryos obtained from ovarian stimulation during IVF [18, 26–28]. Munne et al. [27] have concluded that in morphologically and developmentally normal human embryos at cleavage stages, aneuploidy significantly increases with maternal age. Through the use of fluorescent in situ hybridization (FISH), Munne et al. have convincingly demonstrated that more than 40% of apparently normal developing embryos are aneuploid. This is a primary reason for implantation failure in older women. Less surprisingly, the majority of morphologically abnormal oocytes and embryos are aneuploid [19, 29] and 60% of the embryos arresting in the preimplantation stages showed chromosome mosaicism with FISH analysis [30]. However, it is difficult to assess the true number of abnormal oocytes and their relationship with age, although multifluorescent FISH can reveal the karyotype of human oocytes and their polar bodies [31]. Studies in IVF may not be representative of natural fertility because the stimulation of the ovary of infertile patients might influence the rate of meiotic nondisjunction. Moreover, slight alterations in the culture conditions can impact the meiotic process and the rate of aneuploidy may be underestimated by FISH, which gives information only on the chromosomes for which specific probes are available. However, it is certain that polyploidy, aneuploidy, monosomy, and mosaicism are all common in stimulated cycles from women of all ages. It is also becoming apparent that oocytes from unstimulated ovaries have similar levels of aneuploidy [32].

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Unfortunately, chromosomal normality and viability are not reliably predicted using the morphological grading system used for selecting embryos [27]. When aneuploidy testing is done using FISH analysis, high numbers of embryos in women older than 40 years undergoing IVF are abnormal despite their “good grades” based on embryo grade. We need a simple, noninvasive way to select embryos that is predictive of euploidy and developmental competence.

Assisted Hatching Failure of the embryo to implant may also be the result of zona pellucida hardening, which can be caused by culture conditions and perhaps age [33]. Zona hardening may prevent embryos from hatching. Assisted hatching is most commonly performed with an acidified Tyrode’s solution on day 3 embryos, although it can also be done with laser [34, 35]. With acidified Tyrode’s solution, it is important to reverse the acid quickly after the zona is pierced to avoid the detrimental effects of acid on embryos. In addition, small or narrow gaps may lead to incomplete hatching and possibly increased twinning rates [36]. The benefit of assisted hatching has been demonstrated in a randomized trial in which implantation was studied in more than 100 patients in each group (with or without micromanipulation). The implantation rates were comparable in each group. There was an improvement in the implantation rate of poor prognosis patients, including a subset of women aged 39 or older with thickened zona pellucida and/or elevated FSH levels [34]. In assisted hatching patients, implantation occurred earlier than in patients who did not receive the procedure, and this may also have led to increased implantation rates [37]. Other studies, including prospective randomized studies, have confirmed these results, showing higher pregnancy and delivery rates in selected assisted hatching patients. The benefit was in the poor prognosis patients who were older than 38 years, had elevated FSH levels, and had a history of repeated IVF failure [38–40].

MEIOTIC SPINDLES Overview There has been much debate about the influence of the meiotic spindle apparatus on oocyte viability. The oocyte is a highly specialized cell that must undergo many maturational changes in preparation for fertilization and embryonic development. These changes involve nuclear and cytoplasmic events. At ovulation, mammalian oocytes are arrested at metaphase of the second meiotic division with the meiotic spindle apparatus composed of microtubules with maternal chromosomes attached [41]. Microtubules

