Abstract
There is a need to develop rapid protocols for ovarian stimulation for women who wish to preserve their fertility following diagnosis of cancer. Conventional gonadotropin stimulation protocols are lengthy and are delayed until the start of the next menstrual period, potentially compromising cancer treatments. The development of random start IVF/in vitro maturation has made significant strides for enabling couples undergoing cancer therapy to achieve a family. However, several unanswered questions still remain. What do we know about the endocrinology of stimulating ovarian follicular activity outside the established protocols of stimulation during the follicular phase? This article explores what is known about antral follicle development during the menstrual cycle, novel ovarian stimulation proposals for optimizing assisted reproductive therapies in women, and direction.
Current cancer treatments in women are cytotoxic to the ovaries, leading to reduced fertility [1]. With advancements in cancer therapy, there is an increasing need to develop methodologies to preserve fertility prior to radiation or chemotherapy [2–6]. In some instances of aggressive cancer, cancer treatment must be initiated at the time of diagnosis or as soon as possible thereafter. Historically, women undergoing cytotoxic chemotherapy have had to delay fertility treatment and resort to cryopreservation of ovarian cortical biopsies for use once their cancer treatment was completed. In such cases, conventional IVF strategies may have resulted in an unacceptable delay of several weeks before chemotherapy could begin [2]. In 2010, Bentov and colleagues [7] documented an ongoing pregnancy from two waves of follicles developing during a long follicular phase of the same cycle. Similarly, clinical case reports of successful luteal phase oocyte retrieval and in vitro maturation were reported as an optional procedure for urgent fertility preservation [8,9]. Based on these clinical cases, an increasing body of evidence has developed to support the notion of random start IVF/in vitro maturation (IVM), also referred to as emergency IVF/IVM in cancer patients. Using these approaches, treatment is initiated at any time of the menstrual cycle [10–13], oocytes are collected, fertilized in vitro and resultant embryos are cryopreserved for transfer after completion of cancer therapy.
Folliculogenesis during the menstrual cycle
It has been generally understood that antral ovarian follicles develop during the follicular phase of the menstrual cycle, leading to ovulation of the dominant follicle at mid cycle, while the corpus luteum (CL) functions during the luteal phase in the absence of antral follicle growth. Production of estradiol and progesterone from the CL was thought to have an inhibitory effect on antral follicle development during the luteal phase. In early histologic and endocrinologic studies, antral follicles detected in the luteal phase were thought to be atretic, based on granulosa cell number and oocyte viability [14]. The number of nonatretic follicles was thought to be small (0–4 per ovary). The largest healthy follicles were <5 mm in size and were associated with very low concentrations of estradiol and negligible aromatase activity. The authors concluded that antral follicles observed in the human luteal phase were predominantly atretic and that the number of healthy follicles available for subsequent preovulatory development in women was limited.
By contrast, the use of serial high resolution ultrasonography in both mono-ovular domestic farm animals [15] and women [16,17] has revealed that multiple waves of follicle development occur across the estrous/menstrual cycle. Each wave of follicle growth is characterized by a rise and fall in a number of co-developing antral follicles ≥4–6 mm. Studies have been conducted over an interovulatory interval (IOI) rather an estrous/menstrual cycle, in order to maintain methodologic consistency among species. An IOI is defined as the time point from one ovulation to the subsequent ovulation (i.e., luteal phase followed by follicular phase). In both women and domestic farm animals, 2–3 waves of antral follicle growth were detected across the cycle. In most women (68%), two follicles waves were observed over the IOI, with a minority of women (32%) exhibiting three waves. No ovulatory woman of reproductive age was observed to develop a single wave of follicle growth over the IOI. Major and minor follicle waves have been characterized in women, consistent with studies in mares [18,19]. Major waves are those in which a dominant follicle is selected for preferential growth at the expense of subordinate antral follicles; minor waves are those in which dominant follicle selection does not occur. The final wave of the follicular phase is ovulatory, while all preceding waves (in the follicular or luteal phases) are anovulatory. In other words, the final wave of the IOI leads to ovulation, while all preceding waves are either major anovulatory or minor waves.
