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Prion proteins constitute a major public health concern, which has partly overshadowed their physiological roles in several scenarios. Indeed, these proteins were implicated in male fertility but their role in female fertility is relatively less explored. This study was designed to evaluate the role of SPRN and PRNP prion family genes in bovine follicular steroidogenesis pathways. Post-transcriptional SPRN and PRNP silencing with siRNAs was established in bovine granulosa cell (GC) in vitro culture, and gene expression and progesterone and estradiol concentrations were evaluated. SPRN knockdown, led to a downregulation of CYP11A1 mRNA levels (2.1-fold), and PRNP knockdown led to an upregulation of SPRN mRNA levels (2.3-fold). CYP19A1 expression and estradiol synthesis was not detected in any experimental group. Finally, SPRN knockdown led to a mild reduction in progesterone production in GCs and this was the only experimental group that did not exhibit an increment in progesterone levels after 48 h of culture. As a conclusion, it was possible to detect the expression of the SPRN gene in bovine GCs, a potential interaction between SPRN and PRNP regulation, and the impact of SPRN expression on CYP11A1 and progesterone levels. These findings bring new insights into the role of these genes in ovarian steroidogenesis and female reproductive physiology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In high-yielding dairy cows, the rapidly increasing milk production after parturition can result in a negative nutrient balance, since feed intake is insufficient to cover the needs for lactation. Mobilizing body reserves, mainly adipose tissue (AT), might affect steroid metabolism. We hypothesized, that cows differing in the extent of periparturient lipomobilization, will have divergent steroid profiles measured in serum and subcutaneous (sc)AT by a targeted metabolomics approach and steroidogenic enzyme profiles in scAT and liver. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to a high (HBCS) or normal body condition (NBCS) group fed differently until week 7 antepartum to either increase (HBCS BCS: 3.8 ± 0.1 and BFT: 2.0 ± 0.1 cm; mean ± SEM) or maintain BCS (NBCS BCS: 3.0 ± 0.1 and BFT: 0.9 ± 0.1 cm). Blood samples, liver, and scAT biopsies were collected at week -7, 1, 3, and 12 relative to parturition. Greater serum concentrations of progesterone, androsterone, and aldosterone in HBCS compared to NBCS cows after parturition, might be attributed to the increased mobilization of AT. Greater glucocorticoid concentrations in scAT after parturition in NBCS cows might either influence local lipogenesis by differentiation of preadipocytes into mature adipocytes and/or inflammatory response., (© 2022. The Author(s).)

Oncogenic fusion genes as the result of chromosomal rearrangements are important for understanding genome instability in cancer cells and developing useful cancer therapies. To date, the mechanisms that create such oncogenic fusion genes are poorly understood. Previously we reported an unappreciated RNA-driven mechanism in human prostate cells in which the expression of chimeric RNA induces specified gene fusions in a sequence-dependent manner. One fundamental question yet to be addressed is whether such RNA-driven gene fusion mechanism is generalizable, or rather, a special case restricted to prostate cells. In this report, we demonstrated that the expression of designed chimeric RNAs in human endometrial stromal cells leads to the formation of JAZF1-SUZ12, a cancer fusion gene commonly found in low-grade endometrial stromal sarcomas. The process is specified by the sequence of chimeric RNA involved and inhibited by estrogen or progesterone. Furthermore, it is the antisense rather than sense chimeric RNAs that effectively drive JAZF1-SUZ12 gene fusion. The induced fusion gene is validated both at the RNA and the genomic DNA level. The ability of designed chimeric RNAs to drive and recapitulate the formation of JAZF1-SUZ12 gene fusion in endometrial cells represents another independent case of RNA-driven gene fusion, suggesting that RNA-driven genomic recombination is a permissible mechanism in mammalian cells. The results could have fundamental implications in the role of RNA in genome stability, and provide important insight in early disease mechanisms related to the formation of cancer fusion genes., Competing Interests: The authors have declared that no competing interests exist.

An inappropriate response to progestogens in the human endometrium can result in fertility issues and jeopardize progestin-based treatments against pathologies such as endometriosis. PGRMC1 can mediate progesterone response in the breast and ovaries but its endometrial functions remain unknown. AG-205 is an alleged PGRMC1 inhibitor but its specificity was recently questioned. We added AG-205 in the cultures of two endometrial cell lines and performed a transcriptomic comparison. AG-205 significantly increased expression of genes coding enzymes of the cholesterol biosynthetic pathway or of steroidogenesis. However, these observations were not reproduced with cells transfected with siRNA against PGRMC1 or its related proteins (MAPRs). Furthermore, AG-205 retained its ability to increase expression of selected target genes even when expression of PGRMC1 or all MAPRs was concomitantly downregulated, indicating that neither PGRMC1 nor any MAPR is required to mediate AG-205 effect. In conclusion, although AG-205 has attractive effects encouraging its use to develop therapeutic strategies, for instance against breast cancer, our study delivers two important warning messages. First, AG-205 is not specific for PGRMC1 or other MAPRs and its mechanisms of action remain unclear. Second, due to its effects on genes involved in steroidogenesis, its use may increase the risk for endometrial pathologies resulting from imbalanced hormones concentrations.

Its semi-allogeneic nature renders the conceptus vulnerable to attack by the maternal immune system. Several protective mechanisms operate during gestation to correct the harmful effects of anti-fetal immunity and to support a healthy pregnancy outcome. Pregnancy is characterized by gross alterations in endocrine functions. Progesterone is indispensable for pregnancy and humans, and it affects immune functions both directly and via mediators. The progesterone-induced mediator - PIBF - acts in favor of Th2-type immunity, by increasing Th2 type cytokines production. Except for implantation and parturition, pregnancy is characterized by a Th2-dominant cytokine pattern. Progesterone and the orally-administered progestogen dydrogesterone upregulate the production of Th2-type cytokines and suppress the production of Th1 and Th17 cytokine production in vitro . This is particularly relevant to the fact that the Th1-type cytokines TNF-α and IFN-γ and the Th17 cytokine IL-17 have embryotoxic and anti-trophoblast activities. These cytokine-modulating effects and the PIBF-inducing capabilities of dydrogesterone may contribute to the demonstrated beneficial effects of dydrogesterone in recurrent spontaneous miscarriage and threatened miscarriage. IL-17 and IL-22 produced by T helper cells are involved in allograft rejection, and therefore could account for the rejection of paternal HLA-C-expressing trophoblast. Th17 cells (producing IL-17 and IL-22) and Th22 cells (producing IL-22) exhibit plasticity and could produce IL-22 and IL-17 in association with Th2-type cytokines or with Th1-type cytokines. IL-17 and IL-22 producing Th cells are not harmful for the conceptus, if they also produce IL-4. Another important protective mechanism is connected with the expansion and action of regulatory T cells, which play a major role in the induction of tolerance both in pregnant women and in tumour-bearing patients. Clonally-expanded Treg cells increase at the feto-maternal interface and in tumour-infiltrating regions. While in cancer patients, clonally-expanded Treg cells are present in peripheral blood, they are scarce in pregnancy blood, suggesting that fetal antigen-specific tolerance is restricted to the foeto-maternal interface. The significance of Treg cells in maintaining a normal materno-foetal interaction is underlined by the fact that miscarriage is characterized by a decreased number of total effector Treg cells, and the number of clonally-expanded effector Treg cells is markedly reduced in preeclampsia. In this review we present an overview of the above mechanisms, attempt to show how they are connected, how they operate during normal gestation and how their failure might lead to pregnancy pathologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Piccinni, Raghupathy, Saito and Szekeres-Bartho.)

