Links between age at menarche, antral follicle count, and body mass index in African American and European American women

ORIGINAL ARTICLE: EPIDEMIOLOGY

Links between age at menarche, antral follicle count, and body mass index in African American and European American women Sonya M. Schuh, Ph.D.,a Julia Kadie,a Mitchell P. Rosen, M.D.,b Barbara Sternfeld, Ph.D.,c Renee A. Reijo Pera, Ph.D.,d and Marcelle I. Cedars, M.D.b a Department of Biology, School of Science, Saint Mary's College of California, Moraga, California; b Department of Obstetrics, Gynecology, and Reproductive Sciences and Center for Reproductive Health, University of California, San Francisco, San Francisco, California; c Division of Research, Kaiser Permanente, Oakland, California; and the d Department of Cell Biology and Neurosciences, Montana State University, Bozeman, Montana

Objective: To examine the relationships between age at menarche, antral follicle count (AFC), and body mass index (BMI) in a multiethnic population of women. Design: Community-based, cross-sectional study. Setting: Academic setting. Patient(s): A total of 245 African American women and 273 European American women, aged 25–45 years, with regular menstrual cycles and no reproductive disorders. The ethnicity of these women was self-reported and genetically validated. Intervention(s): The AFCs were measured by transvaginal ultrasound during the early follicular phase. Anthropometric measurements were taken, and age at menarche was gathered by questionnaire. Main Outcome Measure(s): Determination of the associations between age of menarche and adult AFC and BMI. Result(s): Earlier age of menarche was associated with both higher BMIs and higher AFCs in adulthood, with control for female age. The antral follicle difference between early (<12 years) vs. late (R15 years) initiation of menarche in both white and black women was þ3.81 and þ3.34 follicles, respectively, which is equivalent to an approximately 20% difference in AFC. Conclusion(s): This study provides the first evidence that timing of menarche may influence AFC. Because of limited studies on African American women, this work provides additional needed data and may enhance our ability to prospectively screen and better treat various diseases associated with the female reproductive lifespan. (Fertil SterilÒ 2019;111:122–31. Ó2018 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo. Key Words: Antral follicle count, body mass index, menarche, reproductive lifespan, fertility Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertilityand-sterility/posts/38672-26582

W

hen a woman is born, she possesses a fixed number of oocytes, and they are lost throughout life from before birth to

menopause (Supplemental Fig. 1). Unlike men, women have a finite reproductive window that spans from sexual maturation and the first menstruation,

Received June 29, 2018; revised August 14, 2018; accepted September 7, 2018. S.M.S. has nothing to disclose. J.K. has nothing to disclose. M.P.R. has nothing to disclose. B.S. has nothing to disclose. R.A.R.P. has nothing to disclose. M.I.C. has nothing to disclose. This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/National Institute on Aging (grant R01HD044876 to M.I.C., B.S., R.A.R.P.); the National Institutes of Health/National Center for Research Resources and University of California, San Francisco’s Clinical and Translational Science Institute (grant UL1RR024131 to M.I.C.); NICHD grant F32HD061204-01A1 (to S.M.S.); and the Faculty Development Grant of Saint Mary’s College of California (to S.M.S.). Reprint requests: Sonya M. Schuh, Ph.D., Saint Mary's College of California, School of Science, 1928 Saint Mary's Road, Moraga, California 94556 (E-mail: [email protected]). Fertility and Sterility® Vol. 111, No. 1, January 2019 0015-0282/$36.00 Copyright ©2018 American Society for Reproductive Medicine, Published by Elsevier Inc. https://doi.org/10.1016/j.fertnstert.2018.09.007 122

or menarche, to the final menstruation and menopause. Menarche occurs around a mean age of 12.5 years, normally approximately 2 years after the onset of puberty, and menopause takes place around a median age of 51 years (1–3). These complex traits are highly variable among women, both within and between ethnic and racial groups, and are highly heritable (R50%). The female reproductive lifespan and the timing of menarche and menopause likely have numerous underlying genetic, epigenetic, and environmental correlates (2–5). VOL. 111 NO. 1 / JANUARY 2019

