Luteal Phase Concentrations Research Articles

Progesterone profiles in postpartum dairy cows with inflammatory disorders

The objective of this prospective cohort study was to determine if progesterone (P4) profiles differed between dairy cows with or without inflammatory disorders early postpartum. A total of 708 cows from 2 commercial herds were enrolled 3 wk before parturition and examined for clinical health disorders (difficult calving, retained placenta, metritis, displaced abomasum, mastitis, or lameness) until 5 wk postpartum. Serum haptoglobin (Hp) was measured in blood at 2 and 6 DIM (range ±2 d); metritis was assessed at 4, 8, 11, and 15 DIM; and purulent vaginal discharge and endometritis (≥6% PMN in endometrial cytology sampled by cytobrush) were assessed at 35 ± 3 DIM. As Hp ≥0.8 g/L or endometritis were associated with ovarian dysfunction in previous studies, cows with serum Hp ≥0.8 g/L at either time point and endometritis, regardless of clinical disease, were classified as the cohort with inflammatory disorders (INFLAM; n = 139). Clinically healthy cows without difficult calving, with singleton birth, with Hp <0.8 g/L at both sampling times, without endometritis or purulent vaginal discharge, and BCS ≥3.00 (1 to 5 scale) were classified as healthy (n = 133). Cows with only one of the 2 conditions (high Hp or endometritis) were excluded. Cohorts had serum P4 measured twice weekly from 35 to 70 DIM (±3 d), and the first detected luteal phase (LP) during the sampling period was defined as the period from onset of luteal activity (P4 increase to ≥1 ng/mL) until decline of P4 to <1 ng/mL. The odds of prolonged LP (≥21 d long), average LP length, peak P4, and time to P4 decline (hazard rate) were analyzed using multivariable mixed logistic, linear, or Cox proportional hazard regression models including INFLAM status, parity, sampling day (when applicable), and herd as a random effect considering the covariates of season, milk yield at first DHIA test, and DIM at onset of cyclicity or LP length (when applicable). Cows with INFLAM had greater odds of prolonged LP (LSM ± SEM; 67% vs. 37% ± 7%), greater average LP length (17 vs. 15 ± 2 d), lesser P4 at d 4 (4.6 vs. 5.5 ± 0.3 ng/mL) and d 7 (6.0 vs. 7.7 ± 0.3 ng/mL) of the LP, and lesser peak P4 (6.9 vs. 8.2 ± 0.3 ng/mL) during the LP than healthy cows. Status of INFLAM was associated with time to P4 decline in multiparous but not primiparous cows; the LP of INFLAM multiparous cows was less likely to have luteolysis (P4 decline) by d 14 (adjusted hazard ratio [AHR] and 95% CI: 0.54; 0.31 to 0.94) or by d 21 (AHR: 0.32; 0.12 to 0.84) than in healthy multiparous cows. In conclusion, postpartum cows with markers of systemic inflammation at wk 1 and uterine inflammation at wk 5 had altered luteal function (prolonged LP and lower P4 concentrations) before first breeding, which is a possible pathway linking postpartum health disorders and reduced fertility.

Utilising routine non-invasive faecal samples for the detection of oestrus and early gestation in okapi (Okapi johnstoni)

Zoos play a vital role in conservation with an overall aim of preserving species, such as the okapi (Okapi johnstoni), from extinction. To ensure species survival, sustainable ex-situ populations are required, and therefore understanding the reproductive status of individuals is important for population management. In this study, the oestrous cycles and gestations of four okapi (n = 7 pregnancies) were monitored through non-invasive faecal sampling methods to quantify progestagen and oestrogen metabolites. Baseline criteria for determining oestrous cycles was developed using approximately 20 months of sampling from each of two non-pregnant females, before being applied to pre- and early conception data. Pre-conception oestrous cycles were characterised in two females with luteal phase (mean ± SD) was 8.47 ± 1.41 days (n = 59) with an inter-luteal phase of 7.38 ± 1.67 days (n = 58), and overall average cycle length of 15.83 ± 2.02 days (n = 54 full cycles only). Observed in all four females, oestrogen metabolite peaks occurred at the beginning of the inter-luteal phase as progestagen metabolites were decreasing. Gestation was confirmed when progestagen metabolite concentrations remained at luteal phase concentrations for at least 20 days, with concentrations remaining at this level for the first 120 days of gestation without return to baseline values. A secondary increase at around 150 days lasted until parturition. These data demonstrate that okapi exhibit similar oestrous cycles to other members of the giraffidae family, with faecal oestrogens for the first time indicating that ovulation occurs at the beginning of the inter-luteal phase while progestagens are still elevated. Furthermore, we have shown that through routine sample collection and hormone monitoring, gestation can be confirmed within the first trimester, which can benefit the management of the species in ex-situ populations.

Form of dietary selenium affects mRNA encoding interferon-stimulated and progesterone-induced genes in the bovine endometrium and conceptus length at maternal recognition of pregnancy.

