INTRODUCTION
Several million oocytes are residing in each ovary at mid-gestation. Let us look at the journey of primordial follicles from conception to birth and until menopause and thereafter. The resting primordial follicles are initiated in the fetus and some 100–2,000 primordial germ cells enter the massive proliferation process that results in about 70 million potential oocytes at mid-gestation and about 85% of these oocytes are lost before birth. The decline in number of follicles continues throughout the woman's reproductive life and reaching negligible level at menopause, resulting in impaired quality of life, thereafter. The life expectancy of 48.3 years in 1900 has now increased to 80 years or beyond in 2000 due to various advancements in improved healthcare, both in diagnosis and treatment areas. In this chapter, we shall deal with some of these developments in the area of reproduction. In our view, the two major areas are: (1) Young women suffering from cancer being able to produce their own genetic children after treatment and (2) Women being able to postpone menopause by 5–10 years and lead better quality of life through ovarian tissue freezing and reimplantation at convenience.
RECENT ADVANCEMENTS
During the last decade or so, highly significant advances aimed at optimizing results, both on clinical and laboratory sides, with the approach of individualizing diagnosis and treatment rather than adopting “one size fits all” approach, have taken place. This patient-centric approach, aided by advancements in imaging techniques, has satisfied increasing patient requirements and challenging scenario to the clinician and embryologist alike resulting in enhanced patient satisfaction, perhaps with reduced cost in some. Notable advancements have been made in the areas of determining ovarian reserve prior to treatment cycle, reducing length of treatment cycle with enhanced safety and neither under nor over response, greater dependence on laparoscopic intervention for treatment of common ailments associated with infertility like uterine leiomyomas, pelvic and ovarian endometriosis, increasing demand for preimplantation genetic screening, especially beneficial to women of advanced age, women with history of recurrent implantation failure with in vitro fertilization (IVF), women experiencing repeated early pregnancy loss and last but not the least, cryofreezing techniques resulting in higher pregnancy and live birth rates (LBRs) and found beneficial to young women suffering from pelvic cancer desiring future pregnancies with their own genetic children.
In this chapter, we shall deal with selected few developments, viz. determination of ovarian reserve prior to treatment cycle for IVF, refinements in stimulation for optimal mature oocyte recovery, adoption of single embryo transfer (SET) to avoid multiple pregnancy and consequent perinatal morbidity, adoption of freeze all strategy and policy for attaining higher pregnancy rates without complications like ovarian hyperstimulation syndrome (OHSS).
Determination of Ovarian Reserve
The ovarian reserve is typically referred to as population of primordial follicles. The model devised by Wallace and Kelsey in 2010 allows estimation of primordial follicles at any age of a woman. In practice, we find three types of 2response in women undergoing treatment for IVF-ET. They are normal response, poor response, and hyper response. Anticipation of the right response helps devising the most appropriate strategy in terms of the gonadotropin selection and its dosage for maximizing mature oocyte recovery (MII oocytes) with safety from complications and avoidance of suboptimal or poor response. It should be noted that maximum mature oocyte recovery has direct relationship with prospect of pregnancy. Earlier, determination of ovarian reserve used to be on determination of basal follicle stimulating hormone (FSH). However, it was soon realized after multiple studies that the sensitivity and positive predictive value of FSH was far from satisfactory and least reliable for prediction of right type of response. A potential, highly sensitive, and far more reliable is the anti-Müllerian hormone (AMH) or Müllerian inhibitory substance (MIS) test.
Estimation of AMH
Anti-Müllerian hormone is a dimeric glycoprotein belonging to the family of transforming growth factor-beta (TGF-β). AMH is involved in regression of Müllerian ducts during male fetal development and is expressed in Sertoli cells from testicular differentiation up to puberty. In females, it is exclusively produced in granulosa cells from birth to menopause. AMH production starts once the primordial follicles have differentiated to primary stage and it continues until the follicles have reached the antral stage corresponding to diameter of 2–6 mm. The number of small antral follicles is related to the size of the primordial follicle pool and with decrease in number of antral follicles with age, AMH production appears to get diminished and it invariably becomes undetectable at and after menopause. There is increasing accumulated evidence demonstrating usefulness of AMH as a cycle-independent biomarker of ovarian reserve which is in contrast to FSH, serum estradiol (E2), and inhibin B. In women, serum AMH expression can first be observed in granulosa cells of primary follicles and the expression is strongest in preantral and small antral follicles. Expression of AMH disappears in follicles of increasing size and is almost lost in follicles of >8 mm. Another advantage is AMH can be measured at any stage of the menstrual cycle, though recent evidence suggests that AMH attains its peak on Day 7 corresponding to 4–5 days before ovulation of idealized cycle of 28 days. Another observation made is the predictive power of AMH for poor response to controlled ovarian hyperstimulation (COH) and is comparable to that of AFC, another useful biomarker to be dealt with later. AFC is cycle specific and it can concomitantly predict high and low or poor responders. In one study, a strong association was found between mid-luteal, early follicular, and pre-ovulatory AMH levels and number of oocytes retrieved. It was concluded by the authors that mid-luteal and early follicular AMH levels could offer good prognostic value for prediction of clinical pregnancy. The cutoff value observed for prediction of pregnancy in this study was 2.7 ng/mL.
A number of other studies have advocated the level of AMH at below 1 ng/mL for prediction of poor response and even lower chances of pregnancy. Further, as per one study, AMH levels have been shown to be independently associated with occurrence of miscarriage. Higher AMH levels (5.61–35.00 ng/mL) have also been shown to be associated with lowest miscarriage rates and lowest AMH levels (0.08–1.60 ng/mL) with lowest response to COH and negligible chance of pregnancy, regardless of age of women. Overall, it appears from this study and other studies that AMH is not only a useful biomarker quantitatively but also qualitatively, including oocyte/embryo competence. In fact, diminished ovarian reserve has been found to be a cause of recurrent miscarriage defined as 3–4 failed clinical pregnancies of <20 weeks of gestational or fetal weight of <500 g.
Polycystic ovarian syndrome (PCOS) is known to be associated with raised levels of AMH at >4 ng/mL and high AFC.
Estimation of Antral Follicle Count
Ovarian reserve can also be estimated by transvaginal ultrasound by counting number of antral follicles in both the ovaries. AFC consists of counting antral follicles of 2–8 mm. Both AMH and AFC reflect the age-dependent decline in ovarian function. The difference between the two is, whereas AMH indicates ovarian reserve in general, AFC is more cycle specific as it could differ in each cycle. To elaborate further, AMH can be the same in the age range of 10 years in different women, but AFC can be different amongst them and that too different in each cycle in each woman. Hence AFC is more dependable for response in a particular treatment cycle. It should be noted that AFC must be measured in the treatment cycle only on any day from Day 2 to Day 4 and not in the cycle of evaluation or downregulated cycle. AMH has been shown to be a weak marker of pregnancy, but AMH and AFC combined together may indicate higher chance of pregnancy. AMH of <1 ng/mL and/or AFC of <8 has been shown to indicate low response to stimulation suggesting aggressive approach to stimulation in terms of higher doses of gonadotropins and perhaps longer duration of stimulation in some. There is consistent biological evidence suggesting detrimental effect 3of endometrioma and even stripping during surgery, on ovarian reserve resulting from reduced AFC. In cases where the endometriotic or any other functional cyst, unilateral or bilateral, AMH reading becomes debatable, in which case AFC would be the only dependable marker of ovarian reserve. There is high evidence in the literature validating the use of AFC as a surrogate marker of ovarian reserve. However, based on currently accepted recommendations, AFC assessment in women with ovarian endometriosis or in women having undergone previously ovarian surgery has been questioned. Hence, ovarian responsiveness to COH is actually considered the best noninvasive surrogate marker of ovarian reserve in these women.
Conclusively, it can be said and believed that the predictive power of AMH for live birth is higher than that of AFC with the predictive error of 1% to <5% with AMH, both being regarded independent of age. Further, it has been suggested that antral follicles of 5–8 mm size make greatest contribution to AMH levels compared to follicles of 2–6 mm size, providing the most accurate estimate of ovarian response.