Fertility and Sterility® Recently, a multitude of studies have focused on the timing and genetics of menarche and menopause and their associated disease risks. Many genome-wide association and genetic studies have identified a plethora of genetic loci and variants linked with menarche (6–15) and menopause (7, 12, 15–19), underscoring the polygenic nature of these reproductive traits. Not only does the reproductive lifespan's timing and length relate to female fertility, but the lifespan itself is closely associated with risks for various diseases and cancers. Notably, many genetic and epidemiologic studies have found that earlier menarche is linked with great risks for obesity (11, 20–27), type 2 diabetes mellitus or insulin resistance (2, 28, 29), cardiovascular disease and mortality (2, 29, 30), and various cancers, including endometrial cancer (14, 31) and breast cancer (14, 15, 32–36). Conversely, later age of menarche is linked with increased risk for osteoporosis, decreased fertility, and decreased risk for breast and endometrial cancer, type 2 diabetes, and obesity (2, 29, 37). Environmental and anthropometric factors affect the timing of the reproductive lifespan, especially entry into puberty. There has been a marked decline in age at menarche throughout the 20th and 21st centuries in Europe and the United States, which has been mostly attributed to improved nutrition and health. This drop in menarcheal age has an estimated rate of 3 months per decade and has continued to decline over the past couple of decades (1, 2, 29, 38). Because critical height and weight gain precede entry into puberty, the trend toward lower menarcheal age may be occurring and continuing as a result of increasing body fat and the current obesity epidemic (3, 20, 21, 38). This phenomenon may involve common metabolic and physiologic responses, including leptin and insulin, and be due, at least in part, to shared genetic influences or pleiotropy. However, adopted girls from developing countries raised in industrialized countries experience a growth catch-up and earlier age of menarche, compared with their native country peers (1, 39). This fact highlights the role of environmental factors like nutrition and body fat in the timing of sexual maturation and also disease risk. The female reproductive window, reproductive aging, and fertility are also closely associated with several ovarian reserve markers, including antral follicle count (AFC). Several pleiotropic genetic variants have been identified that link some of these reproductive markers and milestones (Supplemental Fig. 1). Antral follicle count can be measured noninvasively by transvaginal ultrasound and reflects the total number of developing follicles (2–10 mm in diameter) within the ovaries (40–43). Indeed, the AFC is proportional to the total primordial follicle pool (44–46). Antral follicle count, like menarche and menopause, is highly variable among women (41, 43, 47) and has high heritability (48). Antral follicle count declines with age, with only a few antral follicles, on average, remaining in women in their late 40s and early 50s (42, 43, 47). This loss of follicles, or atresia, begins even before birth and results in the continual loss of follicles (and eggs within them) throughout life. When all follicles are exhausted, the reproductive lifespan ends (40, 42, 45, 49, 50). Follicle number can be a predictive factor for fertility loss, which occurs approximately VOL. 111 NO. 1 / JANUARY 2019

10 years before menopause (42). Interestingly, follicle number and menopausal age share several underlying genetic variants and are likely strongly genetically programmed traits (47). However, it is not clear whether AFC may also be correlated with the beginning of the reproductive lifespan, or whether age at menarche can predict follicle number later in life. Identification of the phenotypic associations (body, hormonal, and environmental) between the reproductive lifespan and various reproductive parameters will improve our understanding of human female fertility and reproductive and somatic health. Better understanding the variation and timing of the reproductive lifespan can assist with prospective screening, disease risk assessment, and treatment of diseases such as infertility, breast cancer, and other diseases associated with the reproductive lifespan. The relationship between age at menarche and follicle number later in life, and their connections with body mass index (BMI), have yet to be investigated. Further, very few studies have examined multi-ethnic cohorts of normative women. Most work has focused on either women of European ancestry, infertile women, or case–control cancer populations. There is also a paucity of data on non-European and African American populations. Therefore, we aimed to investigate the linkages between menarche, BMI, and AFC in a reproductive population of African American and European American women.

