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Evidence that oocyte quality in younger women with diminished oocyte reserve is superior to those of women of advanced reproductive age
Medical Hypotheses 74 (2010) 264–267
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Evidence that oocyte quality in younger women with diminished oocyte reserve is superior to those of women of advanced reproductive age J.H. Check *, R. Cohen The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School at Camden, Cooper Hospital/University Medical Center, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Camden, NJ, USA
a r t i c l e
i n f o
Article history: Received 1 September 2009 Accepted 10 September 2009
s u m m a r y Evidence is provided supporting the hypothesis that the much improved prognosis for younger women with diminished oocyte reserve compared to women of advanced reproductive age is related to a difference in mechanism for oocyte depletion. For younger women the majority have had destruction of certain portions of their ovarian tissue but the remaining spared ovarian tissue has proportionately the same percentage of normal follicles as their age peers. The hypothesis continues that some factor that is responsible for earlier development of primary to antral follicles persists with the early conceptus and protects it from early programmed cell death. Thus by natural selection most women of advanced reproductive age have oocytes that fertilize normally and produce normal morphologic embryos, but lacking this apoptosis inhibiting factor dies very early between days 6 and 12 from fertilization. Ó 2009 Elsevier Ltd. All rights reserved.
Effect of aging on oocyte quantity and quality There is a decline in fecundity that accelerates between 35 and 40 years of age and approaches zero by age 45 [1]. As one ages, or as menopause approaches, there is a paucity of ovarian follicles [2]. With less total follicles remaining there are less follicles reaching the antral follicle stage (5–9 mm). Biological peptides released from the antral follicles, e.g., inhibin B suppresses the release of follicle stimulating hormone (FSH) from the pituitary. With less antral follicles there is less inhibin B leading to an increase in serum FSH. For women still ovulating the best time to measure serum FSH to use it as an indicator for diminished oocyte reserve is in the early follicular phase when serum estradiol (E2) is also at its lowest. Estradiol also exerts an inhibitory effect on release of FSH. Thus measuring serum FSH at the peak E2 could allow suppression of serum FSH even with diminished antral follicles and thus less inhibin B, thus resulting in wrong conclusions about oocyte reserve. The antral follicles recruited in women of advanced reproductive age are not only fewer in number than younger women but they contain oocytes of inferior quality. These oocytes not only have extra chromosomes but have defective cytoplasmic mitochondria [3]. The defective cytoplasmic mitochondria could lead to apoptosis of the embryo somewhere between days 5 and 12 [4]. One hypothesis is that the presence of more of a certain cytoplasmic mitochondrial factor is what may be responsible for the recruitment of a given number of antral follicles at a given age.
* Corresponding author. Address: 7747 Old York Road, Melrose Park, PA 19027, USA. Tel.: +1 215 635 4400; fax: +1 215 635 2304. E-mail address: [email protected] (J.H. Check). 0306-9877/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2009.09.018
With less of this factor present, the embryo formed is likely to have programmed cell death somewhere between days 5 and 12 of life.
Studies suggesting oocyte quality in younger patients with diminished oocyte reserve is poor resulting in very low chance of pregnancy Since one of the manifestations of diminished oocyte reserve in women of advanced reproductive age is an elevated day 3 serum FSH, it was assumed that a younger woman with an elevated day 3 serum FSH must have similar remaining oocytes to older women. Thus it was concluded that these younger women not only have few remaining oocytes but they are of very poor quality. Indeed in both the early days of in vitro fertilization-embryo transfer (IVF-ET) and in modern times many publications demonstrate very poor pregnancy rates in younger women with increased day 3 serum FSH despite the transfer of normal morphologic embryos [5– 12]. One of the finest IVF centers in the early IVF era concluded that if the serum FSH is ever elevated in one menstrual cycle on day 3 this means that the quality of remaining oocytes are poor similar to women of advanced reproductive age, i.e., >45 [10]. These researchers stated that this conclusion would apply even if the serum FSH was in the normal range indicating at least in that cycle of more antral follicles [10]. These conclusions were mirrored by one of the world’s best IVF centers finding no live pregnancies despite the transfer of embryos of good quality in women of any age using a serum FSH cut-off of 15 mIU/mL [11]. These data supported the concept that younger women with diminished egg reserve must
J.H. Check, R. Cohen / Medical Hypotheses 74 (2010) 264–267
have as the main mechanism of their problem an acceleration of the normal rate of oocyte atresia. Evidence refuting consensus that diminished oocyte reserve in younger women associated with poor quality oocytes A recent study of women aged 39.9 and under with such diminished oocyte reserve that only a single embryo was available for transfer found the clinical pregnancy rate for the 63% who had an embryo with 6, 7, or >8 blastomeres to be 38%, 40%, and 42.4%, respectively, [13]. The respective live delivered pregnancy rates were 31%, 25%, and 36.4% per transfer [13]. The mean day 3 serum FSH (mIU/mL) and ranges for these three groups were 25.3 ± 17 (19.2–31.4), 23.3 ± 9.7 (18.7–27.8), and 18.0 ± 6.9 (15.6– 20.6). Thus in most instances the serum FSH was higher than 15 mIU/mL [13]. A group of women 39.9 with elevated serum FSH (mean 18.9 ±8.3 mIU/mL with a range of 13–43) were treated without IVF-ET for infertility. The infertility treatment included luteal phase support with vaginal progesterone and correcting follicular maturation defects, luteinized unruptured follicle syndrome, cervical and/or male factor, e.g., with intrauterine insemination [14]. The clinical and live delivered pregnancy rates within 6 months of therapy were 46.1% and 34.6%, respectively, [14]. There is evidence that when a woman appears to be in overt menopause with amenorrhea, estrogen deficiency and elevated FSH and failure to menstruate following progesterone withdrawal, that there are still approximately 1000 follicles left [15]. However, all a woman needs is the recruitment and development of one dominant follicle to produce estrogen to allow proliferation of the endometrium, and with ovulation, the additional secretion of progesterone leading to spontaneous menses if successful fertilization did not take place upon the withdrawal of progesterone. The fact that these follicles do not develop has been assumed to be related to very poor quality and the reason why they never developed in the 40 years of menstrual