Progesterone Metabolite Concentrations Research Articles

FRI382 High Androgen And Progesterone Metabolite Concentrations In Naturally Matured Follicles Are Concurrent With High Serum Anti-Müllerian Hormone Concentrations

Abstract Disclosure: T. Du Toit: None. M. Von Wolff: None. A.C. Swart: None. M. Groessl: None. C.E. Flueck: None. Infertility in women is routinely evaluated with the assessment of circulating steroid levels. Profiling steroid levels in follicular fluid (FF) would provide a focused evaluation of the ovarian endocrine milieu and is therefore better suited to identify key hormones potentially involved in dysfunctional follicular growth. The anti-Müllerian hormone (AMH) level in serum is a well-established clinical marker to estimate the ovarian reserve in assisted reproductive technology, while the AMH level in FF serves as a new prognostic marker for oocyte developmental potential. However, AMH-linked changes in the follicular steroid milieu have not been fully explored. We conducted an AMH-dependent analysis of the FF endocrine milieu. Hormones were quantified using LC-MS (n=42) and ELISA (n=4) in FF of 83 women undergoing natural cycle in vitro fertilisation. Patients were age-matched and classified into 4 statistically different (p<0.0001) groups according to serum AMH levels: group A (<1.1 pmol/L, n=20); group B (1 - 7.14 pmol/L, n=21); group C (7.14 - 35.5 pmol/L, n=21); and group D (>35.5 pmol/L, n=21). Group D was designated as our polycystic ovary syndrome (PCOS)-like group, as high serum AMH is a marker for PCOS. We characterised androgen and progesterone metabolic pathways in FF. Androstenedione (A4) and testosterone (T) levels differed significantly between the groups (p<0.01) and were highest in group D at 60 nmol/L and 4.7 nmol/L, respectively. The levels of 11-ketoandrostenedione (11KA4) and 11β-hydroxyandrosterone (adrenal-origin) also differed between the groups (p<0.05), with higher 11KA4 levels (±2.7-fold) quantified in group D. In parallel with serum AMH, FF AMH differed between the groups (p<0.0001), together with luteinizing hormone/FSH, A4/dehydroepiandrosterone, estradiol(E2)/T and E2/A4 ratios. In comparison to groups A, B and C combined, the PCOS-like group D showed significantly increased downstream progesterone metabolite levels –11α-hydroxyprogesterone [OHP4], 17α,20α-diOHP4, 16α-OHP4 and 5α-pregnanolone, with lower FSH levels. Our results show that the follicular milieu is significantly different when high AMH levels are measured compared to when AMH levels are low, marked by: (i) higher progesterone metabolite levels in follicles and (ii) higher A4 and T levels, concomitant with unchanged E2 levels suggesting that androgens were not fully converted to estrogens in these follicles. Our study furthermore corroborates other studies reporting an intrafollicular hyperandrogenic state in women with increased circulatory AMH levels. In summary, our study not only provides data based on a broad steroid profile that the follicular androgen endocrine milieu is affected in women with high serum AMH levels, but it also provides a model for the premature progesterone increase in PCOS women which leads to a reduction in the embryo implantation rate. Presentation: Friday, June 16, 2023

Evaluation of minimally invasive estrus synchronization protocols in brown brocket deer (Subulo gouazoubira)

This study aimed to evaluate minimally invasive protocols for estrus synchronization in the brown brocket deer (Subulo gouazoubira). Females were submitted to Latin square design, in different treatments. All females received 0.25 mg of estradiol benzoate on the first day of treatment, concomitant with one of the following sources of progesterone: (1) DIP: an intravaginal progesterone releasing device for eight days, (2) MGA1x: once a day (in the morning) oral dose of 1 mg melengestrol acetate for eight days, (3) MGA2x: twice a day (morning and afternoon) oral doses of 0.5 mg of MGA for eight days, (4) P4LA: a single i.m. administration of 75 mg of long-acting progesterone (P4LA). Eight days after the beginning of each treatment, females received an i.m. administration of 265 µg of prostaglandin (PGF2α; cloprostenol). Treatment efficacy was evaluated by manifestation of behavioral estrus after treatment and concentration of fecal progesterone metabolites (FPM). The time to onset of estrus in treatment P4LA was significantly longer (180 ± 38.9 h) compared to DIP (63 ± 6.6 h), MGA1x (53 ± 14.4 h) and MGA2x (41 ± 10.1 h) (P = 0.008). According to individual baseline FPM and FPM concentration during the days after estrus, the corpus luteum formation was suggested in all females which responded to the treatments (93.75 %). Low synchrony, longer interval between PGF2α administration and onset of estrus suggest that the P4LA dose (75 mg) is too high and not effective for S. gouazoubira. DIP, MGA 1x and MGA 2x, were effective in estrus synchronization.

