Testing the role of testosterone versus estrogens in mediating reproductive transitions in female rhesus macaques.

In male vertebrates, testosterone is generally known to coordinate reproductive trade-offs, in part by promoting the transition to the next reproduction at the expense of current parental care. The role of testosterone in reproductive transitions has been little tested in female vertebrates, especially in mammals. The present study sought to fill this gap, by first undertaking an experimental study, in which we identified DHT, androstenediol, and in particular etiocholanolone, as fecal androgen metabolites which reflect serum testosterone concentration in female rhesus macaques (Macaca mulatta). Using concentrations of fecal etiocholanolone as proxy for circulating testosterone, we then conducted a field study on 46 free-ranging rhesus macaques of Cayo Santiago, Puerto Rico, to test if testosterone mediates the trade-off between reproductive transition (a higher chance of reproducing in the next year) and current reproduction (providing more care to current offspring). While the evidence for testosterone was weak, the testing of fecal immunoreactive estrogen metabolites suggested a potential role of estrogen in reproductive trade-offs. We found large individual differences in fecal etiocholanolone concentrations during the early postpartum period that were unexplained even after accounting for sociodemographic factors such as age and dominance rank. Further investigation is needed to understand this variation. Our study suggests that the actions of testosterone in females may not have evolved to fulfil the same role in primate reproductive transitions as it does in males, and we encourage more studies to consider the function of testosterone in reproductive behaviors and life history transitions in females of mammalian taxa.

Recognizing normal reproductive biology: A comparative analysis of variability in menstrual cycle biomarkers in German and Bolivian women.

The idealized "normal" menstrual cycle typically comprises a coordinated ebb and flow of hormones over a 28-day span with ovulation invariably shown at the midpoint. It's a pretty picture-but rare. Systematic studies have debunked the myth that cycles occur regularly about every 28 days. However, assumptions persist regarding the extent and normalcy of variation in other cycle biomarkers. The processes of judging which phenotypic variants are "normal" is context dependent. In everyday life, normal is that which is most commonly seen. In biomedicine normal is often defined as an arbitrarily bounded portion of the phenotype's distribution about its statistical mean. Standards thus defined in one population are problematic when applied to other populations; population specific standards may also be suspect. Rather, recognizing normal female reproductive biology in diverse human populations requires specific knowledge of proximate mechanisms and functional context. Such efforts should be grounded in an empirical assessment of phenotypic variability. We tested hypotheses regarding cycle biomarker variability in women from a wealthy industrialized population (Germany) and a resource-limited rural agropastoral population (Bolivia). Ovulatory cycles in both samples displayed marked but nonetheless comparable variability in all cycle biomarkers and similar means/medians for cycle and phase lengths. Notably, cycle and phase lengths are poor predictors of mid-luteal progesterone concentrations. These patterns suggest that global and local statistical criteria for "normal" cycles would be difficult to define. A more productive approach involves elucidating the causes of natural variation in ovarian cycling and its consequences for reproductive success and women's health.

Analytical validation of an Enzyme Immunoassay for the measurement of urinary oxytocin in dogs and wolves.

Assessing changes in oxytocin (OT) levels in response to a variety of social stimuli has become of major interest in the field of behavioral endocrinology. OT is involved in the regulation of various aspects of social behavior such as tolerance, and the formation and maintenance of social bonds but also the regulation of stress. All of these aspects have been identified as potential targets of selection during the domestication process. Therefore, comparing the role of the oxytocinergic system in various aspects of dog and wolf social behavior, might help to understand whether this system was involved in the domestication process. Studies assessing OT levels in dogs and wolves have used invasively collected plasma and serum samples and non-invasively collected urine samples. However, when using an assay system on a new species a careful and complete validation of the method is of crucial importance, and to date no proper validation, to assess urinary OT levels in dogs and wolves, has been reported. We therefore conducted an analytical validation of an Enzyme Immunoassay (EIA) for the measurement of OT in urine of dogs and wolves, using a commercially available EIA. Stability tests revealed that OT levels degrade over time when stored at 4 °C, but are little affected by repeated thawing. In addition, our results indicate that the variance in OT levels is slightly lower when phosphoric acid is added following collection to prevent OT degradation. Long term storage tests revealed that urinary OT levels are least variable when stored as extracts in ethanol at -20 °C, rather than as unextracted urine samples. Validation results were acceptable with regard to parallelism, but values for accuracy and extraction efficiency were not meeting the standard criteria usually applied to steroid EIAs, especially when assessed for the lower range of the assay. The results of this study highlight the importance of an analytical assay validation, since even if validation parameters are not optimal, if published, they allow readers to estimate the relevance of studies using the validated method.

Measuring urinary cortisol and testosterone levels in male Barbary macaques: A comparison of EIA and LC-MS.

The development of methods to quantify hormones from non-invasively collected samples such as urine or feces has facilitated endocrinology research on wild-living animals. To ensure that hormone measurements are biologically meaningful, method validations are strongly recommended for each new species or sample matrix. Our aim was to validate three commonly used enzyme immunoassays (EIA), one for analysis of cortisol and two for analysis of testosterone, to assess adrenocortical and gonadal endocrine activity, respectively, from the urine of male Barbary macaques. We compared EIA and liquid chromatography-mass spectrometry (LC-MS) results to determine if the EIA measurements truly reflect levels of the target hormone and to determine if antibody cross-reactivities with other steroids were potentially confounding results. Furthermore, we conducted a biological validation of testosterone to ensure that both EIA and LC-MS were able to capture physiologically meaningful differences in hormone levels. We found that cortisol measured by EIA correlated strongly with cortisol measured by LC-MS in both adult and immature males, without the need for deconjugation of steroids in the urine. Both testosterone EIAs correlated strongly with LC-MS in adult males, but only if steroids in the urine were deconjugated by enzymatic hydrolysis prior to analysis. However, in immature males, EIA and LC-MS results did not correlate significantly. Further correlation analyses suggest this is likely due to cross-reactivity of the testosterone antibodies with other adrenal steroids such as cortisol, DHEA, and likely others, which are present at much higher concentrations relative to testosterone in immature males. Testosterone levels were significantly higher in adult compared to immature males as measured by LC-MS but not as measured by EIA. Taken together, our results suggest that the testosterone EIAs are suitable to assess gonadal activity in adult but not immature males, and only if a hydrolysis of the urine is conducted prior to analysis.

