Endometrial Progesterone Receptor Research Articles

The adenomyosis/endometriosis IVF patient – call for clinical focus

Endometriosis and adenomyosis are distinct clinical conditions that carry the same pathophysiological features. In recent years the clinical focus on assisted reproductive technology patients with either condition (E/A) has increased, in the recognition that this subgroup of patients might need special attention to obtain reproductive success. Endometriosis and adenomyosis are characterized by a disruption of progesterone and oestrogen signalling pathways, resulting in local oestrogen dominance and progesterone resistance at the receptor level. Recent scientific evidence suggests that the endometrial progesterone receptor resistance encountered in E/A patients can be overcome by a freeze-all policy, followed by down-regulating circulating oestradiol concentrations prior to frozen embryo transfer (FET), in combination with an increase in exogenous luteal phase progesterone supplementation in hormonal replacement therapy (HRT) FET cycles. Specifically, for adenomyosis patients who do not respond to gonadotrophin-releasing hormone agonist down-regulation in terms of a decrease in circulating oestradiol concentrations, a small case series has suggested that the addition of an aromatase inhibitor for 21 days prior to HRT–FET is a valid option. Endometriosis and adenomyosis are hormonally active diseases, which need to be treated by controlling local hyperoestrogenism and progesterone resistance. Based on physiology and recent preliminary clinical data, the authors of this opinion paper wish to stimulate discussion and spark interest in research in E/A patients.

PGRMC1 Regulates Cellular Senescence via Modulating FOXO1 Expression in Decidualizing Endometrial Stromal Cells.

The appropriate differentiation of endometrial stromal cells (ESCs) into decidual cells is required for embryo implantation and subsequent placentation into humans. Decidualization is accompanied by the appearance of senescent-like cells. We recently reported the secretory phase-specific downregulation of endometrial progesterone receptor membrane component 1 (PGRMC1) and enhanced decidualization upon PGRMC1 knockdown and inhibition in cultured ESCs. However, it remains unknown whether PGRMC1 is involved in cellular senescence during decidualization. Here, we showed that the small interfering RNA (siRNA)-mediated knockdown of PGRMC1 and the inhibition of PGRMC1 by AG-205 increased the expression of the transcription factor forkhead box protein O1 (FOXO1) and the senescence-associated β-galactosidase activity in cAMP analog- and progesterone-treated ESCs. Furthermore, the knockdown of FOXO1 repressed the decidual senescence induced by siRNA-based PGRMC1 knockdown or AG-205 treatment. Taken together, the decreased PGRMC1 expression in ESCs may accelerate decidualization and cellular senescence via the upregulation of FOXO1 expression for appropriate endometrial remodeling and embryo implantation during the secretory phase.

P-336 Progesterone receptor is not downregulated in the endometrial epithelial compartment of women without embryo implantation during frozen thawed embryo transfer cycles

Abstract Study question Is the mean endometrial progesterone receptor (PGR) expression in the endometrial epithelial compartment different between women with and without embryo implantation failure? Summary answer PGR expression is higher in epithelial endometrial compartment of women with embryo implantation failure, during mock hormonal endometrial preparation preceding frozen thawed embryo transfer cycles. What is known already PGR downregulation is critical for embryo implantation. Downregulation takes place during the window of implantation in the EEC, while, in stromal cells, it is highly expressed. Embryos are unable to attach to the maternal surface when PGR expression remains expressed in epithelial endometrial compartment (EEC). Limited information is available whether PGR is downregulated during hormonal endometrial preparation for frozen thawed embryo transfer cycles. Study design, size, duration In this cohort study, 47 endometrial biopsies were obtained between 2016-2017 Participants/materials, setting, methods Infertile women (n = 47) submitted to frozen thawed embryo transfer (FET) cycles had an endometrial biopsy after 5 days of intravaginal progesterone supplementation during mock hormonal endometrial preparation (HEP) preceding FET and prospectively followed until confirmation of an intrauterine pregnancy. PGR immunostaining was performed using standard immunohistochemical technique (clone BSB2 - BS2126, BIO SB). PRG expression was quantified using the histologic score (HScore, range from 0 - 4). Ethical approval was obtained from local ethical committee. Main results and the role of chance Age, body mass index, endometrial thickness, and the number of embryos transferred were not significantly different between women who became or not pregnant. PGR Hscore (mean ± SD) in EEC was significantly lower in pregnant women (1.2 ± 0.8; n = 14 ) compared to women without pregnancy (2.8 ± 0.6; n = 33 ) Student t-test p < 0.0001. The AUC was 0.90 ± 0.05 (95% CI: 0.7-1) p < 0.0001 displaying 94.2 % sensitivity and 80.4% specificity for a 1.65 PGR cut-off value. Limitations, reasons for caution This is an observational study to determine if a difference in PGR expression was identified in these cases Wider implications of the findings The mean PGR expression is abnormally higher in EEC of women with embryo implantation failure using HEP for FET cycles. The 1.65 cut-off may be used in future studies to investigate whether high PGR expression is a risk factor for failure of blastocyst implantation in HEP for FET. Trial registration number not applicable