MATERIALS AND METHODS Study Population and Phenotypic Data We analyzed the OVA (Ovarian Aging) Study population, which is a community-based, multi-ethnic cohort of women of the San Francisco Bay Area, aged 25–45 years. These women have been well characterized in terms of various anthropometric (body), reproductive, lifestyle, genetic, and environmental factors. This study cohort has been previously described in detail (43, 47, 48, 51). In brief, blood samples, AFC, and questionnaires were obtained from 245 African American women and 273 European American women. All subjects underwent two-dimensional transvaginal ultrasound assessment of ovarian volumes and AFC, performed on the second to fourth day of the menstrual cycle. A Shimadzu SDU-450XL machine, with a variable 4–8-MHz vaginal transducer, was used to take measurements of the transverse, longitudinal, and anteroposterior diameters of each ovary using electronic calipers. Follicles with a mean diameter (of two dimensions) of 2–10 mm were counted. All echo-free structures meeting these criteria were regarded as follicles. Antral follicle count was determined by summing total AFC for both ovaries. All women were examined at the same time in their cycle, by one of two physicians (M.I.C. and M.P.R.), using the same equipment, to eliminate experimental variability. Unclear ultrasound results were excluded. Interexaminer concordance rates and correlations between repeated measurements exceed 90% (R2 ¼ 0.92). Ten percent of studies were recorded and read by an independent investigator to confirm consistency and validity. The technique used in OVA parallels that advised in a European consensus conference to standardize AFC assessment (52). 123

ORIGINAL ARTICLE: EPIDEMIOLOGY Inclusion criteria in the study required having intact ovaries, not seeking infertility treatments, and being ovulatory with normal predictable menstrual cycles of 22–35 days. Subjects were excluded if they had oligo- or anovulation, surgically diagnosed endometriosis, ovarian failure (loss of all follicles before age 40 years), fibroids that obstructed view of the ovaries, or a history of uterine or ovarian surgery. Subjects were also excluded if they had taken oral contraceptives or estrogen- or progestin-containing medications that alter the menstrual cycle within 3 months before enrollment. Of the recruited 245 African American women, 27 were excluded because of ovarian or reproductive abnormalities, and 15 were excluded because of missing or incomplete data, leaving a final total of 203 women for genotyping and analysis. Of the recruited 273 European American women, 21 were excluded because of ovarian or reproductive abnormalities, and 3 were excluded because of missing or incomplete data, leaving a final total of 249 women for genotyping and analysis. Body measurements, including weight, height, waist and hip circumference, and calculated BMIs, were obtained. Age of menarche was obtained retrospectively, by self-report. One-third to half of these women of the various ethnic groups had demonstrated fertility with a parity of one or more children, and the entire study population had no indications of reproductive disorders or infertility. Therefore, this cohort is an ideal normative population to address various questions of reproductive health, fertility, and the reproductive lifespan. This work was approved by the institutional review boards at Kaiser Permanente, University of California, San Francisco, and Stanford University, and informed consent was obtained for all subjects.

Genotyping and Ethnicity Validation We performed genotyping on a total of 203 African American and 249 European American women using the Genome-Wide Human SNP Array 6.0 (Affymetrix). As previously reported, genomic DNA was extracted and purified from white blood cells using the QIAamp DNA Blood Maxi Kit (Qiagen) according to the manufacturer's instructions (47, 51). The DNA was then scanned with the GeneChip Scanner 3000 7G (Affymetrix). After quality control filtering and ethnicity validation, a total of 200 women of genetically inferred African ancestry and 243 women of genetically inferred European ancestry were included in the final phenotypic analyses (three and six subjects were removed, respectively). Single-nucleotide polymorphisms were genotyped using Genotyping Console (GTC) v.4.0 software (http://www.affy metrix.com). Ethnicity validation and tests of population structure were performed using a principle component analysis with singular value decomposition, as previously described (47). We also genotyped subjects of the HapMap CEU (Utah residents with Northern and Western European ancestry from the CEPH collection) and YRI cohorts (Yoruban in Ibadan, Nigeria) (53) (https://www.sanger.ac.uk/resources/downloads/human/ hapmap3.html) using the same genotyping platform and software, for comparison with our OVA cohorts, as previously 124

described (47). The statistical programs R version 2.11.1 (http://www.rproject.org/) (54) and BEAGLE version 3.0.2 (https://faculty.washington.edu/browning/beagle/beagle.html) (55, 56) were used for these analyses. Using principle component analysis and comparison with CEU and YRI cohorts, fraction of African ancestry, allele composition, and population homogeneity were analyzed. Subjects who selfidentified as one race but were genetically identified as another race were removed from the study. We used only those women whose ethnicity was genetically validated because these women were genotyped and allowed us to have more homogenous, distinct ethnic groups for more accurate phenotypic comparisons and analyses. This was especially important considering our comparative questions between races with respect to AFC, BMI, and age of menarche. Further, with this approach, using data from several larger-scale studies, we have been able to make direct genotypic and phenotypic comparisons with our OVA Study cohort with regard to AFC, menopause, menarche, and BMI. Therefore, using only genetically validated ethnicity for each subject allows us the ability to make very important comparisons both between racial cohorts and with other studies around the world.