Links between breast cancer and birth weight: an empirical test of the hypothesized association between size at birth and premenopausal adult progesterone concentrations.

Some studies have reported that birth size is a risk factor for breast cancer, but the reasons for this observation are unknown. Ovarian hormone concentrations may be a link between birth size and breast cancer, but the few tests of this hypothesis are inconsistent, perhaps because of differences in sample composition, inclusion of anovulatory cycles, or use of one hormonal measurement per woman. We present results from the first study to use daily hormonal measurements throughout a woman's complete ovulatory cycle to test the hypothesized relationship between birth size and adult progesterone concentrations. We used a study sample and accompanying data set previously obtained for another research project in which we had collected daily urine samples from 63 healthy premenopausal women throughout a menstrual cycle. Multivariate regression was used to test for trends of individual progesterone indices (from 55 ovulatory cycles) with birth weight or ponderal index, while controlling for age, adult BMI, and age at menarche. Our main finding was that neither birth weight nor ponderal index was associated with biologically significant variation in luteal progesterone indices; the best-estimated effect sizes of birth size on these progesterone indices were small (3.7-10.2%). BMI was the only significant predictor of mean peak urinary progesterone, but it explained <6% of the variance. Our findings, in light of what is currently known regarding associations of breast cancer risk with birth size and adult size, suggest that environmental factors (particularly those that vary by socioeconomic status and affect growth) may underlie associations between birth size and cancer risks without there being any association of birth size with adult ovarian hormone concentrations.

Simultaneous measurement of endogenous steroid hormones and their metabolites with LC-MS/MS in faeces of a New World primate species, Cebus capucinus.

We developed and validated a method to measure steroid hormones with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in faecal samples of white-faced capuchins. The method includes the measurement of adrenal and gonadal hormones such as cortisol, testosterone, estrone, progesterone and a number of their faecal metabolites. This method can be used for simultaneous routine measurements of steroids in faecal samples and provides a reference method for the validation of new immunoassays in this matrix. The optimised method consists of an extraction of the dried faecal samples with 80% methanol followed by purification of the extracts by solid phase extraction, solvolytic cleavage of conjugates and liquid-liquid extraction. Extracts were measured by LC-MS/MS with an electrospray interface in positive ionisation mode. Out of 19 steroids spiked into methanol extracts, 14 showed a recovery of 79.8-118.5% with an intra-day precision of 2.5-13.0% and an inter-day precision of 7.2-15.1%. Detection limits for these steroids ranged from 0.3 to 27.0 ng/mL of extract. Five steroids did not fulfil our requirements concerning precision and accuracy and we therefore considered these to not be reliably measurable with this method. While there was no indication of considerable amounts of conjugated forms for most metabolites, 87% of the testosterone was found in the solvolysis fraction, which indicates that the majority of testosterone was conjugated. Therefore, solvolysis turned out to be crucial, especially for the quantification of the total amount of testosterone. The physiological validation of this LC-MS/MS method confirmed known physiologically caused differences in faecal steroid concentrations. This indicates the usefulness of the method in investigating variation in the levels of major steroid hormones in faeces of white-faced capuchins. The possibility to simultaneously measure hormones of the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis makes this method suitable for dealing with questions concerning the cross talk between those axes.

Androgen and androgen metabolite levels in serum and urine of East African chimpanzees (Pan troglodytes schweinfurthii): comparison of EIA and LC-MS analyses.

The primary male androgen testosterone (T) is often used as an endocrinological marker to investigate androgen-behaviour interactions in males. In chimpanzees and bonobos, studies investigating the relationship between T levels and dominance rank or aggressive behaviour have revealed contradictory results. The immunoassays used in these studies were originally developed for the measurement of steroids in serum. Their application to non-invasively collected samples, however, can lead to methodological problems due to cross-reacting metabolites, which might occur in urine or faeces but not in blood. The overall aim of this study, therefore, is to clarify whether a T enzyme immunoassay (EIA) is an applicable method to monitor testicular function in adult male chimpanzees. To estimate the impact of cross-reacting androgens on the used T EIA, we compared the results of an EIA measurement with a set of androgen metabolite levels measured by LC-MS. In urine from male chimpanzees, cross-reactivities appear to exist mainly with T and its exclusive metabolites, 5α-dihydrotestosterone (5α-DHT) and 5α-androstanediol (androstanediol). Both urinary and serum T levels of male chimpanzees were significantly higher than female T levels when measured with the T EIA, indicating a reliable measurement of testicular androgens and their exclusive metabolites with the used EIA. In urine from female chimpanzees, the comparison between LC-MS and T EIA results indicated a higher impact of cross-reactions with adrenal androgen metabolites. Therefore, the investigation of urinary T levels in female chimpanzees with a T EIA seems to be problematic. Overall our results show that a T EIA can be a reliable method to monitor testicular function in male chimpanzee urine and that LC-MS is a valuable tool for the validation of immunoassays.