P–619 Oral dydrogesterone (OD) versus micronized vaginal progesterone (MVP) for luteal phase support (LPS) in IVF/ICSI: a double blind, cross-over, pharmacokinetic study

Abstract Study question How does the blood pharmacokinetic (PK) profile of OD/MVP differ after the first and last administration dose when used as LPS for fresh embryo transfer? Summary answer: The PK profile differed strongly between both LPS administration strategies with a more rapid absorption, metabolism and clearance of OD in comparison with MVP. What is known already Adequate LPS is crucial to achieve a successful pregnancy following ovarian stimulation (OS) and fresh embryo transfer. OD has been proven to be non-inferior compared to MVP in two phase III clinical trials. Additionally, a combined individual participant data and aggregate data meta-analysis showed an odds ratio in favor of OD for live birth. Little information is available on the PK of LPS strategies, leaving an important field unexplored. Individualization of LPS has recently gained more interest and insight into the PK of progestogens is essential to correctly interpret the potential impact of circulating hormone levels on reproductive outcomes. Study design, size, duration Twenty oocyte donors underwent two OS cycles followed by one week of LPS (OD or MVP) in a randomized, cross-over, double blind, double dummy fashion. As both dydrogesterone (D) and 20αdihydrodydrogesterone (DHD) are progestogenic, D, DHD and progesterone (P) plasma levels were established using a validated liquid chromatography tandem mass spectrometry assay in each cycle, on the 1st (single dose PK) and 8th day (multiple dose PK) of LPS (9 and 12 harvesting time-points, respectively). Participants/materials, setting, methods All oocyte donors were <35 years, had regular menstrual cycles, no intra-uterine contraceptive device, AMH within normal range and BMI ≤ 29 kg/m2. OS was performed in a GnRH antagonist protocol followed by dual triggering (1000U hCG + 0.2mg triptorelin) as soon as ≥ 3 follicles of 20mm were present. Following oocyte retrieval, subjects initiated LPS consisting of MVP 200 mg (Utrogestan®) or OD 10 mg (Duphaston®), both three times daily. Main results and the role of chance The mean (±SD) age of the subjects was 27.4 (± 3.8) years and BMI was 24.0 (±3.2) kg/m2. The mean (±SD) number of oocytes retrieved was 19.7 ±10. No adverse events were reported during the intake of the study medication. The PK results are best estimates as sampling was reduced compared to a formal PK study. Following the intake of the first dose of OD, the observed maximal plasma concentrations (Cmax) for D and DHD were 2.9 and 77 ng/ml (single dose). The Cmax for D and DHD was reached after 1.5 and 1.6 hours (=Tmax), respectively. On the 8th day of LPS the first administration of that day gave rise to a Cmax of 3.6 and 88 ng/ml for D and DHD (multiple dose). For both, the observed Tmax was 1.5 hours. Following the intake of the first dose of MVP, the Cmax for P was 16 ng/mL with a Tmax of 4.2 hours. On the 8th day of LPS the first administration of that day showed a Cmax for P of 21 ng/mL with a Tmax of 7.3 hours. Although low, the role of chance could be influenced by the relatively low sampling numbers and frequency. Limitations, reasons for caution Peripheral concentrations do not necessarily reflect the steroidogenic effect on endometrial progesterone receptors. Extrapolation to clinical practice is therefore difficult, however, molecular analyses of endometrial tissue harvested within this study protocol are underway to investigate further pharmacodynamics and the progestogenic impact on endometrial receptivity during the embryo implantation period. Wider implications of the findings: This is the first study comparing OD/MVP pharmacokinetics in IVF/ICSI. Results suggest administration frequency to be as important as dose, definitely for OD, showing a rapid absorption/clearance. More studies are needed to investigate blood levels in relation to time of LPS administration, especially in (artificially prepared) FET and LPS individualization. Trial registration number EUDRACT 2018–000105–23

Evaluation of endometrial progesterone receptor expression after 12 weeks of exposure to a low-dose vaginal estradiol insert.