Progesterone is involved in dendritogenesis, synaptogenesis and maturation of cerebellar Purkinge cells, major sites of steroid synthesis in the brain. To study a possible time-relationship between myelination, neurosteroidogenesis and steroid receptors during development of the postnatal mouse cerebellum, we determined at postnatal days 5 (P5),18 (P18) and 35 (P35) the expression of myelin basic protein (MBP), components of the steroidogenic pathway, levels of endogenous steroids and progesterone's classical and non-classical receptors. In parallel with myelin increased expression during development, P18 and P35 mice showed higher levels of cerebellar progesterone and its reduced derivatives, higher expression of steroidogenic acute regulatory protein (StAR) mRNA, cholesterol side chain cleavage enzyme (P450scc) and 5α-reductase mRNA vs. P5 mice. Other steroids such as corticosterone and its reduced derivatives and 3β-androstanodiol (ADIOL) showed a peak increase at P18 compared to P5. Progesterone membrane receptors and binding proteins (PGRMC1, mPRα, mPRβ, mPRγ, and Sigma1 receptors) mRNAs levels increased during development while that of classical progesterone receptors (PR) remained invariable. PRKO mice showed similar MBP levels than wild type. Thus, these data suggests that progesterone and its neuroactive metabolites may play a role in postnatal cerebellar myelination., (Published by Elsevier Ltd.)

Progesterone modulates many processes in the body, acting through nuclear receptors (nPR) in various organs and tissues. However, a number of effects are mediated by membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ (PAQR) receptor family. These receptors are found in most tissues and immune cells. They are expressed in various cancer cells and appear to play an important role in the development of tumors. The role of mPRs in the development of insulin resistance and metabolic syndrome has also attracted attention. Since progesterone efficiently binds to both nPRs and mPRs, investigation of the functions of the mPRs both at the level of the whole body and at the cell level requires ligands that selectively interact with mPRs, but not with nPRs, with an affinity comparable with that of the natural hormone. The development of such ligands faces difficulties primarily due to the lack of data on the three-dimensional structure of the ligand-binding site of mPR. This review is the first attempt to summarize available data on the structures of compounds interacting with mPRs and analyze them in terms of the differences in binding to membrane and nuclear receptors. Based on the identified main structural fragments of molecules, which affect the efficiency of binding to mPRs and are responsible for the selectivity of interactions, we propose directions of modification of the steroid scaffold to create new selective mPRs ligands., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: This study sought to identify the initiation of placental hormonal production as defined by the production of endogenous estradiol (E2) and progesterone (P4) in a cohort of patients undergoing programmed endometrial preparation cycles with single embryo transfers resulting in live-born singletons., Methods: In this retrospective cohort study, patients undergoing either programmed frozen-thawed embryo transfer (FET) with autologous oocytes or donor egg recipient (DER) cycles with fresh embryos were screened for inclusion. Only patients who underwent a single embryo transfer, had a single gestational sac, and a resultant live-born singleton were included. All patients were treated with E2 patches and intramuscular progesterone injections. Main outcome measures were serial E2 and P4, with median values calculated for cycle days 28 (baseline), or 4w0d gestational age (GA), through 60, or 8w4d GA. The baseline cycle day (CD) 28 median value was compared to each daily median cycle day value using the Wilcoxon signed rank test., Results: A total of 696 patients, 569 using autologous oocytes in programmed FET cycles and 127 using fresh donor oocytes, from 4/2013 to 4/2019 met inclusion criteria. Serum E2 and P4 levels stayed consistent initially and then began to increase daily. Compared to baseline CD 28 E2 (415 pg/mL), the serum E2 was significantly elevated at 542 pg/mL (P < 0.001) beginning on CD 36 (5w1d GA). With respect to baseline CD 28 P4 (28.1 ng/mL), beginning on CD 48 (6w6d GA), the serum P4 was significantly elevated at 31.6 ng/mL (P < 0.001)., Conclusion: These results demonstrate that endogenous placental estradiol and progesterone production may occur by CD 36 and CD 48, respectively, earlier than traditionally thought.

Background: Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ 1 -dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq., Results: Although the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex. New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex. Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ 1 -dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76&#95;27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ 1 -dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)-dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D., Conclusion: The results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications.

Purpose: To evaluate whether adjusting timing of modified natural cycle frozen embryo transfer (mNC-FET) 1 day earlier in the setting of a spontaneous LH surge has an impact on pregnancy outcomes., Methods: This retrospective cohort study evaluated all mNC-FET with euploid blastocysts from May 1, 2016 to March 30, 2019, at a single academic institution. Standard protocol for mNC-FET included ultrasound monitoring and hCG trigger when the dominant follicle and endometrial lining were appropriately developed. Patients had serum LH, estradiol, and progesterone checked on day of trigger. If LH was ≥ 20 mIU/mL, trigger was given that day and FET was performed 6 days after surge (LH/HCG+6), with the intent of transferring 5 days after ovulation. If LH was < 20 mIU/mL, FET was performed 7 days after trigger (hCG+7). Primary outcomes included clinical pregnancy and live birth rates. To account for correlation between cycles, a generalized estimating equation (GEE) method for multivariable logistic regression was used., Results: Four hundred fifty-three mNC-FET cycles met inclusion criteria, of which 205 were in the LH/HCG+6 group and 248 were in the HCG+7 group. The overall clinical pregnancy rate was 64% and clinical miscarriage rate was 4.8%, with similar rates between the two groups. The overall live birth rate was 60.9% (61.0% in LH/HCG+6 group and 60.9% in HCG+7 group). After implementing GEE, the odds of CP (aOR 0.97, 95% CI [0.65-1.45], p = 0.88) and LB (aOR 0.98, 95% CI [0.67-1.45], p = 0.93) were similar in both groups., Conclusions: In our study cohort, mNC-FET based on LH/HCG+6 versus HCG+7 had similar pregnancy outcomes.

Wilms' tumor gene WT1 encodes a nuclear transcriptional factor, which has been shown to regulate granulosa cell steroidogenesis in bovine; however, it is not known whether the functions of theca cells are regulated by WT1. Here, we determined the effects of this gene on theca cell proliferation, apoptosis, and steroidogenesis in vitro. In cultured bovine theca cells, the downregulation of WT1 increased the secretion of progesterone but had no effect on proliferation and apoptosis. WT1 includes the variants WT1(+KTS) and WT1(-KTS), which differ by 3 amino acids KTS (lysine, threonine, and serine). WT1(±KTS) upregulation increased the messenger RNA (mRNA) expression of STAR and CYP17A1 and decreased the progesterone secretion and CYP11A1 mRNA expression. In contrast to WT1(+KTS), WT1(-KTS) upregulation also decreased the mRNA expression of 3β-HSD. In both variants, WT1(-KTS) has more obvious effects. In conclusion, WT1 can decrease progesterone secretion, likely due in part to the inhibition of CYP11A1 and 3β-HSD., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cytochromes P450 are key enzymes for steroid hormone biosynthesis in human body. They are considered as targets for the screening of novel high efficient drugs. The results of screening of bile acids and androstane derivatives toward human recombinant steroid 17α-hydroxylase/17,20-lyase (CYP17A1) are presented in this paper. A group of steroids, binding with micromolar or submicromolar affinity (in a range from 9 μM - less than 0.1 μM), was identified. Results presented here showed that these steroidal compounds are able to decrease rate of hydroxylation of essential CYP17A1 substrate - progesterone, while some compounds completely inhibited enzyme activity. Structure-activity relationship (SAR) analysis based on in vitro and in silico studies showed that high affinity of the enzyme to bile acids derivatives is correlated with side chain hydrophobicity and presence of hydroxyl or keto group at C 3 position. From the other side, bile acid-derived compounds with more polar side chain or substituents at C 7 and C 12 positions possess higher K d values. Among androstane-derived steroids couple of Δ 5 -steroids with hydroxyl group at C 3 position, as well as 16,17-secosteroids, were found to be high affinity ligands of this enzyme. The data obtained could be useful for the design of novel highly efficient inhibitors of CYP17A1, since the bile acids-derived compounds are for first time recognized as effective CYP17A1 inhibitors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: In recent years, GATA-binding protein 3 (GATA3) has been indicated as a marker showing good prognosis in breast cancer. In luminal breast cancer, which has good a prognosis, it shows more significant elevation in small-sized and low-grade tumors. In contrast, Ki-67 is defined as a poor prognostic factor. The aim of this study was to emphasise the prognostic importance of GATA3 and the inverse relationship with Ki-67., Materials and Methods: In our study, 90 patients with invasive ductal breast cancer were immunohistochemically evaluated for Ki-67 and GATA3 expression. The relationship between GATA3 and Ki-67 expression was examined. In addition, the relationship between these two factors with estrogen, progesterone, human epidermal growth factor 2 receptor antibodies and other prognostic parameters such as disease-free survival and local recurrence was investigated. We accepted the level of ≥5% nüclear reaction as positive for GATA 3. A Ki-67 cut-off value of 20% was accepted as positive., Results: In GATA3 positive breast cancers, good prognostic parameters were seen including high estrogen receptor (ER) positivity, progesterone receptor (PR) positivity, small tumor size and low histological grade as well as low Ki-67 expression. In breast cancers showing high Ki-67 expression, ER, PR, and GATA3 positivity were lower and there was higher human epidermal growth factor receptor 2 (HER2) positivity and high histological grade while the tumor size was larger., Conclusion: Our study has revealed that GATA3 has an inverse relationship with Ki-67, whereas it has a positive releationship with good prognostic factors., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Emerging evidence indicates that dietary n-3 polyunsaturated fatty acids (PUFA) alter the fatty acid composition of corpus luteum (CL) and directly affect the luteal function in the cow, which is independent of the inhibitory effect on the endometrial PGF 2α production. The present study, thus, investigated the effects of n-3 PUFA rich fish oil (FO) supplementation on the transcriptional modulation of genes involved in the biosynthesis of progesterone (P 4 ) in the CL collected during the luteolytic phase of oestrous cycle in the goat. On the day of synchronized oestrus, goats (n = 6/group) were fed an isocaloric diet supplemented with either FO or palm oil (PO). The dose of oil supplementation was 0.6 mlkg -1 body weight, and the duration was 55-57 days. The FO provided 156 mgkg -1 body weight of n-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The CL was collected by laparotomy on day 16 post-oestrus, and the relative abundance of P450 side-chain cleaving enzyme, steroid acute regulatory protein (StAR) and 3β-hydroxy steroid dehydrogenase (3β-HSD) genes was quantitated by real-time PCR. The results indicated that the dietary FO significantly upregulated the expression of 3β-HSD by 1.13-fold and downregulated StAR by ~2-fold as compared to PO group (p < .05). It is concluded that dietary FO differently affected the expression of genes involved in P 4 synthesis in the CL during the luteolytic window of the oestrous cycle in the goat., (© 2020 Blackwell Verlag GmbH.)