Widespread regions of the southeast United States have soils, and hence forages, deficient in selenium (Se), necessitating Se supplementation to grazing cattle for optimal immune function, growth, and fertility. We have reported that supplementation with an isomolar 1:1 mix (MIX) of inorganic (ISe) and organic (OSe) forms of Se increases early luteal phase (LP) concentrations of progesterone (P4) above that in cows on ISe or OSe alone. Increased early LP P4 advances embryonic development. Our objective was to determine the effects of the form of Se on the development of the bovine conceptus and the endometrium using targeted real-time PCR (qPCR) on day 17 of gestation, the time of maternal recognition of pregnancy (MRP). Angus-cross yearling heifers underwent 45-d Se-depletion then repletion periods, then at least 90 d of supplementation (TRT) with 35 ppm Se per day as either ISe (n = 10) or MIX (n = 10). Heifers were inseminated to a single sire after detected estrus (day 0). On day 17 of gestation, caruncular (CAR) and intercaruncular (ICAR) endometrial samples and the developing conceptus were recovered from pregnant heifers (ISe, n = 6 and MIX, n = 6). qPCR was performed to determine the relative abundance of targeted transcripts in CAR and ICAR samples, with the expression data subjected to one-way ANOVA to determine TRT effects. The effect of TRT on conceptus development was analyzed using a one-tailed Student's t-test. When compared with ISe-treated heifers, MIX heifers had decreased (P < 0.05) abundance of several P4-induced and interferon-stimulated mRNA transcripts, including IFIT3, ISG15, MX1, OAS2, RSAD2, DGAT2, FGF2 in CAR and DKK1 in ICAR samples and tended (P ≤ 0.10) to have decreased mRNA abundance of IRF1, IRF2, FOXL2, and PGR in CAR samples, and HOXA10 and PAQR7 in ICAR samples. In contrast, MIX-supplemented heifers had increased (P < 0.05) mRNA abundance of MSTN in ICAR samples and an increase in conceptus length (ISe: 17.45 ± 3.08 cm vs. MIX: 25.96 ± 3.95 cm; P = 0.05). Notably, myostatin increases glucose secretion into histotroph and contributes to advanced conceptus development. This advancement in conceptus development occurred in the presence of similar concentrations of serum P4 (P = 0.88) and whole blood Se (P = 0.07) at MRP.

New Classification and Management of Abetalipoproteinemia and Related Disorders

New Classification and Management of Abetalipoproteinemia and Related Disorders

Reproductive cycle and pregnancy monitoring in the common hippopotamus (Hippopotamus amphibius) through salivary steroid analyses and trans-abdominal ultrasonography.

The use of an enzyme immunoassay (EIA) was validated to monitor progestogens in common hippopotamus Hippopotamus amphibius saliva and transabdominal ultrasonography was used to diagnose and monitor pregnancy in this species. Both faecal and saliva extracts contained elevated progestogens indicative of luteal phases and gestation. Faecal and saliva progestogen concentrations of six adult female hippos revealed a strong positive correlation between the two sample types (r=0.60–0.85). Salivary and faecal progestogen analysis revealed a cycle length of 31.8±6.9 days and 33.4±2.9 days and average non-pregnant luteal phase duration of 17.3±1.5 days and 14.0±1.5 days, respectively. Progestogen concentrations (faecal and saliva samples collected ~days 0 to 90) from a full-term pregnancy (saliva: 1,167.1±1,269.4 pg/ml; faecal: 2,812.4±1,657.5 ng/g) were higher on average than luteal phase concentrations (saliva: 335.0±358.1 pg/ml; faecal: 1,399.5±613.0 ng/g). Comparatively, progestogen concentrations (saliva samples collected days 0 to 181; faecal samples collected ~days 120 to 181) of a pre-term (premature live birth ~181 days) pregnancy (saliva: 244.3±155.2 pg/ml; faecal: 501.7±492.2 ng/g) were on average lower than concentrations observed during the full-term pregnancy and/or luteal phase. The nulliparous female that gave birth pre-maturely was trained for voluntary transabdominal ultrasound exams conducted weekly. Intrauterine fluid and foetal tissue were observed 79 days following last confirmed mating. Foetal spine, rib cage, beating heart and internal organs were visible at 156 days. In summary, salivary progestogen monitoring and transabdominal ultrasonography appear suitable for tracking reproductive activity and diagnosing and monitoring pregnancy in the common hippo.

Form of Supplemental Selenium in Vitamin-Mineral Premixes Differentially Affects Early Luteal and Gestational Concentrations of Progesterone, and Postpartum Concentrations of Prolactin in Beef Cows.

