Trisomic pregnancy and earlier age at menopause

We tested the hypothesis that the connection between advanced maternal age and autosomal trisomy reflects the diminution of the oocyte pool with age. Because menopause occurs when the number of oocytes falls below some threshold, our hypothesis is that menopause occurs at an earlier age among women with trisomic pregnancies than it does among women with chromosomally normal pregnancies. To determine their menstrual status, we interviewed women from our previous study of karyotyped spontaneous abortions who, in 1993, were age >/=44 years. Premenopausal women completed interviews every 4-5 mo, until menopause or until the study ended in 1997. The primary analyses compare 111 women whose index pregnancy was a trisomic spontaneous abortion with two groups: women whose index pregnancy was a chromosomally normal loss (n=157) and women whose index pregnancy was a chromosomally normal birth (n=226). We used a parametric logistic survival analysis to compare median ages at menopause. The estimated median age at menopause was 0.96 years earlier (95% confidence interval -0.18 to 2.10) among women with trisomic losses than it was among women with chromosomally normal losses and chromosomally normal births combined. Results were unaltered by adjustment for education, ethnicity, and cigarette smoking. Our results support the hypothesis that trisomy risk is increased with decreased numbers of oocytes. Decreased numbers may indicate accelerated oocyte atresia or fewer oocytes formed during fetal development.     

Predisposition to aneuploidy in the oocyte

While the incidence of predisposition to aneuploidy in the oocyte increases with age, there is also evidence of increased incidence in young women with recurrent miscarriage, recurrent aneuploidy, or recurrent implantation failure after in vitro fertilization. There is evidence from mouse models and from observations in humans that follicle-stimulating hormone (FSH) probably has a direct or indirect effect on the occurrence of oocyte aneuploidy. It seems that increased endogenous or exogenous FSH could induce meiotic disruption. Although the effect of FSH may explain the age-related increase in aneuploidy rate, many questions remain regarding young women, even if their FSH level is sometimes increased. Disruption of meiotic gene expression caused by exposure to environmental contaminants or by gene defects could also predispose to oocyte aneuploidy. Such abnormalities could impact on the oocyte pool, recombination and synapsis during fetal life, or oocyte growth.     

Skewed X Chromosome Inactivation and Trisomic Spontaneous Abortion: No Association

Several studies suggest that highly skewed X chromosome inactivation (HSXI) is associated with recurrent spontaneous abortion. We hypothesized that this association reflects an increased rate of trisomic conceptions due to anomalies on the X chromosome that lead both to HSXI and to a diminished oocyte pool. We compared the distribution of X chromosome inactivation (XCI) skewing percentages (range: 50%–100%) among women with spontaneous abortions in four karyotype groups—trisomy (n = 154), chromosomally normal male (n = 43), chromosomally normal female (n = 38), nontrisomic chromosomally abnormal (n = 61)—to the distribution for age-matched controls with chromosomally normal births (n = 388). In secondary analyses, we subdivided the nontrisomic chromosomally abnormal group, divided trisomies by chromosome, and classified women by reproductive history. Our data support neither an association of HSXI with all trisomies nor an association of HSXI with chromosomally normal male spontaneous abortions. We also find no association between HSXI and recurrent abortion (n = 45).     

In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.     

Control of oocyte recruitment: regulative role of follicle cells through the release of a diffusible factor

To determine whether oogonial proliferation and oocyte recruitment are under control of hypophyseal and/or ovarian factors, we carried out a series of investigations using Podarcis sicula, a lizard inhabiting the temperate lowlands of Europe in which oocyte recruitment occurs throughout the year, as animal model. Germinal beds containing oogonia and oocytes in prefollicular stages were cocultured with different ovarian compartments in presence/absence of FSH, and the effects of different treatments were evaluated by counting the number of prelepto-leptotene oocytes. Results revealed that oocyte recruitment from the pool of oogonia is under the control of a factor released by follicle cells while FSH has an indirect effect on modulating oogonial proliferation. SDS-PAGE analyses carried out on media conditioned by follicles suggest that the factor involved in the control of oocyte recruitment may be a small protein (about 21 kDa) and that its release is dependent on the period of the ovarian cycle but apparently not on the circulating levels of FSH.     

Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.     

Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth

While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wild-type follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size.     

Effect of hormonal stimulation on bovine follicular response and oocyte developmental competence in a commercial operation

The widespread use of ultrasonography and IVF over the past decade has provided new tools to evaluate how follicles and oocytes react to different superstimulatory treatments. This information may be used to redefine actual hormonal stimulations to improve results of MOET programs and/or obtain improved responses from the "so-called" poor responders. This retrospective study examined data collected over a 5-year period involving oocyte collections in a commercial embryo transfer unit to determine the stimulation protocol that was most effective in producing competent cumulus oocyte complexes, and to determine a definition of a low responder. Overall, the population of small antral follicles at the time of follicle ablation was the most important factor affecting results. This pool of small antral follicles was significantly correlated with the number of follicles at oocyte collection, and to the number of viable and transferable embryos produced. Varying the superstimulatory treatments in terms of type of FSH in association with a shorter or longer coasting period did not affect ovarian response or embryonic development rates. Low responders (less than 10% of the animals in this study) were defined as animals with a lower than average follicular response following superstimulation. Low potential animals were defined as donors producing a limited number of embryos because of the limited population of small antral follicles present in the ovaries at initiation of FSH treatment. Embryo transfer practitioners must distinguish between low responders and low potential animals as modifications to the stimulation protocol for the latter group is unlikely to result in a higher number of transferable embryos.     

