Markers of ovarian reserve as predictors of future fertility.

Study flow is presented in Figure 1 . Of the 474 TTC participants that consented to future follow-up, 354 (75%) completed the reproductive history questionnaire. Thirteen women had missing or incomplete pregnancy history in the questionnaire. Five women did not have serum values of AMH, FSH, or inhibin B. The final analysis dataset included 336 women. Following their pregnancy attempt during TTC, there were 278 pregnancy attempts among these 336 women. There were 225 pregnancies resulting in live birth and 14 pregnancies without live birth. Ninety-seven women attempted to conceive and did not become pregnant. Seventy-three women met the composite outcome for infertility. Eighty-five women reported zero pregnancy attempts or pregnancies after TTC. Demographic variables and comorbidities of the participants included the follow up study compared to those that only participated in the original TTC are presented in Table 1 . Women that participated in the follow up study tended to be younger at enrollment, white, and had achieved a higher education level compared to women that did not participate in the follow up study. Of the analyzed cohort (n=336 women), most women were non-obese (87%), white (87%), and had a graduate degree (78%). All participants had at least one biomarker available for analysis. AMH values were available for all women in the follow-up cohort. Serum FSH and inhibin B levels were missing from 11.3% of cohort. Regarding AMH levels, 8% of women had levels below < 0.7 ng/mL; 11% of women had levels ≥ 8.5 ng/mL. Nine percent of women had FSH levels ≥10 mIU/mL. Median (Q1, Q3) levels of inhibin B were 70 pg/mL (34.6, 101.5), respectively. The probability of live birth did not differ for women with low AMH (AMH < 0.7 ng/ml) compared to women with normal AMH levels (RR 1.32; 95% CI, 0.95–1.83, Table 2 ). Similarly, the probability of live birth did not differ for women with high FSH levels compared to women with normal values (RR 1.28; 95% CI, 0.97–1.70). Inhibin B levels were not associated with the probability of a future live birth (RR 1.00, 95% CI, 0.98–1.02) ( Table 2 ). Women with low AMH values did not have a significantly higher risk of future infertility (RR 0.65; 95% CI, 0.21–2.07) compared to women with normal AMH values ( Table 3 ). Similarly, there was not a significant association between high FSH levels and risk of future infertility (RR 1.69; 95% CI, 0.86–3.31). There was not a significant association between inhibin B levels and risk of infertility in continuous analysis (RR 0.99; 95% CI, 0.93–1.05) ( Table 3 ). The association between biomarkers of ovarian reserve and fecundability in future pregnancy attempts are presented in Table 4 . Fecundability ratios suggested that women with diminished ovarian reserve, as measured by AMH and FSH levels, did not have reduced fecundability compared to women with normal ovarian reserve (FR 0.97; 95% CI, 0.59, 1.60; and FR 0.86; 95% CI 0.55–1.36, respectively), which contrasts with our hypothesis. Similarly, inhibin B levels were not associated with the probability of conceiving in a given menstrual cycle ( Table 4 ).

