Non-Invasive Reproductive Hormone Monitoring in Endangered Pygmy Hog (Porcula salvania)

The Pygmy hog (Porcula Salvania), till recently, classified as a critically endangered suid, is facing the threat of extinction globally due to habitat degradation. Efforts are being made to protect the pygmy hogs from extinction and breed them in captivity under Pygmy Hog Conservation Pro-gramme (PHCP). However, very little information is available on the reproductive physiology of pygmy hogs. Therefore, the present study aimed to standardize enzyme immunoassays (EIAs) for monitoring pregnancy and reproductive status using progesterone and testosterone metabolites. A total of 785 faecal samples were collected from five females and two males over a period of one year from PHCP Research and Breeding Centre, Guwahati, Assam. High-pressure liquid chro-matography (HPLC) analysis revealed the presence of immunoreactive progesterone and testos-terone metabolites in faeces. Mating was observed in all the five females and four of them gave birth successfully. We were able to detect pregnancy using faecal progesterone metabolites. Based on mating and parturition, the mean gestation period was estimated to be 153.25 days from four females. The breeding centre recorded 172 births between 1996 and 2000 and found strong sea-sonality in births and most of the births were between May and June. Faecal testosterone metabo-lites were significantly higher in the breeding season than the non-breeding season. This is the first study and will help in future breeding programs in other captive breeding centres and reproduc-tive monitoring of reintroduced populations.

Prediction of neonatal morbidity and very preterm delivery using maternal steroid biomarkers in early gestation

Background Preterm delivery is a common pregnancy complication that can result in significant neonatal morbidity and mortality. Limited tools exist to predict preterm birth, and none to predict neonatal morbidity, from early in pregnancy. The objective of this study was to determine if the progesterone metabolites 11-deoxycorticosterone (DOC) and 16-alpha hydroxyprogesterone (16α-OHP), when combined with patient demographic and obstetric history known during the pregnancy, are predictive of preterm delivery-associated neonatal morbidity, neonatal length of stay, and risk for spontaneous preterm delivery prior to 32 weeks’ gestation. Methods and findings We conducted a cohort study of pregnant women with plasma samples collected as part of Building Blocks of Pregnancy Biobank at the Indiana University School of Medicine. The progesterone metabolites, DOC and 16α-OHP, were quantified by mass spectroscopy from the plasma of 58 pregnant women collected in the late first trimester/early second trimester. Steroid levels were combined with patient demographic and obstetric history data in multivariable logistic regression models. The primary outcome was composite neonatal morbidity as measured by the Hassan scale. Secondary outcomes included neonatal length of stay and spontaneous preterm delivery prior to 32 weeks’ gestation. The final neonatal morbidity model, which incorporated antenatal corticosteroid exposure and fetal sex, was able to predict high morbidity (Hassan score ≥ 2) with an area under the ROC curve (AUROC) of 0.975 (95% CI 0.932, 1.00), while the model without corticosteroid and fetal sex predictors demonstrated an AUROC of 0.927 (95% CI 0.824, 1.00). The Hassan score was highly correlated with neonatal length of stay (p<0.001), allowing the neonatal morbidity model to also predict increased neonatal length of stay (53 [IQR 22, 76] days vs. 4.5 [2, 31] days, above and below the model cut point, respectively; p = 0.0017). Spontaneous preterm delivery prior to 32 weeks’ gestation was also predicted with an AUROC of 0.94 (95% CI 0.869, 1.00). Conclusions Plasma levels of DOC and 16α-OHP in early gestation can be combined with patient demographic and clinical data to predict significant neonatal morbidity, neonatal length of stay, and risk for very preterm delivery, though validation studies are needed to verify these findings. Early identification of pregnancies at risk for preterm delivery and neonatal morbidity allows for timely implementation of multidisciplinary care to improve perinatal outcomes.