Controlled Ovarian Hyperstimulation
Since the birth of Louise Brown 39 years before, IVF centers worldwide have been utilizing COH to enhance oocyte recovery to maximize development of good quality viable embryos for transfer to optimize chances of pregnancy. Care is taken in devising the protocol that no woman is excessively stimulated to avoid OHSS and at the same time she is not understimulated to avoid low or poor response. This dual objective calls for individualized patient tailored strategy in devising stimulation. The physician, today, is required to consider a number of parameters, partly based on findings of determination of ovarian reserve, and others like age, BMI, cause and duration of infertility, other existing comorbid illnesses, menstrual irregularity, etc. Care should be taken to see that duration of stimulation is not too long as too long a stimulation could compromise the result. Equal care needs to be taken for choosing single or combination of gonadotropins considering age, BMI, and ovarian reserve. Dyssynchronous growth of follicles is a known phenomenon and this is avoidable by the use of FSH and luteinizing hormone (LH) together right from the start and so also chance of poor response. Minimum possible dosage is used, avoiding chance of over response. Before starting stimulation, uterine status must be evaluated by transvaginal ultrasonography, 3D or 4D, and if necessary, by hysteroscopy. History and findings of sonography should be carefully weighed to rule out need for surgical intervention prior to IVF in case of common illness like uterine leiomyoma, pelvic endometriosis, etc.
Controlled ovarian hyperstimulation is considered to be a key factor for success of IVF, enabling recruitment of multiple dominant follicles to realize maximum mature oocyte recovery. The current standard of care is to use combination of FSH and LH as either human menopausal gonadotropin (hMG) or combination of rFSH and rLH in suitable dosages and to avoid single gonadotropin like FSH alone for reasons to be cited later.
ENDOCRINE REGULATION OF OVARIAN FUNCTION AND FOLLICULOGENESIS
Folliculogenesis is the result of complex and closely integrated series of events which start soon after conception and end about five decades later with menopause. The ovulatory menstrual cycle is the final result of these processes and it relies upon a complex series of physiological mechanisms that link the hypothalamus, the pituitary gland, and the ovary. Although gonadotropins are the key drivers of the menstrual cycle, other compounds such as ovarian steroids and inhibins critically affect and modulate hypothalamic and pituitary hormone selection as well as local gonadal function. Further, folliculogenesis and oocyte maturation depend upon an integration of cellular and endocrine mechanisms which are only in part under gonadotropin control. Much of the germ cell maturation occurs during intrauterine life (Adashi, 1996). These germ cells undergo mitosis and achieve their peak number (6–7 million) by mid-gestation; thereafter, due to initiation of meiosis and follicular atresia, the number of female germ cells drastically declines, so that about 300,000 oocytes are present at puberty.
Gonadotropin levels are low in the prepubertal period, but during fetal life, gonadotropin stimulation may be relevant even in these early stages of development (Gulyas et al., 1977). After attaining antral stage, FSH plays only permissive role in follicular development and ovarian follicles grow slowly. At the recruitable stage which begins in the luteal phase of the cycle, preceding ovulation, follicles can be stimulated by FSH to grow rapidly and enlarge beyond 2 mm diameter. The selective rise of FSH that occurs during luteal-follicular transition is a potent stimulus for follicle recruitment and several early antral follicles respond by beginning to enlarge. Endogenous and exogenous gonadotropins can now exert their full traditional stimulating effects and follicular growth becomes rapid.4
Luteinizing hormone is also critically involved in the physiological events that lead to development of reproductively competent preovulatory follicle. LH is capable of stimulating androgen substrate from theca cells in fetal life, as attested by the elevated steroid levels found in female neonates (Winter et al., 1995). This modular action of gonadotropins has been named “two-cell two-gonadotropin model” (Dorrington and Armstrong, 1979). Estrogens in turn play fundamental role through improving follicle and oocyte maturity, in priming the hypothalamus-pituitary unit in preparation for the preovulatory gonadotropin surge as well as inducing the morphological uterine and endometrial changes needed for embryo implantation. Finally, LH-follicle intersections disrupt theca cell contacts in the cumulus and induce oocyte meiotic maturation to cause follicular rupture (Espey, 1974; Lawrence et al., 1980) and induce granulosa cell luteinization (Brailly et al., 1981).
In the course of follicular phase, FSH stimulates granulosa cells to express LH receptors and this action is facilitated by estrogens (Rani et al., 1981). Thus, in the late stages of follicular development, once antral follicle diameter increases beyond 10 mm, LH receptors are expressed by granulosa cells which become receptive to LH stimulation. LH then is capable of exerting its actions on both granulosa and theca cells (Hillier, 1994). At this stage, LH can exert virtually all physiological actions of FSH on granulosa cells, including stimulation of aromatase system (Zeleznik and Hillier, 1984). Serum FSH levels are reduced during most of the luteal phase, beginning to increase only a few days before menses, and are elevated throughout luteal–follicular transition. FSH thus progressively declines across the follicular phase until the mid-cycle surge is triggered by rising estrogen and progesterone concentrations.
Thus, it is evident that physiological follicular growth and maturation is dependent upon the dynamic interplay between FSH and LH, key physiological events such as follicular recruitment and dominant follicle selection rely upon both the prevalence of FSH and LH activity.
Selective LH activity in Controlled Ovarian Hyperstimulation
Selective LH activity administration in the mid and late stages of ovulation induction, both in COH and anovulatory patients has been demonstrated. Low dose of 75 IU/day of rLH administration, after at least one 14 mm follicle development, has impact on oocyte yield and pregnancy rates in assisted reproductive technology (ART) (Ben-AMOL, 2000). Available data suggests that LH activity can be used to improve and optimize FSH stimulation for COH. LH activity during ovulation induction also causes noticeable and significant modifications in ovarian response. The addition of LH to the FSH therapy during COH consistently and significantly shortens the duration of treatment and reduces the amount of FSH required for final oocyte maturation [Filicori et al., (1999b, 2001)], especially toward the end. Gonadotropin releasing hormone (GnRH) analog type, formulation, and route of administration can undoubtedly contribute to results, when both FSH and LH preparations, individually or in combined form as hMG, are used. GnRH antagonist when started from Day 6 of stimulation or when lead follicle attains 12–14 mm size is known to synergize with exogenous FSH and provide adequate follicle stimulation. In the short flare-up protocol of GnRH agonists (GnRHa), supported by hMG, endogenous FSH and LH are elevated for the first 3 days of administration and thereafter endogenous LH secretion is maintained till the day of trigger. Intranasal administration of GnRHa is associated with low absorption (about 3%) rate and less profound suppression of FSH and LH (Filicori, 1994). In the long mid-luteal suppression protocol, where GnRHa is administered from mid-luteal phase of the previous cycle, downregulation is achieved in about 10–20 days of administration and then switched over to stimulation by hMG or rFSH + rLH till the day of trigger. It is also recognized that during highly purified hMG (HP hMG) administration, estradiol level rise is brisk as compared to slow rise in case of HP FSH.
Effect on Progesterone
Serum levels of progesterone usually remain low during natural cycle or during COH. GnRHa or GnRH antagonist prevents untimely LH surge leading to premature ovulation (Filicori, 1996). Moderate rise of P, around 1.3 ng/mL, is occasionally observed during COH and is equated with premature follicle luteinization (Silverberg et al., 1991; Fanchin et al., 1993). This phenomenon, however, does not affect oocyte or embryo quality (Hofmann et al., 1993a; Legro et al., 1993) but may reduce IVF success through secretory endometrial transformation and impair chance of embryo implantation. Progesterone rise can take place with any preparation like hMG or FSH or rFSH + rLH and in spite of using GnRHa or GnRH antagonist. However, in practice, P rise is observed more often with increased dosage of FSH at the end of stimulation. Hence, it is considered prudent to reduce the dose of FSH and proportionately increase the dose of LH. It is further observed from some studies that if P level on the day of trigger is above 2.5 ng/mL, it is better to avoid fresh transfer and consider frozen embryo transfer (FET) in subsequent natural or modified natural cycle.5
The phenomenon of dyssynchronous follicular growth between both ovaries is well known during the beginning of COH. This is largely due to use of FSH alone, which stimulates unselectively or due to differential uptake of FSH by multitude of follicles. Hence, it is better to start with FSH + LH or hMG, which could provide a means to selectively achieve both large follicles and curtailment of small follicular development (Marco Filicori et al., 2002). In addition to supporting the growth of larger follicles, LH activity appears to exert other important actions on the dynamics of folliculogenesis. In 2001, (Filicori et al., 2001) it was shown that hMG administration leads to progressive decline in number of small follicles (<10 mm) and this was significantly more marked than with HP FSH. This finding was confirmed by significant inverse relationship between the amount of LH administered and the number of small follicles in a study on 120 patients (Filicori et al., 2002a).
