We evaluated genetic associations with ovarian reserve and validated variants associated with menopause, in an independent cohort of women directly assessed for follicle number. Our results indicate that there are underlying variants/genes associated with both ovarian reserve and menopause, providing more evidence for the connection between these two traits. Importantly, we found one of these genes, MCM8, is expressed within follicles of the adult human ovary and is therefore an excellent candidate gene for future studies of human oocyte development and ovarian function. Our work carries population-level genome-wide findings to the cellular level and provides likely functional evidence for a role of MCM8 in ovarian biology. We also identified several additional SNPs associated with follicle number in Caucasian and African American women, with genetic loci that were validated in the different ethnic cohorts. These findings provide further evidence for a genetic basis of the reproductive lifespan and prospective markers and genes for future studies and clinical diagnostics.
Because of the close association between ovarian reserve and female fertility, as well as disease risk, studies have investigated the ability of various non-invasive markers to serve as proxies for ovarian reserve and predictors of reproductive potential. The ability to predict oocyte loss and reproductive lifespan would enable improved reproductive planning and health care decisions.
Our recent work examined genetic variants and environmental factors associated with hormone markers of ovarian reserve, including FSH and AMH (Schuh-Huerta et al. 2012
). As previously reported, there were several variants associated with AMH or FSH, that were also associated with follicle number; four out of the nine top variants were validated for association with AFC. These variants and genes linked with FSH and AMH, including those also significantly associated with AFC are shown in Fig. (asterisks indicate overlapping genes). Because of the close relationships between these serum hormone levels, especially AMH, and total number of antral follicles, this prompted a closer examination of specific genetic factors associated with follicle number. The number of preantral and antral follicles to a large extent drives serum AMH levels, and therefore genetic associations with AMH levels might in large part be driven by follicle number. And this is indeed the case. Here, we report that many of the identified variants associated with AFC are also associated with AMH (Online Resource Table 3; Fig. ). Of the top 16 variants associated with AFC, seven of these are significantly associated with AMH levels, encompassing the GPR12
, and BLK
genes. Therefore, there are both overlapping and independent genetic markers associated with follicle number and other accurate markers of ovarian reserve.
AFC is considered one of the most reliable non-invasive methods for determining the ovarian reserve. Antral follicles are small (millimeters in diameter), fluid-filled structures that contain the developing oocytes and are visible by ultrasound. The assumption that the number of antral follicles is proportional to the total oocyte pool remaining in the ovary is supported by histologic data (Hansen et al. 2008
; Morris et al. 2002
) and has led investigators to compare the relationship between AFC and age (Broekmans et al. 2004
; Faddy et al. 1992
; Rosen et al. 2010b
; Scheffer et al. 1999
). In mathematical models that examined reproductive events in non-contracepting populations, AFC was predictive of age at natural fertility loss (~10 years prior to menopause) and age at menopause (Broekmans et al. 2004
). AFC is also lower in infertile women (Rosen et al. 2011
). Our prior work detected great variability in antral follicle count among Caucasian women (Rosen et al. 2010b
) and a lowered AFC associated with an earlier maternal age of menopause (Rosen et al. 2010a
), suggesting a strong genetic component to ovarian reserve. Indeed, maternal age of menopause is a predictor for AFC such that women whose mothers entered menopause earlier, have lower AFCs at any given age (Rosen et al. 2010a
). However, no previous studies have identified genetic links with follicle number or between follicle number and menopause.
Candidate gene and linkage association studies, mostly in the context of reproductive disorders such as POF (menopause before age 40), have identified a limited number of genes and variants that may be associated with the follicle pool and ovarian aging (Bretherick et al. 2008
; Fassnacht et al. 2006
; Gromoll and Simoni 2005
; Kevenaar et al. 2007
; Simpson 2008
; Suzumori et al. 2007
; Tung et al. 2006
; Yoon et al. 2010
). The X chromosome has a significant role in ovarian function. Monosomy X results in women with Turner syndrome who have accelerated follicle loss, degeneration of ovaries, and generally lack functional oocytes, rendering them infertile (Fassnacht et al. 2006
; Turner 1972
). Various forms of POF, in about 1–4 % of women, are often familial and linked with genetic and structural abnormalities of the X chromosome (Fassnacht et al. 2006
). However, in many POF cases, the underlying genetic variants or mutations are unknown. Putative roles of other female fertility genes have come from transgenic mouse models and include various proteins and transcription factors of oogenesis/folliculogenesis (Fassnacht et al. 2006
; Knight and Glister 2006
; Suzumori et al. 2007
). While POF is rare, the genetic variants responsible for the normal variation in ovarian reserve (oocyte number) in the general population have not been identified.