Assessment of a non-invasive approach to pregnancy diagnosis in gray whales through drone-based photogrammetry and faecal hormone analysis.

Knowledge of baleen whales' reproductive physiology is limited and requires long-term individual-based studies and innovative tools. We used 6 years of individual-level data on the Pacific Coast Feeding Group gray whales to evaluate the utility of faecal progesterone immunoassays and drone-based photogrammetry for pregnancy diagnosis. We explored the variability in faecal progesterone metabolites and body morphology relative to observed reproductive status and estimated the pregnancy probability for mature females of unknown reproductive status using normal mixture models. Individual females had higher faecal progesterone concentrations when pregnant than when presumed non-pregnant. Yet, at the population level, high overlap and variability in progesterone metabolite concentrations occurred between pregnant and non-pregnant groups, limiting this metric for accurate pregnancy diagnosis in gray whales. Alternatively, body width at 50% of the total body length (W50) correctly discriminated pregnant from non-pregnant females at individual and population levels, with high accuracy. Application of the model using W50 metric to mature females of unknown pregnancy status identified eight additional pregnancies with high confidence. Our findings highlight the utility of drone-based photogrammetry to non-invasively diagnose pregnancy in this group of gray whales, and the potential for improved data on reproductive rates for population management of baleen whales generally.

EVALUATING THE EFFICACY OF NONINVASIVE FECAL SAMPLING FOR PREGNANCY DETECTION IN ELK (CERVUS CANADENSIS).

Elk (Cervus canadensis) were reintroduced to Tennessee, USA in the early 2000s, with limited reproductive monitoring since initial release. We assessed the efficacy of noninvasive sampling for determining pregnancy using invasive (capture) and noninvasive (fecal collection in the field) techniques at the North Cumberland Wildlife Management Area (NCWMA), Tennessee. We captured 20 female elk 2019-2020, used pregnancy-specific protein B (PSPB) in blood to determine pregnancy and compared results to fecal progesterone metabolite (FPM) concentrations using two commercially available enzyme immunoassay (EIA) kits. Based on PSPB concentrations, 8/11 and 3/4 of captured adult elk (≥2.5 yr of age) were pregnant in 2019 and 2020, respectively; no 1.5-yr-old elk were pregnant (n=5). Using the progesterone EIA kit, FPM concentrations were x̄=192.84±38.63 ng/g (95% CI, 96.48-289.20) for nonpregnant and x̄=536.17±74.98 ng/g (95% CI, 375.97-696.36) for pregnant captured females. For the progesterone metabolite kit, FPM concentrations were x̄=188.16±43.39 ng/g (95% confidence interval [CI], 76.63-299.69) for nonpregnant and x̄=693.52±126.52 ng/g (95% CI, 407.31-979.72) for pregnant captured females. From February to May 2019, we collected 357 fecal samples in 65 areas across 489.62 km2 of the NCWMA. Using extracted DNA and analysis of 15 microsatellites, we identified 62 unique individuals from 128 female fecal samples collected on the landscape. We categorized females from landscape-collected feces as nonpregnant (35.5-40.3%; Metabolite-EIA kits), undetermined (1.6-6.5%; Metabolite-EIA kits), or pregnant (62.9-53.2%; Metabolite-EIA kits) based on a 95% CI of captured female FPM concentrations, giving an overall pregnancy rate of 53.2% using the recommended EIA kit. The pregnancy rate in sexually mature females may be higher, as it was not possible to distinguish age classes of landscape-collected fecal samples; therefore, some may have been from younger age classes not expected to be pregnant. Analysis of FPM may be useful at a population level to detect pregnancy.