Objective:To evaluate endometrial progesterone receptor (PGR) expression in menopausal women who used vaginal 4-μg and 10-μg estradiol (E2) inserts or placebo.Methods:REJOICE was a randomized, placebo-controlled trial investigating vaginal E2 inserts in women with moderate to severe dyspareunia due to menopause. In this post hoc analysis, 25 eligible women with endometrial biopsies were randomly selected from each treatment group (4-μg and 10-μg E2 vaginal inserts and placebo). Endometrial biopsy sections were immunostained using an anti-PR (A and B) monoclonal antibody. Cell staining was quantified using an artificial intelligence feature-recognition algorithm. Mean PGR expression levels were analyzed between baseline and week 12.Results:PGR expression results were available for 22 women in the 4-μg E2 group, and 25 women each for the 10-μg E2 and placebo groups. Similar PGR expression levels were observed at baseline (0.301-0.470 pmol/mg) and after 12 weeks of treatment (0.312-0.432 pmol/mg) for all treatment groups, with no significant differences between baseline and week 12.Conclusions:No meaningful differences in endometrial PGR expression were observed with the vaginal E2 (4- and 10-μg) inserts at week 12 from baseline, supporting the hypothesis that local exposure to E2 from a low-dose, vaginal insert placed near the vaginal introitus will not be sufficient to upregulate endometrial PGR expression. Coupled with the lack of histologic changes and systemic absorption, our data suggest that these softgel vaginal E2 inserts would not be expected to stimulate endometrial hyperplasia leading to a potential endometrial safety issue in postmenopausal women with moderate to severe dyspareunia, a symptom of vulvar and vaginal atrophy. Further study on the endometrial safety of softgel vaginal E2 inserts is under way.

Therapeutic Potentials of Low-Dose Tacrolimus for Aberrant Endometrial Features in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a major anovulatory infertility affecting a great proportion of women of childbearing age and is associated with obesity, insulin resistance and chronic inflammation. Poor endometrial receptivity and recurrent implantation failure are major hurdles to the establishment of pregnancy in women with PCOS. The accumulating body of evidence obtained from experimental and clinical studies suggests a link between inherent adaptive and innate immune irregularities and aberrant endometrial features in PCOS. The use of conventional therapeutic interventions such as lifestyle modification, metformin and ovarian stimulation has achieved limited clinical success in restoring ovulation and endometrial receptivity in women with PCOS. Unlike other immunosuppressive drugs prescribed in the clinical management of autoimmune and inflammatory disorders that may have deleterious effects on fertility and fetal development, preclinical studies in mice and in women without PCOS but with repeated implantation failure revealed potential therapeutic benefits for the use of low-dose tacrolimus in treating female infertility. Improved systemic and ovarian immune functions, endometrial progesterone receptor and coreceptor expressions and uterine vascular adaptation to pregnancy were among features of enhanced progesterone-receptor sensitivity in the low-dose tacrolimus-treated mouse model of the disease. In this review, we have compiled available experimental and clinical data in literature on endometrial progesterone resistance and current therapeutic options, as well as mechanisms of actions and reported outcomes relevant to the potential therapeutic benefits for the use of low-dose tacrolimus in treating PCOS-associated female infertility.

MON-006 Progesterone Receptor Expression in Endometrial Biopsies After 12 Weeks of Exposure to A 4-µg E2 Softgel Vaginal Insert or Placebo

The softgel, vaginal 17β-estradiol (E2) insert (TX-004HR) significantly improved the maturation index of the vaginal epithelium, dyspareunia and vaginal dryness in menopausal women with vulvar and vaginal atrophy (VVA) and moderate to severe dyspareunia, without histological changes to the endometrium (Constantine G et al, Menopause 2017;24:409-416). The 4-μg and 10-μg E2 doses were FDA approved as Imvexxy® (TherapeuticsMD, Boca Raton, FL), for the treatment of moderate to severe dyspareunia, a symptom of VVA, due to menopause. The progesterone receptor (PR) is an estrogen-regulated gene with expression that is very sensitive to E2 exposure (Xiao CW and Goff AK, J Reprod Fertil.1999;115:101-109). Endometrial PR expression in the biopsies of women using the softgel vaginal 4-µg E2 insert was used as a marker of E2 exposure to determine whether sufficient E2 applied with the vaginal insert reaches the endometrium to upregulate PR expression. Our hypothesis posits that there would be insufficient E2 from the 4-µg E2 insert to stimulate an increase in endometrial PR expression.In this post hoc analysis of the REJOICE trial, endometrial biopsies from 25 women who had a normal baseline biopsy, an on-therapy biopsy after study day 70, and tissue readings from all pathologists were randomly selected from the 4-µg E2 vaginal insert and placebo groups. Endometrial tissue sections were stained to visualize PR expression using PgR1294 (Agilent; Santa Clara, CA). Cell staining was quantified using a trainable feature-recognition algorithm and mean expression levels between baseline and week 12 were analyzed by 2-sided t-tests.Acceptable PR expression results were available for 22 women in the 4-µg E2 group (three had insufficient tissue for analysis) and 25 women in the placebo group. For the 4-µg E2 group, mean ± SD (pmol/mg) PR expression was 0.455 ± 0.203 at baseline and 0.506 ± 0.226 at week 12. For the placebo group, mean PR expression was 0.579 ± 0.196 at baseline and 0.563 ± 0.213 at week 12. Mean PR expression levels at baseline and week 12 were not significantly different from each other within the 4-µg E2 (P=0.438) or placebo (P=0.783) group.No meaningful difference in endometrial PR expression was observed with the vaginal 4-µg E2 insert at week 12. These data support our hypothesis and the assertion that low-dose, local vaginal E2 exposure with the insert placed in the lower part of the vagina, does not pose an endometrial safety issue in postmenopausal women.