Statistical Analyses Descriptive statistics were calculated for age at menarche and all demographic, anthropometric, hormonal, reproductive, and lifestyle variables. The relationship between age of menarche, BMI, follicle number, and age were analyzed in linear regression analyses. Phenotypes and covariates were also independently compared within and between European American and African American women, using the Student t test or Welch 2-Sample t test within R (54). Because female age is associated with AFC and also with BMI, in linear regression analyses of menarcheal age vs. BMI, and menarcheal age vs. AFC, female age was controlled for using the statistical program R. Statistical differences between the slopes and intercepts of the regressions were analyzed using the F test for variable inclusion, with subject age mean-centered. These regressions were tested against the simple null models of an association between only AFC  Age and of no association between menarche and AFC or BMI. A P value of < .05 was considered statistically significant. When calculating the standardized mean differences (SMDs; variation from the cohort mean) for AFC among the different menarcheal age groups and regressions of average AFC vs. menarcheal age, it was found that all of the female ages were statistically similar between all menarcheal age groups (with all women being approximately 35–36 years). Likely because of this with both control for, and no control for female age, the results were nearly identical. Therefore, all graphs are presented with AFC values that have not been adjusted, for better visualization of the data.

RESULTS Characteristics of the Study Population We analyzed the OVA Study cohort for various genotypic and phenotypic parameters of reproductive health and the VOL. 111 NO. 1 / JANUARY 2019

Fertility and Sterility® reproductive lifespan. Body mass index, AFC, and all variables were assessed at the time of subject participation in the study and represent values collected during adulthood, for all subjects from the ages of 25 to 45 years. A total of 243 self-reported and genetically inferred European American women and 200 African American women were included in the final analysis after genotyping and quality control filtering. The relevant demographics of the study population are shown in Table 1. All the variables of this study population were within the normal range and comparable to previous data. The average age was 35.4  0.3 (mean  SEM) and 35.6  0.4 years for the European and African American cohorts, respectively.

Phenotypes and Covariates Among the European American women the mean age at menarche was 12.8  0.09 (SEM) years, whereas that of the African American women was 12.1  0.1 years, and was statistically different (P¼ .015; Table 1). As reported in our previous work, the average AFC for European American women was 15.4  0.6, whereas that of African American women was comparable at 15.6  0.7 (43, 47). In the women genotyped in this study, AFC was negatively associated with age in both ethnic groups (47). The overall average BMIs for both cohorts of women were significantly different, with a greater percentage of overweight (BMI ¼ 25–29.9 kg/m2) and obese (BMI R 30 kg/m2) women among the African American cohort (P< .05). In the genetic analysis of fraction of African ancestry when using the HapMap population data and controlling for female age, BMI was found to be significantly associated with amount of African ancestry (P¼ .014 and .012). Individuals with greater African ancestry had significantly higher BMIs. Body mass index had a small negative association with age at menarche, with control for female age (P< .05). As shown in Figure 1, higher BMIs were associated with earlier ages at menarche in both European and African American women (R2 ¼ 0.045 and 0.033, respectively; Fig. 1A). Note that these menarche data lack the high resolution possible in prospective studies, likely leading to less accuracy and

