Lifestyle factors associated with histologically derived human ovarian non-growing follicle count in reproductive age women.

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 (ß = 0.75, P = 0.04, and ß = 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.

Ovarian primordial and nongrowing follicle counts according to the Stages of Reproductive Aging Workshop (STRAW) staging system.

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.

Diminished ovarian reserve and infertility.

Over the past several decades, social and demographic trends have led to an increased tendency for women to delay childbearing. Owing primarily to abnormalities in the oocyte and resulting embryonic aneuploidy, implantation, clinical pregnancy, and live birth rates decline sharply by the end of the fourth decade. As a result, the incidence of age-related infertility has increased. Improved awareness of the effects of aging on fertility combined with ovarian reserve assessment, patient education, and early infertility evaluation and intervention are important elements in appropriate family planning and prevention of age-related infertility.

A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause.

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.

Estimating human ovarian non-growing follicle number: the application of modern stereology techniques to an old problem.

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.

Assisted reproductive technology practice patterns and the impact of embryo transfer guidelines in the United States.

OBJECTIVE: [1] To evaluate trends in number of embryos transferred and resultant high-order multiple (HOM) pregnancy rates by Society for Assisted Reproductive Technology (SART)-member clinics between 1996 and 2003 and [2] to relate these practice patterns and outcomes to clinic compliance with SART-American Society for Reproductive Medicine (ASRM) embryo transfer guidelines. DESIGN: Retrospective. SETTING: Society for Assisted Reproductive Technology-member fertility centers in the United States. PATIENT(S): Five hundred thirty-six thousand, five hundred twenty-four fresh, nondonor IVF cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Number of embryos transferred; pregnancy rates; implantation rates; and proportion of singleton, twin, and HOM pregnancies. RESULT(S): The number of embryos transferred declined each year. High-order multiple pregnancy rates also declined, whereas the twin rates remained stable. The most pronounced declines in number transferred occurred immediately after publication of SART-ASRM embryo transfer guidelines. After stratifying clinics according to mean and modal number of embryos transferred, clinics transferring the fewest embryos in women <35 years of age had the highest mean implantation and pregnancy rates. Furthermore, the percentage of clinics transferring two embryos to a majority of women <35 years of age increased from 3.3% in 1996 to 49.9% in 2003. CONCLUSION(S): The implementation of SART-ASRM embryo transfer guidelines is associated with significant reductions in the number of embryos being transferred, along with reductions of HOM pregnancies. Initiatives to further reduce twin pregnancies and encourage singleton gestation outcomes are outlined.

Urinary beta-luteinizing hormone and beta-follicle stimulating hormone immunoenzymometric assays for population research.

OBJECTIVE: We developed assays for measurement of urinary betaLH and betaFSH under collection and storage conditions typical of non-clinical research settings. DESIGN AND METHODS: IEMAs for free betaLH and total betaFSH were validated by standard methods. Stability of urinary betaLH and betaFSH was tested across freeze-thaws and stored long term at 4 degrees C or -20 degrees C, or short term at room temperature, and with heating to dissociate the subunits. RESULTS: The IEMAs exhibited acceptable parallelism, specificity, recovery (averaging 100% for betaLH, 97% for betaFSH), imprecision (maximum within-run and between run CVs, respectively, 4.8% and 25.7% for betaLH, 5.6% and 17.0% for betaFSH), and minimum detectable dose (2.5 pmol/L for betaLH, 6.8 pmol/L for betaFSH). Urine and serum measures were highly correlated (r=0.95 for LH, 0.86 for FSH). There was no consistent decline with any storage type. Dissociation of subunits by heating was needed for betaLH, but not betaFSH. CONCLUSION: These IEMAs measure free betaLH and total betaFSH, overcoming inter-individual variability in, and collection and storage effects on, subunit dissociation, without the need for urine preservatives.

Reproductive ageing and ovarian function: is the early follicular phase FSH rise necessary to maintain adequate secretory function in older ovulatory women?

BACKGROUND: Serum FSH elevations and decreases in inhibin B have been consistently demonstrated in the early follicular phase of cycles in women of advanced reproductive age. However, secretory products of the dominant follicle (estradiol and inhibin A) in the serum of older ovulatory women are maintained at levels similar to those of their younger counterparts. The goal of this investigation was to determine if ovarian secretory capacity is dependent on relative FSH levels and if basal measures of ovarian reserve reflect ovarian secretory capacity. METHODS: We administered equivalent low, but effective doses of recombinant FSH for 5 days to a group of older subjects (40-45 years, n=9) and younger controls (20-25 years, n=10) after pituitary suppression with a GnRH agonist. Outcome measures included follicular development as determined by serial transvaginal ultrasound examinations and serum levels of estradiol, inhibin A and inhibin B. RESULTS: Serum levels of estradiol and inhibin A were not statistically different between the two groups, while the number of large follicles formed was greater in the younger subjects. Basal parameters of ovarian reserve were not significantly correlated with ovarian secretory capacity, but did correlate with the number of follicles recruited in response to low-dose FSH. CONCLUSIONS: By providing equivalent serum levels of FSH in older and younger reproductive aged women, this study demonstrates that the secretory capacity of recruited follicles is maintained in older reproductive aged women.