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.     

Genetic markers of ovarian follicle number and menopause in women of multiple ethnicities

Oocyte loss has a significant impact on fertility and somatic health. Yet, we know little about factors that impact this process. We sought to identify genetic variants associated with ovarian reserve (oocyte number as measured by antral follicle count, AFC). Based on recently published genome-wide scans that identified loci associated with age of menopause, we also sought to test our hypothesis that follicle number and menopausal age share underlying genetic associations. We analyzed menopause-related variants for association with follicle number in an independent population of approximately 450 reproductive-aged women of European and African ancestry; these women were assessed for AFC, anthropometric, clinical, and lifestyle factors. One SNP strongly associated with later menopausal age in Caucasian women (+1.07?±?0.11?years) in previous work was also associated with higher follicle counts in Caucasians (+2.79?±?1.67 follicles) in our study. This variant is within the Minichromosome Maintenance Complex Component 8 (MCM8) gene, which we found was expressed within oocytes in follicles of the human ovary. In genome-wide scans of AFC, we also identified one marginally genome-wide and several nominally significant SNPs within several other genes associated with follicle number in both ethnic groups. Further, there were overlapping variants associated with multiple ovarian reserve markers (AFC, serum hormone levels, menopausal age). This study provides the first evidence for direct genetic associations underlying both follicle number and menopause and identifies novel candidate genes. Genetic variants associated with ovarian reserve may facilitate discovery of genetic markers to predict reproductive health and lifespan in women.     

Knockout of luteinizing hormone receptor abolishes the effects of follicle-stimulating hormone on preovulatory maturation and ovulation of mouse graafian follicles

It is considered a dogma that a secretory peak of LH is indispensable as the trigger of ovulation. However, earlier studies on hypophysectomized rodents have shown that stimulation with recombinant FSH, devoid of any LH activity, is able to boost the final stages of follicular maturation and trigger ovulation. As the expression of ovarian LH receptors (LHRs) still persists after hypophysectomy, such studies cannot totally exclude the possibility that LHR activation is involved in the apparently pure FSH effects. To revisit this question, we analyzed in LHR knockout (LuRKO) mice the progression of folliculogenesis and induction of ovulation by human chorionic gonadotropin and human recombinant FSH treatments. The results provide clear evidence that follicular development and ovulation could not be induced by high doses of FSH in the absence of LHR expression. Ovarian histology and oocyte analyses indicated that follicular maturation did not advance in LuRKO mice beyond the antral follicle stage. Neither were ovulations detected in LuRKO ovaries after any of the gonadotropin treatments. The ovarian resistance to FSH treatment in the absence of LHR was confirmed by real-time RT-PCR and immunohistochemical analyses of a number of gonadotropin-dependent genes, which only responded to the treatments in wild-type control mice. Negative findings were not altered by estradiol priming preceding the gonadotropin stimulations. Hence, the present study shows that, in addition to ovulation, the expression of LHR is essential for follicular maturation in the progression from antral to preovulatory stage.     

Antral follicle growth and endocrine changes in prepubertal cattle,sheep and goats

In the growing heifer calve, there is an early post-natal, gonadotrophin driven increase in ovarian antral follicle growth. The endocrine regulation of and reason for this initial stimulation of ovarian follicular development are not fully understood. This initial endocrine activity appears to be later held in check by negative feedback suppression mechanisms until the heifer is of a sufficient body size to initiate oestrous cycles and to reproduce. There is increasing evidence from recent ultrasonographic studies, performed in the same groups of prepubertal heifer calves, that the development of ovarian antral follicles and tubular genitalia occur in parallel. There appear to be two distinct periods of enhanced development of the reproductive organs, from 2 to 14 weeks of age and again from 34 to 60 weeks of age, or just prior to puberty. First ovulation in heifers is preceded by a gradual increase in pulsed LH secretion, which results in enhanced antral follicle development and oestrogen production. It was demonstrated that prepubertal heifers produced recurrent antral follicular waves; maximum sizes and life span of the dominant follicles of waves, as well as periodicity and FSH dependency of wave emergence were similar to those in adult cattle. In does, no Graafian follicles are seen at birth and total follicle numbers increase to 2 months of age, and then decline to 5 months of age. In ewe lambs, studies using transrectal ovarian ultrasonography showed that antral follicle recruitment and growth increased after the first 2 months of age and just before puberty. This bi-phasic pattern of changes in ovarian follicle recruitment and growth is strikingly similar to that in heifer calves, but it contrasts with earlier post-mortem examinations of ovaries in ewe lambs. Unlike in cattle and adult ewes, the rhythmic pattern of follicular wave emergence was not established in pre- and peripubertal ewe lambs. The early increase in antral follicle numbers and size in ewe lambs may be, at least in part, due to changes in FSH release and potency, and enhanced follicle production prior to first ovulation is probably caused by an increase in the frequency of LH pulses.     

Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles

In addition to pituitary gonadotropins and paracrine factors, ovarian follicle development is also modulated by oocyte factors capable of stimulating granulosa cell proliferation but suppressing their differentiation. The nature of these oocyte factors is unclear. Because growth differentiation factor-9 (GDF-9) enhanced preantral follicle growth and was detected in the oocytes of early antral and preovulatory follicles, we hypothesized that this oocyte hormone could regulate the proliferation and differentiation of granulosa cells from these advanced follicles. Treatment with recombinant GDF-9, but not FSH, stimulated thymidine incorporation into cultured granulosa cells from both early antral and preovulatory follicles, accompanied by increases in granulosa cell number. Although GDF-9 treatment alone stimulated basal steroidogenesis in granulosa cells, cotreatment with GDF-9 suppressed FSH-stimulated progesterone and estradiol production. In addition, GDF-9 cotreatment attentuated FSH-induced LH receptor formation. The inhibitory effects of GDF-9 on FSH-induced granulosa cell differentiation were accompanied by decreases in the FSH-induced cAMP production. These data suggested that GDF-9 is a proliferation factor for granulosa cells from early antral and preovulatory follicles but suppresses FSH-induced differentiation of the same cells. Thus, oocyte-derived GDF-9 could account, at least partially, for the oocyte factor(s) previously reported to control cumulus and granulosa cell differentiation.     

Differences in ovarian aging patterns between races are associated with ovarian genotypes and sub-genotypes of the FMR1 gene

ABSTRACT: BACKGROUND: Ovarian aging patterns differ between races, and appear to affect fertility treatment outcomes. What causes these differences is, however, unknown. Variations in ovarian aging patterns have recently been associated with specific ovarian genotypes and sub-genotypes of the FMR1 gene. We, therefore, attempted to determine differences in how functional ovarian reserve (FOR) changes with advancing age between races, and whether changes are associated with differences in distribution of ovarian genotypes and sub-genotypes of the FMR1 gene. METHODS: We determined in association with in vitro fertilization (IVF) FOR in 62 young Caucasian, African and Asian oocyte donors and 536 older infertility patients of all three races, based on follicle stimulating hormone (FSH), anti-Mullerian hormone (AMH) and oocyte yields, and investigated whether differences between races are associated with differences in distribution of FMR1 genotypes and sub-genotypes. RESULTS: Changes in distribution of mean FSH, AMH and oocyte yields between young donors and older infertility patients were significant (all P < 0.001). Donors did not demonstrate significant differences between races in AMH and FSH but demonstrated significant differences in oocyte yields [F(2,59) = 4.22, P = 0.019]: Specifically, African donors demonstrated larger oocyte yields than Caucasians (P = 0.008) and Asians (P = 0.022). In patients, AMH levels differed significantly between races [F (2,533) = 4.25, P = 0.015]. Holm-Sidak post-hoc comparisons demonstrated that Caucasians demonstrated lower AMH in comparison to Asians (P = 0.007). Percentages of FMR1 genotypes and sub-genotypes in patients varied significantly between races, with Asians demonstrating fewer het-norm/low sub-genotypes than Caucasians and Africans (P = 0.012). CONCLUSION: FOR changes in different races at different rates, and appears to parallel ovarian FMR1 genotypes and sub-genotype distributions. Differences in ovarian aging between races may, therefore, be FMR1-associated.     

Progress in understanding ovarian follicular dynamics in cattle

The study of follicular dynamics began in the mid-20th century, but progress has been particularly rapid in the last two decades through the use of tools that have enabled serial, non-invasive examination. A brief overview of early oogenesis and folliculogenesis is provided as a backdrop to the evolution of our understanding of follicular dynamics during the bovine estrous cycle. Studies to date support the concept that the pair of ovaries acts as a single unit and influences follicular development primarily via systemic endocrine routes involving ovarian and uterine products, the gonadotropins, and their receptors. Dominant and subordinate follicles pass through growing, static and regressing phases that have distinct morphologic and biochemical characteristics; these changes are the basis of efforts focused on diagnosing and manipulating follicular status. An update of research progress highlights recent findings on the repeatability (predictability) within individuals of follicle recruitment and wave pattern (two- versus three-wave cycles), the relationship between oocyte competence and follicular status, and the dynamics of small follicles. Recent studies documented that wave emergence and follicular dominance are apparent earlier than previously reported, and on the basis of periodic endogenous FSH surges and the presence of FSH receptors, the hypothesis that follicles become progressively entrained to waves from the earliest stages of development is introduced. Lastly, recent studies comparing old cows and their young daughters provide a new understanding of the effects of aging on gonadotropins and ovarian steroids, follicular dynamics, ovarian response to synchronization, superstimulation, and oocyte competence.     

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.