smaller associations; all menarcheal ages here have resolution of whole years and not fractions of years. However, when the women were categorized and analyzed by BMI class (normal, overweight, and obese), there were statistically significant differences among their average menarcheal ages and AFCs (Fig. 1B). In the European American women there was a positive association between mean AFC and BMI class, with significantly lower mean AFC in the overweight group compared with the obese group (13.2  1.3 vs. 18.8  2.0; P¼ .016), and marginally lower mean AFCs in the normal weight group compared with the obese group (15.3  0.7 vs. 18.8  2.0; P¼ .053). It should be noted, however, that during transvaginal ultrasound examination, it was discovered that many of the women falling into the obese BMI range had a greater number of antral follicles, but these follicles tended to be smaller in size. This trend was reversed in the African American women, however. Higher BMIs were associated with lower mean follicle counts (Fig. 1B, right). There was a significant difference between the normal-weight women and obese women, with higher mean AFCs of 19.3  1.6 in the normal-weight women compared with 13.7  1.0 in the obese women (P¼ .0024). When age at menarche was analyzed among BMI classes there was a clear association between decreasing menarcheal age and increasing BMI, in both ethnic groups (Fig. 1B). In the European American cohort the women with normal BMIs had an average menarcheal age of 13.0  0.1 years, whereas those with obese BMIs possessed a significantly lower average menarcheal age of 12.4  0.3 years (P¼ .045). In the African American cohort the women with normal BMIs had an average menarcheal age of 12.3  0.2 years. Those with overweight BMIs had an average menarcheal age of 12.4  0.2 years, whereas those with obese BMIs had a significantly lower average menarcheal age of 11.9  0.2 years (P¼ .05). Next we analyzed the average AFC for each age at menarche (Fig. 2). In both cohorts there was a small negative association between age at menarche and average adult AFC, with lower AFCs associated with later ages of menarche (R2 ¼ 0.67 and 0.26, in European and African American women, respectively). This significant correlation was small, even when corrected for female age (F test on linear regressions

TABLE 1 Demographics of study population and variables included in association analysis of age at menarche. European Americans Variable Age Age at menarche (y) AFC Normal weight (BMI <25 kg m2) Overweight (BMI 25–29.9 kg m2) Obese (BMI >30 kg m2) Cycle length (d) Parity (%)

African Americans

Mean ± SEM

n

Mean ± SEM

n

35.4  0.3 12.8  0.09a 15.4  0.6 21.8  0.1 (15.3  1.2) 26.8  0.2 (13.2  1.3) 36.2  1.4 (18.8  2.0) 29.1  0.1 36.0

245 243 245 171 43 29 243 243

35.6  0.4 12.1  0.1a 15.6  0.7 22.5  0.3 (19.3  1.6) 27.4  0.2 (16.4  1.5) 38.5  0.6 (13.7  1.0) 29.6  0.2 52.7

203 200 203 45 50 106 200 200

Note: Values are shown as mean  SEM or percentage of the cohort. Values in parentheses denote AFC (mean  SEM). a Significant differences between European American and African American women (P< .001). Schuh. Links between menarche, AFC, and BMI. Fertil Steril 2018.

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ORIGINAL ARTICLE: EPIDEMIOLOGY

FIGURE 1

Associations between age at menarche, BMI, and AFC among European American (left) and African American (right) women. (A) Shown are BMI values vs. menarcheal age for European American and African American women (regression equations and R2 values are indicated). (B) The mean total AFCs for women of each BMI class, and the average age at menarche for each BMI class (shown below x-axes) are plotted for European American (left) and African American (right) women. Asterisks indicate statistically significant differences (*P<.05; **P<.005). Schuh. Links between menarche, AFC, and BMI. Fertil Steril 2018.

with female age mean centered; P< .05), and was further analyzed by examining the SMDs for AFC among the different menarcheal age groups. Interestingly, as menarcheal age went up, average follicle count went down among both European American and African American women (Table 2). Because the African American women had earlier ages of menarche on average, and some of the earliest ages of menarche out of the entire study population, the breakdown of menarcheal age groups was slightly different between African Americans and European Americans, with an earlier menarcheal age group of <11 years in the African American cohort. In the European American women, those with a menarcheal age of %12 years had an average AFC of 17.14 follicles, compared with the mean AFC of the entire cohort of 15.4  0.6 follicles (SMD of þ3.11 follicles). This is equivalent to an 11% increase in follicle number among women with this earlier menarcheal age. Those with a menarcheal age of R15 years had a lower average AFC of 13.33 follicles and an SMD of 3.53, equivalent to a 13% reduction in follicle number (Table 2). It is of note that the average antral follicle difference between early vs. late initiation of 126