Low plasma progestin concentration during the early postovulatory phase impairs equine conceptus development in the late preimplantation phase

In horses, low postovulatory progestin concentrations delay downregulation of endometrial progesterone receptors, impairing histotroph composition. The aim of this study was to evaluate conceptus development until placentation in mares under experimentally reduced progestin concentrations in the early postovulatory phase. Eleven mares were inseminated until ovulation and received the prostaglandin F2α agonist cloprostenol (125 µg) once daily for 4 days after ovulation (treatment) or were left untreated (control). Conceptus growth and development was analysed daily from Day 10 after ovulation by ultrasound. On Day 34 conceptuses were recovered and underwent microcomputed tomography. Statistical comparison was performed by non-parametric Wilcoxon test or Chi2. First visualisation of embryonic vesicle, embryo proper and heartbeat did not differ. Conceptus fixation in the uterus occurred earlier in controls than in conceptuses recovered from treatment pregnancies (P < 0.05). Embryos from treatment pregnancies were smaller (P < 0.05) and lighter (P < 0.05) than controls. In the lung, the mean number of bronchi was lower in embryos from treatment pregnancies than from control pregnancies (P < 0.05). Persistence of communication between the cardiac ventricles was observed more often in embryos from treatment than control pregnancies (P < 0.05). In conclusion, subphysiological progestin concentrations in the early postovulatory period impaired conceptus development before placentation in horses.

61 Low plasma progestin concentration during the early luteal phase impairs equine conceptus development until placentation

During the early luteal phase, low progesterone concentrations delay downregulation of endometrial progesterone receptors. This contributes to impaired histotroph production at Day 14 of pregnancy (Beyer et al. 2019 Theriogenology 125, 236-241). Until the beginning of placentation (i.e. Day 37 of pregnancy), nutritional supply of the equine conceptus depends on histotrophy alone. The aim of the present study was to analyse development of the equine conceptus under reduced plasma progesterone concentrations until shortly before placentation. Fertile Haflinger mares (n=11; 4-11 years old) were examined daily by transrectal ultrasonography, and when in oestrus, they were inseminated every 48h until spontaneous ovulation. Mares were randomly assigned to either the treatment group and received the prostaglandin F2α agonist cloprostenol (PGF2α; 125μg) once daily for 3 days after ovulation (Beyer et al. 2019) or the control group and left untreated. After conceptus collection on Day 34, mares were allowed one oestrous cycle for recovery and subsequently assigned to the opposite treatment, thus serving as their own controls. From Day 10 after ovulation, conceptus development including size, uterine fixation, and heartbeat detection was evaluated daily by transrectal ultrasonography. On Day 34, conceptus and fetal membranes were recovered transcervically. The recovered material was weighed, measured, and stored for further analysis. Conceptuses underwent microcomputed tomography that was evaluated by Amira (Thermo Fisher Scientific). Statistical comparison for differences between control and treatment pregnancies was performed by nonparametric Wilcoxon test or chi-square analysis. The day of first visualisation of the conceptus and of the embryo proper and heartbeat did not differ between treatments. Uterine fixation occurred on Day 19.3±0.5 in conceptuses from PGF2α-treated pregnancies but on Day 16.7±0.4 in controls (P&amp;lt;0.05). At 34 days of gestation, the conceptuses from PGF2α-treated mares were smaller (P&amp;lt;0.05) than control conceptuses when measured with ultrasound callipers in utero (maximal length: PGF2α 17.4±0.3mm, control 19.0±0.4mm). Conceptus weight determined after recovery was less in treated pregnancies (PGF2α 2.5±0.2 g; control 3.5±0.3 g; P&amp;lt;0.05). Microcomputed tomography analysis of selected inner organs showed some differences in development. Seven complete pairs of undestroyed conceptuses were available. Mean numbers of 33.4±3.2 and 18.8±7.5 bronchi were detected in control and PGF2α conceptuses, respectively (P&amp;lt;0.05). Heart volume did not differ, but communication between ventricles was detected in only 1/7 control but 5/7 PGF2α conceptuses (P&amp;lt;0.05). The footpad was present in 6/7 control and 0/7 PGF2α conceptuses (P&amp;lt;0.001). In conclusion, subphysiological progesterone concentration during the early luteal phase delays development of the equine conceptus before placentation. The condition may contribute to early conceptus loss in horses, which occurs in up to 20% of pregnancies.