Statistical correction for non-parallelism in a urinary enzyme immunoassay.

Our aim was to develop a statistical method to correct for non-parallelism in an estrone-3-glucuronide (E1G) enzyme immunoassay (EIA). Non-parallelism of serially diluted urine specimens with a calibration curve was demonstrated in an EIA for E1G. A linear mixed-effects analysis of 40 urine specimens was used to model the relationship of E1G concentration with urine volume and derive a statistical correction. The model was validated on an independent sample and applied to 30 menstrual cycles from American women. Specificity, detection limit, parallelism, recovery, correlation with serum estradiol, and imprecision of the assay were determined. Intra-and inter-assay CVs were less than 14% for high- and low-urine controls. Urinary E1G across the menstrual cycle was highly correlated with serum estradiol (r= 0.94). Non-parallelism produced decreasing E1G concentration with increase in urine volume (slope = -0.210, p < 0.0001). At 50% inhibition, the assay had 100% cross-reactivity with E1G and 83% with 17beta-estradiol 3-glucuronide. The dose-response curve of the latter did not parallel that of E1G and is a possible cause of the non-parallelism. The statistical correction adjusting E1G concentration to a standardized urine volume produced parallelism in 24 independent specimens (slope = -0.043+/-0.010), and improved the average CV of E1G concentration across dilutions from 19.5%+/-5.6% before correction to 10.3%+/-5.3% after correction. A statistical method based on linear mixed effects modeling is an expedient approach for correction of non-parallelism, particularly for hormone data that will be analyzed in aggregate.

Age-related analysis of inhibin A, inhibin B, and activin a relative to the intercycle monotropic follicle-stimulating hormone rise in normal ovulatory women.

Previous studies have reported that the monotropic rise in FSH in older women is associated with decreased inhibin B and/or A levels and increased levels of activin A. Whereas most investigators have found decreased follicular-phase inhibin B, the roles of inhibin A and activin A as modulators of the FSH rise are unclear. The objectives of this study were to determine whether deficiencies in circulating levels of inhibin A, inhibin B, and/or activin A exist during the intercycle interval in ovulatory older (age, 40-45 yr; n = 16), compared with younger women (age, 20-25 yr; n = 13). Blood samples were obtained daily throughout one menstrual cycle and the follicular phase of the subsequent cycle and were analyzed for LH, FSH, estradiol, inhibin A and B, and activin A. Despite significant FSH elevation, no deficiencies in inhibin A, activin A, or estradiol were detected in older subjects. In fact, inhibin A was significantly higher in older participants during the intercycle phase (P = 0.01), whereas inhibin B was significantly lower. Thus, the monotropic rise in FSH does not appear to result from changes in inhibin A or activin A, supporting the concept that inhibin B plays a critical role in mediating the FSH rise in older women.

Comparison of specific gravity and creatinine for normalizing urinary reproductive hormone concentrations.

BACKGROUND: Specific gravity (SG) may perform as well as creatinine (CR) correction for adjusting urinary hormone concentrations, as well as offer some advantages. We compared the two methods and applied them to US and Bangladeshi specimens to evaluate their use in different populations. METHODS: Pearson correlations between serum concentrations and SG, CR, and uncorrected urinary concentrations were compared using paired daily urine and serum specimens from one menstrual cycle from 30 US women. Corrected urinary estrone conjugate and pregnanediol glucuronide concentrations were compared with serum estradiol and progesterone. Urine specimens across one menstrual cycle from 13 Bangladeshi women were used to evaluate the applicability of both methods to a nonindustrialized population. Linear mixed-effects models were used to compare CR and SG values in the Bangladeshi vs US specimens. RESULTS: There was no significant difference between SG-corrected vs serum and CR-corrected vs serum correlations for either assay. Usable CR results were obtained for all US specimens, but 37% of the Bangladeshi specimens were below the CR assay limit of detection. The Bangladeshi sample had significantly lower CR and higher inter- and intrasubject CR variability than the US sample. CONCLUSIONS: SG is a potentially useful alternative to CR correction for normalizing urinary steroid hormone concentrations, particularly in settings where CR values are highly variable or unusually low.

Endocrine assessment of relative reproductive age in normal eumenorrheic younger and older women across multiple cycles.