Simple SummarySoils with inadequate levels of selenium are widespread in the northwest, northeast, and southeast USA. Therefore, dietary supplementation of forage-grazing beef cattle with additional selenium is recommended in these regions for optimal growth, immune function, and fertility. We have reported that the form of selenium provided to Angus-cross cows can affect circulating concentrations of progesterone on day 6 of the estrous cycle, a time when increased progesterone is known to promote fertility. Hence, we sought to confirm this initial finding, determine the effect of the form of selenium on peripheral concentrations of progesterone during gestation, and determine the effect of the form of selenium on circulating concentrations of prolactin during lactation. Cows were supplemented with equimolar amounts of either an inorganic form, or a 1:1 mixture of inorganic and organic forms of selenium throughout this study. We confirmed our original finding that the mixed (1:1 inorganic to organic selenium) supplement increased systemic progesterone in the early luteal phase of the estrous cycle, and determined that cows maintained on this same supplement had elevated concentrations of progesterone throughout gestation. Interestingly, these same cows revealed a treatment-induced decrease in systemic prolactin during late lactation. The form of selenium provided to cows can be manipulated to affect reproductive responses and offers a viable management tool to improve fertility in cows in regions with selenium-deficient soils.Soils with marginal to deficient levels of selenium (Se) are widespread in the northwest, northeast, and southeast US. Supplementation to the diet of forage-grazing beef cattle with a vitamin-mineral mix containing additional Se is recommended in these geographic regions. We have reported that the form of supplemental Se provided to Angus-cross beef cows can affect circulating levels of progesterone (P4) on day 6 of the estrous cycle, a time when increased P4 is known to promote fertility. The objectives of this study were to (1) confirm and expand upon our initial report that the form of Se provided to cows affects early luteal-phase concentrations of systemic P4, (2) determine the effects of the form of Se on concentrations of P4 during gestation, and (3) determine the effects of the form of Se on concentrations of prolactin (PRL) during lactation. Throughout this study, Angus-cross beef cows had ad libitum access to a vitamin-mineral mix containing 35 ppm of Se in either an inorganic form (ISe) or a 1:1 mix of inorganic and organic forms (MIX). We observed a MIX-induced increase (p = 0.006) in systemic concentrations of P4 on day 7 but not on days 4 or 10 of the estrous cycle, consistent with our earlier report. We observed a MIX-induced increase (p = 0.02) in the systemic concentration of P4 at months 1, 3, 5, and 7 of gestation, and a MIX-induced decrease (p < 0.05) in systemic concentrations of PRL at months 5 and 6 of lactation. In summary, the form of Se provided to cows can be manipulated to affect the early luteal phase and gestational concentrations of P4, and postpartum concentrations of PRL.

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.

Plasma steroid profiles and ovarian response in llamas treated with eCG for superovulation combined with exogenous progesterone during early luteal phase

The objective of this study was to evaluate the effects of plasma progesterone (P4) concentrations during eCG-ovarian follicular superstimulatory treatment performed in early luteal phase and estradiol concentrations during peri-ovulatory period on ovarian response, number and embryo quality. On Day −2, females (n = 75) having a follicle ≥7 mm were treated with GnRH to induce ovulation. On Day 0, females that had ovulations (n = 54) were treated with 1000 IU eCG and were assigned to one of two treatments: (1) intravaginal device (ID) containing 0.5 g P4 (P4 group) and (2) no ID (Control group). On Day 5, females were administered PGF2α and the ID was removed. On Day 7 and 8, females were mated and embryo recovery was performed 7 or 8 days later. Blood samples were collected from Day 0 to 9. Number (± SD) of follicles ≥7 mm on day of mating was greater (P = 0.04) in the control (9.7 ± 4.2) than P4-treated (6.7 ± 4.9) group; number of corpora lutea did not differ (5.5 ± 3.1 and 5.2 ± 3.4 respectively). Ovulation rate was greater (P < 0.01) in the P4-group (77.4%; 130/168) than control group (53.3%; 135/253). Number of embryos with an excellent grade (grade 1) tended to be greater (P = 0.07) in the P4-group (82.4%; 42/51) than control group (65.4%; 36/55). It was concluded that supplementation with exogenous P4 during eCG treatment in early luteal phase inhibits excessive follicular growth, increases ovulation rate and improves embryo quality.

Reproductive monitoring of collared peccary females (Pecari tajacu) by analysis of fecal progesterone metabolites

The measurement of fecal progesterone metabolites (fPM) by enzyme immunoassay analysis is a non-invasive technique that permits gathering reproductive information from wildlife without the stress associated with restraint. In the collared peccary (Pecari tajacu), a high correlation between serum progesterone and fPM levels (r2 = 0.783) suggests that fPM can be used to monitor their reproductive function. We monitored fPM during the estrous cycle of 15 collared peccary females. Estrous cycles averaged 27.9 ± 4.5 days (n = 28), ranging from 21 to 36 days. The luteal phase was 22.2 ± 4.8 days and the inter-luteal phase was 4.3 ± 1.4 days. Mean concentration of fPM across pregnancy were not different from those found during the luteal phase (1230 ± 718 and 1265 ± 584 ng/100 mg dried feces, respectively), however, significant differences were found when luteal phase concentrations were compared only against fPM concentrations during late pregnancy. In addition, late pregnancy fPM concentrations (1893 ± 551 ng/100 mg) were also significantly higher than those in the early (639 ± 339 ng/100 mg) and mid (1134 ± 449 ng/100 mg) pregnancy. For females during the early post-partum period, fPM concentrations were significantly increased (243 ± 118 ng/100 mg) than those of non-cycling females (103 ± 89 ng/100 mg). The analysis of fPM is a simple, non-invasive methodology to detect the ovarian activity in the collared peccary; moreover, it provides a husbandry tool, which may be used to help understand how social structure may impact reproduction.