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are dynamic components of the cytoskeleton that have important functions in the processes of cell division and motility, morphogenesis, and organelle transport [42]. In addition, they play important roles in the postfertilization events of spindle rotation, polar body formation, pronuclear migration, and cytokinesis [43–44]. The meiotic spindle of mammalian oocytes is sensitive to environmental changes, notably temperature and pH [45]. Exposure of oocytes to adverse conditions such as cryoprotectants and low temperatures has also been shown to disrupt the microtubules [41, 45–46]. Eroglu et al. [47] reported that cryopreservation of mouse oocytes with DMSO does not impair the function of microtubules postfertilization if a brief incubation period follows the thaw process. Similarly, Baka et al. [48] reported, contrary to some claims, that cryopreservation of prophase I human oocytes did not significantly increase abnormalities in the meiotic spindle. The meiotic spindle human oocytes are highly sensitive to temperature changes and depolymerize when exposed to temperatures less than 37°C even for brief periods [45, 49]. In the bovine, effects of cooling and rewarming on the meiotic spindle of in vitro matured bovine oocytes showed that exposure to temperatures as high as only 25°C resulted in damage to the chromosome alignment. However, rewarming to 39°C enabled the spindle to return to normal in most oocytes [50]. Currently we do not know whether advanced maternal age changes the spindle’s sensitivity to temperature [49]. In addition to spindle disassembly as a result of temperature and pH fluctuations, damage has been reported in the microtubules of the meiotic spindle during manipulation. In the hamster oocyte, Asada et al. [51] demonstrated that ICSI need not result in significant damage to the meiotic spindle if the polar body is oriented away from the injection site. On the other hand, Blake et al. [52] showed that placement of sperm during ICSI relative to the presumed location of the spindle significantly impacts both fertilization and development. The authors concluded that sperm deposition in the area of the meiotic spindle apparatus should be avoided, but debate continues about the optimal orientation of meiotic spindle apparatus and the first polar body position. In the hamster, the first polar body did not accurately predict the spindle location in 25 out of 30 oocytes [53]. Hardarson et al. [54] reported that in human oocytes the majority of the spindles were found in the same hemisphere as the first polar body. Perhaps the orientation of the spindle in relation to the first polar body will be better resolved using the Polscope. Polarization microscopy reveals birefringence, which is an inherent property of highly ordered molecules like microtubules in the meiotic spindle apparatus [34]. Recently, it was reported [55] that the use of the Polscope was a reliable and noninvasive technique to view the meiotic spindle in the hamster oocyte. Similarly, Wang et al. [56] demonstrated that the Polscope can also be safely used with living

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human oocytes and found that visualization of a birefringent spindle is predictive of outcome with ICSI, although fertilization and blastocyst formation also occurred in oocytes without visible spindles.

Spindle and Aneuploidy Age-related aneuploidy may be related to abnormalities in spindle formation and chromosomal alignment in stimulated and unstimulated cycles. Volarcik et al. [32] examined oocytes from unstimulated ovaries by immunofluorescence and FISH to study simultaneously the meiotic stage and the structural aspects of the meiotic spindle apparatus. The overall rate of meiotic maturation after maturation in vitro was only slightly lower among oocytes obtained from donors older than 35 years, but metaphase II (MII)arrested oocytes showed an age-related increase in defects of the second meiotic metaphase spindle formation and chromosome alignment. Battaglia et al. [57] described comparable findings in stimulated cycles: 79% of the oocytes from women aged 40 to 45 exhibited abnormal spindle assembly and chromosome alignment compared with only 17% of younger patients. These two studies demonstrated global defects in the meiotic process in oocytes obtained from reproductively aged donors, and Polscope technology may prove useful in human IVF clinics to help diagnose aneuploidy.

OOCYTE MATURATION AND MITOCHONDRIAL DEOXYRIBONUCLEIC ACID In contrast to the process of nuclear maturation, cytoplasmic changes involved in oocyte maturation are less well understood. In the mouse, the oocyte experiences a stage of rapid growth and metabolic activity following recruitment from the primordial follicle stage. Only fully grown oocytes have the capability to resume meiosis and ovulation, and during this growth period, there are striking changes in the ultra structure of the oocyte, including changes in morphology and number of mitochondria changes in structure and activity of the Golgi apparatus [58]. Recent evidence suggests that physiological factors in the preovulatory follicle can have developmental consequences in the ovary. Factors related to chromosomal normality of the oocyte, embryo cleavage, and embryo transfer outcome include perifollicular vascularity and intrafollicular P02, pH, and biochemistry [59–61]. Van Blerkom et al. [62] investigated the differential mitochondrial distribution in human pronuclear embryos to address the question of what begins to make a good oocyte/embryo. Their findings demonstrated that specific perinuclear mitochondrial aggregation patterns and microtubular organization occur synergistically. Some blastomeres in embryos evidently