Extreme plasticity of reproductive state in a female rodent

Successful sexual reproduction relies on the coordination of multiple biological systems, yet traditional concepts of biological sex often ignore the natural plasticity in morphology and physiology underlying sex. Most female mammals develop a patent (i.e., opened) vaginal entrance (introitus) prenatally or postnatally before or during puberty, usually under the influence of estrogens, and remain patent for the remainder of their lifespan1. An exception is the southern African giant pouched rat (Cricetomys ansorgei), whose vaginal introitus remains sealed well into adulthood2. Here, we explore this phenomenon and report that the reproductive organs and the vaginal introitus can undergo astounding and reversible transformation. Non-patency is characterized by reduced uterine size and the presence of a sealed vaginal introitus. Furthermore, the female urine metabolome shows that patent and non-patent females profoundly differ in their urine content, a reflection of differences in physiology and metabolism. Surprisingly, patency state did not predict fecal estradiol or progesterone metabolite concentrations. Exploring the plasticity that exists in reproductive anatomy and physiology can uncover that traits long considered 'fixed' in adulthood can become plastic under specific evolutionary pressures. Moreover, the barriers to reproduction that such plasticity creates present unique challenges to maximizing reproductive potential.

Comparative analysis of gestation in three rhinoceros species (Diceros bicornis; Ceratotherium simum; Rhinoceros unicornis)

This comparative analysis evaluated endocrine profiles and gestation length data of captive pregnant black rhinoceros (Diceros bicornis), white rhinoceros (Ceratotherium simum), and greater one-horned (GOH) rhinoceros (Rhinoceros unicornis). Hormone profiles were collected over three decades as part of pregnancy diagnoses. After the third month of gestation, the luteo-placental shift in progesterone production in pregnant rhinoceroses causes a significant increase in the concentration of faecal progesterone metabolites. We defined a laboratory-specific value of 1000 ng/g faeces as a threshold for incipient feto-placental progesterone production. Using this value allowed a comparison between species and revealed significant individual differences within a species. The mean ± SEM gestation days for reaching the 1000 ng/g faeces threshold were 89.5 ± 2.9 (range 56–138 days; n = 39) in black, 96.0 ± 2.6 (58–138; n = 39) in white, and 117.8 ± 5.3 (74–173; n = 19) in GOH rhinoceroses. For the calculations of gestation length, we complemented our results from three decades of reproductive monitoring with data from the literature, resulting in about 70 values for each species. Gestation length in the black, the white and the GOH rhinoceros was 460.6 ± 1.5 (range: 436 – 486), 503.8 ± 1.3 (range: 480 – 525) and 480.5 ± 1.1 (range: 453 – 505) days, respectively. Daylight length significantly affected gestation length, while the sex of offspring had no effect. On average, pregnancies with parturitions in spring and summer were one week shorter than those in autumn and winter. Although rhinoceroses are non-seasonal breeders, most parturitions in captivity occur in autumn and winter. We also analysed preconception endocrine profiles in the white rhinoceros. Conceptions in this species occurred after oestrous cycles of approximately 35 days (n = 18), 70 days (n = 3), 15 days (n = 1), after periods of ovarian inactivity (n = 5), and during a foal heat within one month after stillbirth parturition (n = 1). In conclusion, this study provides a comprehensive overview of gestational parameters in three rhinoceros species.