Effects of GABA active steroids in the female brain with a focus on the premenstrual dysphoric disorder.

Premenstrual dysphoric disorder (PMDD) afflicts 3%-5% of women of childbearing age, and is characterised by recurrent negative mood symptoms (eg, irritability, depression, anxiety and emotional lability) during the luteal phase of the menstrual cycle. The aetiology of PMDD is unknown, although a temporal association with circulating ovarian steroids, in particular progesterone and its metabolite allopregnanolone, has been established during the luteal phase. Allopregnanolone is a positive modulator of the GABAA receptor: it is sedative in high concentrations but may precipitate paradoxical adverse effects on mood at levels corresponding to luteal phase concentrations in susceptible women. Saccadic eye velocity (SEV) is a measure of GABAA receptor sensitivity; in experimental studies of healthy women, i.v. allopregnanolone decreases SEV. Women with PMDD display an altered sensitivity to an i.v. injection of allopregnanolone compared to healthy controls in this model. In functional magnetic resonance imaging (fMRI) studies, women with PMDD react differently to emotional stimuli in contrast to controls. A consistent finding in PMDD patients is increased amygdala reactivity during the luteal phase. Post-mortem studies in humans have revealed that allopregnanolone concentrations vary across different brain regions, although mean levels in the brain also reflect variations in peripheral serum concentrations. The amygdala processes emotions such as anxiety and aggression. This is interesting because allopregnanolone is detected at high concentrations within the region into which marked increases in blood flow are measured with fMRI following progesterone/allopregnanolone administration. Allopregnanolone effects are antagonised by its isomer isoallopregnanolone (UC1010), which significantly reduces negative mood symptoms in women with PMDD when administered s.c. in the premenstrual phase. This was shown in a randomised, placebo-controlled clinical trial in which the primary outcome was change in symptom scoring on the Daily Rating of Severity of Problems (DRSP): the treatment reduced negative mood scores (P<.005), as well as total DRSP scores (P<.01), compared to placebo in women with PMDD. In conclusion, the underlying studies of this review provide evidence that allopregnanolone is the provoking factor behind the negative mood symptoms in PMDD and that isoallopregnanolone could ameliorate the symptoms as a result of its ability to antagonise the allopregnanolone effect on the GABAA receptor.

Follicular flushing in natural cycle IVF does not affect the luteal phase – a prospective controlled study

In contrast to multifollicular IVF, follicular flushing seems to increase the efficacy of monofollicular IVF treatments such as natural cycle IVF (NC-IVF). However, because follicular flushing causes loss of granulosa cells, it might negatively affect luteal phase length and endocrine function of the luteal body. A prospective cohort Phase II study was performed in 24 women undergoing NC-IVF. Women underwent a reference cycle with human chorionic gonadotrophin-induced ovulation without follicle aspiration and analysis of the length of the luteal phase and luteal concentrations of progesterone and oestradiol. In addition, they underwent a NC-IVF cycle which was performed identically but follicles were aspirated and flushed three times. The luteal phase was shorter in 29.2%, equal in 16.7% and longer in 50.0% of cases following flushing of the follicles. Overall, neither difference in luteal phase length was significant [median duration (interquartile range) in reference cycle: 13 (12; 14.5), IVF (flushing) cycle: 14 (12.5; 14.5), median difference (95% CI): 0.5 (–0.5 to 1.5)] nor median progesterone and oestradiol concentrations. In conclusion, follicular flushing in NC-IVF affects neither the length of the luteal phase nor the luteal phase concentrations of progesterone and oestradiol, questioning the need for luteal phase supplementation.

Characterization and longitudinal monitoring of serum progestagens and estrogens during normal pregnancy in the killer whale (Orcinus orca)

The secretory patterns of progestagens and estrogens were characterized throughout 28 normal pregnancies until two month post-partum in eleven killer whales. HPLC analysis of serum from different reproductive stages (luteal phase, EARLY, MID, and LATE pregnancy) identified three major immunoreactive progestagen peaks; progesterone (P4), 5α-pregnane-3,20-dione (5α-DHP) and pregnanediol, with 5α-DHP approximately half of that for P4 in the luteal phase, and EARLY, but approximately 2/3 of P4 during MID and LATE pregnancy. At birth, 5α-DHP was the only significant (>10% immunoreactivity) immunoreactive progestagen detected in placental (umbilical cord) serum. Maternal recognition of pregnancy appears to occur between day 21 and 28 post-ovulation when a significant deviation in progestagen concentrations between conceptive and non-conceptive cycles was detected. Progestagen concentrations during pregnancy displayed a bimodal pattern with significant peaks (P<0.05) in EARLY (indexed month post-conception [IMPC] 2, 3, 4) and MID (IMPC 9, 10) before decreasing (P<0.05) over an 11day interval to luteal phase concentrations on the day of parturition. Among estrogens, estriol was secreted in the highest concentrations but only estrone (free and conjugated) and estradiol increased (P<0.001) during pregnancy, with peaks observed during the final month of gestation, and an influence (P<0.05) of fetal sex on estradiol production was detected. Collective findings indicate that P4 derived from the corpus luteum is the major biologically active progestagen during the luteal phase and pregnancy, and that 5α-DHP production, possibly from both luteal and placental sources, increases during the second half of pregnancy.