receive reduced quotas of mitochondria and are therefore prone to diminished adenosine triphosphate (ATP) generation. The authors concluded that this could be an epigenetic basis for the variation in developmental ability observed in morphologically normal IVF cleavage stage embryos. Preexisting oocyte mitochondrial deoxyribonucleic acid (mtDNA) defects or accumulation of age-related mtDNA mutations could result in reduced meiotic competence and ability to fertilize and undergo development [63]. This may explain early pregnancy failure in women of advanced reproductive age [64].

GRANULOSA CELL COMMUNICATION Ovarian physiology is underpinned by the dynamics of primordial follicle growth, development, and atresia during reproductive life [65]. Ovarian senescence is linked with this decreasing pool of primordial follicles. The follicles develop through successive stages through primary and secondary stages until an antral cavity forms. It is at the antral stage when a dominant ovulatory follicle emerges and most follicles undergo atresia [66]. Endocrine and paracrine factors control the fate of the follicles. The process of primordial follicle recruitment is independent of gonadotropins, and intraovarian factors determine which follicles grow and when they start. This recruitment process could be because of the release of inhibitory stimuli that maintains the follicles at rest or a stimulatory factor that triggers growth, or a combination of factors. The existence of oocyte factors influencing follicle development has been established. Oocyte-derived factors stimulate the proliferation of granulosa cells but inhibit LH receptor expression [67]. Growth differentiation factor 9 (GDF-9) is a member of the transforming growth factor-b superfamily and a product of the oocyte that has been implicated in the diversification of the granulosa cell phenotypes [68, 69]. Animals deficient in GDF9 have follicle development arrested at the primary stage [70]. Treatment with recombinant GDF-9, but not FSH, stimulated thymidine incorporation into cultured granulosa cells in early antral and preovulatory follicles [71]. The data suggest that GDF-9 is a proliferative factor for granulosa cells and plays a role in their differentiation. Secondary effects occur in the absence of GDF-9 including the failure of the thecal layer to form and defects in the oocyte, such as abnormal germinal vesicle breakdown, spontaneous parthenogenetic activation, and increased growth rate [72]. Because follicles in knockout mice are blocked at the type 3b stage, GDF-9 is not a principal factor for recruitment of primordial follicles and, so far, research has failed to identify the key triggers/inhibitors. Factor in the germline, alpha, (FIGa) mRNA is required for germ cell development in mice, and the FIGa protein has been implicated in the transcription of zona pellucida genes [73]

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and is expressed in human oogenesis [73a]. The granulosa cell secreted kit ligand has also been implicated in early follicular development [74]. Driancourt et al. [75] showed that kit-kit ligand interaction is involved in the initiation of follicular growth from the primordial pool and progression beyond the primary follicle stage. The protein controls growth and theca cell differentiation during early folliculargenesis, protects preantral follicles from apoptosis, and is required for antral cavity formation. In large antral follicles, it modulates the ability of the oocyte to undergo cytoplasmic maturation and maximizes thecal androgen output [75]. It is not until the onset of follicle growth and zona pellucida formation that full oocyte-granulosa cell bidirectional exchange of signals occurs. This communication system is facilitated by transzonal projections (TZPs) at the oocyte-granulosa cell boundary [76]. TZPs are follicle cell extensions that transverse the zona pellucida and terminate on the oocyte cell surface. The studies of Motta et al. [77] conducted on human ovarian follicles were the first to indicate that TZPs may facilitate the coordination or exchange of signals between oocyte and follicle cells. These structures mediate directed transport, secretion, and possibly selective uptake of factors secreted by the oocyte and the granulosa cells, as well as provide physical anchorage. The exchange via gap junctions includes small molecules such as amino acids, sugar, and nucleotides [78]. Gap junctions occur at places of close cell contact and allow small molecules (<1000 Da) to pass from cell to cell [79]. Connexin is the fundamental unit of gap junction forming a hemichannel in the membrane. Each connexin is a hexamer of protein subunits called connexins [80]. In addition, there is communication via other growth factor– ligand interactions such as oocyte-derived factors (e.g., GDF-9) that affect cumulus mucification [81, 82]. The ovarian follicle is therefore essentially a functional syncytium with the oocyte communicating with the surrounding cumulus cells and the cumulus cells also communicating to the mural cells through gap junctions.