Investigating the utility of using fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas (Tachyglossus aculeatus)

This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesterone metabolite concentrations were elevated above baseline values (448.0 ± 156.3 ng/g) during pregnancy and the luteal phase. However, compared to plasma progesterone the rise in fecal progesterone metabolite concentrations after copulation was delayed (3.3 ± 0.4 versus 8.3 ± 0.6 days, respectively), such that pregnancy was more reliably detected in its latter half when using fecal samples. Mating and oviposition were observed for 14 of the 19 pregnancies resulting in an estimated gestation of 16.7 ± 0.2 days (range 16.0–18.1 d). The estrogen enzyme-immunoassays tested (n = 3) in this study were not suitable for the fecal samples of the echidna. Fecal progesterone metabolites are an effective tool for confirming the timing and occurrence of estrous cycles in captive echidna colonies and can assist zookeepers in identifying possible causes of sub-optimal reproductive success without the unnecessary stress of repeated capture and anaesthesia for blood collection.

Optimizing captive short-beaked echidna (Tachyglossus aculeatus) fecal sample identification and hormonal analysis.

The objectives of this study were to develop a fecal marking protocol to distinguish male from female samples during the echidna breeding season and to determine if normalizing fecal progesterone metabolite data for inorganic content improves the detection of biologically relevant changes in metabolite concentrations. Over a period of 6 weeks, four echidnas were provided with green food coloring powder mixed into 20 g of their regular feed with the dose adjusted weekly by 0.05 g. The proportion of organic (feces) versus inorganic matter (sand) in the fecal samples of three echidnas was determined by combustion of organic matter. Hormonal data was then expressed as metabolite concentration per total dry mass (with sand) of extracted sample versus metabolite concentration per total mass of organic material (without sand). The optimal dose of food coloring powder was 0.30 g: this was excreted in the feces of all echidnas within 24 h of consumption with color present for two consecutive days. Correction for inorganic content (sand) did not significantly affect variability of fecal progesterone metabolite levels (mean CV ± SE with sand: 142.3 ± 13.3%; without sand: 127.0 ± 14.4%; W = 6, p = .2500), or the magnitude of change from basal to elevated fecal progesterone metabolite concentrations (mean ± SE with sand: 8.4 ± 1.7; without sand: 6.6 ± 0.5, W = 10, p = .1250). Furthermore, progesterone metabolite concentrations before and after correction for sand contamination correlated strongly (r = .92, p = < .001). These methods will facilitate future reproductive endocrinology studies of echidna and other myrmecophagous species.

Linking socio-sexual and vocal behaviour with faecal progesterone and oestrogen metabolite levels in Southern white rhinoceros females.

Progesterone and oestrogen are the main gonadal steroid hormones that regulate the ovarian activity and induce the fertile oestrus period in females. The monitoring of this receptive phase is particularly decisive for captive breeding and is commonly based on the observation of female behavioural patterns that coincide with their hormonal oestrus. However, in the white rhinoceros (WR), a species that is well known for its impaired reproductive rate in captivity, the female behavioural and vocal indicators of receptivity have not been systematically investigated or linked to their hormonal states so far. In order to close this gap, we combined behavioural and acoustic recordings, conducted over an average period of 32days, with the analysis of faecal progesterone and oestrogen metabolite concentrations (fPM and fEM) in 27 adult Southern white rhinoceros (Ceratotherium simum simum; SWR) females from 10 European zoos. For eight of the study females, we were able to detect a receptive period indicated by their acceptance of sexual behaviour from the bulls. The comparison of behaviour and vocalization between receptive and non-receptive periods in these females demonstrated that particularly presenting and marking behaviour distinctly peaked during the receptive period, indicating the significance of olfactory signalling for female reproductive behaviour. Based on the analysis of fPM profiles, we were able to identify different reproductive states (cycling, non-cycling, pregnant) in 21 of 27 study females. In contrast, fEM profiles proved to be unsuitable for the detection of ovarian activity. For the majority (five of six females for which behavioural receptivity and endocrine cyclicity were determined), a coincidence of their receptive period and the hormonal oestrus, indicated by a nadir in fPM levels, could be detected. Conclusively, this study revealed a comprehensive behavioural repertoire that reflects the hormonal oestrus in SWR females and can therefore be reliably used for non-invasive ex situ reproduction monitoring.

