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Introduction: BRCA1/2 gene mutations increase risk of breast and/or ovarian cancer and may have implications for reproductive health. Indirect biomarkers of the ovarian primordial follicle pool (anti-Müllerian hormone (AMH)) and one small study in female cadavers suggest that ovarian reserve may be reduced in BRCA mutation carriers, but findings are conflicting and association between circulating AMH and primordial follicle number is not established. The aim of this study is to measure primordial follicle density in premenopausal ovarian tissue samples from women with BRCA1/2 gene mutations versus age-matched comparison group., Methods and Analysis: Prospective observational study measuring associations between BRCA gene mutation status, premenopausal ovarian primordial follicle density and serum AMH concentrations versus age-matched premenopausal women from the general population. Primordial follicle density will be measured in cortical sections from ovarian tissue collected at the time of risk-reducing bilateral salpingo-oophorectomy (RRBSO) in 88 BRCA1 gene mutation carriers, 65 BRCA2 gene mutation carriers and 157 non-mutation carriers. Primordial follicle density will be determined by counting follicles in a known volume of ovarian cortical tissue using light microscopy. Follicles will be identified by immunohistochemical staining for oocyte marker mouse vasa homologue. To inform the mechanisms underlying reduced ovarian reserve, the proportion of follicles containing oocytes with DNA damage will be determined by immunohistochemical staining for phosphorylated histone H2AX and terminal deoxynucleotidyl transferase dUTP nick end labelling assay to identify apoptotic cells. Follicle density will be correlated with circulating AMH concentrations quantified in the same cohort, using an electrochemiluminescence immunoassay on an automated platform., Ethics and Dissemination: Ethics approval has been granted by Peter MacCallum Cancer Centre to access biobanks, including; The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab-HREC#97_27) and the What Happens after Menopause? (HREC12PMCC24-12/90) and Melbourne IVF., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Study Question: Are lifestyle factors (smoking, BMI, alcohol use and oral contraceptive pill use) associated with the human ovarian reserve as determined by the total ovarian non-growing follicle number?, Summary Answer: Light to moderate alcohol use was significantly associated with greater ovarian non-growing follicle (NGF) count, whereas other lifestyle factors were not significantly related., What Is Known Already: A single previous investigation has suggested that smoking and alcohol use are associated with lower ovarian follicle density. However, this investigation utilized follicle density as the outcome of interest rather than the estimated total ovarian NGF count., Study Design, Size, Duration: This cross-sectional investigation included a convenience sample of premenopausal women from two different academic sites, the University of Washington (n = 37, from 1999-2004) and the University of Oklahoma (n = 73, from 2004-2013), undergoing incidental oophorectomy at the time of hysterectomy (total n = 110, age range 21-52 years)., Participants/materials, Setting, Methods: Prior to undergoing oophorectomy, participants completed detailed questionnaires regarding lifestyle exposures. Following surgery, total ovarian NGF counts were determined with systematic random sampling rules and a validated fractionator/optical dissector technique. Associations between lifestyle factors and log-transformed ovarian follicle counts were determined using multivariable linear regression., Main Results and the Role of Chance: After controlling for age, BMI, oral contraceptive pill (OCP) use, tobacco use and site of collection, cumulative alcohol use (measured in alcoholic drinks per day multiplied by years of drinking) was associated with ovarian NGF count. Women reporting light (>0 to <1 drink-years) and moderate (1-3 drink-years) alcohol use had greater NGF counts (β = 0.75, P = 0.04, and β = 1.00, P = 0.03; light and moderate use, respectively) as compared with non-users. Neither heavier alcohol use (>3 drink-years), BMI, OCP use, nor tobacco use were significantly associated with the ovarian NGF