GONADOTROPIN STIMULATION PROTOCOLS Use of Gonadotropin-Releasing Hormone Agonist or Antagonist Gonadotropin-releasing hormone (Gn-RH) is decapeptide secreted by the hypothalamus in a pulsatile pattern that binds to a specific receptor in the pituitary cells to regulate the secretion and synthesis of gonadotropins. After binding with the receptor, the Gn-RH-receptor complex forms a microaggregation that leads to the release of pituitary hormones LH and FSH. Gn-RH agonists and antagonists both exert their specific effects via the binding to the

Amino Acid Sequence 1to10 of Gn-RH and Gn-RH Antagonists Pyro Glu

His

Trp

Ser

Tyr

Gly

Leu

Arg

Pro

Gly Nh2

AcDNal

D4Ci Phe

D-Pal Ser

Tyr

D-Cit Leu

Arg

Pro

DAla

AcDNal

D4Ci

D-Pal Ser

Tyr

DAph (Et2)

LAph (Et2)

Pro

DAla

FIGURE 27.1 nists.

Leu

Specific effects of Gn-RH agonists and antago-

transmembrane receptor according to their structure (Figure 27.1). Gn-RH agonists differ from the antagonists in that initially there is a flare response, followed by a long period of downregulation suppression [83]. With the GnRH antagonist, there is a competitive block of the receptor leading to a direct blockade of FSH and LH release. Because of the prolonged half-life of FSH and perhaps other factors, the effect on LH is more profound than FSH. Gn-RH agonists have gained popularity because of their convenience on controlling stimulation cycles. In addition, metaanalysis of studies comparing the “long protocol” of midluteal Gn-RH-agonist (Gn-RH-a) suppression followed by hMG treatment compared with hMG treatment alone have shown a twofold increase of the pregnancy rate with the use of the drug [84]. With the long Gn-RH-a/hMG regimen, more follicles and oocytes are produced, and there is an improvement in oocyte quality [85]. The purpose of the Gn-RH-a is to suppress bioactive LH to prevent premature luteinization. In addition, increased pregnancy rates with Gn-RH-a are possible because of a larger number of oocytes and embryos [86]. The disadvantage of its use is the increased length of treatment, the greater amount of gonadotropin needed, and the risk of ovarian hyperstimulation syndrome (OHSS). It is possible to prevent the LH surge without a desensitization period if Gn-RH antagonists are used. These medications induce an inhibition of LH secretion without a flare-up period. The new antagonists, Citrorelix and ganirelix, are devoid of the histaminergic reactions of the previous medications, and the treatment period is shorter because the antagonist can be added in the middle of the follicular phase when the leading follicle is already between 14 to 16 mm in diameter. Moreover, there is no flare-up and the risk of ovarian cysts is lessened. The disadvantage of the antagonist is that too early administration can lead to FSH suppression as well, and a poor response and late administration can allow a premature LH surge.

