Role of CYP27A1 in progesterone metabolism in vitro and in vivo

In the kidney, progesterone is inactivated to 20alpha-dihydro-progesterone (20alpha-DH-progesterone) to protect the mineralocorticoid receptor from progesterone excess. In an attempt to clone the enzyme with 20alpha-hydroxysteroid activity using expression cloning in CHOP cells and a human kidney expression library, serendipitously cDNA encoding CYP27A1 was isolated. Overexpression of CYP27A1 in CHOP cells decreased progesterone conversion to 20alpha-DH-progesterone in a dose-dependent manner, an effect enhanced by cotransfection with adrenodoxin and adrenodoxin reductase. Incubation of CHOP cells with 27-hydroxycholesterol, a product of CYP27A1, increased the ratio of progesterone/20alpha-DH-progesterone in a concentration-dependent manner, indicating that the effect of CYP27A1 overexpression was mediated by 27-hydroxycholesterol. In order to analyze whether these observations are relevant in vivo, progesterone and 20alpha-DH-progesterone were measured by GC-MS in 24-h urine of CYP27A1 gene knock out (ko) mice and their control wild type (wt) and heterozygote (hz) littermates. In CYP27A1 ko mice, urinary progesterone concentrations were decreased, 20alpha-DH-progesterone increased and the progesterone/20alpha-DH-progesterone ratio decreased threefold (p<0.001). Thus, CYP27A1 modulates progesterone concentrations. The underlying mechanism is inhibition of 20alpha-hydroxysteroid dehydrogenase by 27-hydroxycholesterol. Key words: Progesterone, sterol 27-hydroxylase, 27-hydroxycholesterol, 20a-steroid dehydrogenase, 20a-DH-progesterone.

Effect of Continuous Infusion of Relaxin on Progesterone, Oxytocin, and Relaxin Blood Concentrations and Time of Parturition in Beef Heifers

These studies were designed to determine whether continuous intravenous infusion of increasing dosages of porcine relaxin during late pregnancy in beef heifers would influence circulating blood concentrations of relaxin, progesterone, and oxytocin, and time of onset of parturition. Beef heifers were bred by artificial insemination and, on Day 277, fitted with indwelling jugular cannulas for hormone infusion and blood sampling from Day 277 to 286. Intravenous infusion of purified porcine relaxin (pRLX, 3000 U mg-1) was started in heifers (n = 8) at increasing dosages (200 U h-1 on Days 277 and 278, 300 U h-1 on Days 279 and 280, 500 U h-1 on Day 281, 600 U h-1 on Day 282, and 700 U h-1 on Days 283 to 286). Phosphate buffer saline (PBS, 10 ml h-1) was infused during these same times to control (n = 6) animals. Relaxin treatment steadily increased the circulating plasma concentration of immunoreactive relaxin to more than 120 ng ml-1 compared with less than 0.5 ng ml-1 in PBStreated controls. Relaxin infusion in increasing dosages over the treatment time was associated with a significant decrease (P < 0.01) in plasma progesterone concentration compared with the PBS controls. Plasma levels of oxytocin at 4- hour intervals remained similar (P > 0.05) during the pretreatment period and throughout continuous infusion of pRLX and PBS. Although continuous intravenous infusion of relaxin resulted in a decrease in circulating blood levels of progesterone, it did not significantly reduce the interval between the beginning of pRLX treatment and parturition compared with the PBS-infused control heifers. These results indicate that continuous intravenous infusion of high levels of porcine relaxin resulted in a decrease in progesterone secretion in late pregnant beef heifers.

Conception Rate Using the Select-Synch Protocol in Combination with a Lower Dose Progesterone-Releasing Intravaginal Insert (1.38 g) in Swiss Dairy Cows