Chemerin is a recently discovered adipokine that participates in the regulation of many physiological and disorder-related processes in mammals, including metabolism, inflammatory reactions, obesity, and reproduction. We investigated how chemerin affects the transcriptome profile of porcine luteal cells. The luteal cells were acquired from mature gilts. After the in vitro culturing with and without chemerin, the total RNAs were isolated and high-throughput sequencing was performed. Obtained datasets were processed using bioinformatic tools. The study revealed 509 differentially expressed genes under the chemerin influence. Their products take part in many processes, important for the functions of the corpus luteum, such as steroids and prostaglandins synthesis, NF-κB and JAK/STAT signal transducing pathways, and apoptosis. The expression of the CASP3 , HSD3B7 , IL1B , and PTGS2 genes, due to their important role in the physiology of the corpus luteum, was validated using the quantitative real-time polymerase chain reaction (qPCR) method. The qPCR confirmed the changes of gene expression. Chemerin in physiological concentrations significantly affects the expression of many genes in luteal cells of pigs, which is likely to result in modification of physiological processes related to reproduction.

Purpose: To examine the expression of MUC16 in the endometrium peri-implantation period in three different cohort studies., Methods: This was a retrospective observational cohort study. A total of 245 participants were recruited in three separate cohort studies: (1) women with recurrent miscarriage (n = 50) and fertile controls (n = 29); (2) women who had high (n = 20) or normal (n = 20) progesterone on the day of hCG trigger in ovarian stimulation cycle for IVF; and (3) women who did (n = 95) or did not (n = 31) conceive following frozen embryo transfer in HRT cycles. All subjects had archived endometrial samples precisely taken on LH+7 in natural cycles, or hCG+6 in ovarian stimulation cycles, or P+5 in HRT cycles. The H-score (median, range) of MUC16 in the luminal epithelium and glandular epithelium was determined by using immunohistochemistry., Results: The median (range) of H-score of MUC16 in the luminal epithelium (1) in women with recurrent pregnancy loss was 23.7 (0-300), which was significantly (P < 0.05) lower than that of 118.4 (7.7-300) in fertile controls; (2) in women with elevated progesterone on the day of hCG administration (147.8, 18.0-230.1), significantly (P < 0.05) higher than that of women with normal progesterone (61.0, 2.3-205.3); (3) in women who conceived (23.1, 0-250.3), significantly (P < 0.001) lower than that in women who did not conceive (58.4, 0-300)., Conclusion: The expression of MUC16 in all three cohort studies is consistent with it being an inhibitor of implantation.

Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) plays a pivotal role in the regulation of adrenal and gonadal steroid hormone biosynthesis. More recent studies highlighted the enzyme's role in the backdoor pathway leading to androgen production. Increased CYP17A1 activity in endocrine disorders and diseases are associated with elevated C 21 and C 19 steroids which include 17α-hydroxyprogesterone and androgens, as well as C11-oxy C 21 and C11-oxy C 19 steroids. We previously reported that 11β-hydroxyprogesterone (11OHP4), 21-deoxycortisol (21dF) and their keto derivatives are converted by 5α-reductases and hydroxysteroid dehydrogenases yielding C 19 steroids in the backdoor pathway. In this study the 17α-hydroxylase and 17,20-lyase activity of CYP17A1 towards the unconventional C11-oxy C 21 steroid substrates and their 5α- and 3α,5α-reduced metabolites was investigated in transfected HEK-293 cells. CYP17A1 catalysed the 17α-hydroxylation of 11OHP4 to 21dF and 11-ketoprogesterone (11KP4) to 21-deoxycortisone (21dE) with negligible hydroxylation of their 5α-reduced metabolites while no lyase activity was detected. The 3α,5α-reduced C11-oxy C 21 steroids-5α-pregnan-3α,11β-diol-20-one (3,11diOH-DHP4) and 5α-pregnan-3α-ol-11,20-dione (alfaxalone) were rapidly hydroxylated to 5α-pregnan-3α,11β,17α-triol-20-one (11OH-Pdiol) and 5α-pregnan-3α,17α-diol-11,20-dione (11K-Pdiol), with the lyase activity subsequently catalysing to conversion to the C11-oxy C 19 steroids, 11β-hydroxyandrosterone and 11-ketoandrosterone, respectively. Docking of 11OHP4, 11KP4 and the 5α-reduced metabolites, 5α-pregnan-11β-ol-3,20-dione (11OH-DHP4) and 5α-pregnan-3,11,20-trione (11K-DHP4) with human CYP17A1 showed minimal changes in the orientation of these C11-oxy C 21 steroids in the active pocket when compared with the binding of progesterone suggesting the 17,20-lyase is impaired by the C11-hydroxyl and keto moieties. The structurally similar 3,11diOH-DHP4 and alfaxalone showed a greater distance between C17 and the heme group compared to the natural substrate, 17α-hydroxypregnenolone potentially allowing more orientational freedom and facilitating the conversion of the C11-oxy C 21 to C11-oxy C 19 steroids. In summary, our in vitro assays showed that while CYP17A1 readily hydroxylated 11OHP4 and 11KP4, the enzyme was unable to catalyse the 17,20-lyase reaction of these C11-oxy C 21 steroid products. Although CYP17A1 exhibited no catalytic activity towards the 5α-reduced intermediates, once the C4-C5 double bond and the keto group at C3 were reduced, both the hydroxylation and lyase reactions proceeded efficiently. These findings show that the C11-oxy C 21 steroids could potentially contribute to the androgen pool in tissue expressing steroidogenic enzymes in the backdoor pathway., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Progesterone receptor (PR) is expressed in a wide variety of human tissues, including both reproductive and non-reproductive tissues. Upon binding to the PR, progesterone can display several non-reproductive functions, including neurosteroid activity in the central nervous system, inhibition of smooth muscle contractile activity in the gastrointestinal tract, and regulating the development and maturation of the lung. PR exists as two major isoforms, PRA and PRB. Differential expression of these PR isoforms reportedly contributes to different biological activities of the hormone. However, the distribution of the PR isoforms in human tissues has remained virtually unexplored. In this study, we immunolocalized PR expression in various human tissues using PR (1294) specific antibody, which is capable of detecting both PRA and PRB, and PRB (250H11) specific antibody. Tissues from the uterus, ovary, breast, placenta, prostate, testis, cerebrum, cerebellum, pituitary, spinal cord, esophagus, stomach, small intestine, colon, pancreas, liver, kidney, urinary bladder, lung, heart, aorta, thymus, adrenal gland, thyroid, spleen, skin, and bone were examined in four different age groups (fetal, pediatric, young, and old) in male and female subjects. PR and PRB were detected in the nuclei of cells in the female reproductive system, in both the nuclei and cytoplasm of pituitary gland and pancreatic acinar cells, and only in the cytoplasm of cells in the testis, stomach, small intestine, colon, liver, kidney, urinary bladder, lung, adrenal gland, and skin. Of particular interest, total PRB expression overlapped with that of total PR expression in most tissues but was negative in the female fetal reproductive system. The findings indicate that progesterone could affect diverse human organs differently than from reproductive organs. These findings provide new insights into the novel biological roles of progesterone in non-reproductive organs., Competing Interests: Declaration of Competing Interest Authors report no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Medroxyprogesterone acetate (MPA) is a first generation progestin that has been in clinical use for various hormonal conditions in women since the 1960s. Although developed as a progesterone receptor (PR) agonist, MPA also has strong binding affinity for other steroid receptors. This promiscuity confounds the mechanistic action of MPA in target cells that express multiple steroid receptors. This study is the first to assess the relative contribution of progesterone, androgen and glucocorticoid receptors in mediating the transcriptional activity of MPA on endogenous targets in breast cancer cells that endogenously express all three receptors at comparable levels. Gene expression profiling in estrogen receptor positive (ER+) ZR-75-1 breast cancer cells demonstrated that although the MPA-regulated transcriptome strongly overlapped with that of Progesterone (PROG), 5α-dihydrotestosterone (DHT) and Dexamethasone (DEX), it clustered most strongly with that of PROG, suggesting that MPA predominantly acts via the progesterone receptor (PR) rather than androgen receptor (AR) or glucocorticoid receptor (GR). Subsequent experiments manipulating levels of these receptors, either through specific culture conditions or with lentiviral shRNAs targeting individual receptors, also revealed a stronger contribution of PR compared to AR and GR on the expression of endogenous target genes that are either commonly regulated by all ligands or specifically regulated only by MPA. A predominant contribution of PR to MPA action in ER+ T-47D breast cancer cells was also observed, although a stronger role for AR was evident in T-47D compared to that observed in ZR-75-1 cells. Network analysis of ligand-specific and commonly regulated genes demonstrated that MPA utilises different transcription factors and signalling pathways to inhibit proliferation compared with PROG. This study reaffirms the importance of PR in mediating MPA action in an endogenous breast cancer context where multiple steroid receptors are co-expressed and has potential implications for PR-targeting therapeutic strategies in ER+ breast cancer., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