Conclusively, therefore administration of rFSH + rLH or hMG promotes synchronized growth of follicles which in turn could help realizing greater yield of mature oocytes. In case of hyper responders, care has to be taken especially in case of younger women. In case of women of advanced age (≥ 35 years) and/or women with high BMI (>29 kg/m2), this procedure may be found equally useful. In women with PCOS, both GnRHa long protocol and fixed protocol of GnRH antagonist can be used. In the case of young lean women with PCOS (BMI < 28 kg/m2 and basal LH >5 mIU/mL), GnRH antagonist in the dose of 0.25 mg/day and FSH 150 or 225 IU from Day 1 of cycle may be preferred, the antagonist continued till the day of trigger. In these patients in whom antagonist is started on Day 1 of cycle, more rapid follicular development and earlier rise in E2 levels can be expected; another advantage being, LH can be suppressed within a matter of hours of starting the antagonist, whereas in case of GnRHa long protocol, suppression takes nearly 2 weeks, not to speak of higher gonadotropin requirement.
Oral contraceptive pill (OCP) pretreatment is used by some physicians for suppression in women with normal basal levels of FSH and LH for not more than 15 days. Women with OCP intake of >15 days are known to consume more gonadotropins and have longer duration of treatment. The antagonist usage is known for much lesser or negligible prevalence of OHSS compared to usage of long GnRHa protocol, both in normal and hyper responders.
UNEXPECTED LOW OR POOR RESPONSE IN WOMEN WITH NORMAL BASELINE HORMONAL LEVELS
Adequate follicular development in response to gonadotropic treatment is a prerequisite to successful IVF program. However, many women respond poorly or not at all to the standard COH protocols. Such patients account for 10–25% of IVF population. Some of these patients have age-related decline in their reproductive performance. Also, a low response is one of the most problematic issues in reproductive medicine.
It has been reported that the follicular fluid (FF) secretes multiple autocrine and paracrine factors that affect follicular development (Weng et al., 2006; Choi YS et al., 2006). There is evidence suggesting that ovarian insulin-like growth factor (IGF-II), insulin-like growth factor binding protein 4 (IGFBP4), and pregnancy-associated plasma protein-A (PAPP-A) system plays a major role in follicular development (Keay SD et al., 2003; Choi YS et al., 2006). Thus, FF levels are significantly lower in poor responders. A correlation is also reported between AMH levels in FF and the responsiveness to stimulation (Fallet ME et al., 1997). In addition, other follicular markers like soluble Fas and vascular endothelial growth factors (VEGFs) could be altered in ovarian response to stimulation (Hasyakor D, 2004; Neulan J et al., 2001). Soluble Fas is anti-apoptotic molecule reported to be positively correlated with oocyte maturation (Malamutsi-Puckner A et al., 2004). A negative correlation has also been suggested between VEGF in FF and the number of follicles (Tokuyama O et al., 2002). In one study (Young AG; Koo et al., 2009), multiple follicular development was found associated with higher levels of VEGF expression in FF and the same was observed in unexpected low responders and normal responder patients. Several other studies have also reported that regulation of follicular angiogenesis is important for normal development of follicles (Fauser BCJM, 1997; Schimizu T et al., 2003; Taylor PD et al., 2004).
EXPLORING UNCHARTED TERRITORIES IN CONTROLLED OVARIAN HYPERSTIMULATION
There are several situations we come across in practice during COH, considered uncommon, but of practical importance and without knowledge of which, they could become hazardous and/or not cost-effective to implement, though they are evidence-based. Let us deal with them individually.
- Pretreatment with OCP: OCP is being used by some in antagonist cycle for the dual purpose of suppression and planning the cycle, both for convenience of patient and the physician. A systematic review and meta-analysis conducted revealed no difference in pregnancy rates between patients who used OCP and those who did not. But gonadotropin consumption and duration of treatment cycle were increased significantly after OCP pretreatment (Georg Griesinger et al., 2008).6
- Importance of LH supplementation: Utilizing beneficial effect of LH activity routinely helps realizing greater yield of mature oocytes and preventing P rise on the day of trigger, thus protecting favorable implantation prospect in fresh ET cycle, as P rise is known to impair implantation by disturbing the window of implantation.
During mid and late follicular phase of natural menstrual cycle, preovulatory follicular development proceeds despite progressive fall in serum FSH level. Studies conducted in women of reproductive age, using rFSH and rLH after downregulation of pituitary in the long protocol with leuprolide acetate (LA) demonstrated that LH sustains follicular E2 production in presence of falling FSH and E2 levels, which were used as index of follicular development (Michael Sullivan et al., 1999). The E2 levels of women receiving LH toward the final stage of stimulation increase throughout despite declining FSH level. It was also observed that thecal androgen production was exquisitely sensitive to LH.
The hypothesis is LH protects the maturing follicles from declining FSH levels. In this study, the FSH treatment was arbitrarily discontinued, when lead follicles reached 14 mm or more in diameter and LH treatment introduced till the end of the cycle. In practical terms it can be stated that when lead follicles reached 14 mm size or so, rFSH dose could be reduced by 75 IU or more depending on size of follicles lagging behind and rLH in the proportionate dose of 75 IU or more introduced and continued till the end of the cycle.
- Inadequate response to FSH in the beginning of COH is a frequent occurrence: After 5 days of stimulation with FSH alone. This phenomenon is observed in about 12–14% of young normogonadotropic women treated with long mid-luteal GnRHa protocol. In such an event, rLH can be introduced from Day 6 of stimulation in the dose of 75 IU or more, depending upon size of growing follicles on Day 6, which are usually <11 mm in diameter. With this strategy, good response can be expected on Day 8 or Day 9 in terms of increased size of follicles coupled with rising level of E2 and this combination continued till the day of trigger. In one study with this strategy, it was found that LH supplementation was more effective than increasing the dose of FSH in terms of mature oocyte outcome (G De Placido et al., 2005).
There are a number of situations where gonadotropin-deficient women do not respond to FSH alone adequately in the initial stage, and the E2 level on Day 6 of stimulation is lower than expected (<250 pg/mL), the follicles stimulated with FSH alone do not consistently rupture after hCG administration and they luteinize poorly and their oocytes may have lower fertilization rates. In this population of women, addition of rLH from Day 6 of stimulation in the dose of 75 IU/day or 150 IU/day has been shown to be effective to achieve adequate follicular development and steroidogenesis (European rLH study group, 1998).
- After pituitary downregulation with GnRHa in the long protocol, suppression of endogenous LH is more profound than of FSH. These are young normogonadotropic women in whom, if FSH is used alone, the oocyte yield is not seen to be adequate and their developmental competence is compromised. They develop poorly as zygotes and their embryos show lower implantation rates compared to those in whom rLH was administered along with rFSH during COH. A number of studies have shown higher implantation and pregnancy rates with combined administration of rFSH and rLH for COH (Jan Tesarik and Carman Mendoza, 2002; Westrgard LG et al., 1996).
- The phenomenon of premature LH surge is known to occur in many women, regardless of age, during COH, it is known to occur even in natural cycle resulting in premature ovulation. This is more commonly encountered in women of advanced age (>35 years). In such women, when GnRHa or GnRH antagonist is used, suppression of LH is seen to be more profound and if FSH is used alone for stimulation, the cycle outcome in terms of oocyte yield becomes suboptimal and consequently number of viable embryos may be less for transfer. Several studies performed to investigate whether the cycle outcome improves if rLH is added to rFSH during initial stage of stimulation. One such study has demonstrated that addition of rLH is essential for oocyte maturation (Mochtiar, 2011). Even in natural cycle, LH is shown to be essential for oocyte maturation (Hillier, 2001). Low LH levels in IVF cycles have been found to be associated with availability of fewer embryos for cryopreservation and transfer (Fleming et al., 1998; Marrs et al., 2004). Bosch et al., 2011, in their study in GnRH antagonist cycles further confirmed this finding and found higher clinical and ongoing pregnancy rates in women of 36–39 years.
- Faultless completion of meiosis I and II is crucial to chromosomal integrity of oocyte and hence oocyte diploidy (Verlinsky and Kuliev, 1996). If meiosis, a process extremely sensitive to disruption, was to be influenced by the presence or lack of appropriate LH level, LH supplementation in the course of fertility treatment could indeed influence diploidy rates in cleavage stage embryos. It has also been observed that diploid embryos are more likely to implant (Santiago Munne et al., 2005) and hence higher percentage 7of diploid embryos would ultimately lead to higher pregnancy chances. These data have also been confirmed by studies by Lisi et al., 2005, and Anderson et al., 2006, who reported higher pregnancy rates in women receiving LH supplemented stimulation rather than stimulation with FSH alone.
