Progestin Metabolites Research Articles

Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching

Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5β-THF, 3β,5α-THF, 3β,5β-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5β-THE, 3β,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20β-DHP, 3α-DHP, 3β-DHP, 5α-DHP, 5β-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5β-THP, 3β,5α-THP, 3β,5β-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 μL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL−1 was achieved for all steroids suitable for the analysis of authentic samples.

Periovulatory changes in behavior and fecal hormone metabolite concentrations but not vaginal cytology or vaginoscopy are indicative for the fertile period in female African wild dogs (Lycaon pictus)

Artificial insemination (AI) can aid conservation of African wild dogs (AWD), but methods to determine the appropriate timing of AI in females are not feasible without immobilization. This study determined whether certain behaviors coupled with fecal estrogen (fEM) and progestogen (fPM) metabolite concentrations, could be used as non-invasive parameters to predict the fertile period in female AWDs. Behavior was observed in three alpha females before, during and after the mating period, and feces analyzed for hormone metabolites. During the periovulatory period, females were immobilized 2–3 times to evaluate vulvar condition, blood hormone concentrations and vaginal cytology, and to conduct vaginoscopy. Late estrus (fertile period) could be distinguished from pro-estrus, early estrus, and diestrus using behavior; with a 2- to 5-fold higher rate of male-female affiliative behavior, sexual and non-sexual follow, alpha male initiating behavior, ride-up and copulation (P ≤ 0.05). Sexual behaviors, and male-female resting patterns declined significantly or ceased the day after last mating. Two females showed a 2.5- to 3-fold increase in fPM concentrations during late estrus compared to the pro-estrus period (P ≤ 0.05) and elevated fEM levels that rose in pro-estrus, declined to baseline by late estrus. The one anovulatory female showed no distinct pattern in fPM or fEM concentrations. Vaginal cytology and vaginoscopy could not discriminate between different phases. In summary, behavioral observations coupled with rising fPM and declining fEM can determine the fertile period in African wild dog females, whereas infrequent measurement of blood hormone concentrations, vaginal cytology and vaginoscopy are unreliable.

Refining the moose serum progesterone threshold to diagnose pregnancy.

Pregnancy determination is necessary for sound wildlife management and understanding population dynamics. Pregnancy rates are sensitive to environmental and physiological factors and may indicate the overall trajectory of a population. Pregnancy can be assessed through direct methods (rectal palpation, sonography) or indicated using hormonal assays (serum progesterone or pregnancy-specific protein B, fecal progestogen metabolites). A commonly used threshold of 2ng/ml of progesterone in serum has been used by moose biologists to indicate pregnancy but has not been rigorously investigated. To refine this threshold, we examined the relationship between progesterone concentrations in serum samples and pregnancy in 87 moose (Alces alces; 64 female, 23 male) captured from 2010 to 2020 in the Grand Portage Indian Reservation in northeastern Minnesota, USA. Pregnancy was confirmed via rectal palpation (n = 25), necropsy (n = 2), calf observation (n = 25) or characteristic pre-calving behavior (n = 6), with a total of 58 females determined pregnant and 6 not pregnant; 23 males were included to increase the non-pregnant sample size. Using receiver operating characteristic analysis, we identified an optimal threshold of 1.115ng/ml with a specificity of 0.97 (95% confidence interval [CI] = 0.90-1.00) and a sensitivity of 0.98 (95% CI = 0.95-1.00). Progesterone concentrations were significantly higher in cases of pregnant versus non-pregnant cows, but we did not detect a difference between single and twin births. We applied our newly refined threshold to calculate annual pregnancy rates for all female moose (n = 133) captured in Grand Portage from 2010 to 2021. Mean pregnancy rate during this period was 91% and ranged annually from 69.2 to 100%. Developing a reliable method for determining pregnancy status via serum progesterone analyses will allow wildlife managers to assess pregnancy rates of moose without devoting substantial time and resources to palpation and calf monitoring.