It was recently shown [20] that 50% of healthy reproductive age women developed luteal phase dominant follicles (LPDFs). The development of a LPDF was associated with a 46% increase in antral follicle count (AFC) 2–10 mm, a 197% increase in AFC ≥6 mm, a 260% increase in serum inhibin B and a 77% increase in serum estradiol, relative to women without a LPDF. Luteal phase serum FSH, LH, progesterone, inhibin A and AMH were similar among groups. Mean follicular and endocrine profiles of women with and without a LPDF are presented in Figures 1 & 2. An ultrasonographic image of a LPDF developing simultaneously with a CL is presented in Figure 3. The prevalence of LPDFs did not change with age, although the growth dynamics of LPDFs differed in older compared with younger women. In women of advanced-reproductive age, LPDFs emerged earlier relative to ovulation, developed over a longer period of time (often persisting into the subsequent follicular phase), and grew to a larger diameter (∼33 mm, often exceeding a preovulatory diameter) compared with younger women [20]. The atypically large and persistent LPDFs were associated with a 188% increase in mean luteal phase estradiol and a 47% reduction in luteal progesterone compared with younger women with LPDFs. A case example is presented in Figure 2. In most cases, LPDFs in the older women were anovulatory; however, in one woman the LPDF ovulated during menses, resulting in three ovulations during her cycle [21].
Conventional ovarian stimulation protocols
IVF procedures are becoming increasingly streamlined and efficient [22]. The conventional and most common approach is to stimulate the ovaries to provide sufficient numbers of mature oocytes to maximize pregnancy outcomes. Typically, gonadotropin therapy is initiated on day three post menses to stimulate the growth of multiple antral follicles. When a predetermined number of follicles reach a preovulatory diameter of >16 mm, hCG (a potent natural luteinizing hormone agonist) is administered to induce final oocyte maturation. Approximately 35 h post hCG, mature follicles are aspirated. Oocytes are collected and fertilized in vitro, with the resultant fresh embryos either transferred or cryopreserved for transfer at a later date.
A number of variations to this basic design have been used. These include: treatment with GnRH agonists beginning in the mid-luteal phase of the cycle preceding FSH ovarian stimulation, downregulation of endogenous pituitary gonadotropin secretion which results in an improved response and prevents premature ovulation [23]; use of GnRH antagonists concurrently with FSH stimulation to inhibit premature ovulation [24]; and use of estradiol antagonists (e.g., clomiphene citrate) or aromatase inhibitors (e.g., letrozole) to amplify endogenous gonadotropin secretion by reducing estradiol negative feedback on gonadotropin secretion [25]. All three of these variations in stimulation protocol involve starting FSH, clomiphene or letrozole during the early-mid follicular phase.
Regardless of the protocol used, serial follicle development is monitored throughout stimulation using transvaginal ultrasonography and serum estradiol concentrations. The gonadotropin dose required may be adjusted throughout the stimulation, according to the ovarian response. Excessive gonadotropin doses can lead to growth of many antral follicles with highly elevated circulating concentrations of estradiol and VEGF. Increased capillary leakage may follow, with accumulation of ascitic fluid into the peritoneal, pleural and pericardial spaces and concurrent intravascular hemoconcentration. This phenomenon of ovarian hyperstimulation syndrome is potentially life threatening.
Conventional ovarian stimulation protocols have been developed based on the generalized view that FSH is rate-limiting in the natural cycle, and that exogenous gonadotropins rescue antral follicles that would otherwise undergo atresia due to insufficient exposure to FSH [26]. The extent to which the induced growth of multiple preovulatory follicles reproduces the natural processes of follicle and oocyte development during the natural ovarian cycle remains debatable. Nonetheless, standard protocols involve initiating stimulation in the early follicular phase. Using this approach, implantation occurs following endometrial exposure to supra-physiologic concentrations of estradiol for approximately 2 weeks.