Reproductive hormones monthly variation in free‐ranging European wildcats: Lack of association with faecal marking

Integrating ecophysiological and behavioural discoveries in conservation and management plans is essential to preserve scarce and elusive species such as the European wildcat (Felis silvestris). The purpose of this study was to characterize the monthly variation in the steroid reproductive hormone metabolite levels (oestradiol, progesterone and testosterone) in this species and to test its possible association with a monthly pattern of faecal marking. By collecting fresh faecal samples in Montes do Invernadeiro Natural Park (Galicia, Northwest Spain) each month, we obtained a total of 110 samples belonging to 25 different individuals. We conducted enzyme immunoassays which allowed us to track the annual variation in reproductive hormone excretion patterns in wildcat scats. Furthermore, we also evaluated the possible relation between the faeces used as marks and the reproductive hormone levels. We found that oestradiol and progesterone metabolite levels exhibited a distinct pattern, both increasing during the breeding months. Oestradiol metabolite larger peaks were found during March and April, whereas the highest concentration of progesterone metabolites appeared in July. On the contrary, testosterone metabolite levels did not significantly change depending on the month. Moreover, we did not find any evidence that the faecal marking behaviour pattern was associated with reproductive hormone metabolite levels. It seems that other factors related to habitat and food resources could be more important in the performance of this behaviour.

Fecal progesterone and estrogen metabolite monitoring for cyclicity and pregnancy in southern tamandua (Tamandua tetradactyla).

Reproductive management of the southern tamandua (Tamandua tetradactyla) should include timed introductions for breeding to minimize aggression and pregnancy monitoring. Since serial blood sampling could cause unnecessary stress, and urinary progesterone metabolites are found in very low concentrations, this study sought to validate progesterone and estradiol enzyme immunoassays for measuring fecal progesterone metabolite (FPM) and fecal estrogen metabolite (FEM) concentrations in two females. Peaks in FEM concentrations coincided with breeding and conception, were 5-6 times higher than baseline concentrations, and were followed by clear luteal phases distinguished by FPM concentrations 5-6 times higher than baseline concentrations. FPM concentrations during the first 30-53 days of gestation overlapped with luteal phase concentrations, thereafter increasing to 8-25 times higher than baseline concentrations. FEM concentrations during the first 41-44 days of gestation remained near basal values for one female, whereas concentrations were 1.8 times higher than baseline for the second. FEM concentrations became elevated for the former by 44 days of gestation and increased further for the latter after 53 days, ultimately averaging four times higher than baseline for both females. The biphasic increase in FPM and FEM concentrations, follicular and luteal phase durations (follicular: 7 ± 1 days, luteal: 25 ± 1 days), total cycle length (41 ± 1 days), and gestation (161-165 days) documented in this study were consistent with previous reports from serum and urine analyses. Monitoring FPM and FEM is a reliable noninvasive method for tracking reproductive cycles and pregnancy in southern tamandua that overcomes the challenges associated with serum or urinary hormone analysis.

Milk production in Saanen goats treated with repeated low doses of intermediate-release insulin during early lactation

ABSTRACT: The effect of insulin administration on the productive responses of Saanen goats during early lactation was investigated. Ten of 20 adult females were subjected to subcutaneous administration of intermediate-acting insulin (0.14UI/kg body weight) at 2, 9, and 14 days postpartum. Milk yield was measured twice daily for 13 weeks and milk samples were collected to measure protein and fat contents. Plasma levels of progesterone, insulin, non-esterifies fatty acids, glucose and other metabolites were measured. Results showed a significantly increased effect of insulin treatment on the content of milk fat and protein; moreover, milk production in the first and second postpartum weeks were higher than control group. The peak of lactation in the insulin group was achieved one week earlier in comparison to the control group. In addition, the milk production rate showed lower persistency (milk yield 13 week/milk yield at peak) in the same group. During the first four weeks of postpartum, treated animals showed greater weight loss and higher non-esterified fatty acid concentration, whereas no effect was observed on the concentration of progesterone and other metabolites. The above results indicated that repeated administration of insulin in dairy goats during early lactation increase yield and qualitative components of milk, but has substantial consequences on animal productive rate and metabolic response.