Effects of hormone replacement therapy on endometrial hormone concentrations and progesterone receptor expression in recurrent pregnancy loss: a self-controlled study

Aim To investigate the differential effects of endometrial preparation approaches, natural cycle (NC) and hormone replacement therapy (HRT), on the endometrial hormone conditions in patients with recurrent pregnancy loss (RPL). Methods 42 RPL patients were recruited, and a self-controlled trial was conducted to compare the effectiveness of NC to HRT on the same patient. The estradiol (E2) and progesterone (P) levels in both serum and endometrial tissue were measured in NC and HRT, respectively. Meanwhile, the expression levels of endometrial progesterone receptor A (PRA) and progesterone receptor B (PRB) were also compared between NC and HRT cycles during the window of receptivity. All statistical analyses were performed using GraphPad Prism 5, and data were averaged across subjects and tested for significance by pair-samples t-test. Results Hormone replacement slightly decreased serum E2 and P levels (0.9- and 0.8-fold in average, respectively), significantly elevated endometrial E2 and P (2.1- and 14.5-fold in average, respectively), and reduced endometrial E2/P ratio (0.34-fold in average) compared to the NC condition. However, no correlation was found between serum and endometrial hormone concentrations. Further, the PRA/PRB ratios of both stromal and glandular cells were comparable between HRT and NC conditions, even though stromal PRA expression was slightly elevated by HRT compared to NC (1.63±0.96 vs 1.18±0.99, p =.0173). Conclusion The effects of HRT for endometrial preparation are similar or slightly superior to NC in RPL patients.

193 Melatonin implants in spring improve embryo production of aged ewes after superovulation regardless of endometrial progesterone receptor expression

Twenty-three Rasa Aragonesa aged ewes (average age: 10.3±0.3 years) were used to determine the effect of melatonin on ovulatory response, embryo production, and endometrial expression of progesterone receptors (PR) after superovulation. Ewes were treated (M, n=13) or not (control, C, n=10) with melatonin implants in March (Day 0, Northern Hemisphere autumn), and received intravaginal progestogen sponges for 14 days on Day 77. Superovulatory treatments consisted of 8 doses in decreasing concentrations (2 mL×2 and 1 mL×6) of 176 NIH-FSH-S1 units of NIADDK-oFSH-17 (Ovagen, ICPbio Reproduction, Auckland, New Zealand) administered twice daily starting 72h before sponge removal. Seven days after oestrus, embryos were recovered by laparotomy, ewes were killed, and uterine horns were processed to study PR expression by immunohistochemistry. The amount of PR was estimated subjectively by 2 independent observers in 5 endometrial compartments: luminal epithelium (LE), superficial (sGE) and deep (dGE) glandular epithelia, and superficial (sS) and deep (dS) stroma. The extent of staining was expressed on a scale from 0 to 100. Data were analysed with a 2×2 factorial ANOVA. Melatonin implants improved fertilization (92v. 57%, for M and C groups, respectively; P&amp;lt;0.01), blastocyst (47v. 9%; P&amp;lt;0.01), viability (88v. 31%; P&amp;lt;0.0001), and freezability (69v. 21%; P&amp;lt;0.001) rates. Specifically, melatonin induced a significant reduction of the number of non-viable (degenerate and retarded) embryos (0.3v. 1.5; P&amp;lt;0.05) and increased blastocysts (2.8v. 0.8; P&amp;lt;0.05) per ewe. Melatonin treatment decreased PR staining intensity (47v. 55%; P&amp;lt;0.05), but this effect was not observed when the individual cell types were compared (Table 1). Because the number of corpora lutea (CL) was responsible for different PR expression in both groups (P&amp;lt;0.0001), animals were divided into 2 ovulation rate categories: &amp;lt;10 CL and ≥10 CL, with lesser PR expression in the ≥10 CL group (P&amp;lt;0.0001); this lower PR immunostaining in ≥10 CL is consistent with progesterone down-regulation of its own receptor. An interaction among number of CL and treatment was found for embryo quality (P&amp;lt;0.05); thus, the positive effect of melatonin on this parameter was particularly effective in the low-ovulation-rate group. These results demonstrate that melatonin treatment in the autumn improves embryo quality in aged ewes, and that this effect is not explained by a differential endometrial sensitivity to progesterone. Table 1.Embryo production (mean±s.e.m.) in melatonin-treated (M) and control (C) ewes after superovulation in autumn, and staining intensity of progesterone receptors in the endometrium (CL=corpora lutea)