Endocrine correlates of female reproductive activity in the roan antelope (Hippotragus equinus)

AbstractAlthough ovarian endocrine activity has been described in most members of the tribe Hippotragini, little is known about the reproductive endocrine correlates of the roan antelope (Hippotragus equinus), an endangered antelope species of southern Africa. This study characterised the endocrine pattern of the reproductive activity (oestrous cycle, pregnancy and post‐partum period) in the female roan antelope by monitoring faecal progestogen metabolite (fPM) concentrations and oestrogen metabolite (fEM) concentrations for cyclicity and observing reproductive behaviours. The study was carried out over the course of 12 months. At the beginning of this study, all 18 focal females (nulliparous, primiparous and multiparous) were monitored for cyclicity (for between 3 and 4 months). Following observed copulation, pregnancy in multiparous females (n = 10) was subsequently monitored until parturition and 21 days post‐partum. Individual faecal samples were collected three times per week during the presumed period of cyclicity, twice per week during pregnancy and daily for 21 days post‐partum. Mating births, courtship events, and copulations were recorded when observed. In eight of the 18 females monitored, the patterns of fPM and fEM concentrations indicated oestrous cycle lengths (n = 10) of approximately 30.8 ± 2.1 days. The determined lengths of luteal and follicular phases were approximately 15.8 ± 1.5 days and 14.6 ± 2.1 respectively. Pregnancy was marked by a pronounced increase in fPM concentrations until parturition, and lasted approximately 280.4 ± 4.8 days; whereas the intercalving period ranged between 306 and 380 days (n = 9; mean: 333.2 ± 7.4 days). Twenty‐one day after parturition, 78% of the focal females (n = 5) did not show a resumption of the ovarian activity. This study demonstrated that monitoring faecal reproductive hormone metabolite patterns is a valuable approach for estimating ovarian activity in roan antelope and may be used to assist respective conservation breeding programmes by improving management practices.

Medroxyprogesterone acetate positively modulates specific GABAA-receptor subtypes - affecting memory and cognition

Medroxyprogesterone acetate (MPA) is a progestin widely used in humans as hormone replacement therapy and at other indications. Many progestin metabolites, as the progesterone metabolite allopregnanolone, have GABAA-receptor modulatory effects and are known to affect memory, learning, appetite, and mood. In women, 4 years chronic treatment with MPA doubles the frequency of dementia and in rats, MPA causes cognitive impairment related to the GABAergic system. Activation of the membrane bound GABAA receptor results in a chloride ion flux that can be studied by whole-cell patch-clamp electrophysiological recordings. The purpose of this study was to clarify the modulatory effects of MPA and specific MPA metabolites, with structures like known GABAA-receptor modulators, on different GABAA-receptor subtypes. An additional aim was to verify the results as steroid effects on GABA response in single cells taken from rat hypothalamus. HEK-293 cell-lines permanently expressing the recombinant human GABAA-receptor subtype α1β2γ2L or α5β3γ2L or α2β3γ2S were created. The MPA metabolites 3α5α-MPA,3β5α-MPA and 3β5β-MPA were synthesised and purified for electrophysiological patch-clamp measurements with a Dynaflow system. The effects of MPA and tetrahydrodeoxycorticosterone were also studied. None of the studied MPA metabolites affected the responses mediated by α1β2γ2L or α5β3γ2L GABAA receptors. Contrary, MPA clearly acted both as a positive modulator and as a direct activator of the α5β3γ2L and α2β3γ2S GABAA receptors. However, in concentrations up to 10 μM, MPA was inactive at the α1β2γ2L GABAA receptor. In the patch-clamp recordings from dissociated cells of the preoptic area in rats, MPA increased the amplitude of responses to GABA. In addition, MPA alone without added GABA, evoked a current response. In conclusion, MPA acts as a positive modulator of specific GABAA receptor subtypes expressed in HEK cells and at native GABA receptors in single cells from the hypothalamic preoptic area.