Random-start or emergency IVF protocols
A number of random-start methodologies have been evaluated (Table 1). In the follicular phase, both short GnRH agonist and GnRH antagonist protocols have been initiated on various days [27,28]. In the luteal phase, GnRH-antagonist has been administered simultaneous with FSH initiation. The latter protocol is referred to as the ‘modified GnRH-antagonist protocol’; the administration of GnRH-antagonist is thought to induce luteolysis and prevent potential inhibitory effects of progesterone on follicle development [28,29]. Estradiol receptor antagonists (e.g., letrozole) have been further used in combination with gonadotropins during the luteal phase. Co-treatment with letrozole is designed to minimize the increase in circulating levels of estradiol seen during luteal phase FSH stimulation and minimize any inhibitory effects of luteal progesterone on endogenous gonadotropin secretion. Reduction of circulating concentrations of estradiol levels is particularly important in treating women with estrogen sensitive cancers [25,27,30–31]. In both follicular and luteal phase stimulations, final oocyte maturation was induced using a GnRH agonist or hCG. Oocytes were aspirated, fertilized and cultured as per standard IVF methods. Resulting embryos were cryopreserved for transfer following cancer treatment.
Comparison of protocols used in random start IVF, in vitro maturation and conventional ovarian stimulation procedures and their outcomes.
The stimulation protocol (stages 1, 2, 3) refers to the initial stimulation followed by treatment with GnRH-antag (when follicles reach 12–14 mm to prevent premature ovulation) with a maturation dose of hCG or GnRHa.
Letrozole was administered when the patient had an estrogen-sensitive malignancy.
Clinical pregnancy rate.
Ongoing pregnancy rate. ## FSH added to low E2 responders in FP.
FP: (early, late) follicular phase; GnRHa: GnRH agonist; GnRH-antag: GnRH antagonist; hCG: Human chorionic gonadotropin; hMG: Human menopausal gonadotropin; IVM: In vitro maturation; LP: Luteal phase.
Compared with conventional follicular phase ovarian stimulation, luteal phase stimulation resulted in comparable fertilization, implantation and pregnancy rates (see Table 1 references). However, a longer FSH treatment period and higher FSH doses have been required using luteal phase protocols. Interestingly, the co-administration of GnRH antagonist or aromatase inhibitors in the luteal phase did not influence success rates (Table 1). The consequences of luteal phase stimulation and the proportion of fertilized embryos that resulted in live births and the incidence of live birth defects were comparable with conventional IVF [32].
In comparison to women, ovarian stimulation in domestic farm animals is initiated at multiple times during the estrous cycle in order to optimize IVF outcomes for the cattle breeding industry [33–37].
Random start or emergency IVM protocols
Random-start stimulation approaches are also relevant to oocyte in vitro maturation (IVM). IVM is a variation on conventional IVF, which involves minimal or no gonadotropin stimulation [38]. Oocytes are collected at immature stages of antral follicle growth and matured in vitro prior to IVF or ICSI [38,39]. As oocyte maturation does not occur in vivo, the controlled generation of large preovulatory follicles achieved through exogenous gonadotropin treatment is not needed. Random-start IVM is in widespread use for domestic animal breeding, particularly in cows, where approximately 500,000 offsprings are produced each year. Animals typically receive no gonadotropin stimulation and immature oocytes are collected at any stage of the cycle, including the luteal phase. Such an approach would be very attractive to cancer patients requiring urgent oocyte collection.
The clinical IVM protocol most commonly employed involves 3 days of FSH beginning on day 3 post menses. Follicle growth is monitored ultrasonographically and immature oocytes are collected at a diameter of approximately 8–10 mm. Following 24–36 h of IVM, mature oocytes are fertilized and cultured using standard IVF/ICSI procedures. The first IVM pregnancies were reported in 1991 [40]. However, low efficiencies of IVM compared with conventional IVF limited its uptake [41]. Recent improvements in IVM success rates [42,43] have increased its appeal, as it is simpler, safer, cheaper and less invasive than IVF. Phenotypic and molecular examinations of resultant embryos and liveborn children have provided evidence that IVM is a safe practice [44–46].
