Kathleen A. O'Connor

Declining fecundity and ovarian ageing in natural fertility populations

Worldwide, human fertility declines with increasing maternal age, after contraceptive-use patterns and behavioral factors are taken into consideration. Here, we summarize some of our theoretical and empirical work examining the biological factors contributing to this age pattern of fertility. We undertook an 11 month prospective endocrinological study in a natural fertility (non-contracepting) population (rural Bangladesh) to estimate the contributions of fetal loss and fecundability (the probability of conception) to declining fecundity with age. Prospective interviews and urine samples for pregnancy tests were collected twice weekly from up to 700 women. These data were used to test mathematical models of the underlying biological processes contributing to changing fecundability and fetal loss risk with maternal age. The results indicate that much of the decline in fecundity can be attributed to an increasing risk of fetal loss with maternal age. Much of this fetal loss is due to chromosomal abnormalities--a result of ageing oocytes. Fecundability, on the other hand, does not begin to decline until the early 40s. We hypothesize that this is also a result of ageing at the ovarian level, namely follicular atresia, in the years just prior to menopause. The irregularity of menstrual cycles--longer cycles and increasingly variable hormonal patterns--at these ages may be a direct result of the small and rapidly dwindling remaining pool of follicles. We present a simple mathematical model of this process, and some preliminary laboratory results that support the model.

Serum, plasma, and dried blood spot high-sensitivity C-reactive protein enzyme immunoassay for population research

C-reactive protein (CRP) is used as a biomarker of morbidity and mortality risk in studies of population health, and is essential to interpretation of several micronutrient biomarkers. There is thus a need for a robust high-sensitivity CRP (hsCRP) measurement method for large-scale, non-clinical studies. We developed an efficient, inexpensive assay suitable for quantifying CRP across the physiological range using any blood specimen type. The ELISA uses readily available monoclonal antibodies to measure CRP in serum, plasma, or dried blood spots (DBS) made from venous or capillary blood. Assay performance was evaluated by standard methods, including comparison with a previously described assay. Effects of specimen type were tested by measuring CRP in 52 matched serum, plasma, and venous and capillary dried blood spot specimens. Long- and short-term CRP stability were evaluated. Assessments of assay limits of detection, linearity, recovery, imprecision, and concordance with an established method (Pearson correlation=0.988, n=20) demonstrated the validity of the new assay. CRP measurements in serum, plasma, and DBS had Pearson correlations from 0.974 to 0.995, n=52, but CRP in serum was on average 1.6 times (SD 0.37) higher than in DBS. CRP was stable in frozen serum for up to 34 months, but DBS CRP declined quickly with exposure to ambient temperatures, and across long-term storage at -20°C. This hsCRP assay is a robust and inexpensive tool designed for use in large-scale population health research. Our results indicate that DBS CRP is less stable than previously reported.

C‐reactive protein across the menstrual cycle

C-reactive protein (CRP) is a widely used, sensitive biomarker of inflammation. Studies conducted among users of exogenous hormones suggest that estrogen increases CRP, whereas progesterone decreases CRP. Examinations of CRP in normally cycling women suggest the opposite: CRP is negatively associated with endogenous estrogen and positively associated with endogenous progesterone. This work evaluates the association between menstrual cycle-related hormone changes and events (menstruation and ovulation) and CRP. Eight female subjects gave urine and blood samples from twelve days across the menstrual cycle, for a total of eleven cycles. Blood samples were assayed for CRP; urine samples for beta-follicle stimulating hormone (betaFSH), pregnanediol 3-glucuronide (PDG), and estrone glucuronide (E1G). Ovulation day was estimated using hormone levels. Presence or absence of menses was reported by subjects. Analyses were conducted with random-effects linear regression. All cycles were ovulatory; day of ovulation was identified for nine cycles. A ten-fold increase in progesterone was associated with a 23% increase in CRP (P = 0.01), a ten-fold increase in estrogen was associated with a 29% decrease in CRP (P = 0.05), and menses was associated with a 17% increase in CRP (P = 0.18); no association between ovulation or FSH and CRP was found. Hormone changes across the menstrual cycle should be controlled for in future studies of inflammation in reproductive-age women.