Background: Synchronization programs have become standard in the dairy industry. In Switzerland, these programs are used but newly. The objective of this study was A) to estimate the pregnancy rate after a Select-Synch protocol in- cluding a low dosage of progesterone in CIDR (1.38 g). As a second step B) this pregnancy rate should be compared to cows from another Swiss study that used a Select-Synch protocol with the 1.9 g insert (Rudolph et al., 2011). Methods: A) 196 cows were included in the study. Cows received a CIDR 1.38 g and 2.5 ml of buserelin i.m. on d 0. On d 7, the CIDR insert was removed and 5 ml of dinoprost was administered i.m. On d 0 a milk sample for progesterone analysis was taken. Pregnancy was determined at or more than 35 days after artificial insemination. B) The 1.38 g group and the 1.9 g group were compared as to cow and farm factors, number of preceding AI’s, gynecological and uterine pretreat- ment and treatment itself. A forward selection procedure was used (test result considered significant if p-value  0.05). Results: A) The pregnancy rate, using the Select-Synch protocol with the CIDR 1.38 g was 44.4%. B) The CIDR 1.9 g Select-Synch group revealed a pregnancy rate of 50.4% (Rudolph et al., 2011). Significant differences between the groups were not found. Conclusion: The 1.38 g CIDR-Select-Synch protocol may be recommended for multiparous dairy cows. The pregnancy rate compared to the 1.9 g CIDR-Select-Synch protocol was 8% lower, but this difference was not significant.

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

Peripartal progesterone and prolactin have little effect on the rapid transport of immunoglobulin G into colostrum of dairy cows

Colostrum formation and lactogenesis in the mammary gland and the timing of parturition are regulated by endocrine signals. Changes in progesterone (P4) and prolactin (PRL) are considered key events that inhibit colostrum formation, trigger parturition, and signal the onset of lactation. The goal of our study was to determine if colostrum yield and composition and immunoglobulin transfer are affected by prepartum milking relative to the decrease in P4, peak of PRL, or occurrence of parturition. Twenty-three multiparous cows were randomly assigned to 1 of 2 groups: (1) control with first milking at 4h postcalving (CON, n=11), and (2) treatment group with first milking approximately 1d before calving and second milking at 4h after parturition (APM, n=12). Colostrum yields were recorded and proportional samples were analyzed for immunoglobulin G (IgG) concentration. Blood plasma samples for the analyses of P4 and PRL were collected 3 times daily at 8-h intervals for 4d prepartum and again taken at 4h after parturition. Total colostrum mass of APM cows was higher than that of CON cows. Immunoglobulin G concentration and protein content did not differ between antepartum milking in APM cows and postpartum milking in CON cows. Colostrum IgG concentration and protein content in APM cows at the postpartum milking were lower compared with the IgG concentration established at the prepartum (APM) and postpartum milkings of CON cows. Immunoglobulin G mass did not differ in first and second colostrum collection in APM cows but was lower compared with that of CON cows. The sum of IgG mass in APM cows (prepartum + postpartum collections) did not differ from that of CON cows. Lactose and fat in milk (concentration and mass) increased from first to second milking in APM cows. Total mass of lactose and fat in APM cows (prepartum + postpartum collections) was greater compared with that of CON cows. The finding that the time of milking relative to parturition, P4 decrease, and PRL peak slightly affected yield and quality of colostrum emphasizes the complex interactions of numerous endocrine and morphological changes occurring during colostrogenesis and lactogenesis in dairy cows. The considerably rapid transfer of immunoglobulins into colostrum of prepartum-milked cows within a few hours leads to the hypothesis that the transfer of IgG can be very fast and-contrary to earlier findings-persist at least until parturition.

Detection and functional portrayal of a novel class of dihydrotestosterone derived selective progesterone receptor modulators (SPRM).

In early pregnancy, abortion can be induced by blocking the actions of progesterone receptors (PR). However, the PR antagonist, mifepristone (RU38486), is rather unselective in clinical use because it also cross-reacts with other nuclear receptors. Since the ligand-binding domain of human progesterone receptor (hPR) and androgen receptor (hAR) share 54% identity, we hypothesized that derivatives of dihydrotestosterone (DHT), the cognate ligand for hAR, might also regulate the hPR. Compounds designed and synthesized in our laboratory were investigated for their affinities for hPRB, hAR, glucocorticoid receptor (hGRα) and mineralocorticoid receptor (hMR), using whole cell receptor competitive binding assays. Agonistic and antagonistic activities were characterized by reporter assays. Nuclear translocation was monitored using cherry-hPRB and GFP-hAR chimeric receptors. Cytostatic properties and apoptosis were tested on breast cancer cells (MCF7, T-47D). One compound presented a favorable profile with an apparent neutral hPRB antagonistic function, a selective cherry-hPRB nuclear translocation and a cytostatic effect. 3D models of human PR and AR with this ligand were constructed to investigate the molecular basis of selectivity. Our data suggest that these novel DHT-derivatives provide suitable templates for the development of new selective steroidal hPR antagonists.