Early luteal phase progestin concentration influences endometrial function in pregnant mares

In the horse, it is still unclear if and to what extent low progestin concentration contributes to early conceptus loss. In the present study, we have investigated if reduced or elevated progestin concentration in the early luteal phase influences endometrial function and conceptus development. We hypothesized that reduced progestin concentration via delayed downregulation of endometrial progesterone receptors (PR) influences endometrial function in healthy fertile mares while progestin substitution does not. Genitally healthy estrous mares (n = 8; age 4–14 years) were inseminated and treated with either altrenogest (0.044 mg/kg once daily orally) on days 5–10 after ovulation (ALT), cloprostenol (125 μg once daily intramuscularly) on days 0–3 after ovulation (CLO) or left untreated (CON). ALT and CLO treatment were chosen to increase and decrease total peripheral progestin concentration, respectively. Each treatment was given to every mare in consecutive cycles. On day 14 after ovulation, endometrial fluid was collected with a cotton roll inserted into the uterus and an endometrial biopsy for immunohistological demonstration of progesterone (PR) receptor distribution was collected. In endometrial fluid, free amino acid concentrations were analyzed by ion exchange liquid chromatography with an amino acid analyzer. Cell nuclei staining positive for the PR were determined in the luminal and glandular epithelium as well as in the stroma. Pregnancy rate tended to differ among treatments. The percentage of luminal epithelial cells staining positive for PR differed among treatments (p < 0.05) and was higher in CLO (84.1 ± 1.9%) than in ALT (70.7 ± 4.7%) and CON cycles (72.8 ± 4.1%). Concentrations of the amino acids isoleucine (CON 0.17 ± 0.03, CLO 0.14 ± 0.02, ALT 0.23 ± 0.04 μmol) and lysine (CON 0.27 ± 0.08, CLO 0.18 ± 0.05, ALT 0.44 ± 0.13 μmol) were influenced by treatment (p < 0.05) and lower in CLO than in ALT and CON cycles. In conclusion, impaired luteal function due to CLO treatment during the early luteal phase of pregnant mares delayed downregulation of progesterone receptors in the endometrial epithelium on day 14. This influenced endometrial function as reflected in lower concentrations of the amino acids lysine and isoleucine in endometrial secretions. Enhanced progestin concentration had less clear effects in healthy fertile mares.

Maternal N-Carbamylglutamate Supply during Early Pregnancy Enhanced Pregnancy Outcomes in Sows through Modulations of Targeted Genes and Metabolism Pathways.

Reducing pregnancy loss is important for improving reproductive efficiency for both human and mammalian animals. Our previous study demonstrates that maternal N-carbamylglutamate (NCG) supply during early pregnancy enhances embryonic survival in gilts. However, whether maternal NCG supply improves the pregnancy outcomes is still not known. Here we found maternal NCG supply during early pregnancy in sows significantly increased the numbers of total piglets born alive per litter ( P < 0.05) and significantly changed the levels of metabolites in amniotic fluid and serum involved in metabolism of energy, lipid, and glutathione and immunological regulation. The expression of endometrial progesterone receptor membrane component 1 (PGRMC1) was significantly increased by NCG supplementation ( P < 0.05) as well as the expression of PGRMC1, endothelial nitric oxide synthesases (eNOS), and lamin A/C in fetuses and placentae ( P < 0.05). Among the NCG-associated amino acids, arginine and glutamine, markedly increased PGRMC1 and eNOS expression in porcine trophectoderm cells ( P < 0.05), whereas glutamate could stimulate the expression of vimentin and lamin A/C in porcine trophectoderm (pTr) cells ( P < 0.05) and proline stimulated lamin A/C expression ( P < 0.05). Collectively, these data reveal the mechanisms of NCG in reducing early embryo loss. These findings have important implications that NCG has great potential to improve pregnancy outcomes in human and mammalian animals.

