James S. Kesner

Application of a method for estimating day of ovulation using urinary estrogen and progesterone metabolites.

Longitudinal epidemiologic studies of menstrual and reproductive function are more informative if one can identify day of ovulation. We previously developed a method for estimating day of ovulation that is feasible for epidemiologic studies. The method relies on the relative concentrations of estrogen and progesterone metabolites in daily first-morning urine specimens and does not require creatinine adjustment. This paper describes results of applying this method to a large study with 724 menstrual cycles from 217 women. The method estimated a credible day of ovulation in 88% of cycles. Missing data accounted for most of the failures. When we excluded anovulatory cycles (1%) and cycles with missing data, the method estimated a day of ovulation in 97% of cycles. Variance in luteal phase length was small for our sample, suggesting that this method of identifying a day of ovulation introduces no more measurement error than when day of ovulation is determined by plasma luteinizing hormone (LH), the standard clinical method.

Antimüllerian Hormone as a Predictor of Natural Fecundability in Women Aged 30-42 Years

OBJECTIVE: To generate estimates of the association between markers of ovarian aging and natural fertility in a community sample at risk for ovarian aging. METHODS: Women aged 30–44 years with no history of infertility who had been trying to conceive for less than 3 months provided early-follicular phase serum and urine (N=100). Subsequently, these women kept a diary to record menstrual bleeding and intercourse and conducted standardized pregnancy testing for up to 6 months. Serum was analyzed for estradiol, follicle-stimulating hormone (FSH), antimüllerian hormone, and inhibin B. Urine was analyzed for FSH and estrone 3-glucuronide. Diary data on menstrual cycle day and patterns of intercourse were used to calculate day-specific fecundability ratios. RESULTS: Sixty-three percent of participants conceived within 6 months. After adjusting for age, 18 women (18%) with serum antimüllerian hormone levels of 0.7 ng/mL or less had significantly reduced fecundability given intercourse on a fertile day compared with women with higher antimüllerian hormone levels (fecundability ratio 0.38; 95% confidence interval [CI] 0.08–0.91). The day-specific fecundability for women with early-follicular phase serum FSH values greater than 10 milli-international units/mL compared with women with lower FSH levels was also reduced, although nonsignificantly (11% of women affected; fecundability ratio 0.44; 95% CI 0.08–1.10). The association with urinary FSH was weaker (27% women affected; fecundability ratio 0.61; 95% CI 0.26–1.26), and the associations for the other markers were weaker still. CONCLUSION: Early-follicular phase antimüllerian hormone appears to be associated with natural fertility in the general population. LEVEL OF EVIDENCE: II

Effects of Gastric Bypass Surgery on Female Reproductive Function

CONTEXT Reproductive function may improve after bariatric surgery, although the mechanisms and time-related changes are unclear. OBJECTIVE The objective of the study was to determine whether ovulation frequency/quality as well as associated reproductive parameters improve after Roux en Y gastric bypass surgery. DESIGN This was a prospective cohort study that enrolled female subjects from 2005 to 2008 with study visits at baseline and then 1, 3, 6, 12, and up to 24 months after surgery. SETTING The study was conducted at an academic health center. PATIENTS Twenty-nine obese, reproductive-aged women not using confounding medications participated in the study. MAIN OUTCOME MEASURES The primary outcome was integrated levels of urinary progestin (pregnanediol 3-glururonide) from daily urinary collections at 12 months postoperatively. Secondary outcomes were changes in vaginal bleeding, other biometric, hormonal, ultrasound, dual-energy x-ray absorptiometry measures, and Female Sexual Function Index. RESULTS Ninety percent of patients with morbid obesity had ovulatory cycles at baseline, and the ovulatory frequency and luteal phase quality (based on integrated pregnanediol 3-glururonide levels) were not modified by bariatric surgery. The follicular phase was shorter postoperatively [6.5 d shorter at 3 months and 7.9-8.9 d shorter at 6-24 months (P < 0.01)]. Biochemical hyperandrogenism improved, largely due to an immediate postoperative increase in serum SHBG levels (P < 0.01), with no change in clinical hyperandrogenism (sebum production, acne, hirsutism). Bone density was preserved, contrasting with a significant loss of lean muscle mass and fat (P < 0.001), reflecting preferential abdominal fat loss (P < 0.001). Female sexual function improved 28% (P = 0.02) by 12 months. CONCLUSIONS Ovulation persists despite morbid obesity and the changes from bypass surgery. Reproductive function after surgery is characterized by a shortened follicular phase and improved female sexual function.