TTC was the first prospective, time-to-pregnancy study designed to determine the extent to which biomarkers of ovarian reserve predicted a woman’s current reproductive capacity independent of age. TTC recruited women 30 to 44 years of age and cohabitating with a male partner who had been attempting to conceive for less than 3 months and followed them for up to 12 months of pregnancy attempt. Women were excluded if they had a known infertility diagnosis (tubal factor, polycystic ovarian syndrome, surgically diagnosed endometriosis, current or prior infertility treatment, prior sterilization) or a partner with infertility. Participants completed a web-based intake questionnaire, online daily and monthly questionnaires, and a study visit in which they provided blood and urine samples. Study methodology and results are available in the prior publication( 8 ). From April 2008 to August 2015, 1189 women were screened and deemed eligible to participate, 831 had a study visit and provided blood and urine samples. Of the 831, 71 (9%) were subsequently lost to follow-up; 73 (9%) withdrew from the study or initiated fertility treatment prior to the end of the study; 154 (18%) did not conceive; and 533 (62%) conceived. Eighty-eight percent of women (n=469) had a known pregnancy outcome with 349 (74%) pregnancies resulting in a live singleton or multiple birth, 111 (24%) in miscarriage, and 9 women with ectopic pregnancy, abortion, or stillbirth after 20 weeks. At the conclusion of the original cohort study, all participants who provided a blood or urine sample were provided their biomarker results and invited to remain active in the cohort. Those that agreed provided consent for continued identification of data and samples, provided updated contact information, and allowed for future contact for research. Of the original 831 participants, 474 agreed to continued participation in the TTC cohort. From October 6, 2020 to February 10, 2021, active participants were asked to complete a web-based, reproductive history questionnaire including questions about their pregnancy attempts and outcomes since participating in TTC. If they were unable to complete the questionnaire electronically, the questionnaire was completed via telephone. Women were provided with an electronic gift card as compensation for their time and participation. Women were contacted up to 3 times to encourage participation. This study was approved by the Institutional Review Board (IRB) of Duke University. Data collected as part of the original TTC study were used in this analysis. Specifically, biomarker levels as measured in the serum obtained at enrollment in TTC.( 8 ) AMH, FSH, and inhibin B levels were measured using enzyme linked immunosorbent assays (ELISA) (Ansh labs)( 8 ). The primary outcome measures were: (1) probability of future live birth; (2) probability of future infertility; and (3) future pregnancy attempt time. A woman was defined as having a live birth if she had one or more live births conceived naturally or with fertility treatment at least 3 years from the TTC enrollment date, to exclude any outcomes associated with original TTC pregnancy attempt. “Infertility” was defined as having a formal infertility diagnosis, a pregnancy attempt of ≥ 12 months duration, or use of fertility medication at any time after TTC. Attempt time was defined as the reported number of months (or menstrual cycles) women attempted to conceive in each pregnancy attempt, whether or not the attempt resulted in a pregnancy. All pregnancies with last menstrual period dates that occurred ≥ 6 months from the end of the original TTC study were included in the analysis to ensure that outcomes were not associated with the original TTC attempt. Primary outcome results were calculated using two denominators: (1) total number of women; and (2) number of women who attempted to conceive after their original attempt in TTC. The biomarkers were categorized corresponding to the following clinical cut-points: AMH (3 categories: 8.4 ng/mL)( 8 , 9 ) and serum FSH (2 categories < 10 or ≥ 10 mIU/mL)( 10 ). The referent groups for AMH and FSH were 0.7–8.4 ng/mL and < 10 mIU/mL, respectively. Inhibin B was modeled continuously given the lack of a clinically relevant cut-off value. Demographic variables, covariates, and ovarian reserve biomarker levels among women who participated in the study were summarized using mean (standard deviation, SD), median (25 th percentile [Q1], 75 th percentile [Q3]), or frequency (percent). Modified Poisson regression models( 11 ) were used to model the association between biomarkers of ovarian reserve and future live birth and future infertility. Risk ratios (RRs) and 95% confidence intervals (CIs) were presented. The association between ovarian biomarkers and future fecundability, the probability of conceiving in a given month, was modeled using discrete-time survival models, specifically, generalized estimating equations (GEE) with a binomial distribution and a complimentary log-log link. Since an individual could have multiple pregnancy attempts, AR( 1 ) variance-covariance matrix was used to account for this correlation and selected based on quasilikelihood under the independence model criterion (QIC). Fecundability ratios (FRs) and 95% CIs were calculated. A FR less than one suggests that the “exposed” are less likely to conceive in a given menstrual cycle compared to the “unexposed” group. All models were adjusted for age at blood draw as a continuous variable, obesity (yes or no; body mass index [BMI] measured at baseline calculated as ≥30; calculated as weight in kilograms divided by height in meters squared) at enrollment in TTC, race (white or non-white), use of hormonal contraception preceding enrollment in TTC (yes or no), and year of enrollment. For the future fecundability outcome, models also included age at pregnancy attempt. All analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC). All testing was two-sided with p < .05 considered statistically significant. An a priori power calculation was used to determine study sample size. Baseline probabilities of live birth were 59% and 42%, respectively, for participants with normal AMH and low AMH (difference = 0.17)( 8 , 12 ). The study required a total of 230 women (115 in each group) to detect a difference of 0.17 at 80% power and a 0.05 level of significance. Power calculations were performed in PASS 2020( 13 ) simulation program.