Recently, a few large-scale GWASs have discovered SNPs associated with age of menarche (Elks et al. 2010
; He et al. 2009
; Ong et al. 2009
; Sulem et al. 2009
) and age of menopause (He et al. 2009
; Murray et al. 2010
; Stolk et al. 2009
). In these large cohorts of post-menopausal European women, several SNPs led to increases or decreases of about 3 months to 1 year in age at menopause, further implicating genetic factors in the timing of follicle loss. Menopausal age is an indirect, retrospective marker of follicle loss; direct measurements of follicle number were not performed in these studies. Direct studies of follicle number and menopausal age in the same women have not been possible. Moreover, most previous work has focused on infertile populations, small observational studies, or post-menopausal women. Much data are lacking on genetics and reproductive parameters in women of reproductive age, without ovarian disorders, and not seeking infertility treatments. There is also little data on women of non-European ancestry. Therefore, our community-based cohort of reproductive-aged women of multiple ethnicities is an ideal population to investigate genetic loci associated with follicle number, and in combination with previous cohorts, menopause. GWA studies in this population can also determine whether additional specific variants may be associated with ovarian reserve markers and various fertility and somatic disorders in different ethnic groups.
We found that antral follicle number declined with female age, showing a similar pattern in both Caucasian and African American women, although with a trend for slightly greater follicle counts, greater variability, and a slower extrapolated rate of follicle loss in the African Americans compared to Caucasians (0.76 vs. 0.93 follicles/year, respectively). To our knowledge there are no other publications on follicle number in fertile African American women, so these results cannot be compared to any previously examined cohorts. It has been reported that African American women may have an earlier age at menopause, 6–12 months sooner than Caucasians (Bromberger et al. 1997
), while other studies have found no difference in menopausal age between the two racial groups (Gold et al. 2001
). Our results on follicle number indicate that this African American population does not appear to have a reduced ovarian reserve or increased risk for entering menopause earlier than the Caucasian population.
There were many differences in anthropometric, hormonal, and reproductive assessments between the Caucasian and African American women. The African American women had greater average weight, shorter height, greater BMI, an earlier age at menarche, greater parity, and several other clinical/hormonal differences compared to the Caucasian cohort. In both ethnic groups, AFC was most closely associated with serum levels of AMH and FSH, independent of female age (Caucasians: R2 = 0.67 and −0.16, respectively; African Americans: R2 = 0.49 and −0.14, respectively; P < 0.001). AMH levels increased and FSH levels decreased with antral follicle count. While we found that BMI was not associated with the total number of follicles, interestingly BMI was associated with total fraction of African ancestry. Although BMI and follicle counts were higher in the African American cohort this cannot be explained by higher rates of PCOS, as women with PCOS and hyperandrogenism, as well as other reproductive disorders were excluded. It is likely that many of these confounding variables, including ancestral genetic background, may influence the oocyte pool, follicular atresia, and reproductive lifespan, implicating several causative factors in differences between racial groups.
To examine the hypothesis that the ovarian reserve and reproductive lifespan are closely associated and affected by multiple genetic factors, we tested the previously identified variants associated with menopausal age in European women, for association with follicle number, AMH and FSH in our population of women living in the US. We found that variants in the region immediately flanking the top menopausal SNP, rs16991615, were associated with AMH and FSH, in both ethnic groups. Notably, the majority of these variants were linked with AMH, FSH, and/or AFC, and were replicated in both Caucasian and African American women. Together, these results indicate there are genetic associations underlying ovarian reserve and menopausal age (reproductive lifespan) in women of multiple ethnicities. We further found that directions and magnitudes of the effects of all tested menopausal variants were highly similar between age at menopause and follicle number among the Caucasian women—variants associated with later menopausal age were associated with higher follicle counts and vice versa. There were ±0.3 to 1-year differences in menopausal age, which were correlated with ±2 to 3-follicle differences in AFC, providing evidence that these reproductive traits are functionally and genetically related. We found that variant rs16991615 within the MCM8
gene was significantly associated with later menopausal age of +0.92 to 1.07 years in previous studies (He et al. 2009
; Murray et al. 2010
; Stolk et al. 2009
) and greater follicle numbers of +2.79 ± 1.67 follicles in the Caucasian cohort of this study. This significant non-synonymous SNP in exon 9 of MCM8
results in an amino acid change from glutamic acid to lysine, which may alter protein function.