H3K27me3 is an epigenetic modification that results in the repression of gene transcription. The transcription factor RUNX1 (the runt-related transcription factor 1) influences granulosa cells' growth and ovulation. This research uses ELISA, flow cytometry, EDU, ChIP-PCR, WB and qPCR to investigate steroidogenesis, cell apoptosis, and the proliferation effect of RUNX1 in porcine granulosa cells (pGCs) as regulated by H3K27me3. Decreased H3K27me3 stimulates the expression of steroidogenesis-related genes, including CYP11A1 , PTGS2 , and STAR , as well as prostaglandin. H3K27me3 transcriptionally represses RUNX1 here, whereas RUNX1 acts as an activator of FSHR , CYP11A1 , and CYP19A1 , promoting the production of androgen, estrogen, and prostaglandin, as well as increasing anti-apoptotic and cell proliferation activity, but decreasing progesterone. Both the complementary recovery of the H3K27me3 antagonist with the siRUNX1 signal, and the H3K27me3 agonist with the RUNX1 signal to maintain RUNX1 lead to the activation of CYP19A1 , ER1 , HSD17β4 , and STAR here. Androgen and prostaglandin are significantly repressed but progesterone is markedly increased with the antagonist and siRUNX1. Prostaglandin is significantly promoted with the agonist and RUNX1. Furthermore, H3K27me3-RUNX1 affects the anti-apoptotic activity and stimulation of proliferation in pGCs. The present work verifies the transcriptional suppression of RUNX1 by H3K27me3 during antral follicular development and maturation, which determines the levels of hormone synthesis and cell apoptosis and proliferation in the pGC microenvironment.

Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2), in the granulosa cells of preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here, we show that the PGR plays an additional essential role: it attenuates ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the nuclear factor κB (NF-κB), a transcription factor required for Ptgs2 expression. When the expression of PGR is ablated in granulosa cells, the ovary undergoes a hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. The PGR-driven termination of PTGS2 expression may protect the ovary from ovulatory inflammation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Toll-like receptors (TLRs) are present in the ovaries and reproductive tract of various mammals. The biological function of TLR during ovulation is one of the main contents in the research of reproductive immunology. In this study, we found that messenger RNA levels of TLR1-TLR10 in granulosa cells were different, and TLRs and high mobility group box 1 (HMGB1) in granulosa cells of large follicles were significantly higher than those of small and middle follicles. Coimmunoprecipitation results showed that HMGB1 interacts with TLR2 in granulosa cells, especially large follicles. The result of immunohistochemistry showed that TLRs and HMGB1 were present in granulosa cell layer of ovarian follicles. We also found 25 mIU/ml follicle-stimulating hormone (FSH) significantly upregulated the expression of TLRs and HMGB1. These results suggest that TLR2/4 and HMGB1 in granulosa cells may be involved in the ovarian innate immune and ovarian follicular maturation, regulated by FSH. However, further research of the function and mechanisms of TLRs and HMGB1 in granulosa cells are needed., (© 2019 Wiley Periodicals, Inc.)

Background: The development efficiency of cloned cattle is extremely low (< 5%), most of them were aborted at late gestation. Based on our previous studies, some recipient cows with a cloned fetus would present as engorged uterine vessels and enlarged umbilical vessels randomly. Abortion involves both maternal and fetal factors., Objective: Our aim was to explore this phenomenon by microRNAs expression profile analysis of maternal corpus luteum (CL), which was related to pregnancy maintenance., Methods: The present study provided the comparison of maternal CL miRNAs expression of abnormally and normally developed cloned bovine fetus at late gestation (~ 210 days) using RNA-Seq technology., Results: We selected two abnormally pregnant cows (abnormal group, AG) and three normally pregnant cows (normal group, NG) and acquired valid reads of 9317,261-12,327,185 (~ 84.53-91.28%) from five libraries. In total, we identified 981 conserved miRNAs and 223 novel miRNAs. 1052 miRNAs were co-expressed, 124 miRNAs were uniquely expressed in AG, and 93 miRNAs were uniquely expressed in the NG. Compared with NG, 11 were significantly overexpressed, and 22 were downregulated (p < 0.05) at AG among 1052 co-expressed miRNAs. The differentially expressed miRNAs-targeted genes were further analyzed by Gene Ontology and KEGG pathway analysis. Notably, the steroid biosynthesis pathway was a significantly enriched term (p < 0.01), which may affect the secretion of progesterone., Conclusion: Our research suggested that abnormal miRNAs expression of bovine maternal CL may affect the pregnant status at late gestation.

The aim was to evaluate in female roe deer: (a) PAG mRNA relative abundance in endometrial uterine tissue for determination of the duration of embryonic diapause, (b) mRNA relative abundance of progesterone, estradiol, and prolactin (P4, E2, and PRL) receptors (PGR, ESR, and PRLR) during diapause and after implantation in the endometrium; (c) concentration of P4, E2, and PRL in the blood, and (d) a noninvasive method of hormone detection by measurement of P4 and E2 concentrations in feces. A total of fifteen individuals were obtained post mortem during hunting seasons and divided into three experimental groups (November, December, January). The results did not reveal mRNA relative abundance for PAGs in the endometrium or detectable PAG concentrations in the serum of all examined females. Concentration of PRL and mRNA relative abundance for PRLR long isoform in the endometrium was the highest in January (p < .01). mRNA relative abundance for PGR, P4 concentration in the endometrium, serum, and feces was the highest in January (p < .01). Endometrial origin PRL and P4 may be responsible for the termination of this process and pregnancy development after implantation., (© 2019 Japanese Society of Animal Science.)