In this cohort study, women with biomarkers suggestive of diminished ovarian reserve did not have reduced future fertility: lower live birth rates, higher incidence of infertility, or reduced fecundability in future pregnancy attempts. These findings suggest that diminished ovarian reserve is not associated with reduced future reproductive capacity. Given the lack of association, women should be cautioned regarding use biomarkers of ovarian reserve as predictors of their future reproductive capacity.

In this cohort study which examined subsequent pregnancy attempts among women enrolled in TTC, ovarian reserve biomarkers indicating diminished ovarian reserve (AMH < 0.7 ng/mL or FSH ≥ 10 mIU/mL) were not associated with reduced future fertility. Probability of achieving a live birth, being diagnosed with infertility, and probability of conceiving in a given menstrual cycle during future pregnancy attempts did not differ by AMH or FSH value. Early follicular phase inhibin B levels were not associated with future fertility outcomes. This is the first study to examine the value of biomarkers of ovarian reserve, which are commonly used as fertility tests, to predict future fertility. These findings contribute to a growing body of literature regarding the role of ovarian reserve biomarkers in clinical practice. Few studies have examined the association between biomarkers of ovarian reserve and current fertility. Steiner et al . (2011) in a prospective pilot study of 100 women aged 30 to 45, found that women with AMH levels < 0.7 ng/mL experienced significantly lower fecundability that women with higher AMH levels. Women with FSH ≥ 10 mIU/mL had lower fecundability, but the result was not statistically significant( 9 ). However, a subsequent analysis of 750 women followed prospectively in TTC showed that diminished ovarian reserve (AMH < 0.7 ng/mL) was not associated with a statistically significant difference in probability of conceiving in 6 or 12 cycles of attempt compared to women with normal AMH. Again, high FSH levels (≥10 mIU/mL) were not associated with fecundability. Another prospective study of younger women aged 20–35 did not show reduced fecundability among women with low AMH (< 1.96 g/mL)( 14 ). The role for the use of AMH in patient counseling should be clarified. The association between AMH levels and response to controlled ovarian stimulation has been well-established( 5 , 15 ). AMH may be useful in predicting time to natural menopause, which can be of benefit to patients. However, some discrepancies in this association may be related to older AMH assays, limited detection and very low levels associated with perimenopause( 7 , 16 – 18 ). There are data suggesting an association between AMH and menstrual cycle length( 19 ). The association between AMH and polycystic ovarian syndrome (PCOS) has been studied extensively( 20 – 28 ), with some consideration of its use for diagnostic purposes, though this remains controversial. Outside of assisted reproductive technology, a clear role for AMH in counseling women attempting to conceive naturally remains limited. This study has several strengths. First, our analysis sought to evaluate a very important and highly controversial clinical question regarding the ability of ovarian reserve biomarkers to predict future fertility capacity. Women who present for evaluation of fertility seek information regarding their ability to conceive in the future, as this can provide guidance regarding timeline, plans to delay childbearing, and/or consideration of fertility preservation through oocyte cryopreservation. Providing evidence-based recommendations and a clear understanding of the limitations of ovarian reserve testing for this purpose is of utmost importance for patient-centered care. Second, we were able to continue a large, prior prospective time-to-pregnancy cohort with baseline markers of ovarian reserve to facilitate development of a longitudinal analysis. Third, our study conclusions are based on a cohort that provided a moderate sample size (> 300 participants). Fourth, we were able to evaluate rate of live birth, which is the most important clinical outcome for counseling women on future fertility. Fifth, the available women for this cohort study provided a robust response rate, limiting the role of non-response bias. Sixth, this is the first cohort study designed to evaluate future fertility. Most studies examining factors affecting