Concentrations of steroid hormones, estrous, ovarian and reproductive responses in sheep estrous synchronized with different prostaglandin-based protocols

To determine estrous, ovarian and reproductive responses after different prostaglandin (PG)-based protocols, ewes were assigned to groups PG10, PG12, PG14 or PG16 (twoPG injections administered 10, 12, 14 or 16days apart; respectively). Experiment I (n=132) was conducted to evaluate the estrous response, ovulation rate (OR), conception and fertility. Experiment II (n=24) was conducted to evaluate ovarian follicle growth, steroid concentrations and the interval from the second PG injection to estrus (PG-estrus) and ovulation (PG-ovulation). Estrous response was less with the PG16 (P<0.05) treatment, and the extent of estrous synchrony was greater with the PG10 and PG12 treatments. Ovarian follicle growth and the intervals for the variables PG-estrus, PG-ovulation and OR were similar among groups (P>0.05). From 8 to 4 days before estrus, progesterone (P4) concentrations were greater for the PG14 and PG16 than for the PG10 and PG12 (P<0.05) groups. There were more days where concentrations of P4 were above 3.18nmol/L with the PG14 and PG16 than PG10 and PG12 (P<0.05) treatments. Use of the PG14 and PG16 treatments resulted in greater estradiol (E2) at estrus and 12h later than use of the PG10 and PG12 treatments. A positive correlation was observed between the duration of the luteal phase and maximum E2 concentrations, and between duration of the luteal phase and days with E2 concentrations above 10pmol/L. Conception and fertility were greater with use of the PG14 compared with PG10 and PG12 (P<0.05) treatments. The administration of two PG injections 10, 12, 14 or 16 days apart resulted in different durations of the luteal phase that were positively associated with E2 concentrations and the reproductive outcome. The shorter luteal phases were associated with greater synchrony in time of estrus. The intervals for the variables PG-estrus, PG-ovulation and OR were similar among groups.

Abstract P6-07-07: Low dose of CDB-2914 and CDB-4124 efficiently inhibit the growth of T47D spheroid induced by pre-menopausal and post-menopausal concentrations of estrogen and progesterone

Abstract Background: The anti-progestins (RU-486, CDB-2914 and CDB-4124) may have potential to prevent estrogen receptor (ER) and progesterone receptor (PR) positive breast cancer. Physiological estradiol (E2), and progesterone (P4) levels are different in pre- and post-menopausal women. The purpose of this study was to determine: 1) Whether the physiological female hormones at pre- and postmenopausal concentrations affect the growth of an in vitro model of breast cancer, i.e., T47D spheroids, and 2) Whether anti-progestins at pharmacological concentrations work as growth inhibitors in high and low hormonal environments. Methods: T47D cells were grown as spheroids in the presence of serum concentrations of E2 and P4 consistent with pre- and postmenopause. Premenopausal luteal phase concentrations were 262 pM (71.3 pg/mL) of E2 + 18 nM (5.66 ng/mL) of P4; postmenopausal concentrations were 122 pM (33.2 pg/mL) of E2 + 3 nM (1.08 ng/mL) of P4. T47D cells (5000 per well) were seeded in 1.5% agarose coated 96 well plates, and grown in phenol red-free mammary epithelial cell growth basal medium (Lonza) supplemented with 10% double charcoal -stripped FBS. Hormones and anti-progestin treatments started 24 hrs after cell seeding. Three concentrations (50, 250, 1000 nM) of the anti-progestins were tested. Images of each spheroid were taken daily for 14 days, and the sizes of spheroids were analyzed by area (Pixel) using ImageJ software. Results: Results are presented in Table 1. Table 1. Relative spheroid growth compared to the hormone treatment aloneHormone treatmentsDrug treatments (nM)RU-486CDB-2914CDB-4124Premenopausal condition+0111+500.80.560.7+2500.720.630.68+10000.840.640.6Postmenopausal condition+0111+500.950.761+2500.90.830.74+10000.960.850.71 Premenopausal and postmenopausal hormone concentrations stimulated spheroid growth by two- fold and 1.7 fold higher, respectively, when compared to the vehicle control (0.1% DMSO) at14 days. In the premenopausal condition, RU-486 showed moderate inhibition of spheroid growth at all three concentrations; CDB-2914 was more efficient at inhibition than CDB-4124 at 50 nM. At 250 nM and 1000 nM, CDB-2914 and CDB-4124 showed similar efficacy. In the postmenopausal condition, RU-486 showed very minor inhibition on spheroid growth at all three concentrations; CDB-2914 showed significantly higher inhibition than CDB-4124 at 50 nM. At 250 nM and 1000 nM, CDB-4124 was more efficient than CDB-2914. Conclusions: Our results indicate that T47D spheroids will grow in postmenopausal hormone concentrations, but growth is enhanced in premenopausal hormone conditions. RU-486 did not produce effective inhibition at postmenopausal hormone levels; however pharmacological concentrations (50, 250 and 1000 nM) of both CDB-2914 and CDB-4124 efficiently decrease the spheroid growth induced by both premenopausal and postmenopausal hormone levels. These data suggest that low doses of CDB-2914 and CDB-4124 should be further investigated for ER/PR positive breast cancer prevention and therapy. Citation Format: Oukseub Lee, Mi Ran Choi, Susan E Clare, Seema A Khan. Low dose of CDB-2914 and CDB-4124 efficiently inhibit the growth of T47D spheroid induced by pre-menopausal and post-menopausal concentrations of estrogen and progesterone [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-07.

