Metabolism Of Progesterone Research Articles

P–293 Ovarian endometriomas are heterogenous for the steroidogenic function and the expression of estrogen and progesterone receptors

Abstract Study question Do all ovarian endometriomas have steroidogenic function and express estrogen and progesterone receptors? Summary answer No, they are heterogenous for the steroidogenic function and the expression of the estrogen and progesterone receptors. What is known already Excessive ectopic estrogen production and up-regulation of estrogen receptor- β, which drives inflammation together with aberrant progesterone signaling leading to impaired decidualization and establishment of ectopic endometrial implants together with down-regulated progesterone receptor (PR) expression are the cardinal molecular features of the disease. However, several fundamental questions still remain to be answered as to whether all ovarian endometriomas carry these molecular aberrations and are steroidogenically active; and if so, the amount of sex steroids they produce correlate with the level of expression of steroidogenic enzymes. We aimed to address these questions in the current study. Study design, size, duration A molecular research study on the surgical specimens collected between April 2020 and December 2020. Seven histopathologically confirmed benign endometriotic cyst capsules obtained from the patients undergoing laparoscopic excision of unilateral ovarian endometriomas without deep infiltrating endometriosis during early follicular phase were used in the study. Participants/materials, setting, methods The mean age±SD (range) of the patients were 32.8±4.9 (30–39). The mean endometrioma size was 5±1.2cm (5–7.5 cm). The samples were cut into equal size pieces of 0.5x0.5cm size and cultured for one day to measure their E2and P4production; and analyzed for the expression of steroidogenic enzymes with quantitative immunoblotting and for the expression of FSH-R, ER and PR with real-time qRT-PCR methods. Luteinized granulosa cells and ovarian cortex were set as references. Main results and the role of chance StAR expression was consistently observed in all samples. However, we noticed significant discrepancies among the samples regarding their steroidogenic function and the expression of aromatase and 3 β-HSD enzymes. E2 production exhibited significant variation (from 5 to 1177pg/mL) from sample to sample despite comparable levels of aromatase expression. ER- βup-regulation as a cardinal molecular feature of endometriosis, was observed in all but one samples (1.46 to 5.48 folds, p < 0.0001). However, its expression level did not correlate with either aromatase expression or the amount of E2 the samples produced. A similar phenomenon was observed in P4 arm of steroidogenesis. Even though 3 β-HSD was expressed by all but one samples detectable amount of P4 was produced only by two samples (up to 15ng/mL). PR expression was down-regulated only in two samples (0.3 to 0.07 folds, p < 0.0001), and significantly up-regulated in the other samples (1.2 to 4.7 folds, p < 0.001). No correlation was found among the samples regarding the expression of PR, 3 β-HSD and P4 output. FSH-R was detected in all samples at the levels comparable to ovarian cortex but its expression level did not show any correlation with ER, aromatase expression and E2 production. Limitations, reasons for caution These results need to be confirmed in studies with larger sample size and different types of endometriotic lesions. Wider implications of the findings: The regulation of steroidogenic activity of endometriomas cannot simply be explained by the expression level of the steroidogenic enzymes, underscoring the importance of other mechanisms that post-translationally regulate their enzymatic activity and metabolism of estrogen and progesterone. PR is not always down-regulated and FSH-R is commonly up-regulated in ovarian endometriomas. Trial registration number Not applicable

Pro‐inflammatory signals induce 20α‐HSD expression in myometrial cells: A key mechanism for local progesterone withdrawal

Metabolism of progesterone (P4) by the enzyme 20α hydroxysteroid dehydrogenase (20α‐HSD) in myometrial cells is postulated to be a mechanism for P4 withdrawal, which occurs concomitant to uterine inflammation (physiologic or infection‐induced) and associated activation of transcription factors: NF‐кB and AP‐1, common to term and preterm labour. We found that 20α‐HSD protein is significantly increased in human myometrium during term labour, and in mouse uterus during term and preterm labour. Treatment of human myometrial cells with the pro‐inflammatory mediators, lipopolysaccharide (LPS, mimicking infection) and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA, mimicking inflammation), induced 20α‐HSD gene expression and increased 20α‐HSD protein abundance. LPS treatment decreased P4 release into the culture medium and resulted in up‐regulation of GJA1 in the hTERT‐HM cells. The NF‐кB /AP‐1 transcription factors mediated effects of LPS and TPA on 20α‐HSD gene transcription. Both pro‐inflammatory stimuli induced 20α‐HSD promoter activity in LPS/TPA‐treated cells which was significantly attenuated by inhibition of NF‐кB (JSH: 20 µM) or AP‐1 signalling (T5224: 10 µM). Deletion of NF‐кB consensus sites abrogated LPS‐mediated promoter induction, while removal of AP‐1 sites reversed the TPA‐mediated induction of 20α‐HSD promoter. We conclude that inflammatory stimuli (physiologic or pathologic) that activate NF‐кB or AP‐1 induce 20α‐HSD transcription and subsequent local P4 withdrawal resulting in up‐regulation of GJA1 and activation of myometrium that precedes labour.