is a member of an evolutionarily conserved family of proteins that are involved in whole genome replication. However, MCM8 does not associate with the other MCMs and likely has a role in DNA elongation (after replication licensing). It is expressed in a variety of human cell lines and tissues (Gozuacik et al. 2003
) and localizes specifically to the chromatin in the nucleus in S phase of the cell cycle (Gozuacik et al. 2003
; Maiorano et al. 2005
). Notably, the fly ortholog of MCM8
), has a pivotal role in the formation of meiotic crossovers during female gamete development with its loss leading to chromosomal non-disjunction, aneuploidy and diminished fertility (Blanton et al. 2005
mutants are specifically unable to copy enough DNA to form linkages between chromosomes during egg meiosis. MCM8 is also expressed in oocytes of the frog, Xenopus
(Maiorano et al. 2005
). Analysis of human EST databases indicates MCM8
may have moderate expression in both the developing embryo and the ovary (http://www.ncbi.nlm.nih.gov/UniGene
), but there are no reports on the expression or role of this highly conserved gene in human gametes.
We discovered that MCM8 is expressed within the human ovary and colocalizes with the germ cell marker, VASA, within the oocytes of several stages of follicle growth. VASA
is expressed specifically within germ cells and developing oocytes and sperm and is therefore a good marker of human oocytes. MCM8 was also expressed at low levels in peritubular cells, but not in developing sperm within the human testis. Interestingly, in some tissues MCM family members are functionally regulated by estrogen and progesterone (Pan et al. 2006
), providing another plausible link between MCMs and ovarian function. Taken together these findings present the first genetic links underlying oocyte number and menopause. There may be direct effects of MCM8
and other genes on oocyte meiosis, development or depletion, and ultimately, menopausal age providing a mechanism by which they influence the reproductive lifespan. Future in vitro studies can examine the functional roles of MCM8
, as well as other identified candidate genes in germ cell and follicle development.
If a gene impacts the initial number of oocytes with which a woman is endowed or their rate of depletion, that gene might be expressed in oocytes, follicles and/or the ovary and vary in the human population. Most of the identified genes containing or near by the variants associated with AFC have no previously demonstrated roles in female reproduction, but like MCM8
, several are expressed in the human ovary. LRRC61
, proximal to the most significant variant and associated with +7 to 8 follicles, encodes a protein-binding gene and is widely expressed, with highest expression in the human ovary, cervix, and adrenal gland. However, there has been no examination of function in female biology. Other variants are within the MACROD2
gene at 20p12.1 and nearby the KLRAP1
gene at 12p13.2. Little is known about the function of MACROD2
, although it appears to be expressed in several tissues of the body including the ovary and testis. The TAF4B
gene, nearby an associated variant is expressed in granulosa cells of the follicle in both female mice and women (Freiman et al. 2001
; Wu et al. 2005
). It is of interest that TAF4B
null mice are female infertile with defects in folliculogenesis and oocyte maturation and resulting POF (Lovasco et al. 2010
) (Ovarian Kaleidoscope database: http://ovary.stanford.edu/
). Future functional studies assessing gene expression and genetic silencing and overexpression studies in female cell lines and ovarian tissues are needed to explore the roles of these genes in human oocyte biology.
As the ability of this work to validate many of the variants associated with menopausal age, as well as discover new genome-wide variants associated with follicle number is limited by sample size, future studies may aim for validation of these findings. As this study is the first to perform a GWA on antral follicle number in fertile, normo-ovulatory women and also to examine multiple ethnic groups, there are no other cohorts for independent validation. It is hoped that future work will collect data on AFC to enable cross-validation and multi-site meta-analyses.
In this and previous work we identify a set of candidate genes that are associated with one or more of the human ovarian reserve markers—AFC, AMH, FSH, and menopause (Fig. ). It is noteworthy that several variants associated with both follicle number and menopause localized to the same genomic regions of 20p12.1–12.3 and 13q (Online Resource Figure 3). Our previous work found common regions on 13q and 12p linked with several ovarian reserve markers (Schuh-Huerta et al. 2012
). Further, other work has identified loci on chromosomes 13, 19, and 20 that are associated with age at menopause (Stolk et al. 2009
). Therefore, these chromosomal regions and associated genes may be hot spots for causative alleles or genes associated with ovarian reserve and reproductive lifespan. Replication of these findings in independent larger cohorts, as well as longitudinal studies directly measuring follicle number over time and the prospective onset of menopause in this cohort could test the actual phenotypic and genotypic associations between rates of follicle loss and entry into menopause within the same population of women.
In conclusion, we present the first evidence for common variants and new candidate genes underlying follicle number (ovarian reserve), menopause, and the reproductive lifespan. A better understanding of the genes and environmental factors that impact ovarian reserve may provide greater insight on the process of reproductive aging and the genetic requirements of human fertility. Prediction of follicle loss could ultimately enhance reproductive potential and overall health. It is hoped that this work may help form a foundation for clinical applications in identifying women at risk for early ovarian failure and hence associated reproductive and somatic diseases.