menarche in European American women was 3.81 follicles, which is equivalent to a 22% difference in AFC. In the African American women, those with a menarcheal age of <11 years had an average AFC of 17.67 follicles, compared with the mean AFC of the entire cohort of 15.6  0.7 follicles (SMD of þ2.79 follicles). This is equivalent to a 13% increase in follicle number among women with this earlier menarcheal age. Those African American women with a menarcheal age of R15 years had a lower average AFC of 14.33 follicles and an SMD of 1.72, equivalent to an 8% reduction in follicle number (Table 2). Notably, the average antral follicle difference between early vs. late initiation of menarche in African American women was 3.34 follicles, which is equivalent to a 19% difference in AFC.

DISCUSSION We evaluated phenotypic and ovarian reserve associations with age at menarche in a multiethnic cohort of women directly assessed for antral follicle number and various anthropometric variables. Our results indicate that the VOL. 111 NO. 1 / JANUARY 2019

Fertility and Sterility®

FIGURE 2

Association between age at menarche and mean AFC among European American and African American women. Shown are menarcheal ages vs. average total AFC for European American (A) and African American (B) women. Regression equations and R2 values are indicated. Schuh. Links between menarche, AFC, and BMI. Fertil Steril 2018.

timing of menarche may influence both adult BMI and antral follicle number among white and black women. The antral follicle difference between early vs. late initiation of menarche in both white and black women was þ3.81 and þ3.34 follicles, respectively, which is equivalent to an approximately 20% difference in AFC. There was a statistically significant clear association for lower menarcheal age with higher adult BMIs in both African Americans and European Americans. Earlier age at menarche was predictive of both higher BMIs and higher follicle counts among all women. As found here and in our previous genetic association studies, BMI was also associated with amount of African ancestry (47). The connection between BMI and menarche is similar to many previous findings of higher body fat and BMI in females who experienced an earlier age of menarche (2, 3, 5, 20–22, 29). Girls who are ‘‘early bloomers’’ tend to be taller and with more body fat as girls, and shorter and with more body fat and higher BMIs as women (20, 21, 38, 57). At any given chronological age, females who had an earlier age at menarche tend to have greater adiposity and BMIs than females who experienced menarche later. Further, as found here, African American women have an earlier age of menarche and greater BMIs on average, compared with VOL. 111 NO. 1 / JANUARY 2019

white women (15, 38, 58). Because earlier age of menarche is a risk factor for obesity, type 2 diabetes, and cardiovascular disease among other chronic diseases, future studies on these at-risk populations are of upmost importance. Because we did not have information on anthropometric variables, including BMIs, at the time of menarche for each woman, we do not know for certain whether adolescent BMIs were similar or consistent with adult BMIs for the women in this study. However, many epidemiologic studies have reported close association between adolescent BMI and adult BMI and strong correlations between earlier menarche and both higher adolescent BMI and adult BMI/adiposity (20, 21). Because the characteristics of our study population are similar to those of many other studies, we postulate that the same associations between adolescent BMI and adult BMI are likely present in this study cohort. Additionally, self-reported reproductive milestones, such as menarche and menopause, have been found to have excellent accuracy, whereas height, weight, and body fat are much more difficult to recall retrospectively and would likely not provide additional meaningful data. The relationship between menarcheal timing and antral follicle number in adulthood has not been previously 127

ORIGINAL ARTICLE: EPIDEMIOLOGY

TABLE 2 Mean AFCs and SMD for different ages of menarche in European American and African American women. Variable European Americans All women Menarcheal age % 12 y Menarcheal age < 14 y Menarcheal age R 14 y Menarcheal age R 15 y African Americans All women Menarcheal age < 11 y Menarcheal age < 12 y Menarcheal age < 14 y Menarcheal age R 14 y Menarcheal age R 15 y

Mean

n

Rangea

SMDb

15.36 17.14 15.70 14.51 13.33

243 106 173 70 27

1–53 1–53 1–53 1–38 2–31

n/a þ3.11 þ0.60 1.47 3.53

15.60 17.67 15.89 15.79 14.75 14.33

200 30 72 163 36 21

2–52 2–48 2–49 2–52 2–39 2–39

n/a þ2.79 þ0.39 0.25 1.15 1.72

a

Antral follicle count range for given menarcheal age group. Standardized difference in AFC between specific menarcheal age group and that of the specific ethnic cohort.