Effects of menopausal hormone therapy-based on the role of estrogens, progestogens, and their metabolites in proliferation of breast cancer cells.

Menopausal hormone therapy (MHT) has been widely used for the clinical treatment of symptoms associated with menopause in women. However, the exact nature of the relationship between MHT and the increased risk of breast cancer has not been fully elucidated. The results of the Women’s Health Initiative’s randomized controlled clinical studies showed that estrogen monotherapy was associated with a lower incidence of breast cancer as compared to estrogen-progesterone combined therapy, with an elevated risk of breast cancer. The evidence currently available from randomized trials and observational studies is based on data from different populations, drug formulations, and routes of administration. Even though the risks of MHT and breast cancer have received a great deal of attention, information regarding the unpredictable toxicological risks of estrogen and progestogen metabolism needs to be further analyzed. Furthermore, the diversity and complexity of the metabolic pathways of estrogen and different progestogens as well as the association of the different estrogen and progestogen metabolites with the increased risk of breast cancer need to be adequately studied. Therefore, this review aimed to describe the biological effects of estrogen, progesterone, and their metabolites on the proliferation of breast cancer cells, based on relevant basic research and clinical trials, to improve our understanding of the biological functions of estrogen and progestogen as well as the safety of MHT.

Progestogen metabolites for use in pregnancy monitoring of 13-lined ground squirrels (Ictidomys tridecemlineatus).

This research was conducted to discover whether pregnancy prediction is possible in female 13-lined ground squirrels (TLGS; a small hibernating ground squirrel named for their number of stripes). Pregnancy status in this species, we postulated, could be anticipated by generating profiles for individuals via a non-invasive technique known as fecal endocrine hormone profiling. Fecal samples were collected from 13 females thrice weekly for 4 weeks post-hibernation in the breeding season of 2016. Fecal samples were then processed and run through an assay known as an ELISA giving concentrations of hormone metabolites excreted through feces. We then set these samples against time points to develop a profile for each female. We have ascertained that elevated progesterone (potential pregnancy) can be detected by a commercially available assay kit. Understanding hormone patterns in animals gives researchers a better idea of best husbandry practices, including breeding in managed care.

Reduced concentrations of vaginal metabolites involved in steroid hormone biosynthesis are associated with increased vulvar vestibular pain and vaginal muscle tenderness in provoked vestibulodynia: An exploratory metabolomics study.

Provoked vestibulodynia (PVD) is a chronic vulvar pain disorder characterized by hypersensitivity and severe pain with pressure localized to the vulvar vestibule. Knowledge regarding pathophysiological mechanisms contributing to the etiology and production of symptoms in PVD remains incomplete but is considered multifactorial. Using a cross-sectional observational study design, data from untargeted metabolomic profiling of vaginal fluid and plasma in women with PVD and healthy women was combined with pain testing and brain imaging in women with PVD to test the hypotheses that women with PVD compared to healthy women show differences in vaginal and plasma metabolites involved in steroid hormone biosynthesis. Steroid hormone metabolites showing group differences were correlated with vulvar vestibular pain and vaginal muscle tenderness and functional connectivity of brain regions involved in pain processing in women with PVD to provide insight into the functional mechanisms linked to the identified alterations. Sensitivity analyses were also performed to determine the impact of hormonal contraceptive use on the study findings. Women with PVD compared to healthy controls had significant reductions primarily in vaginal fluid concentrations of androgenic, pregnenolone and progestin metabolites involved in steroidogenesis, suggesting localized rather than systemic effects in vagina and vulvar vestibule. The observed reductions in androgenic metabolite levels showed large effect size associations with increased vulvar vestibular pain and vulvar muscle tenderness and decreases in androgenic and progestin metabolites were associated with decreased connectivity strength in primary sensorimotor cortices. Women with PVD showed symptom-associated reductions in vaginal fluid concentrations of metabolites involved in the biosynthesis of steroid hormones previously shown to affect the integrity of vulvar and vaginal tissue and nociceptive processing. Deficiency of certain steroids may be an important mechanism contributing to the pathophysiology of symptoms in PVD may provide potential diagnostic markers that could lead to new targets for therapeutic intervention.