Prevention of preterm delivery with vaginal progesterone in women with preterm labour (4P): randomised double-blind placebo-controlled trial.

OBJECTIVE To evaluate the effectiveness of 200 mg of daily vaginal natural progesterone to prevent preterm birth in women with preterm labour. DESIGN Multicentre, randomised, double-blind, placebo-controlled trial. SETTING Twenty-nine centres in Switzerland and Argentina. POPULATION A total of 385 women with preterm labour (24(0/7) to 33(6/7) weeks of gestation) treated with acute tocolysis. METHODS Participants were randomly allocated to either 200 mg daily of self-administered vaginal progesterone or placebo within 48 hours of starting acute tocolysis. MAIN OUTCOME MEASURES Primary outcome was delivery before 37 weeks of gestation. Secondary outcomes were delivery before 32 and 34 weeks, adverse effects, duration of tocolysis, re-admissions for preterm labour, length of hospital stay, and neonatal morbidity and mortality. The study was ended prematurely based on results of the intermediate analysis. RESULTS Preterm birth occurred in 42.5% of women in the progesterone group versus 35.5% in the placebo group (relative risk [RR] 1.2; 95% confidence interval [95% CI] 0.93-1.5). Delivery at <32 and <34 weeks did not differ between the two groups (12.9 versus 9.7%; [RR 1.3; 95% CI 0.7-2.5] and 19.7 versus 12.9% [RR 1.5; 95% CI 0.9-2.4], respectively). The duration of tocolysis, hospitalisation, and recurrence of preterm labour were comparable between groups. Neonatal morbidity occurred in 44 (22.8%) cases on progesterone versus 35 (18.8%) cases on placebo (RR: 1.2; 95% CI 0.82-1.8), whereas there were 4 (2%) neonatal deaths in each study group. CONCLUSION There is no evidence that the daily administration of 200 mg vaginal progesterone decreases preterm birth or improves neonatal outcome in women with preterm labour.

Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF.

BACKGROUND The study was designed to compare the effect of in vitro FSH stimulation on the hormone production and gene expression profile of granulosa cells (GCs) isolated from single naturally matured follicles obtained from natural cycle in vitro fertilization (NC-IVF) with granulosa cells obtained from conventional gonadotropin-stimulated IVF (c-IVF). METHODS Lutein granulosa cells from the dominant follicle were isolated and cultured in absence or presence of recombinant FSH. The cultures were run for 48 h and six days. Messenger RNA (mRNA) expressions of anti-Müllerian hormone (AMH) and FSH receptor were measured by quantitative polymerase chain reaction (qPCR). AMH protein and progesterone concentration (P4) in cultured supernatant were measured by ELISA and RIA. RESULTS Our results showed that the mRNA expression of AMH was significantly higher in GCs from NC- than from c-IVF on day 6 after treatment with FSH (1 IU/mL). The FSH stimulation increased the concentration of AMH in the culture supernatant of GCs from NC-IVF compared with cells from c-IVF. In the culture medium, the AMH level was correlated significantly and positively to progesterone concentration. CONCLUSIONS Differences in the levels of AMH and progesterone released into the medium by cultured GC as well as in AMH gene expression were observed between GCs obtained under natural and stimulated IVF protocols. The results suggest that artificial gonadotropin stimulation may have an effect on the intra-follicular metabolism. A significant positive correlation between AMH and progesterone may suggest progesterone as a factor influencing AMH secretion.

17-beta estradiol and progesterone-induced alterations in human type-1/type-2 cytokine balance and the role of costimulatory and apoptotic mechanisms