Younger women, especially those with normal ovarian reserve or function, are more likely to have low basal LH level and are therefore at higher risk of profound LH suppression during pituitary downregulation (Flamming et al., 1998). The data on beneficial influence LH supplemented embryo diploidy are particularly evident in women who lack adequate endogenous LH level. This interpretation has been supported by Van Waly et al., 2003a, who reported in younger women, significantly higher clinical pregnancy rates after long agonist protocol for COH, using hMG in comparison to using FSH alone. The likely beneficial effect of hMG stimulation in poor responders has also been supported by Cochrane working group (Van Waly et al., 2003b).
In conclusion, the data presented provides overwhelming evidence that in selected patient population, LH supplemented ovarian stimulation protocols could beneficially affect diploidy rates in preimplantation embryos.
- Beneficial effect of androgens in COH: In some cases of poor response to FSH alone or rFSH + rLH or hMG, androgenic agents like hCG and testosterone (T) do impart beneficial effect to folliculogenesis in patients who fail to show adequate response to conventional therapy. Even these agents can be straight away incorporated in stimulation protocol in patients with documented or clinical poor response. This alternative can be deployed in all types of cases except in women with PCOS. In this alternative, downregulation is carried out with long mid-luteal GnRHa protocol. Once downregulation is confirmed by clinical and laboratory criteria, testosterone in the form of intradermal patch in the dose of 1.2 mg/day for 5 days is administered followed by hMG or rFSH + rLH in the appropriate doses is given till lead follicles of minimum 17 mm are seen (Lossi et al., 2006). If T patch is not available, hCG in a small dose of 1,250 IU/day could be used at least for 8 days in the same manner.
- Does growth hormone (GH) has a place in poor responders?: The incidence of poor ovarian response to COH varies from 9% to 24% (Surrey ES et al., 2000).
How do we define poor response?
A consensus study organized by European Society of Human Reproduction and Embryology (ESHRE) 2011 defined poor ovarian response (POR) to include any two of the following three features. They are also referred to as Bologna criteria.
- Maternal age (≥ 40 years) or any other risk factor for POR.
- Clinical evidence of POR [Three or fewer oocytes with ovulation induction in earlier cycle(s)].
- Documented POR by ovarian reserve testing. AMH <0.5–1.1 ng/mL and/or AFC of <8 between both ovaries.
Deficiency of GH causes delay in onset of puberty in addition to growth retardation during childhood. Treatment of these patients with human GH induces sexual maturation (Zachman et al., 2003). These findings suggest that GH influences gonadal function. GH is reported to enhance responsiveness of follicles to gonadotropins leading to stimulation of folliculogenesis in infertile women who fail to respond to exogenous gonadotropins (Burger HG et al., 1991). The action of GH is partly mediated by IGF-1 production from the liver (Homburg R et al., 1996). Both GH and IGF-1 are detected in FF and their receptors are also expressed in the ovary (Givdice LC, 1992; Johnson MC et al., 1996). The role of GH and IGF-1 in follicular development has been examined in humans (Givdice LC, 1992; AdashI EY, 1985).
Growth hormone is also known to influence luteal function, directly or indirectly, to affect P production by luteal cells in vitro (Bergh C et al., 1991).
One of the options to improve the situation of poor response is addition of GH as an adjuvant to other ovulation inducing agents like FSH, LH, etc. (Kucvk T et al., 2008). The efficacy of GH in improving cycle outcome has been controversial till today. A recent, parallel, open-label, randomized study conducted from 2014 to 2016 (Yasmin Ahmed Bassionny, 2016) incorporated GH in antagonist cycles of proven poor responders. The protocol in the study group consisted of hMG 300–450 IU/day from Day 1 or Day 2 of the cycle depending on age and ovarian reserve markers like FSH, AMH, and AFC. GnRH antagonist added when lead follicles were 12–14 mm and GH added from Day 6 of stimulation in the dose of 2.5 mg/day SC, until the day of trigger. Norditropin (Novo Nordisk) 2.5 mg is equivalent to 7.5–8.0 IU/day or 0.1 IU/kg/day. The control group consisted of hMG + antagonist given in the same manner as in the study group patients. The total number of patients was 140 and the results were as below:
- Duration of treatment in both groups, study group and control group, was 10.77 days and 12.02 days, respectively.
- Total dose of GT was 3,900 IU and 4,906 IU.
- E2 on day of trigger was 1,862 and 1,854.
Reproductive outcomes were as below:
- Implantation rates were 11.98% and 9.88%.
- Clinical pregnancy rate/cycle was 22.1% and 15%.
- Live birth rate was 17.5% and 13.6%.
Several studies to improve results in poor responders have reported increase in number of retrieved and MII oocytes (Kucuk T et al., 2008; Effthekar M et al., 2013; Bergh C et al., 1994). One meta-analysis suggested that GH administration is associated with increased proportion of patients who reach ET stage and are thus exposed to chance of pregnancy (Kolibianaskis EM et al., 2009). This finding, in our view, appears illusory. In another systematic review and meta-analysis, different interventions to improve outcome in poor responders were investigated and two interventions with a favorable impact and increased pregnancy rates were the use of GH and Day 2 transfer (Kyton D et al., 2009). The data provided above shows that GH cotreatment with antagonist results in improved parameters assessed, including LBR, and the differences between study group patients and control group patients did not reach statistical significance. Although these data are encouraging, the use of GH as an adjunct treatment in poor responders should be implemented cautiously till further larger and conclusive systematic studies, reviews, and meta-analysis are available.
Additional useful information on poor responders: In view of increasing prevalence of poor or low response to COH, more and more studies are forthcoming leading to availability of beneficial information on the subject, which are as below:
- Poor ovarian response is observed more in younger women of <35 years than in women of advance age.
- Poor ovarian response can be anticipated through determination of ovarian reserve; AMH of <1 ng/mL and/or AFC of <8 is indicative.
- Ultrashort protocol of GnRHa for first 3 days with added hMG from Day 1 till the day of trigger works well in some women.
- GnRH antagonist is known not to act in many of these cases in whom AMH is <1 ng/mL and AFC <8. Hence, use of progestogens like medroxyprogesterone acetate (MPA), Utrogestan, and Duphaston have been tried and found successful. In this protocol, Utrogestan 100 mg/day or MPA 10 mg/day or Duphaston 10 mg/day along with hMG 375–450 IU/day are started from Day 1 or Day 2 or Day 3 till the day of trigger.
- The protocol of double stimulation can be attempted in some. In this protocol, start stimulation with any of aforesaid formulations from Day 1 or Day 2 till follicles of 17–18 mm are seen and proceed with oocyte pickup (OPU) after the trigger. Freeze the embryos and after 3–5 days, assess E2 and P levels and E2, if found at 35 pg/mL or less, and P of up to 2.5 ng/mL, resume stimulation again as earlier, at once and proceed with 2nd OPU, and freeze the additional embryos for transfer of good quality selected embryos in natural or modified natural cycle.
NEWER DEVELOPMENTS IN GONADOTROPIN RESEARCH FOR CONTROLLED OVARIAN HYPERSTIMULATION
There are two new drugs in the category of rFSH under clinical trials for stimulation during COH for IVF patients. They are follitropin delta and corifollitropin. Both appear to be promising. Corifollitropin is long acting rFSH with single dose administration from Day 1 having duration of 7 days, after which current rFSH with one dose/day in the aqueous form is to be administered till the day of trigger. Follitropin delta is rFSH, uniquely expressed in fetal retinal cell line, which owing to differences in glycosylation profile, has a lower clearance and induces higher ovarian response than the existing rFSH preparations, when administered in equal doses of biological activity (IU).