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There have been several controlled, multicenter, randomized trials with hundreds of patients in Europe and North America comparing treatment regimens with GnRH antagonist with the long protocol using Gn-RH-a. Similar numbers of embryos, fertilization rates, and pregnancy rates were obtained for a shorter stimulation duration and fewer side effects with the antagonist protocols in normal responders [87, 88, 89]. Further large-scale studies are now needed for patients with polycystic ovarian syndrome (PCOS) and for patients who are poor responders.

The Role of Follicle-Stimulating Hormone and Luteinizing Hormone on Oocyte and Embryo Quality The gonadotropins, LH and FSH, play critical roles in follicular development. The rise in serum FSH that occurs during the luteal-follicular transition is a stimulus for follicular recruitment and development of early antral follicles. FSH stimulates estrogen production from the granulosa cells through aromatization of the androgens produced by the thecal cells (“two cell theory”). This increase in estradiol suppresses FSH secretion in the late follicular phase, inhibiting maturation of the less mature follicles. Granulosa cells develop their own LH receptors in the mid- to late follicular phase as a result of FSH action. At this stage, LH can synergize with FSH to sustain follicular development and to prepare it for a midcycle LH surge that triggers ovulation. During the normal menstrual cycle, LH levels rise before ovulation along with estradiol levels. LH is also the stimulus for androgen secretion by the theca cells. A role for androgens and LH in follicle atresia and poor oocyte quality in PCOS patients is suspected. IVF protocols that involve Gn-RH-a or antagonists in IVF by suppressing the endogenous LH levels can prevent early luteinization in high LH level preparations, such as HMG. The debate continues whether recombinant FSH is on its own sufficient when bioactive LH levels are suppressed. If the increase in LH in the late follicular phase is important for oocyte development, excessive LH suppression might compromise oocyte quality, whereas pure FSH would lead to a poorer stimulation. Addition of a low dose of LH should increase estradiol levels and follicular growth. In hypothalamic-hypogonadotropic patients, Shoham et al. [90] examined the use of recombinant FSH and found fewer developing follicles, lower estradiol levels, and decreased fertilization and embryo survival rates compared with HMG treatment cycles. However, in ovulatory patients, two studies comparing pure FSH with hMG showed no difference in response parameters [91, 92]. Metaanalyses of studies comparing the use of recombinant FSH with urinary HMG have shown little difference between the two. One showed a higher pregnancy rate with pure FSH compared with hMG (odds ratio 1.7), but Gn-

RH-a was not consistently used [93]. A second metaanalysis comparing FSH and hMG in women using agonist protocols showed a trend toward higher pregnancy rates with HMG [94]. Patients with decreased LH levels demonstrated lower estradiol production, decreased oocyte yield, and increased days of stimulation [95]. Weston et al. [96] found that the cryopreservation survival and implantation rates of blastocysts decreased when pure FSH was used alone to stimulate oocyte production, perhaps because of poorer cytoplasmic maturation. In contrast, a randomized prospective trial of highly purified FSH compared with FSH plus recombinant LH in a long leuprolide (Lupron) protocol found a trend toward better outcomes in the former group [97]. Patients in this study were less suppressed and had higher baseline LH levels (0.5 mg in the midluteal phase that decreased to 0.25 mg with the initiation of the gonadotropins). Therefore the type and dose of Gn-RH-a may be important when using pure FSH because this will have an effect on the extent of suppression of endogenous LH. In the United States the most common dose of leuprolide (Lupron) has been 1 mg daily subcutaneously until the onset of menses, and then the dose is decreased to 0.5 mg until human chorionic gonadotropin (hCG) is given. This protocol suppresses LH at a higher level than Buserelin, which is the most common agonist used in Europe. There appears to be a threshold level of LH required for adequate follicular development and maturation and oocyte quality, as revealed in patients with hypothalamic hypogonadism with baseline LH levels less than 1 mIU/ml. However, the biological threshold may be extremely low because only 1% of LH receptors need to be occupied for normal steroidogenesis to occur [98]. What the threshold level is and whether endogenous LH is sufficient or should be supplemented is still debatable. Studies are required with a more consistent supplement such as minidoses of hCG or recombinant LH because the role of LH is still controversial.