Evidence suggests that sex-based differences in immune function may predispose women to numerous hypersensitivity conditions such as Systemic lupus erythematosus (SLE), Hashimoto's thyroiditis and asthma. To date, the exact mechanisms of sexual dimorphism in immunity are not fully characterized but sex hormones such as 17-β estradiol (E2) and progesterone (PR) are believed to be involved. Since E2 and PR may modulate the production of critical regulatory cytokines, we sought to characterize their effects on the in vitro human type-1/type-2 cytokine balance. We hypothesized that E2 and/or PR vary cytokine production and influence costimulatory molecule expression and apoptosis. We first described the effect of E2 and/or PR on type-1 (IFN-γ and IL-12) and type-2 (IL-4 and IL-10) cytokine production by human peripheral blood mononuclear cells (PBMC) treated with various T-lymphocyte and monocyte stimuli. E2 and/or PR were each used at concentrations similar to those found at the maternal-fetal interface during pregnancy. At this dose, E2 increased IFN-γ and IL-12 production and PR decreased IFN-γ production and tended to increase IL-4 production. Furthermore, the combination of E2+PR decreased IL-12 production. This suggests that E2 shifts the type-1/type-2 cytokine balance towards a type-1 response and that PR and E2+PR shift the balance towards a type-2 response. Next, we used intracellular cytokine detection to demonstrate that E2 and/or PR are capable of altering cytokine production of CD3+ T-cells and the CD3+CD4+ and CD3+CD8+ subsets. In addition, we used the H9 T-lymphocyte cell line and the THP-1 monocyte cell line to show that E2 and/or PR can induce cytokine effects in both T-cells and monocytes independent of their interaction. Lastly, we determined the effect of E2 and/or PR on costimulatory molecule expression and apoptosis as potential mechanisms for the cytokine-induced alterations. E2 increased and PR decreased CD80 expression on THP-1 cells and PR and E2+PR decreased CD28 expression in PBMC and Jurkat cells. Furthermore, E2, PR and E2+PR increased Fas-mediated apoptosis in Jurkat cells and E2 increased FasL expression on THP-1 cells. Thus, E2 and/or PR may alter the cytokine balance by modulating the CD28/CD80 costimulatory pathway and apoptosis. ^

The association between maternal use of spermicides, condoms, intra-uterine devices or progesterone and major structural birth defects

Birth defects occur in 1 of every 33 babies born in the United States, and are the leading cause of infant death. Mothers using contraceptives that become pregnant may continue to use their contraceptives after their first missed menstrual period, thus exposing their baby in utero to the contraceptive product. Progesterone is also sometimes prescribed during the first trimester of pregnancy to mothers with a history of miscarriages or infertility problems. To ensure the safety of these products, it is important to investigate whether there is an increased occurrence of babies born with birth defects to mothers using various contraceptive methods or progesterone in early pregnancy. Using data from the National Birth Defects Prevention Study (NBDPS), an ongoing multi-state, population based case-control study, this study assessed maternal exposures to IUDs, spermicides, condoms and progesterone in early pregnancy. ^ Progesterone used for threatened miscarriage during the first three months of pregnancy was associated with an increased occurrence of hypoplastic left heart (adjusted odds ratios (OR) 2.24, 95% CI 1.13-4.21), perimembranous ventricular septal defects (OR 1.64, 95% CI 1.10-2.41), septal associations (OR 2.52, 95% CI 1.45-4.24), esophageal atresia (OR 1.82, 95% CI 1.04-3.08), and hypospadias (OR 2.12, 95% CI 1.41-3.18). Mothers using progesterone for injectable contraception had increased (OR > 2.5), but insignificant odds ratios for anencephaly, septal associations, small intestinal atresias and omphalocel. Progesterone used for fertility was not associated with an increased occurrence of any birth defects examined. ^ Mothers using progesterone for fertility assistance and threatened miscarriage were very similar with respect to their demographics and pregnancy history. They also both reported similar types of progesterone. Thus, if progesterone was a causal risk factor for birth defects we would have expected to observe similar increases in risk among mothers using progesterone for both indications. Because we predominantly observed increased associations among mothers using progesterone for threatened miscarriage but not fertility assistance, it is possible the increased associations we observed were confounded by indication (i.e. progesterone was administered for vaginal bleeding which occurred as a sequelae to the formation of a congenital anomaly. ^ No significant increased associations were observed between maternal spermicide use during pregnancy and 26 of 27 types of structural malformations. While multiple statistical tests were performed we observed first trimester maternal spermicide use to be associated with a significant increased occurrence of perimembranous ventricular septal defects (OR 2.21, 95% CI 1.16-4.21). A decreased occurrence (OR < 1.0) was observed for several categories of birth defects among mothers who conceived in the first cycle after discontinuing the use of spermicides (22 of 28) or male condoms (23 of 33). ^ Overall the percent of IUD use was similar between mothers of controls and mothers of all cases in aggregate (crude OR 1.05, 95% CI 0.61-1.84). Power was limited to detect significant associations between IUD use and birth defects, however mothers using an IUD in the month immediately prior to conception or during pregnancy were not associated with an increase of birth defects. Limb defects and amniotic band sequence previously reported to be associated with IUD use during pregnancy were not found to occur among any mothers reporting the use of an IUD during pregnancy.^