Additional useful information on prevention of premature LH surge: The phenomenon of premature LH surge has been known for a number of years, not only in COH but even in natural cycle. It can take place anywhere from early follicular phase to the point where the follicles of 12–14 mm, inducing ovulation before trigger, thus impairing oocyte recovery and culminating into empty follicle syndrome in some cases. It is also known that P inhibits E2-induced positive feedback by reduced GnRH pulse frequency. It has also been seen that GnRH antagonist does not work in some cases where AMH is <1 ng/mL in which case, as per early preliminary studies, both MPA and Utrogestan can be used as they have been validated as effective oral alternatives to GnRH antagonist for prevention of this phenomenon. Didrogestral (Duphaston), a synthetic P, differing from Utrogestan in its molecular structure and pharmacological effects, has also been found useful for this purpose but not to have androgenic effects, even at high doses. Utrogestan, a natural micronized P, is fit for both oral and vaginal administration. Duphaston 20 mg has been found to be equivalent to Utrogestan 200 mg. Both have been found to be effective in preventing premature LH surge in a randomized controlled trial (RCT). In the same study, it was also observed that high doses of progestins could swiftly decrease circulating LH levels and low doses could achieve the same effect by prolonged duration. Such a “dose–time” effect has been 9elucidated in large scale investigations for endometrial transformation or ovulatory inhibition. In another similar study, Utrogestan 100 mg/day was found to be as effective as Utrogestan 200 mg/day in preventing premature LH surge.
Further, it is suggested that for optimal effect of preventing premature LH surge, both Utrogestan and Duphaston must be given simultaneously with hMG or rFSH + rLH from Day 1 of stimulation till the day of trigger. MPA in 10 mg/day dosage is also effective in preventing premature LH surge.
Conclusively, it can be said that for poor responder women in whom GnRH antagonist may not work for the purpose of preventing premature LH surge, we have a choice of three progestins. Besides, oral progestins may be more cost-effective than the antagonist.
Application of GnRHa as a Trigger during Controlled Ovarian Hyperstimulation
In the history of IVF over nearly four decades, extraordinary advances have been made by physicians and scientists alike to increase its efficiency and safety. Equally important are the techniques introduced. One such technique is the use of GnRHa as a trigger instead of hCG in the antagonist based protocol to reduce or to avoid the likelihood of OHSS. Initial concerns over altered luteal phase E2 levels in the antagonist cycles with GnRHa as trigger were largely alleviated by administering aggressive luteal support. However, despite its demonstrated efficacy and safety, some clinicians hesitate to use it widely because a small subset of women do not respond with adequate LH rise, which is the obligatory finding or signal for final oocyte maturation. The following information would adequately explain what is the optimal LH rise level desirable for attaining oocyte maturity and what are the factors or reasons why some patients do not adequately respond to GnRHa trigger for oocyte maturity. The contribution made on this subject by Weill Cornell Medical Center, New York and George Washington University, Washington DC has been more than significant. Their study involving 500 fresh IVF cycles over a period of 6 years has suggested the optimal LH level, post-trigger with GnRHa on the day of trigger, required for adequate response and the risk factors present in women not responding adequately to post-GnRHa trigger for oocyte maturity. A number of outcome variables were assessed in this study, viz. suboptimal response to GnRHa trigger in the morning after the trigger, optimal response in the morning after the trigger, age, BMI, trigger type, i.e. single versus double trigger, history of irregular menses or amenorrhea, insignificant basal FSH and LH levels, and OCP pretreatment. The cycle evaluated showed that those patients who had single GnRHa trigger and who exhibited LH level of ≥15–30 mIU/mL had at least eight mature oocytes retrieved with an oocyte maturity rate of 68–100% and had a LBR of 57%. But those who had post-trigger LH of <15 mIU/mL in the morning after trigger, had either three or fewer mature oocytes or had OPU cancelled. Statistical analysis further revealed that suboptimal responders had post-trigger LH of <15 mIU/mL and normal responders, some of them having been given dual GnRHa trigger, had ≥15 mIU. The GnRHa used was LA 2 mg or 4 mg or triptorelin 0.2 mg (decapeptyl). The dual trigger consisted of GnRHa + hCG 1,500 IU immediately after post-trigger LH level was available.
The authors suggested that certain demographic parameters like irregular menses, BMI of < 29 kg/m2, FSH level of <0.1 IU/mL, LH level of <4 mIU/mL on the day of trigger, and OCP intake of >2 weeks, should be regarded as risk factors for optimal response. The authors contend that suboptimal group of patients almost always occurs and advance identification of this group should be guided by the risk factors present, so that precautionary measure like use of double trigger can be taken to avoid cancellation of the cycle. The GnRHa trigger has an added advantage of inducing mid-cycle FSH surge which also contributes to oocyte maturity. It is noteworthy to consider that though LH surge induced by GnRHa trigger effectively secures oocyte maturity, low circulating level of endogenous LH level during early luteal phase could result in corpus luteum demise and poor reproductive outcome, unless the standard luteal phase support is modified and made more aggressive.
Luteal Phase Support by GnRH Agonist as a Sole Support in Antagonist Cycles
Luteal phase deficiency is being observed as an unfavorable sequela to OPU in all IVF–intracytoplasmic sperm injection (ICSI) cycles. Therefore luteal support is being offered routinely in all IVF cycles in the form of P or P + E. Administration of a single or multiple boluses of luteal GnRHa has been gaining popularity in recent years as it has been observed to improve pregnancy rates and LBR. The beginning was made in 2004–2005 by Pirard et al., who investigated use of GnRHa as a substitute to P for luteal support. The same authors conducted a feasibility study followed by pilot study in 2006 and in 2015. All the above studies demonstrated efficiency of GnRHa as a sole luteal support in non-downregulated cycles. Subsequently, a large randomized control study on 2,529 antagonist cycles on 1,479 women, aged 25–41 years by Itai Bar Hava et al., conducted over a period of 6 years from 2009–2015, showed a higher LBR in the entire GnRHa group as compared with 10the P group. GnRHa administration was by intranasal route and P by vaginal route. In the GnRHa group, significantly higher levels of mid-luteal E2 and P were observed. Clinical pregnancy rates were also higher in GnRHa group (27.9% vs. 19.8%). In our opinion, luteal support with intranasal GnRHa spray has several advantages over P by vaginal or P by IM or SC route. It is much more convenient without any irritation and not at all painful unlike P by IM or SC.
The above large study also illustrates that GnRHa as luteal support not only rescues corpus luteum function but is also sufficient to support embryo implantation and further development without any other luteal phase support. The luteal support needs to be given only implantation and on embryonic β-human chorionic for 2 weeks after ET. The observed effects of GnRHa on implantation and on embryonic β-human chorionic gonadotropin (β-hCG) secretion are attributed to direct effect on embryo and on endometrium mediated by LH, in accordance with the previous observations on the effects of LH activity on endometrial receptivity, independent of ovarian function. Further studies have also shown similar beneficial effects of GnRHa administration on implantation. At present, many centers worldwide have been using GnRHa as an effective luteal support. As per our knowledge, GnRHa for luteal support is likely to replace currently used P administration, given higher pregnancy rates and LBRs.
FROZEN VERSUS FRESH EMBRYO TRANSFER
In vitro fertilization with FET has become increasingly common with IVF centers the world over. The number of FETs increased by 80% in the US from 2006–2012, which far outpaced the rate of increase of fresh ETs over the same period. Advances in cryopreservation of embryos have contributed immensely to this trend, as newer vitrification techniques have improved embryo survival rates as compared with slow freezing. In addition, there is increasing evidence of FET leading to more favorable perinatal and live birth outcomes including a lower risk of preterm birth (PTB), low birth weight (LBW), placenta previa, and placenta abruption. One large multicenter matched cohort study on over 2,900 cycles found significantly higher implantation rates and ongoing pregnancy rates with FET cycles compared with fresh transfers. The ongoing PRs with FET cycles were 52% and with fresh cycles it was 45.3%. This effect was most pronounced with P >1 ng/mL on the day of trigger and also in women of advanced age. There are a number of RCTs reporting higher PRs in normal and high responders.
Frozen embryo transfer protocols are thought to have several potential advantages over fresh transfer protocols. Multiple mechanisms have been suggested for the negative impact of COH on implantation and on pregnancy. Hormone regulation plays a large role in endometrial receptivity and high E2 and P levels from COH may affect hundreds of genes involved in implantation. High E2 levels during COH have also been hypothesized to interfere with endometrial perfusion. On the other hand, endometrium in an unstimulated cycle is more receptive to early placentation and embryogenesis than endometrium during COH.
It is believed by most centers that FET is most likely to yield higher implantation and clinical pregnancy rates than fresh transfers, regardless of age, infertility cause, duration of infertility, etc. In fact, many have adopted FET as policy in their centers. In our center, we have adopted FET as a policy for all patients since a year and we have observed more than double the PRs with FET than what it was with fresh transfers.