Physical competition increases testosterone among Amazonian forager-horticulturalists: a test of the ‘challenge hypothesis’

The challenge hypothesis posits that acute increases in testosterone (T) during male–male competition enhance performance and survivability while limiting the physiological costs of consistently high T. Human challenge hypothesis research focuses on young men in industrial populations, who have higher baseline T levels than men in subsistence populations. We tested whether the Tsimane, pathogenically stressed forager-horticulturalists of the Bolivian Amazon, would express acute T increases in response to physical competition. Saliva was collected from 88 Tsimane men (aged 16–59 years) before and after a competitive soccer match. Tsimane men had significantly lower baseline levels of T (β = −0.41, p < 0.001) when compared with age-matched United States (US) males. Linear mixed-effects models were used to establish that T increased significantly immediately following competition (β = 0.23, p < 0.001), remaining high 1 h later (β = 0.09, p = 0.007); equivalent to 30.1 and 15.5 per cent increases in T, respectively. We did not find larger increases in T among winners (p = 0.412), although T increases were positively associated with self-rated performance (β = 9.07, p = 0.004). These results suggest that despite lower levels of T than US males, Tsimane males exhibit acute increases in T at the same relative magnitude reported by studies in industrialized settings, with larger increases in T for those who report better individual performance.

Progesterone and ovulation across stages of the transition to menopause

Objective: Detailed characterization of progesterone and ovulation across the menopausal transition provides insight into conception risk and mechanisms of reproductive aging. Methods: Participants (n = 108, aged 25-58 y) collected daily urine specimens for 6-month intervals in each of 5 consecutive years. Specimens were assayed for pregnanediol glucuronide (PDG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrone glucuronide (E1G). Reproductive stage was determined using cycle length variance. A hierarchical algorithm was used to identify ovulation. Linear mixed-effects models estimated (1) the frequency and day of ovulation by age and stage; (2) differences in FSH, LH, and E1G levels between ovulatory (O) and anovulatory (AO) cycles; and (3) total PDG levels and PDG levels in O cycles by age and stage. Results: The probability of AO cycles increased across the perimenopause (P < 0.0001); reproductive stage was a stronger predictor than age of the probability of anovulation. Most cycles in late perimenopause were AO (>60%), but one quarter of cycles longer than 60 days were O. Average day of ovulation was later in the late perimenopause (mean [SD] cycle day, 27 [25] d) compared with the premenopause. FSH and LH levels were higher and E1G levels were lower in AO than O cycles (P < 0.0001 for each). Total PDG decreased in the late perimenopause, but 95th percentile PDG in O cycles declined steadily across the transition. Conclusions: Exposure to the risk of conception in women experiencing cycles long enough to classify them as late perimenopausal is far from negligible. Reproductive stage is more informative than age about PDG levels and the likelihood of anovulation.

Statistical Correction for Non‐parallelism in a Urinary Enzyme Immunoassay

Abstract 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, paral‐lelism, 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 17β‐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.

Total and Unopposed Estrogen Exposure Across Stages of the Transition to Menopause

Detailed characterization of estrogen dynamics during the transition to menopause is an important step toward understanding its potential implications for reproductive cancers developing in the transition years. We conducted a 5-year prospective study of endogenous levels of total and unopposed estrogen. Participants (n = 108; ages 25-58 years) collected daily urine specimens for 6 months in each of 5 consecutive years. Specimens were assayed for estrone-3-glucuronide (E1G) and pregnanediol-3-glucuronide. Linear mixed-effects models were used to estimate exposure to total and unopposed estrogen by age and reproductive stage. Reproductive stage was estimated using menstrual cycle length variance. E1G mean area under the curve and mean E1G 5th and 95th percentiles represented total estrogen exposure. An algorithm identifying days of above-baseline E1G that coincided with the days of baseline pregnanediol-3-glucuronide was used to identify days of unopposed estrogen. Mean E1G area under the curve increased with age in the pretransition and early transition and decreased in the late transition. Ninety-fifth percentile E1G levels did not decline until after menopause, whereas 5th percentile levels declined from the early transition to the postmenopause. The number of days of unopposed estrogen was significantly higher during the transition compared with the pretransition. Given the length of time women spend in the transition, they are exposed to more total and unopposed estrogen than has been previously appreciated. Coupled with epidemiologic evidence on lifetime exposure to estrogen, these results suggest that variation in the amount of time spent in the transition may be an important risk factor for reproductive cancers. (Cancer Epidemiol Biomarkers Prev 2009;18(3):828–36)