life. There is evidence that this may be related to apoptosis of the granulosa cells surrounding the oocyte [16]. It is assumed because of the paucity of case reports of pregnancies after ‘‘menopause” that even if a rare spontaneous ovulation does occur pregnancy is highly unlikely because of poor oocyte quality. This thought process would apply to not just women of advanced reproductive age with natural menopause but younger women with premature ovarian failure [5–12]. However, there may be an alternative explanation as to why these few remaining primary or pre-antral follicles do not develop into dominant follicles. The possibility exists that they do have potential to develop into dominant follicles but they have become insensitive to FSH. It is well known that most hormones require binding to a receptor for that hormone to provide its function. When too much hormone exists, as a protective mechanism, frequently the target cell will demonstrate down-regulation of the receptors (a prime example is insulin and its receptor). We hypothesized that chronically elevated FSH levels could down-regulate FSH receptors in the granulosa theca cell layer. This could cause gonadotropin-resistance and thus inhibit follicular development. The hypothesis continued that this insensitivity should be potentially reversible by lowering the chronically elevated serum FSH and thus restoring down-regulated FSH receptors. Evidence to support the hypothesis that lowering FSH can restore sensitivity of the remaining follicles in women with diminished oocyte reserve Support of this hypothesis was provided in 1984 by attempting to lower FSH with estrogen to theoretically restore down-regulated
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FSH receptors and thus restoring sensitivity of the follicles followed by stimulation with human menopausal gonadotropins in five women in apparent premature ovarian failure. These five women aged 27, 32, 26, 26, and 22 had peak serum FSH levels (mIU/ mL) of 52, 65.4, 120, 58, and 112. Three of the five ovulated and two conceived and delivered live babies [17]. Though some of these women had failed to respond to high dose gonadotropins before, one possibility is that at a given moment some follicles were selected that just were sensitive to gonadotropins and the response had nothing to do with the suppression of serum FSH. Alternatively, another possibility is that the estrogen had a direct effect on the granulosa-theca cells making them more sensitive to gonadotropin but again lowering the FSH may not have been necessary. In fact some experimental data in rats have suggested that E2 might enhance FSH binding to receptors [18,19]. However in support of the hypothesis of restoring down-regulated FSH receptors on granulosa cells by lowering the serum FSH is the fact that a modification of the ethinyl estradiol technique was made so that no longer was the ethinyl estradiol given for 2 weeks minimum before exogenous gonadotropins were given but observation for a spontaneous rise in E2 while just on the ethinyl estradiol was made [20]. In a larger series of women in apparent ovarian failure 40 women were able to generate a spontaneous rise in estradiol before any gonadotropins were given [20]. In fact some ovulated and successful pregnancies were achieved with ovulation induction by simply lowering the elevated FSH with ethinyl estradiol alone with follicular development related solely to endogenous gonadotropins [21,22]. Further evidence that lowering the FSH rather than restoring sensitivity of the FSH receptors by estrogen is the operating mechanism for temporarily reversing the menopausal state was provided by case reports where ovulation was induced with hypergonadotropic amenorrhea with estrogen deficiency merely by lowering the FSH with either a gonadotropin releasing hormone agonist or antagonist [20,23,24]. In the aforementioned study of 100 consecutive women with infertility related to hypergonadotropic amenorrhea and estrogen deficiency with a mean serum FSH of 68 mIU/mL, there were 61 ovulations in 311 attempts (19.6%) and yet those who responded had not had spontaneous menses on average for 4 years when the diagnosis of ovarian failure was made [20]. With the leuprolide-hCG technique there were seven ovulations in 43 attempts (16.3%) [20]. In fact six of the 37 women who ovulated at least once in four treatment cycles ovulated in all four treatment cycles [20]. More support to the importance of lowering elevated FSH levels to restore sensitivity was provided by a case report of a 25 year old with premature ovarian failure with 2 years of amenorrhea with serum FSH levels (mIU/mL) of 150, 146, and 164 with serum E2 < 10 pg/mL who was made to ovulate in six of 10 attempts by just using ethinyl estradiol alone without any exogenous FSH stimulation [25]. Evidence that these oocytes are not of such poor quality that pregnancies are not possible is the fact that 19 of 91 (20.8%) women treated with ethinyl estradiol and hMG conceived and eight delivered a live baby [20]. The aforementioned 25 year old had a chemical pregnancy in her ninth treatment cycle and had one live delivered full term baby from cycle 10 [25].
Paucity of follicles vs. multiple follicles unable to reach antral stage The antral follicle secretes inhibin B which inhibits the release of FSH. When there are insufficient antral follicles it would follow that there would be insufficient inhibin B and thus an increase in
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serum FSH. Thus if there were a paucity of primary follicles there would be less follicles advancing to the antral stage. But there is also the theoretical possibility that there are an abundance of primary follicles but they are impeded from development into the antral stage, thus providing less inhibin B, and thus an increase in serum FSH. However, there are data that another mechanism for hypergonadotropic amenorrhea with estrogen deficiency could be a circumstance where there are an abundance of primary follicles but they are damaged by an autoimmune mechanism by the release of some trigger antigen at some stage of follicular development but before the maturity level needed to make inhibin B. This circumstance would lead to an increase in serum FSH [26,27]. One might hypothesize that maybe those who ovulate and became pregnant are those who somehow overcame the immunological reaction against the trigger antigen and thus allow some of the plethora of potentially healthy primary follicles to reach the antral stage and those who do not conceive have a paucity of follicles and the ones remaining are poor quality. However, against the hypothesis is the demonstration of successful pregnancies where from direct observation there was hardly any ovarian tissue left [28,29]. By ultrasound these ovaries are generally much smaller than ovaries with a normal number of follicles even without follicles reaching the antral stage. Frequently some pre-antral 1–2 mm follicles are seen. Most evidence suggests that the autoimmune oophoritis mechanism is very uncommon and the majority of women with elevated day 3 serum FSH really do have diminished oocyte reserve [30].