Prolonged luteal lifespan and pseudopregnancy in Asian elephants (Elephas maximus)

Pseudopregnancy is a physiological occurrence in mammals which have copulation induced ovulation, but is rarely described in spontaneous ovulating species. In this study, three cases of prolonged luteal lifespan are reported in non-pregnant Asian elephants (Elephas maximus). Case 1 was a 25-year-old female that had produced three calves previously; Case 2 was a nulliparous and 32-year-old at the start of the pseudopregnancy episode; and Case 3 occurred in a 49-year-old nulliparous elephant. Serum progesterone metabolite concentrations remained elevated for 10 months in Case 1. Urinary progestagens were high for >16 months in Case 2 and for five months in Case 3. In Case 1, multiple persistent corpora lutea were visualized monthly by ultrasonography. In all three cases, uterine leiomyoma were present and progestagen concentrations decreased spontaneously. In Case 1, the elephant became pregnant 3 years later, whilst with Case 2, the female resumed estrous cycling normally, and for the Case 3 female, there was continuation with another prolonged luteal phase before ovarian function was purposely suppressed. These examples indicate that persistently elevated progestagen concentrations may not always be indicative of pregnancy in elephants. The reasons for prolonged luteal lifespan are not understood, although serum prolactin concentrations quantified in the Case 1 female were elevated compared to values from previous reports and two other herd mates. Furthermore, all three elephants had varying degrees of uterine pathologies. It is believed that the resulting damage to the endometrium may have led to a reaction similar to implantation, which includes prolactin secretion. Prolactin may exert luteotropic properties and is thought to initiate luteal rescue during pregnancy in elephants.

FECAL PROGESTERONE METABOLITES IN POSTPARTUM SIBERIAN FLYING SQUIRRELS.

The Siberian flying squirrel ( Pteromys volans) produces up to two litters a year. To deliver second litters in breeding season, P. volans may have a postpartum estrus similarly to that of a variety of small mammals. If this were the case, females would have periods of elevated progesterone levels because of the formation of corpora lutea (CL) after postpartum ovulation. To test this hypothesis, fecal progesterone metabolite dynamics was investigated during lactation in this species using an enzyme immunoassay. In five of the six lactating females, periods of high fecal progesterone metabolite concentration were observed, and, furthermore, progesterone secretion patterns were periodic. Therefore, the source of progesterone during lactation could be arising CL from postpartum ovulation, indicating that ovarian activity was reinitiated after parturition and the CL that formed began secreting progesterone. This study thus showed that P. volans likely has the physiologic potential to mate during lactation.

Fecal steroid hormones reveal reproductive state in female blue whales sampled in the Gulf of California, Mexico

Steroid hormone assessment using non-invasive sample collection techniques can reveal the reproductive status of aquatic mammals and the physiological mechanisms by which they respond to changes in their environment. A portion of the eastern North Pacific blue whale (Balaenoptera musculus) population that seasonally visits the Gulf of California, Mexico has been monitored using photo-identified individuals for over 30 years. The whales use the area in winter-early spring for nursing their calves and feeding and it therefore is well suited for fecal sample collection. Using radioimmunoassays in 25 fecal samples collected between 2009 and 2012 to determine reproductive state and stress, we validated three steroid hormones (progesterone, corticosterone and cortisol) in adult female blue whales. Females that were categorized as pregnant had higher mean fecal progesterone metabolite concentrations (1292.6 ± 415.6 ng·g-1) than resting and lactating females (14.0 ± 3.7 ng·g-1; 23.0 ± 5.4 ng·g-1, respectively). Females classified as pregnant also had higher concentrations of corticosterone metabolites (37.5 ± 9.9 ng·g-1) than resting and lactating females (17.4 ± 2.0 ng·g-1; 16.8 ± 2.8 ng·g-1, respectively). In contrast, cortisol metabolite concentrations showed high variability between groups and no significant relationship to reproductive state. We successfully determined preliminary baseline parameters of key steroid hormones by reproductive state in adult female blue whales. The presence of pregnant or with luteal activity and known lactating females confirms that the Gulf of California is an important winter-spring area for the reproductive phase of these blue whales. The baseline corticosterone levels we are developing will be useful for assessing the impact of the increasing coastal development and whale-watching activities on the whales in the Gulf of California.