Random-start IVM procedures have been developed based on the premise that a wave of mid-sized antral follicles is available for collection and subsequent IVM at any stage of the menstrual cycle. There are a few reports of IVM without the use of exogenous gonadotropins in women [47,48]. In comparison, ‘hCG-primed IVM’ has been developed to retrieve oocytes in the luteal phase. In a study by Maman [9], follicles of 8.2 mm (luteal phase) versus 11.5 mm (follicular phase) were identified for IVM. On average, 13 oocytes were aspirated in the luteal phase compared with 17 oocytes in the follicular phase; similar oocyte maturation, fertilization rates and oocyte/embryo numbers were reported between the luteal and follicular phase protocols. More recently, IVM has been reported in women requiring emergency oophorectomy for fertility preservation prior to chemotherapy [49,50]. In both reports, oocytes were aspirated from unstimulated antral follicles within the excised ovary and matured in vitro; the remaining ovarian cortex was cryopreserved. The resultant embryos were cryopreserved, thawed and transferred upon completion of chemotherapy. Such ex vivo IVM has led to pregnancies in cancer survivors [49,50]. Based on human IVF and IVM studies to date as well as long held applications of domestic animal breeding, it is clear that oocytes obtained from either the follicular or luteal phases of the menstrual/estrous cycle are competent for fertilization, implantation and pregnancy.
Future directions
It is recognized that random start IVF/IVM technologies are relatively new with few comprehensive studies available. However the proposition that oocytes can be readily obtained from ovaries throughout the menstrual cycle with comparable outcomes to conventional IVF procedures is well supported. Continued research is required to address the following unanswered questions:
Future research is required to elucidate the physiologic associations between follicle and luteal dynamics throughout the human menstrual cycle. Initial random-start IVF methodologies were developed with cryopreservation of the oocyte/embryo in mind. Cryopreservation is the preferred outcome, since fresh embryo transfer would be unlikely to lead to pregnancy as the timing of endometrial receptivity would not be synchronized with embryo development. In comparison to women, it is common practice to induce luteolysis for initiating stimulation in association with follicle wave emergence during the luteal phase in domestic farm animal species [34,35]. It is plausible that random start IVF may also be of benefit to women with a failed response to ovarian stimulation, wishing to reinitiate therapy without waiting for the onset of their next menstrual cycle. Continued investigations are needed to evaluate the efficacy of luteal phase stimulation in women with a poor response to IVF treatment; It is not currently known whether the presence of a LPDF will have a deleterious effect on random IVF/IVM start outcomes. Typically, if a large dominant follicle is present at the time when conventional stimulation is planned, one of the following strategies may be used: wait until it regresses before initiating stimulation; if the follicle is persistent, administer hormonal contraceptives to cause the follicle to regress before initiating FSH; if the follicle is persistent, aspirate it before initiating FSH; or try to stimulate additional smaller follicles around the persistent follicle. With this in mind, timing of initiation may be important. Will outcomes be similar if we initiate stimulation in the absence of a LPDF? Futher research is needed to test this hypothesis. Furthermore, one may expect that gonadotropin stimulation in the luteal phase of women with a LPDF would lead to higher serum estradiol compared with women without LPDFs. If so, will there be a differential effect on follicle or endometrial development? Is a greater serum concentration of estradiol likely to be exacerbated in women with low ovarian reserve, as occurs frequently in women of older reproductive age? Is it likely to be similar in women with premature ovarian failure or in women who already have experienced gonadotoxic drugs? The co-administration of letrozole to suppress serum estradiol levels in such women does appear to be a sound precaution, particularly those women with estradiol-sensitive cancers; Continued research is required to study the efficacy of gonadotropin treatment initiated at various stages across the menstrual cycle. The concept of follicular waves as seen in domestic animals and humans suggests that optimal FSH stimulation may require treatment at specific times of the cycle. Certainly, in mono-ovular animals [51,52] protocols for synchronizing stimulation with follicle wave emergence have been widely employed for optimizing assisted reproductive outcomes. Bovine oocyte quality has been shown to be reduced when oocytes are collected in the dominance phase of a wave compared with the growth phase of a wave, irrespective of which wave of the cycle [53]. Baerwald and colleagues [54] investigated the effects of synchronizing follicle wave emergence with ovarian stimulation on IVF outcomes in women. Synchronization of stimulation with wave emergence (i.e., day 1 vs day 4) resulted in an increase in the number of dominant follicles and serum estradiol concentrations; however, improvements in oocyte, embryo or pregnancy outcomes did not occur. These results suggest that oocyte number, but perhaps not quality, is influenced by the timing of FSH treatment. Increasing importance is being placed on oocyte quality versus quantity. Are low oocyte recoveries necessarily a poor index of pregnancy outcome? Fewer oocytes collected at the correct phase of follicular dominance may yield oocytes of higher quality leading to better pregnancy rates. Thus higher oocyte quantity may not translate into better oocyte quality. The notion of oocyte quality as a key factor in optimizing assisted reproduction outcomes is apparent in the implementation of minimal ovarian stimulation protocols over the past 5 years;