Evidence that Orphanin FQ Mediates Progesterone Negative Feedback in the Ewe

Orphanin FQ (OFQ), a member of the opioid family, is found in many areas of the hypothalamus and, when given centrally OFQ inhibits episodic LH secretion in rodents and sheep. Because GnRH neurons are devoid of the appropriate receptors to mediate steroid negative feedback directly, neurons that release OFQ may be involved. Using immunocytochemistry, we first determined that most OFQ neurons in the arcuate nucleus (ARC) and other hypothalamic regions of luteal phase ewes contained both estrogen receptor α and progesterone (P) receptor. Given a similar high degree of steroid receptor colocalization in other ARC subpopulations, we examined whether OFQ neurons of the ARC contained those other neuropeptides and neurotransmitters. OFQ did not colocalize with kisspeptin, tyrosine hydroxylase, or agouti-related peptide, but all ARC OFQ neurons coexpressed proopiomelanocortin. To test for a role for endogenous OFQ, we examined the effects of an OFQ receptor antagonist, [Nphe1,Arg14,Lys15]Nociceptin-NH2 (UFP-101) (30 nmol intracerebroventricular/h), on LH secretion in steroid-treated ewes in the breeding season and ovary-intact ewes in anestrus. Ovariectomized ewes with luteal phase concentrations of P and estradiol showed a significant increase in LH pulse frequency during infusion of UFP-101 (4.5 ± 0.5 pulses/6 h) compared with saline infusion (2.6 ± 0.4 pulses/6 h), whereas ewes implanted with only estradiol did not. Ovary-intact anestrous ewes displayed no significant differences in LH pulse amplitude or frequency during infusion of UFP-101. Therefore, we conclude that OFQ mediates, at least in part, the negative feedback action of P on GnRH/LH pulse frequency in sheep.

Fecal estrogen, progestagen and glucocorticoid metabolites during the estrous cycle and pregnancy in the giant anteater (Myrmecophaga tridactyla): evidence for delayed implantation

BackgroundDeclining numbers of wild giant anteaters highlight the importance of sustainable captive populations. Unfortunately, captive reproductive management is limited by the lack of external physical indicators of female reproductive status and the aggressive behavior of males. We examined the endocrinology of the estrous cycle and pregnancy, and whether delayed implantation is a gestational strategy for giant anteaters as described for other xenarthrans.MethodsFeces were collected from seven captive females 3–5 times weekly and mating was recorded. Concentrations of estrogen (estrone–glucuronide, E1, and estradiol–17β, E2), progestagen (20–oxo–progestagens, P4), and glucocorticoid (GC) metabolites were examined in fecal extracts by enzyme immunoassay.ResultsEstrous cycles for nulliparous females (6 cycles, n = 2) compared to the multiparous female (6 cycles, n = 1) were shorter (47.3 +/− 4.3 days versus 62.5 +/− 2.6 days) with relatively lower luteal phase concentrations of P4 (49.4 +/− 2.9 ng/g versus 136.8 +/− 1.8 ng/g). The four remaining females had unclear ovarian activity: two females exhibited apparent luteal activity but unclear fluctuations in estrogens, while the other two females had parallel fecal P4 and estrogens concentrations. Pregnancy ranged 171–183 days with females returning to estrus post–partum as early as 60 days (n = 3, 1.8-4 years of age at mating). Delayed implantation was indicated by a biphasic elevation in fecal P4 metabolites: the initial 4–fold increase occurred for 81–105 days and was followed by a 26–fold secondary rise in P4 metabolites lasting 66–94 days prior to parturition. Fecal GC was correlated with fecal estrogens and greatest during estrus, late pregnancy, and six days prior to parturition (estrous cycle GC, 14.4-62.8 ng/g; pregnancy GC, 13.6-232.7 ng/g).ConclusionsEstrous cycles of giant anteaters occurred year–round, but were shorter and more intermittent in younger nulliparous animals compared to a multiparous female. A pronounced elevation in fecal P4, estrogen, and GC occurred during late gestation after an initial post-mating delay providing evidence for delayed implantation. Adrenocorticoid activity indicated impending parturition. Differences in estrous cycle characteristics with age and the protracted but variable gestation length must be considered to improve reproductive success and neonatal survival in giant anteaters.