Relationship of Serum Progesterone and Progesterone Metabolites with Mammographic Breast Density and Terminal Ductal Lobular Unit Involution among Women Undergoing Diagnostic Breast Biopsy

The association of progesterone/progesterone metabolites with elevated mammographic breast density (MBD) and delayed age-related terminal duct lobular unit (TDLU) involution, strong breast cancer risk factors, has received limited attention. Using a reliable liquid chromatography-tandem mass-spectrometry assay, we quantified serum progesterone/progesterone metabolites and explored cross-sectional relationships with MBD and TDLU involution among women, ages 40–65, undergoing diagnostic breast biopsy. Quantitative MBD measures were estimated in pre-biopsy digital mammograms. TDLU involution was quantified in diagnostic biopsies. Adjusted partial correlations and trends across MBD/TDLU categories were calculated. Pregnenolone was positively associated with percent MBD-area (MBD-A, rho: 0.30; p-trend = 0.01) among premenopausal luteal phase women. Progesterone tended to be positively associated with percent MBD-A among luteal phase (rho: 0.26; p-trend = 0.07) and postmenopausal (rho: 0.17; p-trend = 0.04) women. Consistent with experimental data, implicating an elevated 5α-pregnanes/3α-dihydroprogesterone (5αP/3αHP) metabolite ratio in breast cancer, higher 5αP/3αHP was associated with elevated percent MBD-A among luteal phase (rho: 0.29; p-trend = 0.08), but not postmenopausal women. This exploratory analysis provided some evidence that endogenous progesterone and progesterone metabolites might be correlated with MBD, a strong breast cancer risk factor, in both pre- and postmenopausal women undergoing breast biopsy. Additional studies are needed to understand the role of progesterone/progesterone metabolites in breast tissue composition and breast cancer risk.

Alterations in endogenous progesterone metabolism associated with spontaneous very preterm delivery