Hypothesis as to why there seems to be different conclusions about the quality (poor vs. good) of oocytes in younger women with diminished oocyte reserve The infertility history of one woman vividly demonstrates that follicle maturing drugs may create an adverse uterine environment that can negatively affect successful implantation [31,32]. This woman with polycystic ovarian syndrome who was anovulatory but was replete with follicles failed to conceive despite 6 years of ovulation induction with clomiphene citrate or exogenous gonadotropins and progesterone supplementation in the luteal phase with meticulous attention paid to subtle ovulatory factors, e.g., follicular maturation, oocyte release from the follicles, and using sufficient progesterone to acquire in phase late luteal phase endometrial biopsies. Post-coital tests were normal, semen analysis was normal, fallopian tubes were patent and four laparoscopies over 6 years failed to reveal any endometriosis or other infertility factors. After 6 years of treatment she decided to do IVF-ET. However she failed to conceive despite 10 IVF-ET cycles from three of the world’s top IVF centers and in her last six ETs she had 12 embryos transferred each time [31]. Thus she failed to conceive despite 92 embryos transferred [31]. She was successful with her first embryo transfer at our facility [31]. We purposely froze all the embryos, waited a month, and transferred frozen-thawed embryos (n = 5) with the assumption that the previous failed IVF-ET cycles were related to an adverse effect on the endometrium [31]. There are data suggesting this adverse effect may be related to premature trophoblast invasion [33]. More support of this hypothesis was provided by the same woman who conceived on her first cycle of progesterone support naturally at the age of 40 and had another successful delivery [32]. For women with normal serum FSH this adverse effect may occur in about 15–20% of women treated with follicle maturing drugs especially those who were hyperstimulated [34]. If one looks at the difference between our IVF center claiming good success with IVF in women younger than 40 despite diminished oocyte reserve vs.
the highly rated other IVF centers claiming very poor results, the one glaring difference is in the dosage of gonadotropins used. In contrast to the other centers who frequency even increased the amount of gonadotropins above normal controlled ovarian hyperstimulation in order to create more oocytes we consistently used a lower dosage of gonadotropins [10,11,13,35]. In contrast to the aforementioned woman with excellent ovarian reserve who was successful following her first and only frozen-thawed ET having failed 10 previous fresh transfers, our unpublished experience suggests that embryo freezing is not a successful alternative for the embryos obtained from women given normal controlled ovarian hyperstimulation despite an elevated day 3 serum FSH. Thus we hypothesized that for women with diminished oocyte reserve the adverse effect of the controlled ovarian hyperstimulation is on the embryo itself and not the endometrium. We hypothesize that some FSH dependent implantation protein is down regulated by the high levels of FSH generated producing a normal appearing embryo with very little implantation potential. The hypothesis continues that low dose FSH protocols allow adequate production of this FSH dependent implantation protein.
Hypothesis why advanced age makes a difference, i.e., much lower pregnancy rates despite equal or greater oocyte reserve than younger women As previously mentioned the pregnancy rate within 6 months of therapy in women aged <39.9 with a mean duration of infertility of 2.3 years with an average day 3 serum FSH of 18.9 mIU/mL was 46% clinical and 34.6% live delivery rate [14]. In that same study women aged >40 with a similar average serum FSH of 20.8 with 2.6 years of infertility they only had a 6 month pregnancy rate of 10.5% and a live delivery rate of 5.3% [14]. In another study of the effect of age on pregnancy rates following IVF-ET in women with normal serum FSH (<12 mIU/mL) the clinical pregnancy rate per transfer was 48.3% for ages <35, 31.6% for 36–39, 21.6% for women aged 40–42 and 5.9% for women aged >43 [35]. The live delivered pregnancy rates were 45.6%, 29.1%, 14.4%, and 3.4%, respectively, [35]. Interestingly in a study of women with diminished oocyte reserve using minimal ovarian stimulation the clinical and live delivered pregnancy rate was 21.7% for age 40–42 [36]. In contrast in women aged <35 no differences in pregnancy rates were noted in pregnancy rates per transfer in women with a normal day 3 serum FSH (mIU/mL) of <10 (43.7% clinical, 39.7% live delivered) vs. FSH of 11–16 (52.5% clinical, 47.3% live delivered [35]. Even with younger women with an FSH of >17 the clinical rate was 33% vs. 5.9% for women aged >43 with a normal day 3 serum FSH [35]. Though these data show that as long as low dose stimulation protocols are used younger women with diminished oocyte reserve not only have a much better prognosis compared to women of advanced reproductive age with diminished oocyte reserve but also to women of advanced reproductive age with normal oocyte reserve. One hypothesis to explain these observations is that a destructive process is responsible for the depletion of oocytes in younger women so that certain portions of the ovaries have been damaged. However what ovarian tissue is left has the same percentage of good oocytes left as their age peers with normal oocyte reserve. In contrast by this hypothesis, women of advanced reproductive age have been recruiting the best follicles each month for the cohort of antral follicles. Thus they may have not less follicles or even more than younger women with diminished oocyte reserve but the oocytes do not produce embryos that are nearly as likely to result in successful implantation.
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Embryos transferred on day 2, 3 or 5 from women of advanced age do not have any morphologic differences than their younger counterparts. Similarly there is no difference in fertilization rates or the percentage of fertilized oocytes making it to transferable embryos [37]. Thus it seems that the main reason for very low pregnancy conception rates in women of advanced reproductive age is death of the early conceptus somewhere between day 6 from fertilization to day 12 (the time of many pregnancy tests). Thus one hypothesis to explain these observations is that whatever oocyte factor is responsible for a primary follicle not undergoing atresia and developing into an antral follicle persists with the embryo and prevents subsequent apoptosis of the early conceptus. The hypothesis continues that the higher the concentration of this protective factor in the primary follicle the more likely that primary follicle will develop earlier in life into an antral follicle. Thus with advanced reproductive age with natural selection the follicles that develop will be the ones with the least amount of this protective factor and thus ever obtaining a positive pregnancy test is unlikely. Exceptions where pregnancy after age 45 are found are more likely related to a genetic predisposition for higher concentrations of this factor in all primary follicles [4,38]. A greater chance of aneuploidy may partially explain the aging process but one would think if this was the entire answer there would be more conceptions but a very high rate of miscarriage. Instead, however conception itself is very rare. Conflicts of interest statement None declared. References [1] Menken J, Trussel J, Larsen U. Age and infertility. Science 1986;233:1389–94. [2] Goldenberg RL, Grodin J, Rodbard D, Ross GT. Gonadotropins in women with amenorrhea: the use of follicle stimulating hormone to differentiate woman with and without ovarian follicles. Am J Obst Gyn 1973;11:1003–12. [3] Muasher SJ, Oehninger S, Simonetti S, et al. The value of basal and/or stimulated serum gonadotropin levels in prediction of stimulation response and in vitro fertilization outcome. Fertil Steril 1988;50:298–307. [4] Laufer N, Simon A, Samueloff A, Yaffe H, Milwidsky A, Gielchinsky Y. Successful spontaneous pregnancies in women older than 45 years. Fertil Steril 2004;81:1328–32. [5] Toner JP, Philput CB, Jones GS, Muasher SJ. Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril 1991;55:784–91. [6] Scott RT, Toner JP, Muasher SJ, Oehninger S, Robinson S, Rosenwaks Z. Follicle stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril 1989;51:651–4. [7] Fenichel P, Grimaldi M, Oliverao J-F, Donzeau M, Gillet J-Y, Harter M. Predictive value of hormonal profiles before stimulation for in vitro fertilization. Fertil Steril 1989;51:845–9. [8] Tanbo T, Dale PO, Abyholm T, Stokkke KT. Follicle-stimulating hormone as a prognostic indicator in clomiphene citrate/human menopausal gonadotropinstimulated cycles for in vitro fertilization. Hum Reprod 1989;4:647–50. [9] Cahill DJ, Prosser CJ, Wardle PG, Ford WCL, Hull MGR. Relative influence of serum follicle stimulating hormone, age and other factors on ovarian response to gonadotrophin stimulation. Br J Obst Gyn 1994;101:999–1002. [10] Scott Jr RT, Hofmann GE, Oehninger S, Muasher SJ. Intercycle variability of day 3 follicle-stimulating hormone levels and its effects on stimulation quality in in vitro fertilization. Fertil Steril 1990;54:297–302. [11] Roberts JE, Spandorfer S, Fasouliotis SJ, Kashyap S, Rosenwaks Z. Taking a basal follicle stimulating hormone history is essential before initiating in vitro fertilization. Fertil Steril 2005;83:37–41. [12] Kolibianakis E, Zikopoulos K, Camus M, Tounaye H, Van Steirteghem A, Devroey P. Modified natural cycle for IVF does not offer a realistic chance of parenthood in poor responders with high day 3 FSH levels, as a last resort prior to oocyte donation. Hum Reprod 2004;19:2545–9.