Gas chromatography-mass spectrometry-based metabolic profiling of androgens, progestins and glucocorticoids in women with polycystic ovary syndrome

Hypothesis/aims of study. Polycystic ovary syndrome (PCOS) is a common disease. Depending on the diagnostic criteria, the disease is seen in 10-20% of women of reproductive age and accounts for 70-80% of all forms of hyperandrogenic syndrome. PCOS is a heterogeneous condition of multifactorial etiology characterized by various clinical, endocrine and metabolic disorders. Therefore, it is important to clarify the specific features of steroid hormone biosynthesis and metabolism and steroidogenesis enzyme activity, as well as to search for new laboratory criteria for early diagnosis and prompt treatment. The aim of this study was to perform metabolic profiling of androgens, progestins and glucocorticoids using gas chromatography-mass spectrometry (GC-MS) in obese and non-obese women with PCOS.
 Study design, materials and methods. We examined 53 women of reproductive age diagnosed with PCOS. The first group included 30 women aged 22 to 29 years with normal body weight. The second group comprised 23 obese patients aged 25 to 33 years with an average body mass index (BMI) of 35.3 0.4 kg/m2. The control group consisted of 25 healthy women aged 26 0.6 years having a normal BMI without clinical and biochemical signs of hyperandrogenism. Immunoassay methods were used to determine the serum levels of luteinizing hormone, follicle-stimulating hormone, free testosterone, 17-hydroxyprogesterone, and sex hormone-binding globulin. A glucose tolerance test was performed to determine glucose and insulin levels before and after load. Urine steroid profiles were studied by GC-MS with the optimization of the sample preparation schedule. Statistical data processing was performed using the STATISTICA for WINDOWS software system (version 10). The main quantitative characteristics of the patients are presented as the median (Me), the 25th percentile and the 75th percentile (Q25Q75). To compare the results obtained in the study groups, the nonparametric Mann-Whitney test was used. The 95% confidence interval was considered statistically significant.
 Results. The article presents a metabolomics analysis of androgens, glucocorticoid hormones and progestins in women with PCOS compared to the control group. It was revealed that non-obese patients with PCOS had increased urinary excretion of androstenedione metabolites, dehydroepiandrosterone and its metabolites, 17-hydroxypregnanolone, pregnantriol, and 5-ene-pregnenes, while obese patients with PCOS had increased that of androsterone and dehydroepiandrosterone metabolites (16-oxo-androstenediol and androstenediol-17) compared to the control group findings. Decreased ratios of cortisol and cortisone tetrahydro metabolite amount to the levels of 11-oxo-pregnanetriol, pregnanetriol and 17-hydroxypregnenolone, when compared to the control group, was obtained in non-obese patients with PCOS, which indicates 21-hydroxylase deficiency. In obese patients with PCOS, four signs of increased 5-reductase activity were obtained, and in PCOS patients with a normal BMI, three signs were obtained, which indicates varying 5-reductase activity in PCOS patients depending on the BMI.
 Conclusion. Quantitative evaluation of androgen and progestin metabolites, as well as 5- and 5-metabolites of androstenedione and glucocorticoids in the study of urine steroid profiles by GC-MS method opens new opportunities for PCOS diagnostics.

Impacts of the season and reproductive status on fecal reproductive and adrenocortical steroid metabolites in zoo Cuban crocodiles (Crocodylus rhombifer).