Proliferation index in ductal carcinoma in situ of the breast: Relation to estrogen, progesterone and HER2/neu receptors

Background. Ductal carcinoma in situ (DCIS) is the most prevalent precursor to invasive breast cancer (IBC), the second leading cause of death in women in the United States. The three most important prognostic markers for IBC are Estrogen receptor (ER), Progesterone receptor (PR) and HER2/neu. The four groups (IBC) defined as (1) ER and/or PR positive and HER2/neu negative, (2) ER and/or PR positive and HER2/neu positive (3) ER and/or PR negative and HER2/neu positive and (4) negative for all three of these receptors (Triple negative). However, they have not been well studied in DCIS. This is an exploratory study with a primary objective to examine the prevalence of ER, PR, and HER2/neu in DCIS, to explore if the defined groups of IBC occur in DCIS and to consider the biological relationship between these four groups and the proliferative activity of the tumor. A secondary goal of this study is to examine the relationship between grade and proliferative activity. Methods. Using immunohistochemistry, I have measured Ki-67, ER, PR and HER2/neu positivity for a series of cases of DCIS. Results. 20 ER and/or PR positive and HER2/neu negative (50%) with average PI of 0.05, 7 ER and/or PR positive and HER2/neu positive (17.5%) with average PI of 0.14, 10 ER and/or PR negative and HER2/neu positive (25%) with average PI of 0.18, and three triple negative (7.5%) with average PI of 0.18. ER and/or PR positive and HER2/neu positive group has the highest PI (p<0.001). Further, the ER and/or PR positive and HER2/neu positive group show a linear relationship between PI and average ER/PR positivity (R=0.6). PI increases with higher grades. Conclusion. PI appears to depend upon the average fraction of positive ER/PR tumor cells, possibly with a synergistic dependence when HER2/neu is positive. If ER/PR is negative, then both HER2/neu positive and the triple negative cases appear to cluster around an average PI that is higher than the average PI in HER2/neu negative ER/PR positive negative cases. In the triple negative tumors there must be another driver of proliferation.^

Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor α knockout mouse uterus

Estrogen is known to increase progesterone receptor (PR) levels in the wild-type mouse uterus, and this estrogen induction was thought to be important for progesterone action through the PR. The estrogen receptor α knockout (ERKO) mouse uterus was observed to express PR mRNA that cannot be induced by estrogen. Progesterone action was characterized to determine whether it was diminished in ERKO mice. The PR protein is present in the ERKO uterus at 60% of the level measured in a wild-type uterus. The PR-A and PR-B isoforms are both detected on Western blot, and the ratio of isoforms is the same in both genotypes. Although the level of PR is reduced in the ERKO uterus, the receptor level is sufficient to induce genomic responses, since both calcitonin and amphiregulin mRNAs were increased after progesterone treatment. Finally, the ERKO uterus can be induced to undergo a progesterone-dependent decidual response. Surprisingly, the decidual response is estrogen independent in the ERKO, although it remains estrogen dependent in a wild type. These results indicate that estrogen receptor α modulation of PR levels is not necessary for expression of the PR or genomic and physiologic responses to progesterone in the ERKO uterus.

Vaginal progesterone prophylaxis for preterm birth (the OPPTIMUM study) : a multicentre, randomised, double-blind trial

Acknowledgments We thank the members of the Trial Steering and Data Monitoring Committee and all the people who helped in the conduct of the study (including the OPPTIMUM collaborative group and other clinicians listed in the appendix). We are grateful to Paul Piette (Besins Healthcare Corporate, Brussels, Belgium) and Besins Healthcare for their kind donation of active and placebo drug for use in the study, and to staff of the pharmacy and research and development departments of the participating hospitals. We are also grateful to the many people who helped in this study but who we have been unable to name, and in particular all the women (and their babies) who participated in OPPTIMUM. OPPTIMUM was funded by the Efficacy and Mechanism Evaluation (EME) Programme, a Medical Research Council (MRC) and National Institute of Health Research (NIHR) partnership, award number G0700452, revised to 09/800/27. The EME Programme is funded by the MRC and NIHR, with contributions from the Chief Scientist Office in Scotland and National Institute for Social Care and Research in Wales. The views expressed in this publication are those of the author(s) and not necessarily those of the MRC, National Health Service, NIHR, or the Department of Health. The funder had no involvement in data collection, analysis or interpretation, and no role in the writing of this manuscript or the decision to submit for publication.