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[13] Check JH, Summers-Chase D, Yuan W, Horwath D, Wilson C. Effect of embryo quality on pregnancy outcome following single embryo transfer in women with a diminished egg reserve. Fertil Steril 2007;87(4):749–56. [14] Check JH, Peymer M, Lurie D. Effect of age on pregnancy outcome without assisted reproductive technology in women with elevated early follicular phase serum follicle-stimulating hormone levels. Gynecol Obstet Invest 1998;45:217–20. [15] Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992;7:1342–6. [16] Vaskivuo TE, Anttonen M, Herva R, et al. Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4. J Clin Endocrinol Metab 2001;86:3421–9. [17] Check JH, Chase J. Ovulation induction in hypergonadotropic amenorrhea with estrogen and human menopausal gonadotropin therapy. Fertil Steril 1984;42:919–22. [18] Louvet JP, Vaitkuaitis JL. Induction of follicle-stimulating hormone (FSH) receptors in rate ovaries by estrogen priming. Endocrinology 1976;99:758–64. [19] Richards JS, Ireland JJ, Rao MC. Ovarian follicular development in the rat: hormone receptor regulation by estradiol follicle-stimulating hormone and luteinizing hormone. Endocrinology 1976;99:1562–70. [20] Check JH, Nowroozi K, Chase JS, Nazari A, Shapse D, Vaze M. Ovulation induction and pregnancies in 100 consecutive women with hypergonadotropic amenorrhea. Fertil Steril 1990;53(5):811–6. [21] Check ML, Check JH, Choe JK, Berger GS. Successful pregnancy in a 42-year-old woman with imminent ovarian failure following ovulation induction with ethinyl estradiol without gonadotropins and in vitro fertilization. Clin Exp Obst Gyn 2002;29:11–4. [22] Check JH, Katsoff B. Successful pregnancy with spontaneous ovulation in a woman with apparent premature ovarian failure who failed to conceive despite four transfers of embryos derived from donated oocytes. Clin Exp Obst Gyn 2006;33:13–5. [23] Check JH, Wu CH, Check M. The effect of leuprolide acetate in aiding induction of ovulation in hypergonadotropic hypogonadism: a case report. Fertil Steril 1988;49(3):542–3. [24] Check JH, Katsoff B. Ovulation induction and pregnancy in a woman with premature menopause following gonadotropin suppression with the gonadotropin releasing hormone antagonist, cetrorelix – a case report. Clin Exp Obst Gyn 2008;35(1):10–2. [25] Check ML, Check JH, Kaplan H. Pregnancy despite imminent ovarian failure and extremely high endogenous gonadotropins and therapeutic strategies: case report and review. Clin Exp Obst Gyn 2004;31:299–301. [26] Irvine WJ, Barnes EW. Addison’s disease and autoimmune ovarian failure. J Reprod Fertil 1974;21(Suppl. 1):1–6. [27] Sedmak DD, Hart WR, Tubbs RR. Autoimmune oophoritis: a histopathologic study of involved ovaries with immunologic characterization of the mononuclear cell infiltrate. Inj J Gynecol Pathol 1987;6:73–81. [28] Check JH, Chase JS, Wu CH, Adelson HG. Case report: ovulation-induction and pregnancy using an estrogen-gonadotropin stimulation technique in a menopausal woman with marked hypoplastic ovaries. Am J Obst Gyn 1989;160:405–6. [29] Shanis BS, Check JH. Spontaneous ovulation and successful pregnancy despite bilateral streaked ovaries. Infertility 1992;15:70–7. [30] Check JH, Stegonshek J, Wilson C. No association found between decreased ovarian reserve and low thyroid function. Clin Exp Obst Gyn 2009;36:85–6. [31] Check JH, Choe JK, Nazari A, Summers-Chase D. Ovarian hyperstimulation can reduce uterine receptivity. A case report. Clin Exp Obst Gyn 2000;27(2):89–91. [32] Check JH, Check ML. A case report demonstrating that follicle maturing drugs may create an adverse uterine environment even when not used for controlled ovarian hyperstimulation. Clin Exp Obst Gyn 2001;28:217–8. [33] Check JH, Check ML. Evidence that failure to conceive despite apparent correction of ovulatory defects by follicle-maturing drugs may be related to premature trophoblast invasion. Med Hypoth 2002;59(4):385–8. [34] Check JH. Progesterone therapy versus follicle maturing drugs – possible opposite effects on embryo implantation. Clin Exp Obst Gyn 2002;29:5–10. [35] Check JH. Mild ovarian stimulation. J Assist Reprod Genet 2007;24:621–7. [36] Check ML, Check JH, Wilson C, Choe JK, Krotec J. Outcome of in vitro fertilization-embryo transfer according to age in poor responders with elevated baseline serum follicle stimulation hormone using minimal or no gonadotropin stimulation. Clin Exp Obst Gyn 2004;31:183–4. [37] Check JH, Nazari P, Check ML, Choe JK, Liss JR. Prognosis following in vitro fertilization-embryo transfer (IVF-ET) in patients with elevated day 2 or 3 serum follicle stimulating hormone (FSH) is better in younger vs older patients. Clin Exp Obst Gyn 2002;29:42–4. [38] Gielchinsky Y, Mazor M, Simon A, Mor-Yossef S, Laufer N. Natural conception after age 45 in Bedouin women, a uniquely fertile population. J Assist Reprod Genet 2006;23:305–9.