There are two major considerations for FET as a policy: (1) Premature rise in P is known to occur in most cases, making difficult the determination of opening and closing of the window of implantation (WOI), affecting adversely the chance of fresh embryo implantation. (2) It is much easier for the clinician to determine WOI in natural or modified natural cycle to facilitate FET and to maximize the pregnancy rates.
CORRELATION BETWEEN EMBRYO DEVELOPMENTAL STAGE AND ENDOMETRIAL STATUS
Asynchrony between embryo developmental stage and endometrium can result from COH leading to advanced histology and downregulation of P receptors. Prior to this event, COH may lead to premature rise in P, which is more due to FSH than to LH. Hence higher dependence on LH than on FSH toward the end of stimulation may prevent rise in P on the day of trigger, which can induce premature decidualization disrupting or advancing WOI, which is known to last for about 48 hours. It has also been hypothesized that the freeze-thaw process in freeze-only cycles serves as filter for embryos of poor quality, which may not survive the thaw process, FET procedure can also reduce the risk of OHSS.
Failure of implantation is the biggest challenge to IVF today. Uterine contribution to implantation success should never be underestimated. Uterine receptivity, 11which facilitates implantation besides embryo quality, is controlled by hormonal milieu during COH and the appropriate timing of embryo transfer, which in turn depends upon whether the WOI is open. The WOI opens with the appearance of P (including endogenous P) in serum, which is difficult to determine in stimulated cycles but not in unstimulated ones.
Another important factor for effective implantation is the endometrial thickness (EnT) on the day of ET influenced by rising E2 level. However, contribution of EnT to implantation is considered controversial. EnT of 6–7 mm or >10–14 mm has been reported to affect implantation adversely. Other studies have shown no association between EnT and implantation process. In view of this controversy, some studies have advocated using endometrial pattern on the day of ET for implantation. The reduced implantation rate in patients with a particular type of endo pattern suggests that premature luteinization leading to uterine-embryo asynchrony is a significant contributor to implantation failure.
MAXIMIZING LIVE BIRTH WITH SINGLE EMBRYO TRANSFER
Today, every couple desires birth of one healthy child at a time. However, in India, a woman willingly and gracefully accepts twins or triplets under compelling circumstances. So is the gentle nature of an Indian woman. The increase in multiple births observed in the last 25 years or so is largely attributed to ART. Many consider multiple births as an adverse outcome because of the associated maternal and neonatal morbidity as well as adverse economic impact. The risks with multiple pregnancies include prematurity, intrauterine growth restriction, LBW, cerebral palsy, learning disabilities, and developmental delays. Strategies to curb multiple births, to improve implantation by transfer of single blastocyst transfer, preferably FET, are being increasingly adopted all over the world, including India, today. In the US, the practice of SET increased from 2% in 2002 to 12% in 2011. However, it appears underutilized compared to European countries, particularly Sweden, which was 73.3%. The number of SETs performed in the US increased by 82.5% from 2006 to 2012. It is well established and acknowledged now that SET reduces multiple births and improves neonatal end points, while providing acceptable LBRs.
In Japan, in early 2000s, conflict developed between ART specialists and obstetricians. The neonatal intensive care units (NICUs) throughout the country were occupied by babies born after infertility treatment. ART specialists were accused of producing multiple pregnancies by their ignorance. Since then, the need for SET has been seriously contemplated. The study by Takeshima et al. highlights the success of SET policy for improvement of perinatal outcome and mortality rate. SET policy was adopted by the entire country. It is noteworthy that Japanese medical practices for ART are self-regulated by the rules of the society and not by legislation. The policy, thereafter, produced SET rate of 82.6% in 2012.
The important pre-requisite for the success of SET policy is the “normal euploid embryo”. There are several methods to ensure euploid embryo morphology, the latest being preimplantation genetic screening (PGS). The study by Kazumi Takeshima et al., reviewed retrospectively the total of 140,718 live births and 510 stillbirths (after 22 weeks of gestation) conceived by ART between 2007 and 2012. The following were the findings, based on analysis of Japanese registry data:
- The practice of SET increased from 52.2% in 2007 to 82.6% in 2012.
- The multiple pregnancy prevalence decreased from 10.7% to 4.1% during the same period.
- The prevalence of PTB, LBW, and small for gestational age (SGA) decreased significantly while that of large for gestational age (LGA) increased.
- Perinatal morbidity decreased from 0.7% to 0.4% in fresh cycles, while that of FET cycles did not change.
The scenario in South Asian countries, as indicated by one British study, showed higher cancellation rates and lower LBRs. Another collaborative study by two centers based in the US demonstrated lower implantation rates in south Asian women versus Caucasian women (28% vs 39%) and lower LBRs among South Asian women versus Caucasians (24% vs 41%).
Summarily, there is enough data to support transfer of single frozen blastocyst, providing excellent LBRs, greatly reduced multiple and preterm births. In our view all patients should be encouraged to opt for frozen thawed blastocyst transfer with autologous oocytes and sperm.
Why euploid blastocysts do not always implant?
Early on, higher number of oocytes harvested could produce higher number of embryos to enable transfer of multiple embryos because of enhanced efficacy of ovarian stimulation to establish implantation and possibly pregnancy.
Today, this practice has changed. Improvements in IVF technology have rendered multiple embryo transfer unacceptable to both, clinicians and patients. The obligation to foster SET for reducing multiple pregnancy 12has underscored the need for ideal endometrial receptivity conditions prior to transfer. To achieve the ideal eutopic endometrial status, the clinician may be required to look at number of abnormalities, both in the woman's pelvis and the uterus itself. There could be several abnormal conditions, such as inflammation of the endometrium arising from primary infection, other conditions distorting the pelvic anatomy such as endometriosis, and/or adenomyosis, asymptomatic endometriosis producing P resistance or P block, leiomyomatosis requiring surgical intervention prior to IVF treatment, numerous endometrial gene expressions known to occur after COH rendering endometrium simply abnormal, etc. In fact, when office hysteroscopy is performed in women undergoing first IVF attempt and in women with history of repeat implantation failures or early pregnancy failures, uterine abnormalities are detected in as much as 11–22% and 26–45%, respectively. Myometrial pathologies like leiomyoma or fibroids, submucosal or intramural myomas are known to encroach uterine cavity with associated reduced implantation and increased miscarriage rates. Adenomyosis, which is steroid dependent and which is identified in 27–79% of infertile women and up to 90% of women with endometriosis, has been shown to significantly reduce pregnancy rates as per recent meta-analysis.
In view of the above conditions, impairing endometrial receptivity, euploid blastocyst, certified by PGS, cannot be expected to implant or to produce pregnancy.
CONCLUSION
Quite often, question is raised, especially in non-medical society, are we moving forward with assisted reproductive technology? Some say Yes and some No.
We shall attempt to answer. Let us look at the journey that began about 30 years back. The process was termed inefficient, full of failures like multiple births, intolerable prevalence of implantation failures and early pregnancy loss, no cost-effectiveness, to name the few. The positive side of the scenario has been contributed by advancements like extended culture of cleavage stage embryos, cryopreservation techniques, especially, the vitrification process, time lapse morphokinetics, preimplantation genetic screening, oocyte and embryo freezing techniques, ovarian tissue freezing and reimplantation, uterine transposition and transplant, preservation of fertility in young women suffering from pelvic and uterine cancers, etc. The future, in our view, appears full of promise. Already, young cancer survivors are having better quality of life and expecting their own genetic children. Shortly, women will be able to postpone menopause by at least 5–10 years, possibly because of depleted ovarian reserve, to have better quality of living, with no possibility of early menopause, reduction in the incidence of aneuploidy in embryos of women of advance age, and so on.
Taking stock of all these and other positive developments and future possibilities, we can confidently say that we have progressed well through the journey and the future is highly promising.
Lastly, we wish all readers a happy reading and hope that they would add to their skills and efficiency. Thank you all.
EDITORS' COMMENTS
- Demographic characteristics, genetic predisposition, and diverse pathologies should also be considered and extensively studied while performing COS and while assessing its response.
- Individualized COS (iCOS) optimizes outcomes and safety issues by adapting patient specific characteristics. With endocrine, paracrine, and genetic biomarkers iCOS can be refined further.
- Natural and modified natural cycle protocols may be discussed to complete knowledge on COS.
- Assisted reproductive technology is such an ever expanding field, that to summarize it in a few words is next to impossible. From discovering the importance of sex hormones, studying impact of age, hormones on eggs, understanding ovum and embryo genetics, to newer advances in ART technology like Embryoscope, Poloscope, etc., the infertility specialist is exposed to wide option to maximise the success.