Stimulation in Patients with Polycystic Ovarian Syndrome Patients with PCOS have excessive secretion of LH and irregular menstrual cycles, an ovulation and infertility, and an increased frequency of miscarriage. LH-stimulated excessive androgen secretion by the thecal cells may promote more atresia and disruption of the dominant follicle, which may account for the reduced quality of oocytes in PCOS. Excessive LH levels might directly or indirectly influence late follicular phase meiotic maturation, disrupting cumulus-granulosa gap junctions and attenuate oocyte maturation inhibitors, all of which lead to poorer quality oocytes and reduced fertilization potential. Hence, an exaggerated response to gonadotropins is at the heart of the problem of oocyte quality in PCOS. Stimulation with gonadotropins leads to multifollicular development, and

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super-elevated estradiol levels are reached earlier in the cycle than is appropriate. As a result, the oocytes retrieved are more likely to be immature and will have been exposed to high estradiol levels, exhibiting low fertilization rates and poor embryonic development. Several groups, including our center, have devised strategies to dampen the response to gonadotropins to improve follicular development, oocyte quality, and embryo quality. This has involved a combination of a stepdown protocol and the use of insulin-sensitizing drugs such as metformin during stimulation. Starting with low-dose gonadotropins for all patients with PCOS is not the best strategy, because there is a critical level of FSH stimulation per follicle that is required for the early stages of follicular development, after which the requirement lessens as follicles grow. Coasting or withdrawing gonadotropin treatment can be successfully done when the follicle size is larger than 14 mm and the estradiol levels are higher than 2000 pg/ml [99–101]. Coasting has been shown to decrease the rate of cycle cancellation and the incidence of OHSS. Other options to reduce OHSS include (1) reducing the hCG dose or (2) using Gn-RH antagonist during stimulation and a Gn-RH agonist or recombinant LH to trigger the surge, both of which are more rapidly cleared from the circulation and produce a shorter duration of exposure of the follicular/luteal cells to hCG [102, 103]. The other option is to cryopreserve all the embryos, which should be done for patients with estradiol levels greater than 7000 pg/ml because these patients have lower pregnancy rates. It should also be done for patients with a high risk for developing OHSS. Suppression of the pituitary gland is achieved with leuprolide (Lupron) (0.5 mg daily subcutaneously) in the luteal phase or, in anovulatory patients, there is an overlap of the oral contraceptive pill (OCP) with the Gn-RH agonist for 1 week with continuation of the agonist until menses. Both the agonist and OCPs suppress the high LH levels in these patients and dampen the response to doses of FSH during the stimulation phase of the cycle. This has lead to an improvement in the stimulation of the ovaries, leading to better egg and embryo quality, higher pregnancy rates, and decreased OHSS. Studies are currently underway to test if the Gn-RH antagonist has any advantage over the agonist in these patients with PCOS. Initial reports with the antagonist reveal a comparable clinical outcome in patients with PCOS [104].

Minimal Ovarian Stimulation and Poor Responders Minimal ovarian stimulation can be done in selected cases to reduce the cost of treatment and decrease the risk of OHSS (Figure 27.2). Suitable candidates are young, normal ovulatory women with tubal or male factor and poor prognosis patients that consistently produce fewer

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Minimal Stimulation Protocol

Gn-RH antagonist Clomiphere citrate 100 mg

Day 3

FIGURE 27.2

rFSH

7

9

Progesterone

hCG ER ET

Minimal ovarian stimulation protocol.