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Evidence that oocyte quality in younger women with diminished oocyte reserve is superior to those of women of advanced reproductive age J.H. Check *, R. Cohen The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School at Camden, Cooper Hospital/University Medical Center, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Camden, NJ, USA
a r t i c l e
i n f o
Article history: Received 1 September 2009 Accepted 10 September 2009
s u m m a r y Evidence is provided supporting the hypothesis that the much improved prognosis for younger women with diminished oocyte reserve compared to women of advanced reproductive age is related to a difference in mechanism for oocyte depletion. For younger women the majority have had destruction of certain portions of their ovarian tissue but the remaining spared ovarian tissue has proportionately the same percentage of normal follicles as their age peers. The hypothesis continues that some factor that is responsible for earlier development of primary to antral follicles persists with the early conceptus and protects it from early programmed cell death. Thus by natural selection most women of advanced reproductive age have oocytes that fertilize normally and produce normal morphologic embryos, but lacking this apoptosis inhibiting factor dies very early between days 6 and 12 from fertilization. Ó 2009 Elsevier Ltd. All rights reserved.
Effect of aging on oocyte quantity and quality There is a decline in fecundity that accelerates between 35 and 40 years of age and approaches zero by age 45 [1]. As one ages, or as menopause approaches, there is a paucity of ovarian follicles [2]. With less total follicles remaining there are less follicles reaching the antral follicle stage (5–9 mm). Biological peptides released from the antral follicles, e.g., inhibin B suppresses the release of follicle stimulating hormone (FSH) from the pituitary. With less antral follicles there is less inhibin B leading to an increase in serum FSH. For women still ovulating the best time to measure serum FSH to use it as an indicator for diminished oocyte reserve is in the early follicular phase when serum estradiol (E2) is also at its lowest. Estradiol also exerts an inhibitory effect on release of FSH. Thus measuring serum FSH at the peak E2 could allow suppression of serum FSH even with diminished antral follicles and thus less inhibin B, thus resulting in wrong conclusions about oocyte reserve. The antral follicles recruited in women of advanced reproductive age are not only fewer in number than younger women but they contain oocytes of inferior quality. These oocytes not only have extra chromosomes but have defective cytoplasmic mitochondria [3]. The defective cytoplasmic mitochondria could lead to apoptosis of the embryo somewhere between days 5 and 12 [4]. One hypothesis is that the presence of more of a certain cytoplasmic mitochondrial factor is what may be responsible for the recruitment of a given number of antral follicles at a given age.
* Corresponding author. Address: 7747 Old York Road, Melrose Park, PA 19027, USA. Tel.: +1 215 635 4400; fax: +1 215 635 2304. E-mail address: [email protected] (J.H. Check). 0306-9877/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2009.09.018
With less of this factor present, the embryo formed is likely to have programmed cell death somewhere between days 5 and 12 of life.
Studies suggesting oocyte quality in younger patients with diminished oocyte reserve is poor resulting in very low chance of pregnancy Since one of the manifestations of diminished oocyte reserve in women of advanced reproductive age is an elevated day 3 serum FSH, it was assumed that a younger woman with an elevated day 3 serum FSH must have similar remaining oocytes to older women. Thus it was concluded that these younger women not only have few remaining oocytes but they are of very poor quality. Indeed in both the early days of in vitro fertilization-embryo transfer (IVF-ET) and in modern times many publications demonstrate very poor pregnancy rates in younger women with increased day 3 serum FSH despite the transfer of normal morphologic embryos [5– 12]. One of the finest IVF centers in the early IVF era concluded that if the serum FSH is ever elevated in one menstrual cycle on day 3 this means that the quality of remaining oocytes are poor similar to women of advanced reproductive age, i.e., >45 [10]. These researchers stated that this conclusion would apply even if the serum FSH was in the normal range indicating at least in that cycle of more antral follicles [10]. These conclusions were mirrored by one of the world’s best IVF centers finding no live pregnancies despite the transfer of embryos of good quality in women of any age using a serum FSH cut-off of 15 mIU/mL [11]. These data supported the concept that younger women with diminished egg reserve must
J.H. Check, R. Cohen / Medical Hypotheses 74 (2010) 264–267
have as the main mechanism of their problem an acceleration of the normal rate of oocyte atresia. Evidence refuting consensus that diminished oocyte reserve in younger women associated with poor quality oocytes A recent study of women aged 39.9 and under with such diminished oocyte reserve that only a single embryo was available for transfer found the clinical pregnancy rate for the 63% who had an embryo with 6, 7, or >8 blastomeres to be 38%, 40%, and 42.4%, respectively, [13]. The respective live delivered pregnancy rates were 31%, 25%, and 36.4% per transfer [13]. The mean day 3 serum FSH (mIU/mL) and ranges for these three groups were 25.3 ± 17 (19.2–31.4), 23.3 ± 9.7 (18.7–27.8), and 18.0 ± 6.9 (15.6– 20.6). Thus in most instances the serum FSH was higher than 15 mIU/mL [13]. A group of women 39.9 with elevated serum FSH (mean 18.9 ±8.3 mIU/mL with a range of 13–43) were treated without IVF-ET for infertility. The infertility treatment included luteal phase support with vaginal progesterone and correcting follicular maturation defects, luteinized unruptured follicle syndrome, cervical and/or male factor, e.g., with intrauterine insemination [14]. The clinical and live delivered pregnancy rates within 6 months of therapy were 46.1% and 34.6%, respectively, [14]. There is evidence that when a woman appears to be in overt menopause with amenorrhea, estrogen deficiency and elevated FSH and failure to menstruate following progesterone withdrawal, that there are still approximately 1000 follicles left [15]. However, all a woman needs is the recruitment and development of one dominant follicle to produce estrogen to allow proliferation of the endometrium, and with ovulation, the additional secretion of progesterone leading to spontaneous menses if successful fertilization did not take place upon the withdrawal of progesterone. The fact that these follicles do not develop has been assumed to be related to very poor quality and the reason why they never developed in the 40 years of menstrual life. There is evidence that this may be related to apoptosis of the granulosa cells surrounding the oocyte [16]. It is assumed because of the paucity of case reports of pregnancies after ‘‘menopause” that even if a rare spontaneous ovulation does occur pregnancy is highly unlikely because of poor oocyte quality. This thought process would apply to not just women of advanced reproductive age with natural menopause but younger women with premature ovarian failure [5–12]. However, there may be an alternative explanation as to why these few remaining primary or pre-antral follicles do not develop into dominant follicles. The possibility exists that they do have potential to develop into dominant follicles but they have become insensitive to FSH. It is well known that most hormones require binding to a receptor for that hormone to provide its function. When too much hormone exists, as a protective mechanism, frequently the target cell will demonstrate down-regulation of the receptors (a prime example is insulin and its receptor). We hypothesized that chronically elevated FSH levels could down-regulate FSH receptors in the granulosa theca cell layer. This could cause gonadotropin-resistance and thus inhibit follicular development. The hypothesis continued that this insensitivity should be potentially reversible by lowering the chronically elevated serum FSH and thus restoring down-regulated FSH receptors. Evidence to support the hypothesis that lowering FSH can restore sensitivity of the remaining follicles in women with diminished oocyte reserve Support of this hypothesis was provided in 1984 by attempting to lower FSH with estrogen to theoretically restore down-regulated
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FSH receptors and thus restoring sensitivity of the follicles followed by stimulation with human menopausal gonadotropins in five women in apparent premature ovarian failure. These five women aged 27, 32, 26, 26, and 22 had peak serum FSH levels (mIU/ mL) of 52, 65.4, 120, 58, and 112. Three of the five ovulated and two conceived and delivered live babies [17]. Though some of these women had failed to respond to high dose gonadotropins before, one possibility is that at a given moment some follicles were selected that just were sensitive to gonadotropins and the response had nothing to do with the suppression of serum FSH. Alternatively, another possibility is that the estrogen had a direct effect on the granulosa-theca cells making them more sensitive to gonadotropin but again lowering the FSH may not have been necessary. In fact some experimental data in rats have suggested that E2 might enhance FSH binding to receptors [18,19]. However in support of the hypothesis of restoring down-regulated FSH receptors on granulosa cells by lowering the serum FSH is the fact that a modification of the ethinyl estradiol technique was made so that no longer was the ethinyl estradiol given for 2 weeks minimum before exogenous gonadotropins were given but observation for a spontaneous rise in E2 while just on the ethinyl estradiol was made [20]. In a larger series of women in apparent ovarian failure 40 women were able to generate a spontaneous rise in estradiol before any gonadotropins were given [20]. In fact some ovulated and successful pregnancies were achieved with ovulation induction by simply lowering the elevated FSH with ethinyl estradiol alone with follicular development related solely to endogenous gonadotropins [21,22]. Further evidence that lowering the FSH rather than restoring sensitivity of the FSH receptors by estrogen is the operating mechanism for temporarily reversing the menopausal state was provided by case reports where ovulation was induced with hypergonadotropic amenorrhea with estrogen deficiency merely by lowering the FSH with either a gonadotropin releasing hormone agonist or antagonist [20,23,24]. In the aforementioned study of 100 consecutive women with infertility related to hypergonadotropic amenorrhea and estrogen deficiency with a mean serum FSH of 68 mIU/mL, there were 61 ovulations in 311 attempts (19.6%) and yet those who responded had not had spontaneous menses on average for 4 years when the diagnosis of ovarian failure was made [20]. With the leuprolide-hCG technique there were seven ovulations in 43 attempts (16.3%) [20]. In fact six of the 37 women who ovulated at least once in four treatment cycles ovulated in all four treatment cycles [20]. More support to the importance of lowering elevated FSH levels to restore sensitivity was provided by a case report of a 25 year old with premature ovarian failure with 2 years of amenorrhea with serum FSH levels (mIU/mL) of 150, 146, and 164 with serum E2 < 10 pg/mL who was made to ovulate in six of 10 attempts by just using ethinyl estradiol alone without any exogenous FSH stimulation [25]. Evidence that these oocytes are not of such poor quality that pregnancies are not possible is the fact that 19 of 91 (20.8%) women treated with ethinyl estradiol and hMG conceived and eight delivered a live baby [20]. The aforementioned 25 year old had a chemical pregnancy in her ninth treatment cycle and had one live delivered full term baby from cycle 10 [25].
Paucity of follicles vs. multiple follicles unable to reach antral stage The antral follicle secretes inhibin B which inhibits the release of FSH. When there are insufficient antral follicles it would follow that there would be insufficient inhibin B and thus an increase in
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serum FSH. Thus if there were a paucity of primary follicles there would be less follicles advancing to the antral stage. But there is also the theoretical possibility that there are an abundance of primary follicles but they are impeded from development into the antral stage, thus providing less inhibin B, and thus an increase in serum FSH. However, there are data that another mechanism for hypergonadotropic amenorrhea with estrogen deficiency could be a circumstance where there are an abundance of primary follicles but they are damaged by an autoimmune mechanism by the release of some trigger antigen at some stage of follicular development but before the maturity level needed to make inhibin B. This circumstance would lead to an increase in serum FSH [26,27]. One might hypothesize that maybe those who ovulate and became pregnant are those who somehow overcame the immunological reaction against the trigger antigen and thus allow some of the plethora of potentially healthy primary follicles to reach the antral stage and those who do not conceive have a paucity of follicles and the ones remaining are poor quality. However, against the hypothesis is the demonstration of successful pregnancies where from direct observation there was hardly any ovarian tissue left [28,29]. By ultrasound these ovaries are generally much smaller than ovaries with a normal number of follicles even without follicles reaching the antral stage. Frequently some pre-antral 1–2 mm follicles are seen. Most evidence suggests that the autoimmune oophoritis mechanism is very uncommon and the majority of women with elevated day 3 serum FSH really do have diminished oocyte reserve [30].