Influence of the length of proestrus on fertility and endocrine function in female cattle

Previous research from our laboratory in beef cattle suggests that ovarian follicle maturity and subsequent fertility is influenced by length of proestrus across a range of follicle sizes. To test this hypothesis an animal model was used in which ovulation from similar sized follicles was induced following either a long (LPE; ∼2.25 days) or short (SPE; ∼1.25 days) proestrus (interval from PGF 2α administration to a GnRH-induced LH surge). Specific objectives were to compare pregnancy rates and luteal phase concentrations of progesterone (Experiment 1) and to characterize preovulatory concentrations of estradiol, the GnRH-induced LH surge, and concentrations of progesterone in the subsequent estrous cycle (Experiment 2) between the LPE and SPE treatments. In Experiment 1, ovulation from follicles that were previously synchronized using follicular aspiration was induced with GnRH (Day 0) after either 2.25 days (LPE; n = 40) or 1.25 days (SPE; n = 38) of proestrus. Lactating and non-lactating cows were inseminated 12 h following GnRH administration. Ovulatory follicle diameter was similar between treatments. Pregnancy rates to AI were greater ( P < 0.01) in the LPE (50.0%) compared to the SPE (2.6%) treatment. The proportion of cows having a short luteal phase in the subsequent estrous cycle was greater ( P < 0.01) in the SPE than LPE treatment. In cows with a luteal phase of normal length, timed-AI pregnancy rates and concentrations of progesterone in the subsequent luteal phase were greater ( P < 0.05) in the LPE than SPE treatment. In Experiment 2, a similar experimental approach was taken with non-lactating beef cows and ovulation was induced following either 2.2 days (LPE; n = 8) or 1.2 days (SPE; n = 8) of proestrus. Ovulatory follicle diameter was similar between treatments. Concentrations of estradiol during the proestrus period were greater ( P < 0.05) in the LPE than SPE treatment from Days −1.9 to Day 0 (GnRH administration). Concentration of LH during the GnRH-induced LH surge and concentrations of progesterone in the subsequent estrous cycle did not differ between treatments although there was a tendency ( P = 0.10) for increased incidence of short luteal phases in the SPE treatment. In conclusion, decreasing the length of proestrus before induction of ovulation of a large follicle resulted in lesser pregnancy rates and an increased incidence of short luteal phases. The impact of a shortened proestrus on concentrations of progesterone in cows with luteal phases of normal length varied among experiments. Decreased circulating concentrations of estradiol during the preovulatory period and/or reduced concentrations of progesterone during the subsequent estrous cycle in cows that do not experience a shortened luteal phase may represent the mechanism responsible for reduced fertility with the SPE treatment.

162 REPRODUCTIVE CYCLES, PREGNANCY AND REVERSAL OF LONG TERM ACYCLICITY IN CAPTIVE SOUTHERN WHITE RHINOCEROS AT HAMILTON ZOO

In 1999, the New Zealand captive population of Southern white rhinoceros (Ceratotherium simum simum) was increased with the addition of six wild-caught founders. We report on the breeding success of two females (est. 5 to 6 years at import) and a female born in 2000 (conceived in the wild). Fecal samples were collected 1 to 3 times per week from females (n = 3) for several periods (5 to 24 months) between 2000 and 2008. Reproductive cycles and pregnancy were characterized using fecal progestogen concentrations, observations of courtship behaviour and parturition dates. The average length of the reproductive cycle was 31.6 ± 0.6 days (range 26–38 days, n = 34 cycles). Four pregnancies were confirmed in one female with a sex ratio of 1 female to 3 males (including 1 male loss at an estimated 5 months of gestation), known gestation lengths were 514 and 507 days. In the non-reproducing adult female, long cycles (67.2 ± 1.3 days, n = 5) were detected in addition to regular cycles (31.9 ± 0.9 days, n = 13) during the first 3 years in captivity, consistent with previous reports (Schwarzenberger F et al. 1998 An. Repro. Sci. 53, 173–190; Brown JL et al. 2001 Zoo Biol. 20, 463–486). The absence of breeding behavior and low progestogen concentrations for an extended 4-year period was cause for concern for the Australasian Species Management Programme managers (Hermes R et al. 2004 An. Repro. Sci. 82–83, 49–60; Hermes R et al. 2006 Theriogenology 65, 1492–1515). In 2007, the social structure of the Hamilton Zoo rhinoceros group was altered by exchanging the breeding male and two male offspring for a wild-caught male from Auckland Zoo. Within two months of arrival the long-term acyclic female had resumed cycling and had conceived six months after introduction of the new male. The young captive-born female continued to have regular length cycles and mating was observed with the new male. Recently, early embryonic loss was determined by ultrasound in the young female having a long 70 day cycle determined by fecal analysis. Fecal progestogen concentrations were useful for diagnosing pregnancy after 3 months gestation with concentrations four times higher than luteal phase concentrations by 9 to 12 weeks after mating (n = 3). Two females exhibited seasonal acyclic periods characterized by low progestogen concentrations corresponding to decreasing daylength (autumn and winter months). Fecal progestogen monitoring of reproductive status and pregnancy combined with behaviour observations has provided valuable information for breeding management decisions and planning for parturition in this population. The reversal of long-term acyclicity and a resulting pregnancy that represents two original founders is particularly significant for the genetic diversity of the Australasian population of white rhinoceros.