Conservation strategies for crocodilians often include captive breeding to create stable assurance populations. Evaluating adrenal and gonadal hormone patterns can provide animal managers with data to more effectively monitor animal welfare and reproductive status. This study evaluated the effects of season (breeding, nesting, or off), sex (male and female), and reproductive status of females (egg-laying/housed with a male or non-laying/housed solo) on concentrations of fecal glucocorticoid metabolite (FGM), fecal androgen metabolite (FAM), and fecal progestogen metabolite (FPM) in seven Cuban crocodiles, Crocodylus rhombifer, at the Smithsonian's National Zoological Park. Overall, seasonal changes in FGM and FPM concentrations were only observed in egg-laying females; FGM and FPM concentrations were both higher during the nesting season compared to the breeding and off seasons. Seasonal changes in FAM concentrations were only observed in males; males had higher FAM concentrations during the breeding and nesting seasons compared to the off season. Future studies investigating the use of fecal hormone metabolites in crocodilians are necessary to understand differences between individuals and species, to further elucidate the interactions between hormones and environmental factors, such as social housing, and to develop long-term datasets for the management of this species.

Immunoglobulin J chain as a non-invasive indicator of pregnancy in the cheetah (Acinonyx jubatus)

The North American cheetah population serves as a reservoir for the species, and acts as a research population to help understand the unique biology of the species. Little is known about the intrauterine physiology of the cheetah, including embryo differentiation, implantation, and the development of the placenta. After mating, cheetah females frequently experience (30–65% of matings) a non-pregnant luteal phase where progestogen metabolite levels match those found in pregnant females for the first ~55 days of gestation, but parturition does not occur. Immunoglobulin J chain (IgJ) is a molecule that is involved in the activation of the secretory immune response and has been found to be indicative of pregnancy in the cheetah using fecal monitoring. In this study, western blotting was employed to track IgJ abundance in pooled weekly fecal samples following natural breeding or exogenous stimulation to ovulate, and IgJ levels were compared between individuals undergoing a pregnant (n = 12) and non-pregnant (n = 19) luteal phase. It was revealed that IgJ abundance was increased in pregnant females compared to non-pregnant females at week 4 and week 8 post-breeding, indicating the potential modulation of maternal immunity in response to sensitive events such as implantation and the increased secretory activity of the placenta. IgJ levels also tended to be higher early after breeding in females that were bred naturally with intact males compared to exogenously stimulated females with no exposure to seminal plasma, potentially indicating a response to the act of intromission or the stress of breeding, or possibly demonstrating an immune response resulting in the promotion of maternal tolerance to seminal antigens present upon embryonic implantation. Monitoring fecal IgJ may be a potential method to determine gestational status in the cheetah and will aid future conservation efforts of the species.

Assessment of fecal steroid and thyroid hormone metabolites in eastern North Pacific gray whales.

Baleen whale fecal samples have high potential for endocrine monitoring, which can be used as a non-invasive tool to identify the physiological response to disturbance events and describe population health and vital rates. In this study, we used commercial enzyme-linked immunosorbent assays to validate and quantify fecal steroid (progestins, androgens and glucocorticoids) and thyroid hormone metabolite concentrations in eastern North Pacific gray whales (Eschrichtius robustus) along the Oregon coast, USA, from May to October of 2016-2018. Higher mean progestin metabolite concentrations were observed in postweaning females, followed by pregnant females. Mean androgen, glucocorticoid and thyroid metabolites were higher in mature males. Progestin, glucocorticoids and thyroid fecal metabolites varied significantly by year, with positive correlations between progestin and androgen, and between glucocorticoid and thyroid metabolites. We also present two case studies of a documented injured whale and a mature male displaying reproductive competitive behavior, which provide reference points for physiologically stressed individuals and adult breeding males, respectively. Our methods and findings advance the knowledge of baleen whale physiology, can help guide future research on whale physiology and can inform population management and conservation efforts regarding minimizing the impact of anthropogenic stressors on whales.

Enzyme immunoassays for water-soluble steroid metabolites in the urine and feces of Japanese macaques (Macaca fuscata) using a simple elution method.