The present in vitro study was conducted to examine the direct action of the plant steroidal sapogenin, diosgenin, on basic farm animal ovarian cell functions. As models, we used cultured porcine ovarian granulosa cells, porcine whole follicles, and rabbit ovarian fragments. The effects of diosgenin (0, 1, 10, or 100 μg/mL medium) on the markers of proliferation, cytoplasmic apoptosis, steroid (progesterone: P4, testosterone: T, and estradiol: E2) release, and peptide hormone (insulin-like growth factor I: IGF-I) release were analyzed by quantitative immunocytochemistry and radioimmunoassay. Diosgenin promoted proliferation, apoptosis, and T and E2 release and inhibited P4 output in cultured porcine granulosa cells. Similarly, cultured porcine ovarian follicles showed diosgenin-induced inhibition of P4 and stimulation of T release. In cultured rabbit ovarian fragments, diosgenin stimulated P4 and IGF-I release. This is the first study showing that diosgenin can promote basic ovarian cell functions such as proliferation, apoptosis, and steroid and peptide hormone release. The similar effects of diosgenin on porcine granulosa cells and ovarian follicles suggest that granulosa cells are the primary ovarian target of diosgenin. The contrasting effects of diosgenin on porcine and rabbit ovarian P4 output suggest that diosgenin functions in a species-specific manner. These observations indicate that diosgenin has potential applications for improving female reproduction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the role of sex steroid hormone receptors in corneal epithelium in etiopathogenesis of keratoconus (KC)., Methods: Thirty patients with KC who were planned for corneal collagen-crosslinking and 20 patients who were planned for excimer laser for refractive errors included in this study. Corneal epitheliums were curated mechanically during surgeries. Right eyes were evaluated immunohistochemically and left eyes were evaluated by quantitative polymerase chain reaction (qPCR) to investigate estrogenα, estrogenβ, progesterone and androgen receptors., Results: Immunohistochemically, staining for progesterone and androgen receptors did not significantly differ between KC and control groups (p > 0.05). None of the cases had staining for estrogenα and estrogenβ receptors. qPCR showed that mRNA expressions of estrogenα and androgen receptors were significantly higher in the KC group (p < 0.001)., Conclusion: A significantly higher rate of estrogenα and androgen receptor expressions in corneal epithelium from patients with KC through qPCR supports a possible relation between KC and sex steroid hormones., (Copyright © 2018 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Introduction: Exogenous progesterone is prescribed for a variety of conditions with endogenous progesterone deficiency, e.g. menstrual alterations, primary or secondary infertility or premenopause. To the best of our knowledge, no pharmacogenetic studies have been published in relation to exogenous progesterone pharmacokinetic safety or progesterone metabolites so far., Methods: Candidate-gene study where we evaluated whether five single-nucleotide polymorphisms (CYP2C9*2, *3, CYP2C19*2, *3 and *17) were related to the pharmacokinetics, safety and metabolism of progesterone in 24 healthy volunteers who received a 200-mg progesterone formulation either orally or vaginally., Results: The vaginal formulation had an average AUCt value approximately 18 times greater than the oral formulation. CYP2C19 intermediate metabolizers (IM) consistently showed higher adjusted AUCt and adjusted C max than extensive metabolizers (EM) (P < 0.05); CYP2C9 EM incongruently exhibited higher adjusted C max and longer half-life than IM (p < 0.05)., Conclusion: This is the first study that reports variability in progesterone disposition according to the CYP2C19 and CYP2C9 phenotype. We suggest that CYP2C19 may condition progesterone disposition and that it may be more relevant than CYP2C9. This study lays the foundations for further in-depth research to evaluate the pharmacogenetics of progesterone., Trial Registration: EudraCT numbers are 2012-005105-43 and 2012-005011-10.

Altered reproductive hormone levels have been associated with the pathophysiology of depressive disorders and this risk may be imparted by their modulatory effect upon hippocampal structure and function. Currently it is unclear whether altered levels of reproductive hormones are causally associated with hippocampal volume reductions and the risk of depressive disorders. Here, we utilize genome-wide association study (GWAS) summary statistics from a GWAS focusing on reproductive hormones, consisting of 2913 individuals. Using this data, we generated polygenic risk scores (PRS) for estradiol, progesterone, prolactin and testosterone in the European RADIANT cohort consisting of 176 postpartum depression (PPD) cases (100% female, mean age: 41.6 years old), 2772 major depressive disorder (MDD) cases (68.6% female, mean age: 46.9 years old) and 1588 control participants (62.5% female, mean age: 42.4 years old), for which there was also a neuroimaging subset of 111 individuals (60.4% female, mean age: 50.0 years old). Only the best-fit PRS for estradiol showed a significant negative association with hippocampal volume, as well as many of its individual subfields; including the molecular layer and granule cell layer of the dentate gyrus, subiculum, CA1, CA2/3 and CA4 regions. Interestingly, several of these subfields are implicated in adult hippocampal neurogenesis. When we tested the same estradiol PRS for association with case-control status for PPD or MDD there was no significant relationship observed. Here, we provide evidence that genetic risk for higher plasma estradiol is negatively associated with hippocampal volume, but this does not translate into an increased risk of MDD or PPD. This work suggests that the relationship between reproductive hormones, the hippocampus, and depression is complex, and that there may not be a clear-cut pathway for etiology or risk moderation., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Parturition involves cellular signaling changes driven by the complex interplay between progesterone (P4), inflammation, and the cyclic adenosine monophosphate (cAMP) pathway. To characterize this interplay, we performed comprehensive transcriptomic studies utilizing eight treatment combinations on myometrial cell lines and tissue samples from pregnant women. We performed genome-wide RNA-sequencing on the hTERT-HM${}^{A/B}$ cell line treated with all combinations of P4, forskolin (FSK) (induces cAMP), and interleukin-1$\beta$ (IL-1$\beta$). We then performed gene set enrichment and regulatory network analyses to identify pathways commonly, differentially, or synergistically regulated by these treatments. Finally, we used tissue similarity index (TSI) to characterize the correspondence between cell lines and tissue phenotypes. We observed that in addition to their individual anti-inflammatory effects, P4 and cAMP synergistically blocked specific inflammatory pathways/regulators including STAT3/6, CEBPA/B, and OCT1/7, but not NF$\kappa$B. TSI analysis indicated that FSK + P4- and IL-1$\beta$-treated cells exhibit transcriptional signatures highly similar to non-laboring and laboring term myometrium, respectively. Our results identify potential therapeutic targets to prevent preterm birth and show that the hTERT-HM${}^{A/B}$ cell line provides an accurate transcriptional model for term myometrial tissue., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Progesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.

Purpose: Patient management of invasive breast cancer (IBC) is to a large extent based on hormone- and HER2-receptor assessment. High-quality, reliable receptor assessment is of key importance as false results may lead to under- or overtreatment of patients. Surveillance of case-mix adjusted positivity rates has been suggested as a tool to identify laboratories with insufficient testing assays, as this covers the whole process of receptor assessment and enables laboratories to benchmark their positivity rates against other laboratories. We studied laboratory-specific variation in hormone- and HER2 positivity rates of 33,046 breast cancer patients using real-life nationwide data., Methods: All synoptic pathology reports of IBC resection-specimens, obtained between 2013 and 2016, were retrieved from the nationwide Dutch pathology registry (PALGA). Absolute and case-mix adjusted receptor positivity rates were compared to the mean national proportion and presented in funnel plots in separate analyses for estrogen (ER), progesterone (PR) and HER2. Case-mix adjustment was performed by multivariable logistic regression., Results: 33,794 IBC lesions from 33,046 patients of 39 pathology laboratories were included. After case-mix adjustment, mean positivity rates were 87.2% for ER (range 80.4-94.3), 71.3% for PR (62.5-77.5%), and 9.9% for HER2 (5.5-12.7%). Overall, 14 (35.9%), 17 (43.6%) and 11 (28.2%) laboratories showed positivity rates outside the 95% confidence interval for ER, PR and HER2, respectively., Conclusion: This nationwide study shows that absolute variation in hormone- and HER2-receptor positivity rates between Dutch pathology laboratories is limited. Yet, the considerable number of outlying laboratories shows that there is still need for improvement. Continuous monitoring and benchmarking of positivity rates may help to realize this.