b

Schuh. Links between menarche, AFC, and BMI. Fertil Steril 2018.

investigated. We found here that antral follicle number may be affected by the timing of menarche. There was an inverse relationship between age of menarche and AFC. This association was independent of female age and crossed racial backgrounds: it was present in both white and black women. Earlier work analyzing follicle numbers from ovarian biopsies from fetuses through menopausal-age women found that the total number of nongrowing follicles is depleted throughout life until there are approximately 1,000, at the time of menopause (45, 49, 50). One of the most complete, consistently performed studies by Hansen et al. (45) found that follicles are lost throughout life, and this loss slightly accelerates over time and is best fit by a simple power model. Our later work on the decline in adult AFC with age was consistent with this finding (43, 47). A closer examination of these data indicates that the greatest losses seem to occur during the late childhood/early adolescent years (10–19 years) and in mid-life (35–40 years) (45, 49). According to these and our current findings, menarche initiation and the early ovarian cycles may slow this follicle loss. Therefore, girls who begin cycling sooner might avoid the bigger follicle loss that happens in the early teenage years. Our findings superimposed on these life history follicle count results seem to align with this hypothesis. There are limited, conflicting data on menarcheal timing and fertility or ovarian reserve. Women with later ages of menarche have been reported to have overall slightly reduced fertility, whereas earlier menarcheal age has been associated with increased fertility (2, 59). As we find here, this observed phenomenon may be due at least in part to slightly reduced follicle counts throughout life in women who experienced later ages of menarche. However, Weghofer et al. (60) reported that earlier age at menarche was associated with reduced functional ovarian reserve later in life, as measured by the ovarian reserve hormone antim€ ullerian hormone (AMH) (60). In contrast to our findings, they reported that age-specific functional ovarian reserve was diminished in women with early menarche, 128

defined as <13 years. They postulated that there might be a relationship between timing of menarche and a woman's follicle pool size and/or speed of follicle recruitment. Indeed, our results support a connection between menarche and the follicular pool. However, there is a major difference between our respective studies that may explain the difference in findings. The women of this previous study were from a diverse infertile population undergoing clinical screening who were analyzed for AMH levels, whereas the women of the present study were a normative population and were directly analyzed for AFC. However, we too collected reproductive hormone data, including that of AMH, on all the women of our study. As previously reported, AMH and AFC were strongly positively correlated in our study cohort (R2 ¼ 0.67; P< .05), with these reproductive phenotypes even sharing common underlying genetic variants (51). Antral follicle count, as well as AMH, were correlated with age of menarche (P< .05). Although the previous study found a decrease in AMH with early menarche, we found an increase of AFC and AMH with early menarche. However, our study populations were very different regarding reproductive and fertility phenotypes, which might at least partially explain the confounding results. Notably, follicular recruitment (the development and growth of primordial follicles; Supplemental Fig. 1) peaks around menarche and declines thereafter (61). Further, follicular recruitment is associated with greater follicle loss. The association between follicular recruitment and follicular survival to the later stages of development is not constant across the reproductive lifespan (61). Therefore, the combination of greater loss of nongrowing follicles in addition to greater follicle recruitment may indeed lead to more rapid follicle loss in these early adolescent years. In light of our findings, it seems likely that females who begin menarche much sooner might avoid or slow some of this increased loss of follicles that occurs during the early teenage years. There are, however, genetic determinants underlying antral follicle number (47, 48), so it is highly likely that the total follicle pool and rate of follicle loss also have genetic determinants and are impacted not only by menarcheal timing but also by overlapping genetic variants and environmental influences. There are numerous interactions between environmental conditions and genetic susceptibility that can influence physiologic and pathological processes, including timing of menarche. The age of menarche, as well as height, weight, and adiposity, all have strong hereditary and nutritional and environmental components. For girls whose mothers had an early age of menarche, their age of menarche mirrors that of their mother's, and vice versa for women having a later age of menarche (4, 62). We now know that many genes are likely at least partially responsible for this inheritance. Interestingly, the genetic loci of age at menarche possess substantial cross-over with genes implicated in BMI, obesity, height, and various diseases, including rare disorders of puberty (3, 10, 11, 25, 27, 57). Additionally, several genetic variants that influence age of menarche also affect other events of puberty, including the age of thelarche, or the beginning of breast development (6, 25, 63, 64). As VOL. 111 NO. 1 / JANUARY 2019