A simple, non-alcoholic extraction method for measuring estrogen and progesterone metabolites in excreta using enzyme immunoassays (EIAs) was developed in Japanese macaques. The obtained detection limits of EIAs using estrone conjugates (E1C), pregnanediol glucuronide (PdG), and estriol glucuronide (E3G) polyclonal antibodies with cross-reactivity to urinary and fecal steroid metabolites were 6.6 pg/ml, 2.1 ng/ml and 0.35 ng/ml, respectively. These assays allowed the determination of E1C, PdG, and E3G from the excreta with good reproducibility and accuracy. Thereafter, urine and fecal samples of two menstrual cycles and six pregnancies from eight female Japanese macaques were assayed. A typical increase in urinary and fecal E1C in follicular phase and PdG in luteal phase were shown during non-conceptive menstrual cycles. Urinary E3G levels also showed a preovulatory increase; however, fecal E3G levels were very low throughout the non-conceptive menstrual cycles. Levels of E1C and PdG in the urine and feces of pregnant females were gradually increased until parturition, while fecal E3G levels were low and reached detectable levels after the mid-pregnancy period. Although the extraction rate of estrogen and progestogen metabolites by our method was lower compared to those of the previous extraction method using an alcohol-containing buffer, our method was simple, and the correlation coefficients for the relationship between two methods were found to be statistically significant. The results presented here are of great practical value for a non-invasive method of monitoring ovarian function and pregnancy in Japanese macaques.

Determination of two progestin metabolites (17α-hydroxypregnanolone and pregnanediol) and different classes of steroids (androgens, estrogens, corticosteroids, progestins) in rivers and wastewaters by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)

A highly sensitive and robust method was developed for routine analysis of two progestin metabolites, 17α-hydroxypregnanolone (17OH-Δ5P) and pregnanediol (PD), and 31 other natural and synthetic steroids and related metabolites (estrogens, androgens, corticosteroids, progestins) in river water, as well as influents and effluents of municipal wastewater treatment plants (WWTP) using HPLC-MS/MS combined with solid-phase extraction. For the various matrixes considered, the optimized method showed satisfactory performance with recoveries of 70–120% for most of target steroids. The method detection limits (MDLs) ranged from 0.01 to 3ng/L for river water, 0.02 to 10ng/L for WWTP effluents, and 0.1 to 40ng/L for influents with good linearity and reproducibility. The developed method was successfully applied for the analysis of steroids in rivers and WWTP influent and effluents. WWTP influents concentrations of 17OH-Δ5P and PD were 51–256ng/L and up to 400ng/L, respectively, along with androstenedione (concentration range: 38–220ng/L), testosterone (11–26ng/L), estrone (2.3–37ng/L), 17β-estradiol (N.D.–8.7ng/L), 17α-hydroxyprogesterone (N.D.–66ng/L), medroxyprogesterone acetate (N.D.–5.3ng/L), and progesterone (2.0–22ng/L), while only androstenedione (ADD), estrone (E1), and estriol (E3) were detected in effluent with concentrations ranging up to 1.7ng/L, 0.90ng/L and 0.8ng/L, respectively. In river water samples, only ADD and E1 were detected with concentrations up to 1.0ng/L and 0.91ng/L. Our procedure represents the first method for analyzing 17OH-Δ5P and PD in environmental samples along with a large series of steroids.

Characterizing the reproductive biology of the female pygmy hippopotamus (Choeropsis liberiensis) through non-invasive endocrine monitoring