count. Similar patterns of association with moderate cumulative alcohol use were observed when evaluating associations with pre-antral follicles and total follicle counts., Limitations, Reasons for Caution: All participants in this convenience sample had a benign indication for hysterectomy, and therefore may not be broadly representative of the population without such an indication. Additionally, lifestyle factors were self-reported, and the sample size of the present investigation limits our ability to detect associations of smaller magnitude., Wider Implications of the Findings: While our findings are in disagreement with a single investigation that utilized human follicle density as the outcome of interest, they are consistent with many studies investigating the relationship between lifestyle factors and the age of spontaneous menopause. Furthermore, they suggest a mechanism that does not involve accelerated follicular atresia to explain the association between smoking and an earlier age of menopause., Study Funding/competing Interests: This investigation was funded by NIA R29-HD37360-04 (N.A.K.) and OCAST HR04-115 (K.R.H.) and by the National Institute of General Medical Sciences, Grant 1 U54GM104938 (J.D.P.). There is no conflict of interest., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To validate recently proposed models of ovarian nongrowing follicle (NGF) decay associated with aging within the context of an independent data set., Design: Prospective investigation., Setting: Academic medical center., Patient(s): Normal appearing ovaries collected from 52 women (age 28-51 years) undergoing oophorectomy for benign gynecologic indications., Intervention(s): Determining ovarian NGF counts with systematic random sampling rules and a validated fractionator/optical disector technique. The goodness-of-fit of predicted NGF counts based on the power and double Gaussian models and those observed in the validation set was assessed with the calculation of the Akaike information criterion and R(2) values., Main Outcome Measure(s): The goodness-of-fit between observed and expected ovarian NGF counts., Result(s): The power model was an excellent fit to the observed data. The average difference between the observed and expected NGF count was 0.161 (95% CI, -0.058, 0.327). In the present study population, the power model was a superior fit to the observed data compared with the double Gaussian model., Conclusion(s): This prospective investigation with an independent set of ovarian NGF counts validates the power model as an excellent characterization of the ovarian NGF decline associated with aging., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to characterize the ovarian primordial and nongrowing follicle number according to the Stages of Reproductive Aging Workshop (STRAW) staging system as defined by menstrual cycle characteristics., Methods: Normal ovaries were collected from 63 women (age 26-52 y) undergoing oophorectomy for benign indications. Before surgical operation, each participant completed a detailed questionnaire collecting information regarding menstrual cycle characteristics and was classified by bleeding patterns into STRAW stages -4, -3, -2, and -1. A single ovary was selected for the determination of ovarian primordial and total nongrowing follicle number using a validated fractionator/optical disector method. A subset of the participants (n = 43) underwent transvaginal ultrasound examination for the determination of the ovarian antral follicle count and serum measurements of follicle-stimulating hormone, estradiol, antimüllerian hormone, and inhibin B. All measurements were obtained within 2 weeks of surgical operation, irrespective of cycle day., Results: Significant differences were identified in ovarian primordial (P < 0.0001) and nongrowing follicle (P < 0.0001) counts across the STRAW stages. In post hoc testing, the differences in primordial follicle counts were significant between each of the STRAW stages. Significant differences were also identified in serum levels of antimüllerian hormone, follicle-stimulating hormone, and ovarian antral follicle count across the STRAW stages., Conclusions: Progression through the STRAW stages as defined by menstrual cycle characteristics is associated with progressive and significant decreases in the ovarian primordial follicle number.