Hypothesis as to why there seems to be different conclusions about the quality (poor vs. good) of oocytes in younger women with diminished oocyte reserve The infertility history of one woman vividly demonstrates that follicle maturing drugs may create an adverse uterine environment that can negatively affect successful implantation [31,32]. This woman with polycystic ovarian syndrome who was anovulatory but was replete with follicles failed to conceive despite 6 years of ovulation induction with clomiphene citrate or exogenous gonadotropins and progesterone supplementation in the luteal phase with meticulous attention paid to subtle ovulatory factors, e.g., follicular maturation, oocyte release from the follicles, and using sufficient progesterone to acquire in phase late luteal phase endometrial biopsies. Post-coital tests were normal, semen analysis was normal, fallopian tubes were patent and four laparoscopies over 6 years failed to reveal any endometriosis or other infertility factors. After 6 years of treatment she decided to do IVF-ET. However she failed to conceive despite 10 IVF-ET cycles from three of the world’s top IVF centers and in her last six ETs she had 12 embryos transferred each time [31]. Thus she failed to conceive despite 92 embryos transferred [31]. She was successful with her first embryo transfer at our facility [31]. We purposely froze all the embryos, waited a month, and transferred frozen-thawed embryos (n = 5) with the assumption that the previous failed IVF-ET cycles were related to an adverse effect on the endometrium [31]. There are data suggesting this adverse effect may be related to premature trophoblast invasion [33]. More support of this hypothesis was provided by the same woman who conceived on her first cycle of progesterone support naturally at the age of 40 and had another successful delivery [32]. For women with normal serum FSH this adverse effect may occur in about 15–20% of women treated with follicle maturing drugs especially those who were hyperstimulated [34]. If one looks at the difference between our IVF center claiming good success with IVF in women younger than 40 despite diminished oocyte reserve vs.
the highly rated other IVF centers claiming very poor results, the one glaring difference is in the dosage of gonadotropins used. In contrast to the other centers who frequency even increased the amount of gonadotropins above normal controlled ovarian hyperstimulation in order to create more oocytes we consistently used a lower dosage of gonadotropins [10,11,13,35]. In contrast to the aforementioned woman with excellent ovarian reserve who was successful following her first and only frozen-thawed ET having failed 10 previous fresh transfers, our unpublished experience suggests that embryo freezing is not a successful alternative for the embryos obtained from women given normal controlled ovarian hyperstimulation despite an elevated day 3 serum FSH. Thus we hypothesized that for women with diminished oocyte reserve the adverse effect of the controlled ovarian hyperstimulation is on the embryo itself and not the endometrium. We hypothesize that some FSH dependent implantation protein is down regulated by the high levels of FSH generated producing a normal appearing embryo with very little implantation potential. The hypothesis continues that low dose FSH protocols allow adequate production of this FSH dependent implantation protein.
Hypothesis why advanced age makes a difference, i.e., much lower pregnancy rates despite equal or greater oocyte reserve than younger women As previously mentioned the pregnancy rate within 6 months of therapy in women aged <39.9 with a mean duration of infertility of 2.3 years with an average day 3 serum FSH of 18.9 mIU/mL was 46% clinical and 34.6% live delivery rate [14]. In that same study women aged >40 with a similar average serum FSH of 20.8 with 2.6 years of infertility they only had a 6 month pregnancy rate of 10.5% and a live delivery rate of 5.3% [14]. In another study of the effect of age on pregnancy rates following IVF-ET in women with normal serum FSH (<12 mIU/mL) the clinical pregnancy rate per transfer was 48.3% for ages <35, 31.6% for 36–39, 21.6% for women aged 40–42 and 5.9% for women aged >43 [35]. The live delivered pregnancy rates were 45.6%, 29.1%, 14.4%, and 3.4%, respectively, [35]. Interestingly in a study of women with diminished oocyte reserve using minimal ovarian stimulation the clinical and live delivered pregnancy rate was 21.7% for age 40–42 [36]. In contrast in women aged <35 no differences in pregnancy rates were noted in pregnancy rates per transfer in women with a normal day 3 serum FSH (mIU/mL) of <10 (43.7% clinical, 39.7% live delivered) vs. FSH of 11–16 (52.5% clinical, 47.3% live delivered [35]. Even with younger women with an FSH of >17 the clinical rate was 33% vs. 5.9% for women aged >43 with a normal day 3 serum FSH [35]. Though these data show that as long as low dose stimulation protocols are used younger women with diminished oocyte reserve not only have a much better prognosis compared to women of advanced reproductive age with diminished oocyte reserve but also to women of advanced reproductive age with normal oocyte reserve. One hypothesis to explain these observations is that a destructive process is responsible for the depletion of oocytes in younger women so that certain portions of the ovaries have been damaged. However what ovarian tissue is left has the same percentage of good oocytes left as their age peers with normal oocyte reserve. In contrast by this hypothesis, women of advanced reproductive age have been recruiting the best follicles each month for the cohort of antral follicles. Thus they may have not less follicles or even more than younger women with diminished oocyte reserve but the oocytes do not produce embryos that are nearly as likely to result in successful implantation.