Fertility and Sterility® discovered in a study of girls in Copenhagen who were followed throughout puberty and analyzed for genetic variants and clinical stages of puberty, LIN28B (Lin Homolog 28 B), a previously identified height-related gene, was linked with both thelarche and menarche (64). Additionally, many large genome-wide studies have found links between LIN28B and menarche (6–8, 10, 15, 64). Importantly, genetic loci that influence the age of menarche and age of thelarche often also impact breast cancer risk in women (15, 33–36). These studies underscore the tremendous complexity of the regulation of puberty timing and the pleiotropy and environmental interactions that likely exist between menarche, body fat, and several diseases. This work provides new associations between ovarian reserve and age of menarche in a multi-ethnic, normative population of women. Our results show that timing of menarche may influence both BMI and antral follicle number among African American and European American women. Owing to limited studies on African Americans and individuals of non-European ancestry, this work provides additional needed data on this underrepresented and understudied population. Better understanding the phenotypic and genotypic variation and correlates of the female reproductive lifespan may enhance our ability to prospectively screen, assess disease risk, and better treat various diseases, such as infertility and breast cancer. Acknowledgments: The authors thank all of the female study participants who made this work possible; the administrative support at Saint Mary's College, especially Amy Bockman, Julie Curtiss, and Dr. Roy Wensley for various assistance and support; Dr. Nicholas Johnson, of Stanford (now Google), for assistance with custom-made script for data analyses within R and Beagle; Elizabeth Zuo and Natalia Kosovilka of the PAN Facility for their assistance with microarray processing; Marty Flores and Alison Lerner for some assistance with DNA sample maintenance and processing, at Stanford University; all the clinical research staff, especially Deborah Trevithick, Margaret Takeda, Tatiana Carranza, Chim Lau, and Carolyne Addauan-Andersen (at UCSF); and Rosemary Murphy, Mariana Pereyra, Katy Leung, Liliana ValderramosMetzger, Claudia Cruz, and Maila Martin (at Kaiser), for their efforts in the coordination of study participants.

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Fertility and Sterility® Enlaces entre la edad de la menarquia, el recuento de folículos antrales y el índice de masa corporal en mujeres afroamericanas y europeoamericanas. Objetivo: Examinar las relaciones entre la edad de la menarquia, el recuento de folículos antrales (AFC), y el índice de masa corporal (IMC) en una poblaci on de mujeres multietnica. ~o: Estudio transversal basado en la comunidad. Disen Entorno: Academico. Paciente(s): Un total de 245 mujeres afroamericanas y 273 mujeres europeas americanas, de edades entre los 25 y 45 a~ nos, con ciclos menstruales regulares y sin trastornos reproductivos. La etnicidad de estas mujeres fue auto-informada y validada geneticamente. Intervencion(es): El AFCs fue medido por ecografía transvaginal en la fase folicular precoz. Se tomaron medidas antropometricas, y la edad de la menarquia fue recogida mediante cuestionario. Principales medidas de resultados: Determinacion de las asociaciones entre la edad de la menarquia y el AFC e IMC en la edad adulta. Resultados: La edad de la menarquia mas temprana se asoci o tanto con el IMC como con los AFC mas altos en la edad adulta, con control para la edad femenina. La diferencia de folículos antrales entre el inicio precoz de la menarquia (<12 a~ nos) vs. el tardío (R15 a~ nos) en ambos grupos de mujeres blancas y negras, fue +3.81 y +3.34 folículos respectivamente, lo que equivale aproximadamente a un 20% de diferencia en AFC. Conclusion(es): Este estudio proporciona la primera evidencia de que el momento de la menarquia puede influir en el AFC. Debido al limitado n umero de estudios en mujeres afroamericanas, este trabajo suministra datos adicionales necesarios y puede mejorar la capacidad para detectar y tratar mejor de manera prospectiva varias enfermedades asociadas con la vida reproductiva femenina.

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