The pygmy hippopotamus (Choeropsis liberiensis) is endangered in the wild and very little is known about its reproductive biology. In zoological facilities, this species experiences a number of reproductive issues that complicate breeding management, including a high rate of stillbirths and failure of many pairs to reproduce. We conducted a comprehensive study to evaluate reproductive cycles and pregnancy in this species using enzyme immunoassays (EIAs) for fecal hormone metabolite analysis. Fresh fecal samples were collected twice weekly for a one to three year period from 36 female pygmy hippos housed at 24 zoological institutions. Samples were analyzed in three separate laboratories. Three progestogen metabolite EIAs (Pg-diol: 5β-pregnane-3α,20α-diol 3HS:BSA; PdG: pregnanediol-3-glucuronide R13904; mono-P4: Quidel clone 425) and three estrogen metabolite EIAs (E2a: estradiol-17β-OH 17-HS:BSA; E2b: estradiol 17β R0008; E2c: estradiol 17β R4972) accurately reflected reproductive events. Average estrous cycle length was 31.8 ± 7.4 days based on estrogen metabolite peaks and 30.9 ± 7.3 days based on nadir to nadir progestogen metabolite concentrations. Cyclical patterns in both estrogen and progestogen metabolites were detected throughout the year, indicating a lack of seasonality. Estrogen metabolite peaks were also observed during pregnancy and lactation, suggesting that follicular development occurs during both reproductive states. Pregnancy was most reliably demonstrated by elevation in progestogen metabolites (Pg-diol or PdG) in the second half of gestation. Average gestation length based on breeding to calving date was 203 ± 4 days for 15 pregnancies. This comprehensive overview of the reproductive biology of the female pygmy hippo provides valuable data for guiding long-term breeding management for this endangered species and serves as a baseline for future studies addressing the potential influence of social structure, diet, body condition, and other husbandry factors on estrous cycling and reproduction.

Characterization and manipulation of reproductive cycles in the jaguar (Panthera onca)

Basic reproductive information in female jaguars (Panthera onca) is lacking, thus longitudinal fecal samples from seven females were analyzed via enzyme immunoassay to measure estradiol and progestin metabolites throughout the year. Mean estrus length of 194 estrus periods measured hormonally was 6.5±0.3d, mean peak fecal estradiol concentration was 138.7±5.7ng/g; and in one female, estrus resumption occurred approximately 15d post-partum. Ovulation, as indicted by sustained elevated progestin concentrations (>20d), was successfully induced one time by treatment with exogenous hormones in one female and by physical vaginal stimulation in two females a combined total of three times. Elevated fecal progestin was observed outside exogenous stimulation on five occasions, suggesting ovulation occurred spontaneously. Mean length of physically induced and spontaneous pseudopregnancies was 24.7±4.2d and 29.6±2.6d, respectively, and mean length of pregnancy (n=2) was 98.0±0.0d. Mean peak progestin concentration for spontaneous and induced pseudopregnancies, and pregnancy was 7.4±1.4μg/g, 6.4±1.2μg/g, and 13.7±1.0μg/g, respectively. This data suggests jaguars are polyestrous and generally induced ovulators, with a moderate incidence of spontaneous ovulation. Additionally, two protocols to successfully stimulate ovarian activity in jaguars are described.

The use of altrenogest to avoid hyperestrogenism after eCG-hCG ovulation induction in southern tigrina (Leopardus guttulus)

The goal of this study was to optimize an ovulation induction protocol for use with artificial insemination in the southern tigrina (Leopardus guttulus). The specific aims were to report the efficacy of using altrenogest, an oral progestin (Regumate, MSD Animal Health, Merck Animal Health), to suppress ovarian activity and prevent follicular hyperstimulation and hyperestrogenism after the administration of exogenous eCG and hCG. To monitor ovarian responses, fecal estrogen and progestogen metabolites were quantified by enzyme immunoassay in females before and after intramuscular administration of 200-IU eCG and 150-IU hCG in two trials, 4 months apart. During the first trial, there was no use of altrenogest, only the eCG-hCG ovulation induction protocol. In the second trial, the ovulation induction protocol was preceded by the administration of oral altrenogest for 14 days (minimum of 0.192 mg per kg per day). Altrenogest administration resulted in a suppression of follicular activity in three out of six females before eCG-hCG administration on the basis of lower mean estrogen concentrations (P < 0.05). It also resulted in four out of six females presenting lower fecal estrogen metabolite concentrations (P < 0.05) after ovulation induction, and two out of six individuals showed a reduction (P < 0.05) in postovulatory fecal progestogen metabolite concentrations, all when compared to the same female's cycles without the progestin. Fecal estrogen metabolite concentrations were closer to baseline in 50% of these individuals after altrenogest and eCG-hCG treatments when compared to basal concentrations before gonadotropins without the use of altrenogest. This study reported that use of altrenogest in southern tigrina can suppress ovarian activity and avoid hyperestrogenism after administration of eCG and hCG treatment. However, not all females responded uniformly, so more studies are needed to increase the efficacy of ovulation induction for use with artificial insemination in this species.