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Embryos transferred on day 2, 3 or 5 from women of advanced age do not have any morphologic differences than their younger counterparts. Similarly there is no difference in fertilization rates or the percentage of fertilized oocytes making it to transferable embryos [37]. Thus it seems that the main reason for very low pregnancy conception rates in women of advanced reproductive age is death of the early conceptus somewhere between day 6 from fertilization to day 12 (the time of many pregnancy tests). Thus one hypothesis to explain these observations is that whatever oocyte factor is responsible for a primary follicle not undergoing atresia and developing into an antral follicle persists with the embryo and prevents subsequent apoptosis of the early conceptus. The hypothesis continues that the higher the concentration of this protective factor in the primary follicle the more likely that primary follicle will develop earlier in life into an antral follicle. Thus with advanced reproductive age with natural selection the follicles that develop will be the ones with the least amount of this protective factor and thus ever obtaining a positive pregnancy test is unlikely. Exceptions where pregnancy after age 45 are found are more likely related to a genetic predisposition for higher concentrations of this factor in all primary follicles [4,38]. A greater chance of aneuploidy may partially explain the aging process but one would think if this was the entire answer there would be more conceptions but a very high rate of miscarriage. Instead, however conception itself is very rare. Conflicts of interest statement None declared. References [1] Menken J, Trussel J, Larsen U. Age and infertility. Science 1986;233:1389–94. [2] Goldenberg RL, Grodin J, Rodbard D, Ross GT. Gonadotropins in women with amenorrhea: the use of follicle stimulating hormone to differentiate woman with and without ovarian follicles. Am J Obst Gyn 1973;11:1003–12. [3] Muasher SJ, Oehninger S, Simonetti S, et al. The value of basal and/or stimulated serum gonadotropin levels in prediction of stimulation response and in vitro fertilization outcome. Fertil Steril 1988;50:298–307. [4] Laufer N, Simon A, Samueloff A, Yaffe H, Milwidsky A, Gielchinsky Y. Successful spontaneous pregnancies in women older than 45 years. Fertil Steril 2004;81:1328–32. [5] Toner JP, Philput CB, Jones GS, Muasher SJ. Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril 1991;55:784–91. [6] Scott RT, Toner JP, Muasher SJ, Oehninger S, Robinson S, Rosenwaks Z. Follicle stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril 1989;51:651–4. [7] Fenichel P, Grimaldi M, Oliverao J-F, Donzeau M, Gillet J-Y, Harter M. Predictive value of hormonal profiles before stimulation for in vitro fertilization. Fertil Steril 1989;51:845–9. [8] Tanbo T, Dale PO, Abyholm T, Stokkke KT. Follicle-stimulating hormone as a prognostic indicator in clomiphene citrate/human menopausal gonadotropinstimulated cycles for in vitro fertilization. Hum Reprod 1989;4:647–50. [9] Cahill DJ, Prosser CJ, Wardle PG, Ford WCL, Hull MGR. Relative influence of serum follicle stimulating hormone, age and other factors on ovarian response to gonadotrophin stimulation. Br J Obst Gyn 1994;101:999–1002. [10] Scott Jr RT, Hofmann GE, Oehninger S, Muasher SJ. Intercycle variability of day 3 follicle-stimulating hormone levels and its effects on stimulation quality in in vitro fertilization. Fertil Steril 1990;54:297–302. [11] Roberts JE, Spandorfer S, Fasouliotis SJ, Kashyap S, Rosenwaks Z. Taking a basal follicle stimulating hormone history is essential before initiating in vitro fertilization. Fertil Steril 2005;83:37–41. [12] Kolibianakis E, Zikopoulos K, Camus M, Tounaye H, Van Steirteghem A, Devroey P. Modified natural cycle for IVF does not offer a realistic chance of parenthood in poor responders with high day 3 FSH levels, as a last resort prior to oocyte donation. Hum Reprod 2004;19:2545–9.
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[13] Check JH, Summers-Chase D, Yuan W, Horwath D, Wilson C. Effect of embryo quality on pregnancy outcome following single embryo transfer in women with a diminished egg reserve. Fertil Steril 2007;87(4):749–56. [14] Check JH, Peymer M, Lurie D. Effect of age on pregnancy outcome without assisted reproductive technology in women with elevated early follicular phase serum follicle-stimulating hormone levels. Gynecol Obstet Invest 1998;45:217–20. [15] Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992;7:1342–6. [16] Vaskivuo TE, Anttonen M, Herva R, et al. Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4. J Clin Endocrinol Metab 2001;86:3421–9. [17] Check JH, Chase J. Ovulation induction in hypergonadotropic amenorrhea with estrogen and human menopausal gonadotropin therapy. 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Successful pregnancy with spontaneous ovulation in a woman with apparent premature ovarian failure who failed to conceive despite four transfers of embryos derived from donated oocytes. Clin Exp Obst Gyn 2006;33:13–5. [23] Check JH, Wu CH, Check M. The effect of leuprolide acetate in aiding induction of ovulation in hypergonadotropic hypogonadism: a case report. Fertil Steril 1988;49(3):542–3. [24] Check JH, Katsoff B. Ovulation induction and pregnancy in a woman with premature menopause following gonadotropin suppression with the gonadotropin releasing hormone antagonist, cetrorelix – a case report. Clin Exp Obst Gyn 2008;35(1):10–2. [25] Check ML, Check JH, Kaplan H. Pregnancy despite imminent ovarian failure and extremely high endogenous gonadotropins and therapeutic strategies: case report and review. Clin Exp Obst Gyn 2004;31:299–301. [26] Irvine WJ, Barnes EW. Addison’s disease and autoimmune ovarian failure. J Reprod Fertil 1974;21(Suppl. 1):1–6. [27] Sedmak DD, Hart WR, Tubbs RR. Autoimmune oophoritis: a histopathologic study of involved ovaries with immunologic characterization of the mononuclear cell infiltrate. Inj J Gynecol Pathol 1987;6:73–81. [28] Check JH, Chase JS, Wu CH, Adelson HG. Case report: ovulation-induction and pregnancy using an estrogen-gonadotropin stimulation technique in a menopausal woman with marked hypoplastic ovaries. Am J Obst Gyn 1989;160:405–6. [29] Shanis BS, Check JH. Spontaneous ovulation and successful pregnancy despite bilateral streaked ovaries. Infertility 1992;15:70–7. [30] Check JH, Stegonshek J, Wilson C. No association found between decreased ovarian reserve and low thyroid function. Clin Exp Obst Gyn 2009;36:85–6. [31] Check JH, Choe JK, Nazari A, Summers-Chase D. Ovarian hyperstimulation can reduce uterine receptivity. A case report. Clin Exp Obst Gyn 2000;27(2):89–91. [32] Check JH, Check ML. A case report demonstrating that follicle maturing drugs may create an adverse uterine environment even when not used for controlled ovarian hyperstimulation. Clin Exp Obst Gyn 2001;28:217–8. [33] Check JH, Check ML. Evidence that failure to conceive despite apparent correction of ovulatory defects by follicle-maturing drugs may be related to premature trophoblast invasion. Med Hypoth 2002;59(4):385–8. [34] Check JH. Progesterone therapy versus follicle maturing drugs – possible opposite effects on embryo implantation. Clin Exp Obst Gyn 2002;29:5–10. [35] Check JH. Mild ovarian stimulation. J Assist Reprod Genet 2007;24:621–7. [36] Check ML, Check JH, Wilson C, Choe JK, Krotec J. Outcome of in vitro fertilization-embryo transfer according to age in poor responders with elevated baseline serum follicle stimulation hormone using minimal or no gonadotropin stimulation. Clin Exp Obst Gyn 2004;31:183–4. [37] Check JH, Nazari P, Check ML, Choe JK, Liss JR. 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