Purpose: The estrogen (ER), progesterone (PR), and HER2 status are essential in guiding treatment decisions in breast cancer patients. In daily life, the ER/PR/HER2 status is expected to be commonly tested twice, i.e., at diagnosis using material from tumor needle biopsies, and after tumor resection using full tumor tissue material. This study explored the discordance of ER/PR/HER2 between tumor needle biopsies and full tumor resection material using real-world patient-level data from Dutch breast cancer patients., Methods: Pathology reports of 11,054 breast cancer patients were derived from PALGA (Dutch Pathology Registry). Discordance was calculated for multiple combinations of the ER/PR/HER2 receptor status. The influence of patient and tumor characteristics on the probability of having discordant test results was analyzed using multiple logistic regression models (separately for ER, PR and HER2)., Results: For 1279 patients (14.4%), at least one of the receptors (ER/PR/HER2) was determined on both biopsy and tumor tissue material. The majority had concordant test results for ER (n = 916; 94.8%), PR (n = 1170; 86.7%), and HER2 (n = 881; 98.1%). Patients having an ER- and HER2-positive but PR-negative biopsy classification, BR grade III, and < 10% tumor tissue remaining after neoadjuvant therapy (NAT) have the highest probability of ER discordant test results (OR 4.991; p = 83.31%). The probability of discordance in PR is based on different sets of patient and tumor characteristics. Potential cost savings from omitting multiple tests if concordance can be perfectly predicted can be up to €205,000 yearly., Conclusions: Double testing of ER/PR/HER2 is less common than expected. Discordance in ER/PR/HER2 test results between tumor needle biopsy taken at the time of diagnosis and tumor resection material is very low, especially in patients not receiving any form of neoadjuvant therapy. These results imply that a substantial number of tests can potentially be omitted in specific subgroups of breast cancer patients.

Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.

To better understand the function of oar-miR-150 in the ovine granulosa cells (GCs) during the estrus cycle, the five turpan sheep were selected for detection of the expression of oar-miR-150 in follicular and luteal ovaries, respectively. Then the granulosa cells treated with oar-miR-150 mimics or negative control (NC) were analyzed by qPCR to assess the expression of genes involved in steroidogenic and apoptosis. Expression of oar-miR-150 was increased in follicular phase compared with that in luteal ovaries. STAR was a target gene of miR-150 with oar-miR-150 mimic or inhibitor and luciferase reporter assay. Overexpression of oar-miR-150 promoted expression of bax, bcl2 and Casp3 expression and declined the expression of STAR, Cyp11a1 and HSD3B1 in vitro. Taken together, these results demonstrate that the oar-miR-150 promote GCs apoptosis through modifying the expression of genes involved in progesterone synthesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Granulosa cells (GCs) have many functions in the endocrine system. Most notably, they produce progesterone following ovulation. However, it has recently been proven that GCs can change their properties when subjected to long‑term culture. In the present study, GCs were collected from hyper‑stimulated ovarian follicles during in vitro fertilization procedures. They were grown in vitro, in a long‑term manner. RNA was collected following 1, 7, 15 and 30 days of culture. Expression microarrays were used for analysis, which allowed to identify groups of genes characteristic for particular cellular processes. In addition, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was performed to validate the obtained results. Two ontological groups characteristic for processes associated with the development and morphogenesis of the heart were identified during the analyses: 'Heart development' and 'heart morphogenesis'. The results of the microarrays revealed that the highest change in expression was demonstrated by the lysyl Oxidase, oxytocin receptor, nexilin F‑actin binding protein, and cysteine‑rich protein 3 genes. The lowest change was exhibited by odd‑skipped related transcription factor 1, plakophilin 2, transcription growth factor‑β receptor 1, and kinesin family member 3A. The direction of changes was confirmed by RT‑qPCR results. In the present study, it was suggested that GCs may have the potential to differentiate towards other cell types under long‑term in vitro culture conditions. Thus, genes belonging to the presented ontological groups can be considered as novel markers of proliferation and differentiation of GCs towards the heart muscle cells.

Progesterone (P 4 ) plays a key role in the establishment and maintenance of pregnancy in most mammals. Unravelling the expression of progesterone-regulated genes can expand the understanding of the embryonic mortality. Accordingly, we studied the relative mRNA expression of the P 4 -regulated genes in the buffalo. Uteri were collected from the abattoir and categorized into nonpregnant late luteal phase, stage I (28-38th days of gestation) and stage II (48-56th days of gestation) of pregnancy (n = 6/group). After extraction of total RNA from the endometrial tissues, we carried out qRT-PCR for determining the relative mRNA expression of the P 4 -regulated genes using nonpregnant late luteal phase as calibrator group. The expression of LGALS3BP (essential for maternal recognition of pregnancy) gene was found to be significantly upregulated (p < 0.05), while MUC1 (important for embryo attachment) gene was downregulated in stage I and II of pregnancy. We observed no significant change in the expression of LGALS1, LGALS9 and CTSL genes. The SLC5A11 and SLC2A1 genes (involved in the transport of glucose to endometrium) in early pregnancy were upregulated in the pregnancy stage I (p < 0.05) relative to nonpregnant late luteal phase. The CST3 gene was significantly upregulated in pregnancy stage II (p < 0.01). These results provide molecular insights into the specific pathways involved in foeto-maternal communication during early pregnancy in buffaloes., (© 2018 Blackwell Verlag GmbH.)

Interferon-τ (IFNT) is a major cytokine produced by the ruminant trophoectoderm during the peri-implantation period exerting immunomodulatory actions on various cells including neutrophils. The current in vitro study was undertaken to analyze the concentration-dependent effects of IFNT on neutrophil gene dynamics to understand its possible role in implantation process. The neutrophils were isolated from the blood of heifers and were cultured subjecting them to different IFNT concentrations (1, 5, or 10 ng/mL). The gene-expression patterns of different interferon-stimulated genes, l-selectin, CD31, CD11b, and progesterone-induced blocking factor (PIBF) were analyzed by quantitative real-time PCR. It was observed that at lower concentrations of IFNT, the IFI16, l-selectin, ISG15, and PIBF were upregulated, whereas at higher concentrations the same were down regulated. At all the experimental concentrations, IFI44, OAS1, MX genes were significantly upregulated and CD31, CD11b were significantly downregulated. At lower concentrations of IFNT, the neutrophil activity with respect to chemoattraction is stimulated, whereas at higher concentrations the same is reduced. Hence, it can be concluded that IFNT exerts concentration-dependent actions on neutrophil gene-expression dynamics indicating a fine modulation of its activity depending upon the temporal variation in its destined functions ultimately leading to successful implantation., (© 2018 Wiley Periodicals, Inc.)