Estrogen receptor, progesterone receptor and CD8+ expression in endometrium of women of unexplained infertility

ObjectiveThe present study aimed to investigate the changes of endometrial progesterone and estrogen receptors in luteal phase biopsy specimens of infertile women and find a correlation, if any, between these and CD8+ receptors in the same. MethodsThe study was conducted on luteal phase endometrial biopsy specimens of 30 women of unexplained infertility and 15 age matched controls. Paraffin sections were first H & E stained. A standardized immunohistochemical protocol was then used to localize the estrogen, progesterone and CD8+ receptors in these samples that were expressed as percentage positivity. Unpaired T test was applied between the controls and cases both for epithelial and stromal cells. The data was also analyzed for correlation in cases for the positivity of CD8+ Cells with that of ER and PR. ResultsThe positivity of estrogen receptors (ER) in stromal cells was significantly lower (p<0.001) in the infertile women when compared to controls and in both the epithelial and stromal cells for progesterone receptors (p<0.001).The results were non significant for CD8+ cells (p=0.19) and also showed no significant correlation in the positivity of CD8+ cells with that of ER and PR. ConclusionsThe development of molecular probe like ER and PR positivity in endometrial epithelial and stromal cells allows a new approach to be made to the characterization of normal and defective endometrial function.

Alterations in the Calcitonin and Calcitonin modulated proteins, E-cadherin and the enzyme tissueTransglutaminase II during the window of implantation in a baboon model of endometriosis

Endometriosis is a gynecological condition associated with infertility. We have previously demonstrated dysregulation of several molecular markers of uterine receptivity in a baboon model of induced endometriosis. Specifically, reduced levels of endometrial progesterone receptor (PR) and progesterone (P)-regulated genes, HOXA10 and FKBP52 were observed during the window of uterine receptivity in baboons with endometriosis, suggesting that this disease results in the development of an endometrial P resistance. In this study calcitonin (CALC) and CALC-modulated proteins, E-cadherin (E-Cad) and tissue Transglutaminase (tTgase-2) were evaluated in the eutopic endometrium of endometriotic animals throughout disease progression. Endometriosis was induced in normal cycling baboons by intraperitoneal inoculation of menstrual endometrium. Eutopic and ectopic endometrium was harvested consecutively from each animal at 1, 6 and 15 months of disease, during the window of receptivity. Control eutopic endometrium was s...

Endometrial expression of estrogen and progesterone receptors in non-cyclic mares treated only with long-acting progesterone

Administration of progesterone (P4) after estradiol is usually performed to prepare non-cyclic mares as embryo recipients. However, there are successful pregnancy reports after embryo transfer in non-cyclic mares treated only with progestins. The objective of this study was to evaluate endometrial gene expression and immunostaining for estrogen receptor alpha (ERα), beta (ERβ) and progesterone receptor (PR) in seasonal anestrous mares treated with long acting P4 (LA P4). Endometrial tissue from eight seasonal anestrous mares was collected immediately before administration of 1.5 g of LA P4 and five days after. The receptors protein expression was evaluated by immunohistochemistry and the percentage of the immunostained area was determined by ImageJ software. Transcripts abundance for ERα, ERβ and PR were determined by RT-qPCR. Blood samples were collected daily to measure plasma P4 concentrations. Protein expression for ERα was greater (p < 0.05) after LA P4 administration, although gene expression was not affected by treatment (p > 0.05). There was no difference for ERβ protein expression (p = 0.07) and ERβ gene expression was reduced (p < 0.05) after treatment. Gene and protein expression for PR was not altered (p > 0.05). In conclusion, endometrial PR and ERα expression patterns after LA P4 administration were similar to those previously found in protocols using estradiol prior to LA P4 to prepare non-cyclic mares as embryo recipients.

Polycomb subunit BMI1 determines uterine progesterone responsiveness essential for normal embryo implantation.

Natural and synthetic progestogens have been commonly used to prevent recurrent pregnancy loss in women with inadequate progesterone secretion or reduced progesterone sensitivity. However, the clinical efficacy of progesterone and its analogs for maintaining pregnancy is variable. Additionally, the underlying cause of impaired endometrial progesterone responsiveness during early pregnancy remains unknown. Here, we demonstrated that uterine-selective depletion of BMI1, a key component of the polycomb repressive complex-1 (PRC1), hampers uterine progesterone responsiveness and derails normal uterine receptivity, resulting in implantation failure in mice. We further uncovered genetic and biochemical evidence that BMI1 interacts with the progesterone receptor (PR) and the E3 ligase E6AP in a polycomb complex-independent manner and regulates the PR ubiquitination that is essential for normal progesterone responsiveness. A close association of aberrantly low endometrial BMI1 expression with restrained PR responsiveness in women who had previously had a miscarriage indicated that the role of BMI1 in endometrial PR function is conserved in mice and in humans. In addition to uncovering a potential regulatory mechanism of BMI1 that ensures normal endometrial progesterone responsiveness during early pregnancy, our findings have the potential to help clarify the underlying causes of spontaneous pregnancy loss in women.