SUGGESTED READING
- Alcoba DD, Pimentel AM, Brum IS, et al. Developmental potential of in vitro or in vivo mature oocytes. Zygote. 2015;23(1):93–8.
- Atasever M, Soyman Z, Demirel E, et al. Diminished ovarian reserve: is it a neglected cause in the assessment of recurrent miscarriage? A cohort study. Fertil Steril. 2016;105(5): 1236–40.
- Balaban B, Urman B, Ata B, et al. A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod. 2008;23:1976–82.
- Bar Hava I, Blueshtein M, Ganer Herman H, et al. Gonadotropin-releasing hormone analogue as sole luteal support in antagonist-based assisted reproductive technology cycles. Fertil Steril. 2017;107(1):130–5.e1.
- Benaglia L, Candotti G, Busnelli A, et al. Antral follicle count as a predictor of ovarian responsiveness in women with endometriomas or with a history of surgery for endometriomas. Fertil Steril. 2015;103(6):1544–50.
- Bodri D, Colodron M, Vidal R, et al. Prognostic factors in oocyte donation: an analysis through egg-sharing recipient pairs showing a discordant outcome. Fertil Steril. 2007 88(6):1548–53.
- Broekmans FJ, de Ziegler D, Howles CM, et al. The antral follicle count: practical recommendations for better standardization. Fertil Steril. 2010;94(3):1044–51.
- Broekmans FJ, Kwee J, Hendriks DJ, et al. A systematic review of tests predicting ovarian reserve and in vitro fertilization outcomes. Hum Reprod Update. 2006;12(6):685–718.
- Chabbert-Buffeta N, Skinner DC, Caraty A. Neuroendocrine effects progesterone. Steroids. 2001;65(10-11):613–20.
- Chen ZJ, Shi Y, Sun Y, et al. Fresh versus frozen embryos for infertility in the polycystic ovary syndrome. N Engl J Med. 2016;375(6):523–35.
- Chian RC, Buckett WM, Abdul Jalil AK, et al. Natural-cycle in vitro fertilization combined with in vitro maturation of immature oocytes is a potential approach in infertility treatment. Fertil Steril. 2004;82(6):1675–8.
- Chian RC, Uzelac PS, Nargund G. In vitro maturation of human immature oocytes for fertility preservation. Fertil Steril. 2013 99(5):1173–81.
- Choavaratana R, Thanaboonyawat I, Laokirkkiat P, et al. Outcomes of FSH priming and nonpriming in in vitro maturation of oocytes in infertile women with polycystic ovarian syndrome: a single blinded randomized study. Gynec Obstet Invest. 2015;79(3):153–9.
- Csokmay JM, Hill MJ, Chason RJ, et al. Experience with patient-friendly, mandatory single blastocyst transfer policy: the power of one. Fertil Steril. 2011;96(3):580–4.
- Dain L, Bider D, Levron J, et al. Thin endometrium in donor oocyte recipients: enigma or obstacle for implantation? Fertil Steril. 2013;100(5):1289–95.
- de Ziegler D, Pirtea P, Galliano D, et al. Optimal uterine anatomy and physiology necessary for normal implantation and placentation. Fertil Steril. 2016;105(4):844–54.
- Durlinger AL, Kramer P, Karels B, et al. Control of primordial follicle recruitment by anti-Müllerian hormone in the mouse ovary. Endocrinology. 1999;140:5789–96.
- Durlinger AL, Vissar JA, Themmen AP. Regulation of ovarian function: the role of anti-Müllerian hormone. Reproduction. 2002;124(5):601–9.
- Elgindy EA, El-Haieg DO, El-Sebaey A. Anti-Müllerian hormone: correlation of early follicular, ovulatory and midluteal levels with ovarian response and cycle outcome in intracytoplasmic sperm injection patients. Fertil Steril. 2008;89(6):1670–6.
- Engmann L, Benadiva C. Agonist trigger: what is the best approach? Agonist trigger with aggressive luteal support. Fertil Steril. 2012;97(3):531–3.
- Fitzpatrick LA, Good A. Micronized progesterone: clinical indications and comparison with current treatments. Fertil Steril. 1999;72(3):389–97.
- Grande M, Borobio V, Bennasar M, et al. Role of ovarian reserve markers, antimüllerian hormone and antral follicle count, as aneuploidy markers in ongoing pregnancies and miscarriages. Fertil Steril. 2015;103(5):1221–7.
- Grunfeld L, Walker B, Bergh PA, et al. High resolution endovaginal ultrasonography of the endometrium: a non-invasive test for endometrial adequacy. Obstet Gynecol. 1991;78(2):200–4.
- Harris TG, Dye S, Robinson JE, et al. Progesterone can block transmission of the estradiol-induced signal for luteinizing hormone surge generation during a specific period of time immediately after activation of the gonadotropin-releasing hormone surge-generating system. Endocrinology. 1999;140(2):827–34.
- Hehenkamp WJ, Looman CW, Themmen AP, et al. Anti-Müllerian hormone levels in the spontaneous menstrual cycle do not show substantial fluctuation. J Clin Endocrinol Metab. 2006;91(10):4057–63.
- Homburg R, Ray A, Bhide P, et al. The relationship of serum anti-Müllerian hormone with polycystic ovarian morphology and polycystic ovary syndrome: a prospective cohort study. Hum Reprod. 2013;28(4):1077–83.
- Horcajadas JA, Riesewijk A, Polman J, et al. Effect of controlled ovarian stimulation in IVF on endometrial gene expression profiles. Mol Hum Reprod. 2005;11(3):195–200.
- Huang R, Fang C, Xu S, et al. Premature progesterone rise negatively correlated with live birth rate in IVF cycles with GnRH agonist: an analysis of 2,566 cycles. Fertil Steril. 2012;98(3):664–70.
- Humaidan P, Kol S, Papanikolaou EG; Copenhagen GnRH Agonist Triggering Workshop Group. GnRH agonist for triggering of final oocyte maturation: time for a change of practice? Hum Reprod Update. 2011;17(4):510–24.
- Jayaprakasan K, Deb S, Batcha M, et al. The cohort of antral follicles measuring 2-6 mm reflects the quantitative status of ovarian reserve as assessed by serum levels of anti-Müllerian hormone and response to controlled ovarian stimulation. Fertil Steril. 2010;94(5):1775–81.
- Kansal Kalra S, Ratcliffe SJ, Milman L, et al. Perinatal morbidity after IVF is lower with frozen embryo transfer. Fertil Steril. 2011;95(2):548–53.
- Kasius A, Smit JG, Torrance HL, et al. Endometrial thickness and pregnancy rates after IVF: a systematic review and meta-analysis. Hum Reprod Update. 2014;20:530–41.
- Kiliçdag EB, Haydardedeoglu B, Cok T, et al. Premature progesterone elevation impairs implantation and live birth rates in GnRH-agonist IVF/ICSI cycles. Arch Gynecol Obstet. 2010;281(4):747–52.
- Kitajima M, Defrère S, Dolmans MM, et al. Endometriomas as a possible cause of reduced ovarian reserve in women with endometriosis. Fertil Steril. 2011;96(3):685–91.
- Kitajima M, Dolmans MM, Donnez O, et al. Enhanced follicular recruitment and atresia in cortex derived from ovaries with endometriomas. Fertil Steril. 2014;101(4):1031–7.
- Kunz G, Beil D, Huppert P, et al. Adenomyosis in endometriosis—prevalence and impact on fertility. Evidence from MRI. Hum Reprod. 2005;20(8):2309–16.
- Kwee J, Schats R, McDonnell J, et al. Evaluation of anti-Müllerian hormone as a test for the prediction of ovarian reserve. Fertil Steril. 2008;90;3737–43.
- La Marca A, Stabile G, Artenisio AC, et al. Serum anti-Müllerian hormone throughout the human menstrual cycle. Hum Reprod. 2006;21(12):3103–7.
- La Marca A, Sunkara SK. Individualization of controlled ovarian stimulation in IVF using ovarian reserve markers: from theory to practice. Hum Reprod Update. 2014;20(1):124–40.
- Lambert-Messerlian G, Plante B, Eklund EE, et al. Levels of anti-müllerian hormone in serum during the normal menstrual cycle. Fertil Steril. 2016;105(1):208–13.