Variation in female reproductive traits, such as fertility, fecundity, and fecundability, are heritable in humans, but identifying and functionally characterizing genetic variants associated with these traits have been challenging. Here, we explore the functional significance and evolutionary history of a G/A polymorphism at SNP rs2523393, which is an eQTL for HLA-F and is significantly associated with fecundability (the probability of being pregnant within a single menstrual cycle). We replicated the association between the rs2523393 genotype and HLA-F expression by using GTEx data and demonstrate that HLA-F is upregulated in the endometrium during the window of implantation and by progesterone in decidual stromal cells. Next, we show that the rs2523393 A allele creates a GATA2 binding site in a progesterone-responsive distal enhancer that loops to the HLA-F promoter. Remarkably, we found that the A allele is derived in the human lineage and that the G/A polymorphism arose before the divergence of modern and archaic humans and segregates at intermediate to high frequencies across human populations. Remarkably, the derived A allele is has also been identified in a GWAS as a risk allele for multiple sclerosis. These data suggest that the polymorphism is maintained by antagonistic pleiotropy and a reproduction-health tradeoff in human evolution., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The paired-like homeobox 2B gene (PHOX2B) encodes a key transcription factor that plays a role in the development of the autonomic nervous system and the neural structures involved in controlling breathing. In humans, PHOX2B over-expression plays a role in the pathogenesis of tumours arising from the sympathetic nervous system such as neuroblastomas, and heterozygous PHOX2B mutations cause Congenital Central Hypoventilation Syndrome (CCHS), a life-threatening neurocristopathy characterised by the defective autonomic control of breathing and involving altered CO 2 /H + chemosensitivity. The recovery of CO 2 /H + chemosensitivity and increased ventilation have been observed in two CCHS patients using the potent contraceptive progestin desogestrel. Given the central role of PHOX2B in the pathogenesis of CCHS, and the progesterone-mediated effects observed in the disease, we generated progesterone-responsive neuroblastoma cells, and evaluated the effects of 3-Ketodesogestrel (3-KDG), the biologically active metabolite of desogestrel, on the expression of PHOX2B and its target genes. Our findings demonstrate that, through progesterone nuclear receptor PR-B, 3-KDG down-regulates PHOX2B gene expression, by a post-transcriptional mechanism, and its target genes and open up the possibility that this mechanism may contribute to the positive effects observed in some CCHS patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

It is well known that prostaglandin (PG) E2 and PGF2α are secreted in copious amounts from the menstruating uterus. The aim of the present study was to determine whether PGs affect the growth of uterine leiomyomas (ULs) to the same extent as estrogen or progesterone (P4). The present study evaluated the expression of eight microRNAs (miRNAs) by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) through treatment with estradiol (E2), P4, PGE2, PGF2α and each antagonist or cyclooxygenase‑2 (COX‑2) inhibitor of cultured leiomyoma and myometrial cells (LC and MC, respectively). The eight miRNAs were divided into two groups according to their primary biological action, namely apoptosis‑regulating miRNAs (let‑7a, miR‑21, miR‑26a and miR‑200a) and inflammation‑regulating miRNAs (miR‑29b, miR‑93, miR‑106b and miR‑100b). PGE2 induced significantly higher expression of the 3 anti‑apoptotic miRs, let‑7a, miR‑16a and miR‑200a, in LC when compared with the non‑treated control or E2. PGE2 significantly promoted a greater expression of let‑7a and miR‑26a in LC when compared with P4. Overall, PGE2 exerted the highest anti‑apoptotic and anti‑inflammatory effect in LC, which was comparable with E2. It was not observed among the inflammation‑regulating miRNAs in LC. PGF2α did not exert effects as prominent as those of PGE2. In MC, PGs and sex steroids exerted no similar effects on MC compared with LC. The present study demonstrated that PGE2 levels during menstruation may affect the growth of preexisting ULs without affecting the normal myometrium. Therefore, the control of secretion of PGs from the menstruating uterus or the administration of antagonists may be an alternative therapy for inhibiting the growth of ULs.

Purpose: We have reported a survival benefit of single injection of hydroxyprogesterone prior to surgery for primary tumour in patients with node-positive operable breast cancer. Hydroxyprogesterone was meant to recapitulate the luteal phase of menstrual cycle in these women. We wanted to understand the molecular basis of action of hydroxyprogesterone on primary breast tumours in a peri-operative setting., Methods: We performed whole transcriptome sequencing (RNA-Seq) of primary breast tumour samples collected from patients before and after hydroxyprogesterone exposure and controls. Paired breast cancer samples were obtained from patients who were given hydroxyprogesterone before surgery and a group of patients who were subjected to only surgery., Results: A test of significance between the two groups revealed 207 significantly altered genes, after correction for multiple hypothesis testing. We found significantly contrasting gene expression patterns in exposed versus unexposed groups; 142 genes were up-regulated post-surgery among exposed patients, and down-regulated post-surgery among unexposed patients. Significantly enriched pathways included genes that respond to progesterone, cellular stress, nonsense-mediated decay of proteins and negative regulation of inflammatory response. These results suggest that cellular stress is modulated by hydroxyprogesterone. Network analysis revealed that UBC, a mediator of stress response, to be a major node to which many of the significantly altered genes connect., Conclusions: Our study suggests that pre-operative exposure to progesterone favourably modulates the effect of surgical stress, and this might underlie its beneficial effect when administered prior to surgery.

Problem: Dedicator of cytokinesis (DOCK 180) involved in cytoskeletal reorganization is primarily a cytosolic molecule. It is recently shown to be nuclear in HeLa cells but its nuclear function is not known., Method of Study: The spatiotemporal distribution of DOCK180 in uterus was studied in uterine cytoplasmic and nuclear compartments during the "window of implantation." The functional significance of nuclear DOCK180 was explored by homology modeling, co-immunoprecipitation assays, and mass spectrometric analysis. Dock180's role in early pregnancy was ascertained by Dock 180 silencing and subsequent quantitative real-time PCR and Western blotting analysis., Results: Our study shows a nuclear DOCK180 in the uterus during "window of implantation." Estrogen and progesterone mediate expression and nuclear translocation of DOCK180. The nuclear function of DOCK180 is attributed to its ability to import autoimmune regulator (AIRE) into the nucleus. Silencing of Dock180 inhibited AIRE nuclear shuttling which influenced its downstream targets, thereby affecting decidualization with AIRE and HOXA-10 as the major players as well as lack of implantation site formation due to impact on angiogenesis-associated genes., Conclusion: DOCK180 has an indispensable role in pregnancy establishment as knocking down Dock180 abrogates pregnancy by a consolidated impact on decidualization and angiogenesis by regulating AIRE nuclear entry., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

BACKGROUND Increasing evidence indicates that long noncoding RNAs (LncRNAs) play a key role in multiple pathological processes. It has been shown that LncRNA steroid receptor RNA activator (SRA) is elevated in peripheral blood of patients with polycystic ovary syndrome (PCOS). The aim of this study was to assess the effect of elevated LncRNA SRA on ovarian granular cells of mice in vitro. MATERIAL AND METHODS We firstly isolated granular cells from mouse ovaries and over-expressed the LncRNA SRA by means of lentiviral transfection in this cell line. Then, we assessed the effects of LncRNA SRA on granular cells through real-time PCR, CCK-8 assay, flow cytometry, Hoechst staining, and Western blot assay. RESULTS We demonstrated that elevated LncRNA SRA stimulated cell growth, changed distribution of cell cycle phases with increase of Cyclin B, Cyclin E, and Cyclin D1, and inhibited cell apoptosis with up-regulation of bcl2 and down-regulation of bax, cleaved-caspase 3, and cleaved-PARP. Moreover, the contents of estradiol (E2) and progesterone (PG) and expressions of their key enzymes (CYP19A1 and CYP11A1) were up-regulated following over-expression of LncRNA SRA. CONCLUSIONS Taken together, our results indicate that abnormal LncRNA SRA may be a risk factor for evoking PCOS.

Puberty is a complex biopsychosocial process that can affect an array of psychiatric and medical disorders emerging in adolescence. Although the pubertal process is driven by neuroendocrine changes, few quantitative genetic studies have directly measured puberty-relevant hormones. Hair samples can now be assayed for accumulation of hormones over several months. In contrast to more conventional salivary measures, hair measures are not confounded by diurnal variation or hormonal reactivity. In an ethnically and socioeconomically diverse sample of 1286 child and adolescent twins and multiples from 672 unique families, we estimated genetic and environmental influences on hair concentrations of testosterone, DHEA, and progesterone across the period of 8-18 years of age. On average, male DHEA and testosterone were highly heritable, whereas female DHEA, progesterone, and puberty were largely influenced by environmental components. We identified sex-specific developmental windows of maximal heritability in each hormone. Peak heritability for DHEA occurred at approximately 10 years of age for males and females. Peak heritability for testosterone occurred at age 12.5 and 15.2 years for males and females, respectively. Peak heritability for male progesterone occurred at 11.2 years, while the heritability of female progesterone remained uniformly low. The identification of specific developmental windows when genetic signals for hormones are maximized has critical implications for well-informed models of hormone-behavior associations in childhood and adolescence., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Oestrogen-progesterone signalling is highly versatile and critical for the maintenance of healthy endometrium in humans. The genomic and nongenomic signalling cascades initiated by these hormones in differentiated cells of endometrium have been the primary focus of research since 1920s. However, last decade of research has shown a significant role of stem cells in the maintenance of a healthy endometrium and the modulatory effects of hormones on these cells. Endometriosis, the growth of endometrium outside the uterus, is very common in infertile patients and the elusiveness in understanding of disease pathology causes hindrance in selection of treatment approaches to enhance fertility. In endometriosis, the stem cells are dysfunctional as it can confer progesterone resistance to their progenies resulting in disharmony of hormonal orchestration of endometrial homeostasis. The bidirectional communication between stem cell signalling pathways and oestrogen-progesterone signalling is found to be disrupted in endometriosis though it is not clear which precedes the other. In this paper, we review the intricate connection between hormones, stem cells and the cross-talks in their signalling cascades in normal endometrium and discuss how this is deregulated in endometriosis. Re-examination of the oestrogen-progesterone dependency of endometrium with a focus on stem cells is imperative to delineate infertility associated with endometriosis and thereby aid in designing better treatment modalities.