Objective: To investigate the relationship between clinical markers of ovarian reserve and the true ovarian reserve as determined by the ovarian primordial follicle number., Design: Prospective investigation., Setting: Academic medical center., Patient(s): Forty-two healthy women (aged 26-52 years) undergoing oophorectomy for benign gynecologic indications., Intervention(s): Transvaginal ultrasound examination for the determination of the ovarian antral follicle count (AFC) and serum measurements of clinical markers of ovarian reserve. All measurements were obtained within 2 weeks of surgery, irrespective of cycle day. Ovarian primordial follicle count was then determined using a validated fractionator/optical disector method., Main Outcome Measure(s): Univariate and partial correlations between ovarian reserve markers and ovarian primordial follicle count., Result(s): There were significant correlations between the ovarian primordial follicle count and AFC (r=0.78), anti-Müllerian hormone (AMH; r=0.72), FSH (r=-0.32), inhibin B (r=0.40), and chronological age (r=-0.80). After adjusting for age, significant correlations were identified between the ovarian primordial follicle count and AFC (r=0.53) and AMH (r=0.48)., Conclusion(s): The ovarian AFC and serum levels of AMH correlate with the ovarian primordial follicle number even after adjustment for chronological age., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The primary determinant of reproductive age in women is the number of ovarian non-growing (primordial, intermediate and primary) follicles (NGFs). To better characterize the decline in NGF number associated with aging, we have employed modern stereology techniques to determine NGF number in women from birth to menopause., Methods: Normal human ovaries were collected from 122 women (aged 0-51 years) undergoing elective oophorectomy, organ donation or autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. Models to describe the resulting decay curve were constructed and evaluated., Results: NGF decay was best described by a simple power function: log (y) = ax(b) + c, where a, b and c are constants and y = NGF count at age x (R(2) = 0.84, Sums of Squares Error = 28.18 on 119 degrees of freedom). This model implies that follicles decay faster with increasing age., Conclusions: Unlike previous models of ovarian follicle depletion, our model predicts no sudden change in decay rate, but rather a constantly increasing rate. The model not only agrees well with observed ages of menopause in women, but also is more biologically plausible than previous models. Although the model represents a significant improvement compared with earlier attempts, a considerable percentage of the variation in NGF number between women cannot be explained by age alone.

BACKGROUND Previous published reports on the number of non-growing follicles (NGFs) in the human ovary have employed model-based methods for number estimates. These methods are time-intensive, and require correction factors and assumptions that ultimately limit their accuracy. Here, we describe the modification, application and validation of a modern fractionator/optical disector technique for the estimation of human ovarian NGF number. METHODS Forty-eight pairs of normal human ovaries were collected from women (age 8-51 years) undergoing elective bilateral oophorectomy, organ donation, or from autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. The precision of individual NGF counts was determined by calculating the observed coefficient of error (OCE). Intra-observer variability and variation in NGF number between ovaries within a pair were also determined. RESULTS The mean OCE was 16.6% with larger variations observed at lower follicle counts. In recount experiments of the same ovary, NGF number estimates varied by 15-29%, except at very low follicle counts where variation was greater, but absolute differences were small. There was no significant difference in NGF number between ovaries within a pair (Wilcoxon signed rank test, P = 0.81). CONCLUSIONS Modern stereology methods provide an unbiased, efficient method for estimating NGF number in the human ovary. Both ovaries within a pair contain similar numbers of NGFs.

Objective: To determine the extent of intercycle and interobserver variability in antral follicle (AF) count and their impact on stimulation quality in IVF., Design: Prospective evaluation of the impact on AF count of GnRH agonist down-regulation and interobserver variability. Retrospective evaluation of intercycle variability in AF count., Setting: University ART clinic., Patient(s): Twenty subjects were used to evaluate the effect of GnRH agonist down-regulation upon AF count; six of whom were used to evaluate interobserver variability. Fifty patients experiencing two or three cycles of IVF within a 1-year interval., Intervention(s): Transvaginal ultrasound exams before and after down-regulation with a GnRH agonist. Videotaped day-3 transvaginal ultrasound exams., Main Outcome Measure(s): [1] Intercycle and interobserver variability in antral follicle count. [2] Oocytes retrieved, peak estradiol, gonadotropin dose, duration of stimulation and cancellation rates., Result(s): There is moderate intercycle and interobserver variability in AF counts. GnRH agonist down-regulation does not significantly change AF count. In infertility patients undergoing IVF, paired analysis between the low- and high-AF count cycles did not show a difference in quality of stimulation or cycle cancellation rates., Conclusions: Within an individual patient, higher AF count in a given cycle was not predictive of better stimulation compared with the case of a lower count cycle.