Successful hunting increases testosterone and cortisol in a subsistence population

Controversy over the adaptive significance of male hunting in subsistence societies hinges on the relative importance of familial provisioning and mate-quality signalling. This paper examines the proximate and ultimate motivations of hunting behaviour from a neuroendocrine perspective, using salivary testosterone and cortisol data collected before, during and after hunting focal follows from 31 Tsimane hunters aged 18–82 years. Despite circadian declines in hormone levels, testosterone and cortisol of Tsimane hunters increased at the time of a kill, and remained high as successful hunters returned home. Previous studies of hormonal changes during competitions find that high-stakes and success in the presence of relevant audiences result in increased neuroendocrine arousal. If men hunt primarily to provision their families, then an additional audience would not be expected to impact testosterone or cortisol, nor would the size of the animal killed. However, if signalling male quality by ‘showing off’ was a larger relative driver of mens hunting behaviour, one would expect greater hormonal response in cases where men returned with large sharable kills, especially in the presence of community members. Consistent with provisioning models of male hunting motivation, neither kill size nor encountering an audience of villagers while returning from hunting was associated with hormonal changes for successful hunters.

Responsiveness of the reproductive axis to a single missed evening meal in young adult males

Objectives: The male reproductive axis is responsive to energetic deficits, including multiday fasts, but little is known about brief periods of fasting (<24 hours). Reduced testosterone in low‐energy balance situations is hypothesized to reflect redirection of resources from reproduction to survival. This study tests the hypothesis that testosterone levels decrease during a minor caloric deficiency by assessing the effects of a single missed (evening) meal on morning testosterone in 23 healthy male participants, age 19–36.

The effects of a long-term psychosocial stress on reproductive indicators in the baboon.

Psychosocial stress is thought to negatively impact fecundity, but human studies are confounded by variation in nutrition and lifestyle. Baboons offer a useful model to test the effect of prolonged mild stress on reproductive indicators in a controlled setting. Following relocation from social groups to solitary housing, a previously documented stressful event for nonhuman primates, daily urine samples, tumescence, and menstrual bleeding were monitored in twenty baboons (Papio sp.) for 120-150 days. Specimens were assayed for estrone conjugates (E1C), pregnanediol-3-glucuronide (PDG), follicle-stimulating hormone (FSH), and cortisol. Linear mixed effects models examined (1) the effects of stress on frequency of anovulation, hormone levels, tumescence and cycle length, and (2) the relationship of cortisol with reproductive indicators. Despite cortisol levels indicative of stress, anovulation was negligible (1% in 102 cycles). PDG, FSH, cycle length, and tumescence declined during the first four cycles, but began recovery by the fifth. Cortisol was negatively associated with FSH but not associated with PDG, E1C or tumescence. Ovulation, E1C, and luteal phase length were not affected. Tumescence tracked changes in FSH and PDG, and thus may be a useful indicator of stress on the reproductive axis. Elevated cortisol was associated with reduced FSH, supporting a model of cortisol action at the hypothalamus rather than the gonad. After four to five menstrual cycles the reproductive indicators began recovery, suggesting adjustment to new housing conditions. In conclusion, individual housing is stressful for captive baboons, as reflected by cortisol and reproductive indicators, although ovulation, a relatively direct proxy for fecundity, is unaffected.