Methods of monitoring menstrual function in field studies : efficacy of methods

Efficacy of methods for monitoring female reproductive potential under field study conditions was evaluated. Women (n = 10) were recruited to participate for two menstrual cycles on the bases, in part, of not seeking fertility assistance, working full-time but not in the medical field, and having less than one year of college education. Luteinizing hormone (LH), estrone-3-glucuronide, and pregnanediol-3-glucuronide were measured in daily morning urine and normalized to creatinine concentrations. These urinary measures were parallel to serum LH, estradiol, and progesterone profiles. Based on these urinary measures, 6 of 19 cycles were judged to be atypical. Transvaginal ultrasonography provided insights into ovarian activity during the atypical cycles. Of 13 LH surges detected by radioimmunoassay, 7 were not detected by a semiquantitative dipstick (OvuSTICK), perhaps due to that methods sensitivity to loss of LH immunoactivity caused by sample freezing. While intervals from salivary and vaginal mucous electrical resistance signals to the LH surge during typical cycles were similar to those reported previously, they were not predictive of ovulatory status during atypical cycles. Fifty-three percent of the cycles were misclassified on the basis of the basal body temperature rise. Cervical mucous color, amount, and consistency were not predictive of ovulation under these study conditions. The results from these 19 menstrual cycles provide information about the efficacy of various methods for characterizing menstrual function under field study conditions. In this regard, urinary endocrine measures are the most informative or practical.

Stability of urinary female reproductive hormones stored under various conditions.

Urinary reproductive hormones afford specific and sensitive evaluation of female reproductive potential in epidemiologic and clinical settings. The goal of this study was to characterize the stability of urinary luteinizing hormone, follicle stimulating hormone, estrone 3-glucuronide, pregnanediol 3-glucuronide, and creatinine during storage as functions of time, temperature, and additives. After 2 weeks with no additives, activity of the four analytes, relative to initial concentrations, ranged from 91.9 to 102.8% at 4 degrees C, 35.1 to 89.6% at 25 degrees C, and 7.5 to 66.9% at 37 degrees C. Antimicrobial additives did not consistently improve stability. Analyte activity for samples stored with no additives for 24 weeks at -80 degrees C ranged from 69.0 to 101.2%. Glycerol and bovine serum albumin improved analyte stability; activity ranged from 91.1 to 106.3%. Other additives were ineffective. These results reveal conditions for storing reproductive hormone analytes in urine during epidemiologic field studies.

Validations of time-resolved fluoroimmunoassays for urinary estrone 3-glucuronide and pregnanediol 3-glucuronide

Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.

Assessment of reproductive disorders and birth defects in communities near hazardous chemical sites. II. Female reproductive disorders

Members of the workgroup on female reproductive disorders discussed methods to evaluate five principal functions: menstrual dysfunction, infertility, pregnancy loss, lactation disorders, and pregnancy complications. To test each function, a nested strategy was considered, based on progressive levels of effort available to conduct field investigations. This strategy was analogous to the three-tier classification of biomarkers used by other workshops. The lowest level of effort, corresponding to Tier 1, consists only of questionnaires, diaries, and reviews of maternal and infant medical records. The medium level of effort (Tier 2) collects data from questionnaires and diaries, and some biologic specimens. Suggested laboratory analyses included measurement of progesterone in saliva and several glycoprotein hormones in urine that evaluate menstrual dysfunction, infertility, and pregnancy loss. The highest level of effort (Tier 3) involves prospective collection of diary information and simultaneous collection of biological specimens.

Association Between Biomarkers of Ovarian Reserve and Infertility Among Older Women of Reproductive Age