Sexual Differentiation of the LH Surge Response to Estradiol in Sheep Treated Prenatally with Testosterone Plus an Antiandrogen.

The steroid feedback mechanisms regulating GnRH secretion are sexually differentiated by testicular steroids during prenatal development. In sheep, prenatal testosterone (T) treatment of developing females results in disruption of the GnRH surge response to estradiol (E) positive feedback. Defeminization of the surge mechanism is considered to be defeminized by the estrogenic metabolites of testosterone because the surge mechanism is functional in females treated prenatally with the non-aromatizable androgen, dihydrotestosterone (DHT). However, it is difficult to confirm this premise by studying the organizing actions of prenatal estrogens because of the disruptive effects of exogenous estrogens on the maternal uterus. We developed an approach to study the consequences of excess estrogens in the developing female by co-administering T and the antiandrogen flutamide, and used this approach to test the hypothesis that prenatal estrogens defeminize the GnRH surge response to E. We predicted that the surge would be defeminized by prenatal T and T+flutamide treatments, but not by prenatal DHT. Lambs born to untreated females or females treated with T, DHT, or T+flutamide from Days 30-90 of gestation were neonatally gonadectomized, and steroid levels were standardized with a chronic E implant (30-mm sc). At 8 months of age, the chronic implants were removed and females and T+flutamide males were treated with new implants to maintain luteal phase concentrations of progesterone (P) and E for 9 days. After 9 days, P implants were removed and additional E implants (4 x 30-mm) were inserted to maintain follicular phase concentrations of E. Plasma concentrations of LH were measured in venous blood samples collected every 2 hours from -4 to 72 h from E administration. An LH surge was produced in all untreated, control (C, n=6) and DHT-treated females (n=7), 1 of 9 T-treated females, 4 of 6 T+flutamide-treated females, and 3 of 7 T+flutamide males. The onset of the surge relative to E administration did not differ between C (18.7 ± 1.6 h) and DHT (16.9 ± 0.9 h) females, but it was delayed (p < 0.02) in the T+flutamide groups (females: 40.5 ± 6.9 h; males: 66.7 ± 1.8 h). The peak amplitude of the LH surge was not different between C (175.9 ± 13.7 ng/ml) and DHT (209.6 ± 18.0 ng/ml) females, but was lower in T+flutamide females (84.2 ± 15.3 ng/ml) and males (48.0 ± 14.9 ng/ml). The one LH surge produced by a T-treated female had an onset of 44 h and a peak amplitude of 30.3 ng/ml. The LH surge response in some of the T+flutamide females and males contradicts our prediction, and fails to support our hypothesis that prenatal estrogens, made available to the developing fetus by the aromatization of T, defeminize the positive feedback actions of estradiol. However, the LH surges in the T+flutamide animals were abnormal, particularly in the males. This suggests that endogenous androgens not blocked by flutamide treatment or having organizational actions outside of the treatment period contributed to differentiation of the surge mechanism. Alternatively, individual differences in neural aromatase activity could result in different levels of estradiol exposure during the treatment period. We conclude that prenatal T+flutamide treatment defeminizes the neural circuitry underlying the GnRH surge mechanism, but that both androgens and estrogens may both be necessary for complete ablation of the surge response. Supported by P01 HD-44232.

Differences in Free Estradiol and Sex Hormone-Binding Globulin in Women with and without Premenstrual Dysphoric Disorder

Over the years, different hypotheses involving the ovarian steroid hormones have been proposed to explain the luteal phase occurrence of severe premenstrual syndrome symptoms. Although it had been strongly suspected that differences in the concentrations of the ovarian steroids may underlie the mood and psychological imbalance of this disorder, the evidence for this hypothesis has been inconsistent and remains controversial. Our objective was to measure the ovarian steroid hormones across the menstrual cycle in women with and without luteal phase symptoms consistent with premenstrual dysphoric disorder (PMDD). We measured estradiol (E2), progesterone, and SHBG in women with and without PMDD using a cross-sectional and prospective experimental design. Participating women underwent 2-month self-assessment symptom screening and 1-month hormonal evaluation. Overall means for LH, progesterone, E2, peak E2, and free E2 were not different between groups. Across the menstrual cycle, overall percent free E2 was significantly lower and SHBG significantly greater in the PMDD group compared with controls (1.39 +/- 0.26 vs. 1.50 +/- 0.28, P = 0.03; 61.4 +/- 25.1 vs. 52.4 +/- 21.3 nmol/liter, P = 0.046, respectively). During the luteal phase, free E2 was significantly lower in the PMDD group compared with controls (PMDD 7.6 +/- 7.0 vs. controls 8.9 +/- 8.4 pmol/liter; P = 0.032). For both follicular and luteal phases, SHBG was significantly higher in the PMDD group (follicular phase 60.5 +/- 31.7 vs. 51.4 +/- 38.2 nmol/liter, P = 0.047; luteal phase 65.1 +/- 32.3 vs. 55.1 +/- 38.9 nmol/liter, P =0.03). In both groups, SHBG significantly increased from the follicular to luteal phase. Luteal phase concentrations of free E2, percent free E2, and SHBG differ significantly between women with and without PMDD.