than five follicles with high dosage gonadotropins. We start with 00 mg clomiphene citrate on day 3 of the cycle, followed by gonadotropins on day 8. The Gn-RH antagonist is used to reduce the risk of a premature LH surge, and, although pregnancy rates are slightly lower than after full stimulation, the cost is reduced, which is an attractive option for some patients. There is no uniform definition of a poor responder; various stimulation protocols have attempted to improve IVF success rates in these patients. We define low responders as those patients with diminished ovarian reserve based on age greater than 40 years, FSH levels on day 3 greater than 15 mIU/ml, or those who consistently have peak estradiol concentrations of less than 500 pg/ml with fewer than five dominant follicles on day of hCG. Reports show that in these poor responders, increasing the dosage of medication may lead to increased numbers of oocytes retrieved and embryos, but the pregnancy rates were equally poor [105]. In addition, poor responders are more likely to fail to respond to gonadotropins following Gn-RH downregulation. Although donor egg treatment is the best option for these patients, many are not emotionally ready for this decision and want to try first with their own eggs. We, along with other researchers, have had some success with other protocols for the low responders that do not involve Gn-RH-a downregulation, including (1) “stop Lupron”, (2) “microdose flare,” and (3) “Gn-RH antagonist” protocols (summarized in Figures 27.3 to 27.5). In the “stop Lupron” protocol, Gn-RH agonist (leuprolide [Lupron] 0.5 mg daily subcutaneously) downregulation is initiated in the luteal phase followed by commencement of high dosages of gonadotropins when menses begins (300 IU of pure FSH plus 300 IU of urinary combined FSH and LH). Lupron is stopped at the initiation of gonadotropins, after downregulation [106]. The pro-

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Gn-RH Agonist + Gonadotropins: SL

Oral contraceptive pill/ Microflare/FSH

rFSH

rFSH Progesterone

Progesterone Stop Lupron Day 21

OCP 3

FIGURE 27.3

FIGURE 27.4

Gonadotropins + Gn-RH Antagonist

Gn-RH antagonist Progesterone rFSH

Day 3

FIGURE 27.5

7

2

hCG ER ET

Stop Lupron protocol.

9

hCG ER ET

Gn-RH antagonist protocol.

tocol maximizes the ovarian response without risking a spontaneous, premature LH surge. Faber et al. [106] reviewed 182 low-responder patients treated with this protocol and obtained a clinical pregnancy rate of 32% and cycle cancellation rate of only 12.5%, but there was no control group. Another study showed improved pregnancy rates in poor-responder patients and found a clinical pregnancy rate per cycle of 31.4% [107]. In the “microdose flare” protocol, microdoses of GnRH-a and gonadotropins are initiated in the follicular phase after a month of OCPs [108]. On day 3, we use 50 mg Lupron twice daily and start gonadotropin stimulation on day 5. Success is similar to the “stop Lupron” protocol. Studies show no difference in pure FSH compared with FSH/LH in poor-responder protocols, because significant endogenous LH is released. In fact several centers have

Lupron hCG ER ET

Microdose flare protocol.

found a trend toward better outcomes with recombinant FSH alone. The “antagonist protocol” has also been used in poor responders. Akman et al. [109] compared gonadotropins alone to gonadotropins with Gn-RH antagonist in cycles of poor responders undergoing IVF and found higher implantation, clinical pregnancy rates, and ongoing pregnancy rates in these patients. Studies that compared the Gn-RH antagonist with the microdose flare showed no differences in outcome [110]. However, no large prospective, randomized controlled studies have been done. Each program should evaluate the three protocols in their own poor-responder population. Addition of LH is recommended in the “stop–Lupron” protocol where basal LH levels will be suppressed. Because oocyte donation is a back-up option and unacceptable to some couples, cytoplasmic or germinal vesicle transfer have been explored experimentally for age-related or poor oocyte quality problems [111, 112]. There was an initial flurry of excitement that these approaches may avoid aneuploidy, perhaps as a result of the younger cytoplasm promoting a healthy spindle and hence allowing normal chromosomal segregation during meiosis. However, the implications of this radical strategy for mitochondrial heteroplasmy and imprinting phenomena have yet to be fully investigated and the risks for the child-tobe are unknown.

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