Importance Despite lack of evidence of their utility, biomarkers of ovarian reserve are being promoted as potential markers of reproductive potential. Objective To determine the associations between biomarkers of ovarian reserve and reproductive potential among women of late reproductive age. Design, Setting, and Participants Prospective time-to-pregnancy cohort study (2008 to date of last follow-up in March 2016) of women (N = 981) aged 30 to 44 years without a history of infertility who had been trying to conceive for 3 months or less, recruited from the community in the Raleigh-Durham, North Carolina, area. Exposures Early-follicular-phase serum level of antimüllerian hormone (AMH), follicle-stimulating hormone (FSH), and inhibin B and urinary level of FSH. Main Outcomes and Measures The primary outcomes were the cumulative probability of conception by 6 and 12 cycles of attempt and relative fecundability (probability of conception in a given menstrual cycle). Conception was defined as a positive pregnancy test result. Results A total of 750 women (mean age, 33.3 [SD, 3.2] years; 77% white; 36% overweight or obese) provided a blood and urine sample and were included in the analysis. After adjusting for age, body mass index, race, current smoking status, and recent hormonal contraceptive use, women with low AMH values (<0.7 ng/mL [n = 84]) did not have a significantly different predicted probability of conceiving by 6 cycles of attempt (65%; 95% CI, 50%-75%) compared with women (n = 579) with normal values (62%; 95% CI, 57%-66%) or by 12 cycles of attempt (84% [95% CI, 70%-91%] vs 75% [95% CI, 70%-79%], respectively). Women with high serum FSH values (>10 mIU/mL [n = 83]) did not have a significantly different predicted probability of conceiving after 6 cycles of attempt (63%; 95% CI, 50%-73%) compared with women (n = 654) with normal values (62%; 95% CI, 57%-66%) or after 12 cycles of attempt (82% [95% CI, 70%-89%] vs 75% [95% CI, 70%-78%], respectively). Women with high urinary FSH values (>11.5 mIU/mg creatinine [n = 69]) did not have a significantly different predicted probability of conceiving after 6 cycles of attempt (61%; 95% CI, 46%-74%) compared with women (n = 660) with normal values (62%; 95% CI, 58%-66%) or after 12 cycles of attempt (70% [95% CI, 54%-80%] vs 76% [95% CI, 72%-80%], respectively). Inhibin B levels (n = 737) were not associated with the probability of conceiving in a given cycle (hazard ratio per 1-pg/mL increase, 0.999; 95% CI, 0.997-1.001). Conclusions and Relevance Among women aged 30 to 44 years without a history of infertility who had been trying to conceive for 3 months or less, biomarkers indicating diminished ovarian reserve compared with normal ovarian reserve were not associated with reduced fertility. These findings do not support the use of urinary or blood follicle-stimulating hormone tests or antimüllerian hormone levels to assess natural fertility for women with these characteristics.

Pubertal delay in male nonhuman primates (Macaca mulatta) treated with methylphenidate

Juvenile male rhesus monkeys treated with methylphenidate hydrochloride (MPH) to evaluate genetic and behavioral toxicity were observed after 14 mo of treatment to have delayed pubertal progression with impaired testicular descent and reduced testicular volume. Further evaluation of animals dosed orally twice a day with (i) 0.5 mL/kg of vehicle (n = 10), (ii) 0.15 mg/kg of MPH increased to 2.5 mg/kg (low dose, n = 10), or (iii) 1.5 mg/kg of MPH increased to 12.5 mg/kg (high dose, n = 10) for a total of 40 mo revealed that testicular volume was significantly reduced (P < 0.05) at months 15 to 19 and month 27. Testicular descent was significantly delayed (P < 0.05) in the high-dose group. Significantly lower serum testosterone levels were detected in both the low- (P = 0.0017) and high-dose (P = 0.0011) animals through month 33 of treatment. Although serum inhibin B levels were increased overall in low-dose animals (P = 0.0328), differences between groups disappeared by the end of the study. Our findings indicate that MPH administration, beginning before puberty, and which produced clinically relevant blood levels of the drug, impaired pubertal testicular development until ∼5 y of age. It was not possible to resolve whether MPH delayed the initiation of the onset of puberty or reduced the early tempo of the developmental process. Regardless, deficits in testicular volume and hormone secretion disappeared over the 40-mo observation period, suggesting that the impact of MPH on puberty is not permanent.

Time-resolved immunofluorometric assays for urinary luteinizing hormone and follicle stimulating hormone

Abstract The goal of this effort was to develop and validate non-radioisotopic immunoassays for measuring luteinizing hormone and follicle stimulating hormone in unextracted urine. Towards this goal, commercial time-resolved immunofluorometric assays (IFMAs) were modified. Validation demonstrated that the resultant assays were specific, sensitive, accurate, and precise. Urine matrix was shown not to interfere with the assay. Gonadotropin profiles generated using these assays conformed to those measured in urine and serum by other established immunoassays. These IFMAs afford the collective advantages of non-radioisotopic procedures and urine sample collection (convenience, noninvasiveness, integration of pulsatile secretion), plus the superior sensitivity and specificity of IFMAs. Applications include epidemiology and medicine.