Mental Disorders and Thrombotic Risk

Patients with psychosis, severe depression, or chronic stress are at increased risk for thromboembolism. Evidence suggests that tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) imbalance may play an important role in pathophysiology of mental and thromboembolic disorders. tPA facilitates clot dissolution and participates in several brain functions, including response to stress, learning, and memory. Depression is characterized by high PAI-1 levels, and conditions related to a hypofibrinolytic status, such as polycystic ovary syndrome and metabolic syndrome, are associated with an increased risk for both depression and cardiovascular events. Depression/psychosis may occur when estrogen and progesterone levels decrease. Estrogen inhibits synthesis of transforming growth factor-β (TGF-β), a protein increasing PAI-1 synthesis, whereas progesterone induces brain-derived neurotrophic factor release, stimulating tPA expression in neurons. Synthetic progestogens (progestins) may cause severe depression. Notably, progestin metabolites, which may prevent synthesis of natural progesterone by feedback, do not cross the blood-brain barrier. Intense suprapubic menstrual cramp, which may relate to difficulty to dissolve clots, correlates with severe premenstrual tension. Exercise, which increases tPA synthesis, has antidepressive and antithrombotic effects. We suggest that thromboembolism and some mental disorders are mechanistically related: tPA-PAI-1 imbalance seems to be a common denominator.

Detection of pregnancy and fertility status in big cats using an enzyme immunoassay based on 5α-pregnan-3α-ol-20-one

Development of non-invasive steroid hormone assays using fecal samples is crucial for detection of pregnancy and monitoring of fertility status in big cats and thus facilitates conservation and management of wild animals. Due to changes in metabolism and excretory pattern, animals excrete different steroid metabolites in feces and urine. The present study is an attempt to develop a common enzyme immunoassay for 5α-pregnan-3α-ol-20-one one of the predominant progestogen metabolites in the feces samples of big cats. The developed ELISA showed a high sensitivity and low cross reactivity to other hormones compared to commercially available RIA kits based on progesterone antibody. It could be used in a wide range of animals for monitoring fertility status and pregnancy detection by measuring fecal steroid metabolites.

The Role of Progestogens in Regulating Matrix Metalloproteinase Activity in Macrophages and Microglial Cells

Although the systemic effects of progestogens have been extensively studied, little is known in regards to the cellular effects of these compounds. Using a cellular model for vascular (macrophages) and brain (microglial) cells, we studied the effects of various progestogens, either alone or in combination with 17β-estradiol (E(2)) on the activity of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme involved in vascular remodeling and plaque destabilization in cardiovascular events, blood-brain barrier breakdown in stroke and brain regeneration and neurovascular remodeling during repair phases of brain injury. In the absence of E(2), medroxyprogesterone acetate (MPA), a synthetic progestogen and progesterone (PG) metabolites tended to increase MMP-9 enzyme activity in macrophages and microglial cells, whereas PG decreased such activity in macrophages; exceptions being that MPA and the PG metabolite, pregnanediol (Pdiol) had no effect on macrophage MMP-9 enzyme activity and PG had no effect on microglial cell MMP-9 enzyme activity. In the presence of E(2), an opposite affect was observed whereby MPA and the PG metabolites tended to decrease MMP-9 enzyme activity from macrophages and microglial cells, whereas PG had no effect; exceptions being that MPA and Pdiol had no effect on macrophage MMP-9 enzyme activity. In conclusion, these results demonstrate that the effects of PG, PG metabolites and MPA on MMP-9 enzyme activity differ across vascular and brain cells when administered alone or in combination with E(2) which could have important mechanistic implications for hormone therapy.