Annual ovarian activity monitored by the noninvasive measurement of fecal concentrations of progesterone and 17β-estradiol metabolites in rusa deer (Rusa timorensis).

To clarify the reproductive cycle of female Rusa deer (Rusa timorensis), the fecal concentrations of progesterone and 17β-estradiol metabolites were measured. Fecal samples were collected on a weekly basis for one year (between October, 2012 and September, 2013) from five healthy adult hinds in Thailand. At the beginning of the study, three hinds were pregnant. Two hinds delivered one healthy offspring, and one hind delivered a stillborn calf. The mating period of Rusa hinds in Thailand is from November to April. In pregnant hinds, fecal progesterone metabolite concentration was high in late pregnancy and abruptly declined to the baseline around parturition, suggesting that the placenta secretes a large amount of progesterone. Fecal 17β-estradiol metabolite concentration remained elevated around the day of parturition. Both concentrations of fecal progesterone and 17β-estradiol metabolites in non-lactating hinds were significantly higher than those in lactating hinds, indicating that ovarian activity of lactating hinds is suppressed by the suckling stimulus of fawn during lactation. The present study demonstrated that monitoring of fecal steroid hormones is useful method for assessing ovarian function in this species.

Fecal progestin concentrations as an indicator of reproductive success in American Mink

Efforts to increase mink reproductive success (live births and litter sizes) can be partly assessed by measurement of blood progesterone levels. However, the stress of blood sampling increases the incidence of failed matings, aborted fetuses and death of the dam. We have therefore non-invasively measured fecal progesterone metabolite (progestin) concentrations during the reproductive cycle of mink. We tested the hypothesis that fecal progestin concentrations during the window of implantation (late March–early April) will, (1): be higher for whelping than non-whelping mink, and (2): be higher for mink mated multiple times, compared to single matings. Mink were mated once (March 3), twice (March 3 and 10) or three times (March 3, 10 and 11) and fecal progestin concentrations determined from March 1 to April 30. The percent mink in each group giving birth to live offspring was 42.8%, 80.8% and 92.3% for mink mated once, twice or three times, respectively (P<0.05). Litter sizes did not differ among mink mated once (5.22±0.55), twice (6.29±0.35) or three times (6.08±0.32; P>0.05). Mean fecal progestin concentrations from mating to diapause (March 19) did not differ between mink that whelped or not, nor in response to the number of times mated. However, mean fecal progestin concentrations for mink that whelped were higher on March 25 (peri-implantation) than March 19 after being mated once (51.96±2.96 vs 23.53±1.89nM/g dry wt; P<0.05), twice (66.00±1.60 vs 25.57±1.28nM/g dry wt; P<0.05) or three times (66.48±1.42/g vs 19.16±1.09nM/g dry wt; P<0.05). During implantation (April 5), mean fecal progestin concentrations for mink that whelped after being mated once (146.60±10.02nM/g dry wt), twice (162.10±5.64nM/g dry wt) or three times (188.50±3.92nM/g dry wt) were significantly higher than for those that failed to whelp; 119.30±8.87nM/g dry wt, 77.84±5.86nM/g dry wt. and 118.9±6.55nM/g dry wt., respectively (P<0.05). Our findings suggest that measurement of fecal progestin concentrations during blastocyst reactivation and implantation may be a useful indicator of successful pregnancies in mink.

Reproducibility of an assay to measure serum progesterone metabolites that may be related to breast cancer risk using liquid chromatography-tandem mass spectrometry.