Epidemiological studies have exhibited a positive association between fine particulate matter (PM 2.5 ) exposure and adverse pregnancy outcome (APO). However, source-related effect and the potential mechanism have not been thoroughly elucidated in toxicology. In this study, PM 2.5 was collected during a severe winter haze episode in an energy-base city of China. We coupled this approach with the source appointment by applying the Lagrangian Integrated Trajectory and Concentration Weighted Trajectory model. We observed that the primary trajectory with high polluted air mass came from the northwest of the sampling site. Approximately 90% or more of PM 2.5 was derived from the industry at this haze period. Next, the sampled PM 2.5 was used to study the classical hormone synthesis pathway on trophoblast JEG-3 cells. PM 2.5 induced the secretion of human chorionic gonadotrophin (HCG) and the proliferation of JEG-3 cells at a noncytotoxic concentration. However, the synthesis of progesterone was significantly suppressed, even if both hCG and cyclic adenosine monophosphate (cAMP) were increased, suggesting that PM 2.5 may interfere the downstream of cAMP. As expected, the phosphorylated activity of protein kinase A (PKA) was attenuated. Subsequently, the downstream molecules of steroidogenesis, such as ferredoxin reductase (FDXR), CYP11A1 (encoded P450scc), and 3β-Hydroxysteroid dehydrogenase type 1 (3β-HSD1), were inhibited. Therefore, PM 2.5, primarily derived from industry, may directly inhibit the phosphorylation status of PKA in JEG-3 which, in turn, inhibited the proteins expression in progesterone-synthesis to suppress progesterone levels. Considering the pivotal role of progesterone in pregnancy maintenance, the mechanism on hormone synthesis may provide a better understanding for PM 2.5 -caused APO. Industry-emanated PM 2.5 , though not specific, could threaten the placenta, which needs to be verified by further epidemiological studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes. SIGNIFICANCE STATEMENT The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the female brain. An important role of endogenous progesterone in cerebroprotection was demonstrated by the conditional inactivation of its receptor in neural cells. These results show the importance of endogenous progesterone, its metabolites, and neural progesterone receptors in acute cerebroprotection after stroke. This new concept could be exploited therapeutically by taking into account the progesterone status of patients and by supplementing and reinforcing endogenous progesterone signaling for attaining its full cerebroprotective potential., (Copyright © 2017 the authors 0270-6474/17/3710998-23$15.00/0.)

Half of estrogen receptor-positive breast cancers contain a subpopulation of cytokeratin 5 (CK5)-expressing cells that are therapy resistant and exhibit increased cancer stem cell (CSC) properties. We and others have demonstrated that progesterone (P4) increases CK5+ breast cancer cells. We previously discovered that retinoids block P4 induction of CK5+ cells. Here we investigated the mechanisms by which progesterone receptors (PR) and retinoic acid receptors (RAR) regulate CK5 expression and breast CSC activity. After P4 treatment, sorted CK5+ compared to CK5- cells were more tumorigenic in vivo. In vitro, P4-treated breast cancer cells formed larger mammospheres and silencing of CK5 using small hairpin RNA abolished this P4-dependent increase in mammosphere size. Retinoic acid (RA) treatment blocked the P4 increase in CK5+ cells and prevented the P4 increase in mammosphere size. Dual small interfering RNA (siRNA) silencing of RARα and RARγ reversed RA blockade of P4-induced CK5. Using promoter deletion analysis, we identified a region 1.1 kb upstream of the CK5 transcriptional start site that is necessary for P4 activation and contains a putative progesterone response element (PRE). We confirmed by chromatin immunoprecipitation that P4 recruits PR to the CK5 promoter near the -1.1 kb essential PRE, and also to a proximal region near -130 bp that contains PRE half-sites and a RA response element (RARE). RA induced loss of PR binding only at the proximal site. Interestingly, RARα was recruited to the -1.1 kb PRE and the -130 bp PRE/RARE regions with P4, but not RA alone or RA plus P4. Treatment of breast cancer xenografts in vivo with the retinoid fenretinide reduced the accumulation of CK5+ cells during estrogen depletion. This reduction, together with the inhibition of CK5+ cell expansion through RAR/PR cross talk, may explain the efficacy of retinoids in prevention of some breast cancer recurrences.

Leptin has been found to be involved in the ovarian granulosa cell apoptosis and steroidogenesis. Loss of neuropeptide Y (NPY) can correct the obesity syndrome of mutant mice lacking of leptin (ob/ob). However, the association of NPY and leptin in ovarian granulosa cells and ovarian steroidogenesis has not been investigated. Here, C57BL/6J ob/ob mice and C57BL/6J (control) mice were intraperitoneally injected with PBS, leptin (0.4μg/g bodyweight) or BIIE0246 (NPY2 receptor [NPY2R] antagonist, 30μg/kg bodyweight) every day for 15days. We found that NPY2R mRNA expression in mouse ovary was suppressed by leptin treatment, but increased by leptin deficiency. Leptin or BIIE0246 treatment significantly increased E2, but notably decreased progesterone in both mice. A lower level of E2 and a higher level of progesterone was observed in ob/ob mice than in control mice. Further, we then knocked down leptin expression in human ovarian granulosa cells by siRNA transfection and treated the cells with DMSO or BIIE0246. In vitro experiments confirmed the findings in mice. siLeptin treatment decreased the secretion of E2, anti-Mullerian hormone (AMH), insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β, and the cell proliferation, but increased the secretion of progesterone and cell apoptosis. Western blotting analysis of PCNA, Bcl-2 and Bax confirmed the results of cell proliferation and apoptosis. Activation of JAK2 and STAT3 was also suppressed by knocking down leptin. All the effects of siLeptin on ovarian granulosa cells were partially reversed by BIIE0246. In conclusion, knockdown of leptin significantly affected ovarian steroidogenesis and ovarian function through NPY. siLeptin transfection impaired the activation of JAK2/STAT3 and contributed to ovarian granulosa cell apoptosis partially through up-regulating NPY2R expression., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In ruminants, prostaglandin F2alpha (PGF2α)-mediated luteolysis is essential prior to estrous cycle resumption, and is a target for improving fertility. To deduce early PGF2α-provoked changes in the corpus luteum a short time-course (0.5-4 h) was performed on cows at midcycle. A microarray-determined transcriptome was established and examined by bioinformatic pathway analysis. Classic PGF2α effects were evident by changes in early response genes (FOS, JUN, ATF3) and prediction of active pathways (PKC, MAPK). Several cytokine transcripts were elevated and NF-κB and STAT activation were predicted by pathway analysis. Self-organizing map analysis grouped differentially expressed transcripts into ten mRNA expression patterns indicative of temporal signaling cascades. Comparison with two analogous datasets revealed a conserved group of 124 transcripts similarly altered by PGF2α treatment, which both, directly and indirectly, indicated cytokine activation. Elevated levels of cytokine transcripts after PGF2α and predicted activation of cytokine pathways implicate inflammatory reactions early in PGF2α-mediated luteolysis., (Published by Elsevier B.V.)