- Lashen H, Afnan M, Sharif K, et al. A controlled comparison of ovarian response to controlled stimulation in first generation Asian women compared with white Caucasians undergoing in vitro fertilisation. Br J Obstet Gynaecol. 1999;106(5):407–9.
- Lopata A, Johnston IW, Hoult IJ, et al. Pregnancy following intrauterine implantation of embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril. 1980;33(2):117–20.
- Maheshwari A, Bhattacharya S. Elective frozen replacement cycles for all: ready for prime time? Hum Reprod. 2013;28(1): 6–9.
- Maheshwari A, Pandey S, Shetty A, et al. Obstetric and perinatal outcomes in singleton pregnancies resulting from the transfer of frozen thawed versus fresh embryos generated through in vitro fertilization treatment: a systematic review and meta-analysis. Fertil Steril. 2012;98(2):368–77.e1-9.
- Mahmud G, López Bernal A, Yudkin P, et al. A controlled assessment of the in vitro fertilization performance of British women of Indian origin compared with white women. Fertil Steril. 1995;64(1):103–6.
- Marinov B, Petkova S, Dukovski A, et al. Utrogestan and high risk pregnancy. Akush Ginekol (Sofiia). 2004;43(5):22–4.
- Meyer L, Murphy LA, Gumer A, et al. Risk factors for a suboptimal response to gonadotropin-releasing hormone agonist trigger during in vitro fertilization cycles. Fertil Steril. 2015;104(3):637–42.
- Nelson SM. Biomarkers of ovarian response: current and future applications. Fertil Steril. 2013;99(4):963–9.
- Nelson SM, Fleming R, Gaudoin M, et al. Anti-müllerian hormone levels and antral follicle count as prognostic indicators in a personalized prediction model of live birth. Fertil Steril. 2015;104(2):325–32.
- Pigny P, Jonard S, Robert Y, et al. Serum anti-Müllerian hormone as a surrogate for antral follicle count for definition of the polycystic ovary syndrome. J Clin Endocrinol Metab. 2006;91(3):941–5.
- Pirard C, Donnez J, Loumaye E. GnRH agonist as luteal phase support in assisted reproduction technique cycles: results of a pilot study. Hum Reprod. 2006;21(7):1894–900.
- Pirard C, Donnez J, Loumaye E. GnRH agonist as novel luteal support: results of a randomized, parallel group, feasibility study using intranasal administration of buserelin. Hum Reprod. 2005;20(7):1798–804.
- Pirard C, Loumaye E, Laurent P, et al. Contribution to more patient-friendly ART treatment: efficacy of continuous low-dose GnRH agonist as the only luteal support-results of a prospective, randomized, comparative study. Int J Endocrinol. 2015;2015:727569.
- Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology. In vitro maturation: a committee opinion. Fertil Steril. 2013;99(3):663–6.
- Raffi F, Metwally M, Amer S. The impact of excision of ovarian endometrioma on ovarian reserve: a systematic review and meta-analysis. J Clin Endocrinol Metab. 2012;97(9):3146–54.
- Reh A, Fino E, Krey L, et al. Optimizing embryo selection with day 5 transfer. Fertil Steril. 2010;93(2):609–15.
- Rodin DA, Bano G, Bland JM, et al. Polycystic ovaries and associated metabolic abnormalities in Indian subcontinent Asian women. Clin Endocrinol. 1998;49(1):91–9.
- Roque M, Valle M, Guimarães F, et al. Freeze-all policy: fresh vs. frozen-thawed embryo transfer. Fertil Steril. 2015;103(5):1190–3.
- Schindler AE, Campagnoli C, Druckmann R, et al. Classification and pharmacology of progestins. Maturitas. 2003;46:S7–16.
- Shah MS, Caballes M, Lathi RB, et al. In vitro fertilization outcomes after fresh and frozen blastocyst transfer in South Asian compared with Caucasian women. Fertil Steril. 2016;105(6):1484–7.
- Shahine LK, Lamb JD, Lathi RB, et al. Poor prognosis with in vitro fertilization in Indian women compared to Caucasian women despite similar embryo quality. PloS One. 2009 4(10):e7599.
- Shapiro BS, Daneshmand ST, Garner FC, et al. Clinical rationale for cryopreservation of entire embryo cohorts in lieu of fresh transfer. Fertil Steril. 2014;102(1):3–9.
- Shapiro BS, Daneshmand ST, Garner FC, et al. Freeze all can be a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure. Reprod Biomed Online. 2014;29(3):286–90.
- Shih W, Rushford DD, Bourne H, et al. Factors affecting low birth weight after assisted reproductive technology: difference between transfer of fresh and frozen embryo transfer suggests an adverse effect of oocyte collection. Hum Reprod. 2008;23(7):1644–53.
- Smith LC, Olivera-Angel M, Groome NP, et al. Oocyte quality in small antral follicles in the presence or absence of a large dominant follicle in cattle. J Reprod Fertil. 1996;106(2):193–9.
- Somigliana E, Berlanda N, Benaglia L, et al. Surgical excision of endometriomas and ovarian reserve: a systematic review on serum anti-müllerian level modifications. Fertil Steril. 2012;98:1531–8.
- Stillman RJ, Richter KS, Banks NK, et al. Elective single embryo transfer: a 6-year progressive implementation of 784 single blastocyst transfers and the influence of payment method on patient choice. Fertil Steril. 2009;92(6):1895–906.
- Takeshima K, Jwa SC, Saito H, et al. Impact of single embryo transfer policy in fresh and frozen cycles—analysis of the Japanese Assisted Reproduction Technology registry in Japan between 2007 and 2012. Fertil Steril. 2016;105(2):337–46.
- Tarasconi B, Tadros T, Ayoubi JM, et al. Serum antimüllerian hormone levels are independently related to miscarriage rates after in vitro fertilization-embryo transfer. Fertil Steril. 2017;108;3:518–24.
- Tesarik J, Hazout A, Mendoza C. Enhancement of embryo developmental potential by a single administration of GnRH agonist at the time of implantation. Hum Reprod. 2004;19(5):1176–80.
- Trounson A, Wood C, Kausche A. In vitro maturation and fertilization and developmental competence of oocytes recovered from untreated polycystic ovary syndrome patients. Fertil Steril. 1994;62:353–62.
- van Rooij IA, Broekmans FJ, Scheffer GJ, et al. Serum anti-müllerian hormone levels best reflect the reproductive decline with age in normal women with proven fertility: a longitudinal study. Fertil Steril. 2005;83(4):979–87.
- van Rooij IA, Tonkelaar Id, Broekmans FJ, et al. Anti-müllerian hormone is a promising predictor for the occurrence of the menopausal transition. Menopause. 2004;11:601–6.
- Vercellini P, Consonni D, Dridi D, et al. Uterine adenomyosis and IVF outcome: a systematic review and meta-analysis. Hum Reprod. 2014 29(5):964–77.
- Vigier B, Tran D, Legeai L, et al. Origin of anti-Müllerian hormone in bovine freemartin fetuses. J Reprod Fertil. 1984;70:473–9.
- Wang A, Santistevan A, Hunter Cohn K, et al. Freeze-only versus fresh embryo transfer in a multicenter matched cohort study: contribution of progesterone and maternal age to success rates. Fertil Steril. 2017;108(2):254–61.e4.
- Weissman A, Gotlieb L, Casper RF. The detrimental effect of increased endometrial thickness on implantation and pregnancy rates and outcome in IVF program. Fertil Steril. 1999;71(1):147–9.
- Yding Andersen C, Leonardsen L, Ulloa-Aguirre A, et al. FSH-induced resumption of meiosis in mouse oocytes: effect of different isoforms. Mol Hum Reprod. 1999;5(8):726–31.
- Zhao J, Zhang Q, Wang Y, et al. Endometrial pattern, thickness and growth in predicting pregnancy outcome following 3319 IVF cycle. Reprod Biomed Online. 2014;29(3):291–8.
- Zhu X, Ye H, Fu Y, et al. Use of Utrogestan during controlled ovarian hyperstimulation in normally ovulating women undergoing in vitro fertilization or intracytoplasmic sperm injection treatments in combination with a “freeze all” strategy: a randomized controlled dose-finding study of 100 mg versus 200 mg. Fertil Steril. 2017;197(2):379–86.
- Zhu X, Ye H, Fu Y. The Utrogestan and hMG protocol in patients with polycystic ovarian syndrome undergoing controlled ovarian hyperstimulation during IVF/ICSI treatments. 2016;95(28):e4193.