Androgens Upregulate Endometrial Epithelial Progesterone Receptor Expression: Potential Implications for Endometriosis

Androgenic compounds have been implicated in induction of endometrial atrophy yet the mechanisms of androgen effects on human endometrium have not been well studied. We hypothesized that androgens may promote their endometrial effects via modulation of progesterone receptor (PR) expression. Proliferative phase endometrial samples were collected at the time of hysterectomy. We evaluated the effect of the potent androgen 5α-dihydrotestosterone (DHT) on endometrial PR expression by treating human endometrial explants, endometrial stromal cells, and Ishikawa cells with DHT. Ishikawa cells were also treated with DHT ± the androgen receptor (AR) blocker flutamide. The PR-B, total PR messenger RNA (mRNA), and PR protein expression were assessed. Expression of cyclin D1 and D2 was checked as markers of cell proliferation. As expected, estradiol induced PR expression in isolated stromal cells, endometrial epithelial cells, and tissue explants. The DHT treatment also resulted in increased PR expression in endometrial explants and Ishikawa cells but not in stromal cells. Further, protein levels of both nuclear PR isoforms (PR-A and PR-B) were induced with the DHT treatment. Although flutamide treatment alone did not affect PR expression, flutamide diminished androgen-induced upregulation of PR in both endometrial explants and Ishikawa cells. Although estradiol induced both cyclin D1 and cyclin D2 mRNA, DHT did not induce these markers of cell proliferation. Androgens may mediate endometrial effects through upregulation of PR gene and protein expression. Endometrial PR upregulation by androgens is mediated, at least in part, through AR.

Reproductive Characteristics in Old and Young Subfertile Mares: Are They Really Different?

Physiological and pathological mechanisms that determine subfertility (pregnancy failure, irregular cycles, and abnormalities in reproductive tract) in old mares (OM) have being studied by many authors. However, some young mares also share this reproduction condition although no previous reports have being published. We decided to investigate reproductive parameters of young subfertile mares (YSM) in order to understand the basis of their reproductive behavior. Forty-nine subfertile mares were classified and separated into 2 groups: YSM (3–10 years old; n = 28) and OM (13–23 years old; n = 21). Different number of cycles (1–8) was used for data analysis on the embryo recovery rate (ERR), interovulatory interval (IOI), multiple ovulation rate (MOR) and plasmatic progesterone. Embryo quality was evaluated by gene expression through RNAm analysis. Effluent samples were taken for bacteriological and cytological evaluation and endometrial biopsies were performed to evaluate the presence of inflammatory cells and endometrial progesterone receptors (PR). There was no significant differences in ERR (P = .1230) on the percentage of each embryonic stage found on the different days of flushing (P > .05); on embryo gene expression (P > .05); on MOR (P = .1218); and on plasmatic progesterone at day 8 PO (P = .1230). However, differences were found on the percentage of positive cytologies (P = .0122) and bacteriological cultures (P = .0023); the amount of mononuclear cells in biopsies (P < .05) and distribution of PR on endometrial localization. In conclusion, YSM share some physiopathological mechanisms with OM that could explain their reproductive performance’s similarities.

Uterine Cavity Assessment and Endometrial Hormonal Receptors in Women with Peri and Post Menopausal Bleeding

Objectives: To compare the accuracy of 2D-Transvagfinal ultrasound (TVUS), saline infused sonohysterography (SIS) and hysteroscopy (DH) in assessment of the uterine cavity in women with peri- and postmenopausal bleeding and to study the expression of endometrial estrogen receptors (ER) and progesterone receptors (PR) in them. Study design: 100 women with abnormal uterine bleeding (peri and postmenopausal) were subjected to TVUS, SIS and DH and fractional curettage followed by histopathological examination and immunohistochemical analysis for ER and PR. Results: Measurement of endometrial thickness by TVUS showed a significant difference between normal and atrophic endometrium and between atrophic endometrium and endometrial polyp (P value 0.004 and 0.001 respectively) DH had the best sensitivity, specificity, PPV and NPV as a diagnostic procedure followed by SIS then TVUS (97.7, 100,100,99.4 % vs. 74,91.2,67.3,93.5 and 52.9,89.4,56.3, 88.1 respectively) Both ER and PR scoring among glands and stroma showed a significant difference between normal and abnormal endometrium. ER expression in glands showed a significant difference between endometrial polyp and surrounding endometrium (P value 0.006) Conclusions: Sonohysterography is superior to ultrasound and very close to hysteroscopy, especially with intra-cavitary lesions. Hysteroscopy remains the gold standard for uterine cavity assessment, but cannot replace the histopathology. The expression of endometrial steroid receptors is important in the pathogenesis of endometrial polyps and endometrial hyperplasia.