OBJECTIVE: The aim of this study was to better characterize the ranges and intercycle variability for day 3 follicle-stimulating hormone, estradiol, and inhibin B levels in normal eumenorrheic women. STUDY DESIGN: Healthy eumenorrheic volunteers were recruited, of whom 27 women were 20 to 25 years old (peak reproductive age) and 36 women were 40 to 45 years old (study population). Blood samples were obtained on day 3 of two consecutive menstrual cycles. In some women, an additional blood sample on day 3 was obtained within 1 year. RESULTS: In normal women aged 20 to 25 years versus women aged 40 to 45 years, the day 3 follicle-stimulating hormone geometric mean is 5.6 IU/L (95% CI, 3.3-9.5 IU/L) versus 9.6 IU/L (95% CI, 3.8-23.8 IU/L), the day 3 estradiol geometric mean is 44.0 pg/mL (95% CI, 20.4-95.0 pg/mL) versus 52.4 pg/mL (95% CI, 22.4-122.8 pg/mL), and the day 3 inhibin B geometric mean is 100.4 pg/mL (95% CI, 51.7-195.0 pg/mL) versus 52.4 pg/mL (95% CI, 9.5-289.3 pg/mL). Furthermore, 22% of women in the older age group have a normal day 3 follicle-stimulating hormone and estradiol level in one cycle but an elevated value in a consecutive cycle (P=.008). CONCLUSION: In women of peak reproductive age, the upper limit of day 3 follicle-stimulating hormone and estradiol levels are 9.5 IU/L and 95.0 pg/mL, respectively, and the lower limit of day 3 inhibin B level is 51.7 pg/mL. If the initial day 3 follicle-stimulating hormone and estradiol levels in an older woman are normal, then a second measurement in a subsequent cycle should be obtained before counseling this woman regarding her reproductive potential.

Urinary estrone conjugate and pregnanediol 3-glucuronide enzyme immunoassays for population research.

BACKGROUND: Monitoring of reproductive steroid hormones at the population level requires frequent measurements, hormones or metabolites that remain stable under less than ideal collection and storage conditions, a long-term supply of antibodies, and assays useful for a range of populations. We developed enzyme immunoassays for urinary pregnanediol 3-glucuronide (PDG) and estrone conjugates (E1Cs) that meet these criteria. METHODS: Enzyme immunoassays based on monoclonal antibodies were evaluated for specificity, detection limit, parallelism, recovery, and imprecision. Paired urine and serum specimens were analyzed throughout menstrual cycles of 30 US women. Assay application in different populations was examined with 23 US and 42 Bangladeshi specimens. Metabolite stability in urine was evaluated for 0-8 days at room temperature and for 0-10 freeze-thaw cycles. RESULTS: Recoveries were 108% for the PDG assay and 105% for the E1C assay. Serially diluted specimens exhibited parallelism with calibration curves in both assays. Inter- and intraassay CVs were <11%. Urinary and serum concentrations were highly correlated: r = 0.93 for E1C-estradiol; r = 0.98 for PDG-progesterone. All Bangladeshi and US specimens were above detection limits (PDG, 21 nmol/L; E1C, 0.27 nmol/L). Bangladeshi women had lower follicular phase PDG and lower luteal phase PDG and E1Cs than US women. Stability experiments showed a maximum decrease in concentration for each metabolite of <4% per day at room temperature and no significant decrease associated with number of freeze-thaw cycles. CONCLUSIONS: These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.

Is the short follicular phase in older women secondary to advanced or accelerated dominant follicle development?

This study sought to determine whether the shortened follicular phase in ovulatory older women is secondary to advanced (i.e. earlier) or accelerated (i.e. more rapid) folliculogenesis. Normal ovulatory women, aged 40-45 yr (n = 15) and 20-25 yr (n = 13), underwent daily venipuncture and transvaginal ultrasonography throughout the follicular phase of a spontaneous menstrual cycle (control cycle) and after pituitary down-regulation with a GnRH agonist (study cycle). As expected, the older subjects in the control cycles demonstrated an elevated d 3 FSH and a shortened follicular phase compared with the younger subjects. After release from hypothalamic-pituitary-ovarian axis suppression, the early follicular phase FSH peak occurred earlier (6.8 vs. 9.8 d; P < 0.01) and was of a greater magnitude (12.1 vs. 6.5 mIU/ml; P < 0.01) in the older subjects. The time from release of suppression until the subsequent LH surge was also shorter (17.5 vs. 20.8 d; P < 0.01) in the older group. However, the time from FSH peak to LH surge was similar in the older and younger groups (10.7 vs. 11.0 d; P = 0.74). Compared with younger women, older subjects had normal follicular phase levels of estradiol and inhibin A and lower levels of inhibin B in both control and study cycles. We conclude that the shortened follicular phase observed in older ovulatory women is due to earlier dominant follicle selection, independent of hormonal influences from the preceding luteal phase.