future fertility use case-control study design. Generally, cohort studies are difficult to complete due longer duration of time between exposure and outcome assessments. Seventh, we evaluated future fertility from 4–12 years following baseline biomarker assessment. Women are commonly delaying family-building( 1 ), which provides real-world context. Lastly, this cohort study evaluated fertility outcomes among women at risk for reproductive aging, as all women were age 30 to 44 years at enrollment, and then were followed for 6–12 years. As such, these findings are generalizable to that population. This study has several limitations. First, a pregnancy attempt is not a validated concept. As such, couples may classify pregnancy attempts differently and therefore misclassify intervals of unprotected intercourse. Additionally, some intervals may not have been included due to recall. Second, prospective covariates were not collected for each pregnancy attempt, which could affect estimates and lead to misclassification. For example, successful attempts and pregnancy could be associated with weight gain, which may or may not affect subsequent attempts. Third, although adequately powered, the paucity of studies and available data evaluating the association between baseline markers of ovarian reserve and future fertility limits the ability to perform sample size calculations that are directly applicable to the current study cohort. Fourth, pregnancy attempt time was censored at above 10 months based on the answer choices provided in the reproductive history questionnaire. This could potentially decrease precision among participant responses indicating longer pregnancy attempt time. Fifth, TTC follow-up cohort overall is very similar to those who did not participate. However, there were statistically significant differences in race and education level, which could raise concern for selection bias. Biomarker values did not differ between those that did and did not participate.

Women are delaying attempts to conceive until older ages,( 1 ) and fertility is known to decline with age( 2 ). Currently, the best predictor of a woman’s future reproductive capacity is chronologic age. However, there is no method to differentiate those women that will conceive from those that will not conceive at any given age. Thus, women seek “fertility” tests to predict whether they will have difficulty conceiving should they delay pregnancy attempts. Ovarian reserve, the number of oocytes remaining in the ovary, can be indirectly measured using hormones such as early follicular phase inhibin B, follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH)( 3 ). Ovarian reserve, and therefore AMH, correlates inversely with age as follicular atresia and ovulation deplete oocytes over time as women approach reproductive senescence and menopause. There is considerable variation in ovarian reserve among women of the same chronologic age( 4 ). AMH levels are associated with time to menopause( 5 – 7 ). For these reasons, biomarkers of ovarian reserve have been proposed and marketed as “fertility” tests. Women use these biomarkers to make decisions regarding fertility preservation and timing of their pregnancy attempts. Findings by Steiner et al. ( 8 ) in the Time to Conceive study (TTC), which followed women for one pregnancy attempt for up to one year, demonstrated limited utility of ovarian reserve makers in predicting a woman’s current fertility( 8 ). However, this previous work did not examine the probability of achieving a live birth or being diagnosed with infertility in future pregnancy attempts. The objective of this study was to determine the extent to which biomarkers of ovarian reserve predict future fertility, up to 12 years later, as measured by rates of live birth, infertility, and time-to-pregnancy in conception attempts after participation in TTC. We hypothesized that biomarker values suggesting diminished ovarian reserve would be associated with a lower probability of achieving a live birth >3 years after enrollment, a higher likelihood of an infertility diagnosis, and reduced fecundability in future pregnancy attempts. To date, no cohort studies have determined the extent to which ovarian biomarkers predict a woman’s future reproductive capacity. The TTC study population is uniquely positioned to answer this question.

Supplemental Figure 1. Flow of Participants Through the Time to Conceive Cohort Follow-up Study