Recent data suggest a novel role of progesterone in breast cancer etiology involving the progesterone metabolites 3α-dihydroprogesterone (3αHP), 5α-dihydroprogesterone (5αP), and 20α-dihydroprogesterone (20αHP). Accurate and precise measures of progesterone metabolites are needed for etiologic studies of hormonally related cancers. We have developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to measure five hormones, including progesterone, its precursor pregnenolone, and three progesterone metabolites, 5αP, 3αHP, and 20αHP. Hormone levels were measured in serum from 20 healthy volunteers (7 men, 5 premenopausal women, and 8 postmenopausal women). Two blinded, randomized aliquots per individual were assayed in each of four batches. The coefficients of variation (CV) and intraclass correlation coefficients (ICC) were calculated from the individual components of variance. The overall laboratory CVs were <3% and ICCs were uniformly high (>98%) for all hormones measured across sex/menopausal status groups. Our HPLC-MS/MS assay of progesterone metabolites demonstrated excellent sensitivity, laboratory reproducibility, and interindividual variation, suggesting that this serum assay is suitable for epidemiologic research. The high sensitivity of the assay, and thus the ability to quantify concentrations among postmenopausal women and men, further supports that this novel assay is suitable for studies of serum progesterone metabolite concentrations and risk of breast cancer or other hormonally related cancer.

Non-invasive endocrine monitoring of ovarian and adrenal activity in chinchilla (Chinchilla lanigera) females during pregnancy, parturition and early post-partum period

The chinchilla is a rodent that bears one of the finest and most valuable pelts in the world. The wild counterpart is, however, almost extinct because of a drastic past and ongoing population decline. The present work was developed to increase our knowledge of the reproductive physiology of pregnancy and post-partum estrus in the chinchilla, characterizing the endocrine patterns of urinary progesterone, estradiol, LH and cortisol metabolites throughout gestation and post-partum estrus and estimating the ovulation timing at post-partum estrus.Longitudinal urine samples were collected once per week throughout pregnancy and analyzed for creatinine, cortisol, LH, estrogen and progesterone metabolite concentrations. To indirectly determine the ovulation timing at post-partum estrus, a second experiment was performed using pregnant females subjected to a post-partum in vivo fertilization scheme. Urinary progestagen metabolites increased above baseline levels in early pregnancy between weeks-8 and -11 respectively to parturition, and slightly declined at parturition time. Urinary estrogens showed rising levels throughout mid- and late pregnancy (weeks-9 to -6 and a further increase at week-5 to parturition) and decreased in a stepwise manner after parturition, returning to baseline levels two weeks thereafter. Cortisol metabolite levels were relatively constant throughout pregnancy with a tendency for higher levels in the last third of gestation and after the pups’ birth. Parturition was associated with dramatic reductions in urinary concentrations of sex steroids (especially progestagens). Observations in breeding farms indicated that the females that resulted in a second pregnancy after mating, did so on the second day after parturition. These data were in agreement with an LH peak detected 24h after parturition. Urinary steroid hormone patterns of estrogen and progestagen metabolites provided valuable information on endocrine events during pregnancy and after parturition in the chinchilla. Results presented in this study enhance our understanding of natural reproductive dynamics in the chinchilla and support empirical observations of breeders that post-partum ovulation occurs ∼48h after parturition.

Differences in ovarian hormones in relation to parity and time since last birth

To examine ovarian function in relation to parity and time since last birth. Cross-sectional study. Health-care program in California. 346 naturally cycling women, aged 18 to 39 years. None. Mean follicular urinary estradiol metabolite concentration (E1C) (cycle days -8 to -1), mean luteal progesterone metabolite concentration (PdG) (days 0 to +10), and cycle phase lengths in ovulatory cycles. After the women had collected daily urine samples for up to eight menstrual cycles, we measured the E1C and PdG using enzyme-linked immunoassay. The cycle phase lengths were calculated from the hormone profiles and daily diaries. Women who had given birth within the previous 3 years had lower E1C than the nulliparous women and women who last given birth >3 years earlier. Among the parous women, E1C was positively associated with the time since last birth. Women who last gave birth >3 years earlier had longer follicular phases than the nulliparous women. There were no associations between parity and PdG or luteal phase length. Our cross-sectional data suggest that ovarian function differs in nulliparous and parous women and is positively associated with the time since last birth. Longitudinal research is needed to explore within-woman changes in ovarian function prepartum and postpartum.