Conclusion
In this article, we explored novel approaches for fertility preservation before initiation of gonadotoxic cancer treatments in women. A variety of ovarian stimulation methodologies have been previously explored utilizing GnRH antagonists, hCG/GnRH agonists and aromatase inhibitors. Random start IVF/IVM methodologies have been developed whereby treatment may be initiated in the luteal phase, with similar oocyte, embryo and pregnancy rates compared with those conventional follicular phase ovarian stimulation. Continued research is required to elucidate the effects of the corpus luteum on luteal phase follicle dynamics in women. The presence of estrogenic LPDFs with elevated serum estradiol is more pronounced in older women and may be a complication with some ovarian stimulation protocols. It is thus suggested that follicular development, duration and stage when treatment is initiated, as well as the changes in the serum estradiol levels are monitored when treatment is initiated in the luteal phase
Future perspective
It does appear that in situations where a rapid ART protocol is desirable for fertility reasons, in particular before cancer treatment, random-start IVF or IVM procedures have merit. Research to date has shown that initiation of ovarian stimulation or oocyte collection at variable stages of the menstrual cycle can lead to a comparable pregnancy outcome; however, differences in efficiencies relative to IVF have been noted. Luteal phase dominant follicles develop in 50% of women, and produce considerable amounts of estradiol. With this in mind, careful monitoring of estradiol levels or use of aromatase inhibitors should be considered to minimize the effects of luteal hormone production on luteal phase follicle growth. The use of random-start IVF/IVM has been further suggested as a broader application for use as an appropriate IVF/IVM methodology prior to cryopreservation. Random-start IVF methodologies were developed with cryopreservation of the oocyte/embryo in mind. Cryopreservation is the preferred outcome, since fresh embryo transfer would be unlikely to lead to pregnancy as the timing of endometrial receptivity would not be synchronized with embryo development. Future perspective should concentrate on a more detailed assessment of efficiencies across the cycle and a careful monitoring of ovarian responses, particularly with the presence of large luteal phase follicles.
Executive summary
Random-start IVF/in vitro maturation (IVM) is a rapid oocyte collection procedure for women who wish to preserve their fertility following diagnosis of cancer. Treatment is initiated immediately using IVF or IVM procedures in either the follicular or luteal phase, oocytes are retrieved, and the resulting embryos are cryopreserved for transfer upon completion of cancer therapy. However, the physiologic mechanisms underlying ovarian stimulation, in particular during the luteal phase of the menstrual cycle, are not fully understood.
A number of random-start IVF/IVM protocols have been developed, using gonadotropins in combination with GnRH agonists, GnRH antagonists and/or aromatase inhibitors. Protocols developed to date have resulted in assisted reproductive outcomes comparable to those obtained following conventional follicular phase stimulation procedures.
Two-three coordinated waves of antral follicles develop across the menstrual cycle. Luteal phase dominant follicles (LPDFs) have been identified in 50% of women. Serum estradiol and inhibin B are elevated while progesterone is decreased in association with LPDFs. Other reproductive hormones remain unchanged.
To what extent does the presence of a corpus luteum and/or LPDF influence random start ovarian stimulation or IVM outcomes? Are aromatase inhibitors needed to offset greater estradiol levels in women with LPDFs present at the time of treatment initiation, particularly in those with steroid sensitive cancers?
Random-start IVF/IVM protocols have been developed, and provide comparable fertilization and pregnancy rates to conventional follicular phase assisted reproduction protocols. Continued research is required to characterize the physiologic mechanisms underlying random-start IVF/IVM strategies and factors (e.g., timing of treatment initiation) which may influence outcomes.
Footnotes
This study was supported by research grants from the Canadian Institutes of Health Research; Canadian Foundation for Women's Health; Establishment Grant from the University of Saskatchewan; the National Health and Medical Research Council of Australia (Program Grant # 494802 and Research Fellowships, DM Roberston #169201, RB Gilchrist #441023210); and the Victorian Government's Operational Infrastructure Support Program. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.