STUDY QUESTION Do maternal serum levels of progesterone metabolites early in pregnancy correspond to an increased risk for very preterm delivery prior to 32 weeks? SUMMARY ANSWER Maternal serum levels of 11-deoxycorticosterone (DOC) measured during the late first trimester or early second trimester correlate with an increased risk for preterm delivery prior to 32 weeks, and the correlation becomes stronger when the ratio of DOC to 16-alpha-hydroxyprogesterone was measured. WHAT IS KNOWN ALREADY Progesterone is a pro-gestational steroid hormone that has been shown to decrease the risk of preterm birth in some pregnant women. Progesterone is metabolized by the body into various metabolites including members of the mineralocorticoid and glucocorticoid families. Our group has previously demonstrated that some progesterone metabolites enhance myometrial contractility in an ex vivo system, while others result in myometrial relaxation. The current exploratory study was designed to determine if pre-specified metabolites of progesterone measured early in pregnancy were associated with a woman’s risk for delivery prior to 32 weeks, which is referred to as a very preterm delivery. STUDY DESIGN, SIZE, DURATION The Building Blocks of Pregnancy Biobank (BBPB) is a biorepository at Indiana University (IU) that follows women prospectively through their pregnancy. A variety of biospecimens are collected at various time points during a woman’s pregnancy. Women participating in the IU BBPB who were enrolled after 8 weeks’ gestation with pregnancy outcome data were eligible for participation. PARTICIPANTS/MATERIALS, SETTING, METHODS Women delivering prior to 37 weeks (preterm) and at or after 37 weeks (term) who had blood samples collected during the late first trimester/early second trimester and/or during the early third trimester were identified. These samples were then processed for mass spectroscopy, and the amount of progesterone and progesterone metabolites in the samples were measured. Mean values of each measured steroid metabolite were calculated and compared among women delivering at less than 32 weeks, less than 37 weeks and greater than or equal to 37 weeks. Receiver operating characteristic (ROC) curves were constructed and threshold levels determined for each compound to identify a level above or below which best predicted a woman’s risk for delivery prior to 32 and prior to 37 weeks. Mann–Whitney U nonparametric testing with Holm–Bonferroni correction for multiple comparisons was utilized to identify steroid ratios that could differentiate women delivering spontaneously at less than 32 weeks from all other pregnancies. MAIN RESULTS AND THE ROLE OF CHANCE Steroid hormone levels and pregnancy outcome data were available for 93 women; 28 delivering prior to 32 weeks, 40 delivering between 32 0/7 and 36 6/7 weeks and 25 delivering at or greater than 37 weeks: the mean gestational age at delivery within the three groups was 27.0, 34.4 and 38.8 weeks, respectively. Among women delivering spontaneously at less than 37 weeks, maternal 11-deoxycorticosterone (DOC) levels drawn in the late first trimester/early second trimester were significantly associated with spontaneous preterm delivery prior to 32 weeks; a threshold level of 47.5 pg/ml had 78% sensitivity, 73% specificity and an AUC of 0.77 (P = 0.044). When DOC levels were analyzed as a ratio with other measured steroid hormones, the ratio of DOC to 16-alpha-hydroxyprogesterone among women delivering spontaneously prior to 37 weeks was able to significantly discriminate women delivering prior to 32 weeks from those delivering at or greater than 32 weeks, with a threshold value of 0.2 with 89% sensitivity, 91% specificity and an AUC of 0.92 (P = 0.002). When the entire study cohort population was considered, including women delivering at term and women having an iatrogenic preterm delivery, the ratio of DOC to 16-alpha-hydroxyprogesterone was able to discriminate women delivering spontaneously prior to 32 weeks from the rest of the population at a threshold of 0.18 and 89% sensitivity, 59% specificity and an AUC of 0.81 (P = 0.003). LIMITATIONS, REASONS FOR CAUTION This is a discovery study, and the findings have not been validated on an independent cohort. To mitigate issues with multiple comparisons, we limited our study to pre-specified metabolites that are most representative of the major metabolic pathways for progesterone, and adjustments for multiple comparisons were made. WIDER IMPLICATIONS OF THE FINDINGS Spontaneous preterm birth is increasingly being recognized to represent a common end pathway for a number of different disease phenotypes that include infection, inflammation, premature rupture of the membranes, uterine over distension, cervical insufficiency, placental dysfunction and genetic predisposition. In addition to these phenotypes, longitudinal changes in the maternal–fetal hypothalamic–pituitary–adrenal (HPA) axis also likely contribute to a significant proportion of the disease burden of spontaneous preterm birth. Here, we demonstrate that differential production of steroid metabolites is associated with very early preterm birth. The identified biomarkers may hint at a pathophysiologic mechanism and changes in the maternal–fetal dyad that result in preterm delivery. The early identification of abnormal changes in HPA axis metabolites may allow for targeted interventions that reverse the aberrant steroid metabolic profile to a more favorable one, thereby decreasing the risk for early delivery. Further research is therefore required to validate and extend the results presented here. STUDY FUNDING/COMPETING INTEREST(S) Funding for this study was provided from the Office of the Vice Chancellor for Research at IUPUI, ‘Funding Opportunities for Research Commercialization and Economic Success (FORCES) grant’. Both A.S.P. and C.A.G. are affiliated with Nixxi, a biotech startup. The remaining authors report no conflict of interest. TRIAL REGISTRATION NUMBER Not applicable.

Negligible hormonal response following dehorning in free-ranging white rhinoceros (Ceratotherium simum)

The white rhinoceros (Ceratotherium simum) is experiencing unsustainable poaching losses fuelled by a demand for horn. Increasingly, private and state reserves are dehorning their rhinoceros populations in an attempt to reduce poaching pressure. Rhinoceroses use their horns in social interactions as well as during resource access and so its partial removal as part of reserve management practices may adversely influence these behaviours. Physiological stress can correlate with animal welfare, reproductive state and health and thus acts as a useful indicator of these parameters. To establish whether dehorning causes a physiological stress response, glucocorticoid and gonadal steroid profiles of free-ranging white rhinoceroses were determined through the collection and analysis of faecal steroid metabolites before and after dehorning. Faecal corticoid profiles were not influenced by the number of occasions a rhinoceros had been dehorned or by the number of days that had elapsed since dehorning. Furthermore, there was no apparent suppression in the concentrations of testosterone or progesterone metabolites in males and females, respectively, after exposure to multiple dehorning procedures. These findings should increase wildlife managers’ confidence that dehorning does not negatively impact white rhinoceros physiology as measured hormonally.