Steroid hormone bioavailability is controlled by the lymphatic system

The steroid hormone progesterone accounts for immune tolerance in pregnancy. Enhanced progesterone metabolism to 6α-OH-pregnanolone occurs in complicated pregnancies such as in preeclampsia with preterm delivery or intrauterine growth restriction, and in cancer. As lymphatic endothelial cells (LECs) promote tumor immunity, we hypothesized that human LECs modify progesterone bioavailability. Primary human LECs and mice lymph nodes were incubated with progesterone and progesterone metabolism was analyzed by thin layer chromatography and liquid chromatography-mass spectrometry. Expression of steroidogenic enzymes, down-stream signal and steroid hormone receptors was assessed by Real-time PCR. The placental cell line HTR-8/SV neo was used as reference. The impact of the progesterone metabolites of interest was investigated on the immune system by fluorescence-activated cell sorting analysis. LECs metabolize progesterone to 6α-OH-pregnanolone and reactivate progesterone from a precursor. LECs highly express 17β-hydroxysteroid dehydrogenase 2 and are therefore antiandrogenic and antiestrogenic. LECs express several steroid hormone receptors and PIBF1. Progesterone and its metabolites reduced TNF-α and IFN-γ production in CD4+ and CD8+ T cells. LECs modify progesterone bioavailability and are a target of steroid hormones. Given the global area represented by LECs, they might have a critical immunomodulatory control in pregnancy and cancer.

Abstract PS19-09: Alternative splicing events from progesterone exposure differ based on BRCA1 mutation status

Abstract Background: Progesterone (P) and progestin are instrumental in the breast cancer risk associated with reproductive hormones. Progesterone receptor (PR) signaling has been shown to be inhibited by the wild-type BRCA1 protein and it is hypothesized that if this inhibition is lost, due to gene mutation, potentially tumorigenic consequences may result. Our efforts are focused on understanding the role PR and P play in BRCA1 mutation carriers. The purpose of this study was to determine if the presence of a BRCA1 mutation effects alternative splicing (AS) events. Methods: Mammary organoids from 12 patients, six with a BRCA1 mutation and six without (WT, control), were cultured in vitro. The organoids from each patient were divided into two groups and treated over the course of 28 days to mimic a normal menstrual cycle. One group was treated was estradiol and progesterone (EP) while the other was treated with EP and the PR antagonist telapristone acetate (TPA) to identify PR-mediated effects. After treatment, RNA was extracted from both groups and RNA-sequencing performed. A statistical model, “replicate Multivariate Analysis of Transcript Splicing” (rMATS) was employed to identify AS events. Output from rMATS was then entered into “RNA Map Analysis And Plotting Server” (rMAPS) to identify RNA-binding proteins (RBP) motifs in the region of the splices. Droplet digital PCR (ddPCR) from Bio-Rad was employed, with the use of Evagreen intercalating dye, to validate a subset of the AS events identified by the RNA-Seq data. RNA from eight of the EP treated samples was reverse transcribed and ddPCR performed. The data was analyzed using QuantaSoft Analysis Pro (Bio-Rad). The identified AS events were validated additionally using data generated from solid tissue normals (STN) from premenopausal, BRCA1mut and BRCA1WT patients, who developed breast cancer, which was available for download from The Cancer Genome Atlas (TCGA). Results: Genes involved in the epithelial-mesenchymal-transition (EMT), progesterone metabolism, and cellular proliferation are significantly differentially spliced in the EP treated BRCA1mut tissue (p-value < 0.01 and FDR < 5%) and absent in those with TPA treatment, suggesting the AS is a PR-mediated effect. CD44, associated with cellular proliferation and migration, NCOR2 associated with tamoxifen resistance, and AKR1C2 associated with progesterone metabolism all showed significant skipped exon (SE) events. Exon 11 in CD44 and exon 45 in NCOR2 were skipped in both the BRCA1 organoids as well as in BRCA1 STN from TCGA. Skipping of Exon 4 in AKR1C2 was validated using the ddPCR: the BRCA1mut organoids displayed two isoforms, indicative of a skipped exon. Conclusions: PR-mediated alternative splicing events differ in mammary organoids of BRCA1 carriers compared to non-carriers. We hypothesize that the skipping of Exon 4 of AKR1C2 in BRCA1mut results in a structural alteration that decreases protein activity, leading to increased concentrations of P4 and 5α-pregnane-3,20-dione. This may explain the previously reported high median luteal phase serum P levels (p=0.00034) in BRCA1mut/BRCA2mut (PMID: 24140203). Citation Format: Gannon Cottone, Batzaya Davaadelger, Shivangi Yadav, J Julie Kim, Seema A. Khan, Susan Clare. Alternative splicing events from progesterone exposure differ based on BRCA1 mutation status [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-09.

Developmental expression of genes involved in progesterone synthesis, metabolism and action during the post-natal cerebellar myelination

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 progesteroneʾs 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.

The role of 7-hydroxymatairezinol in modulation of estrogen metabolism and therapy for mastopathy

Quantitative estimates of the effect of 7-hydroxymatairesinol (7-HMR) intake on estrogen balance and clinical aspects of the course of fibrocystic mastopathy have been carried out.
 Aim. Assessment of effects 7-HMR on metabolism of estrogen (16a-OHE1, 2-OHE2, 2-OHE1, 2-OMEE1, 4-OMEE2, 4-OHE1, a ratio 2-OHE1/16a-OHE1, 2-OHE1/2-OMeE1 and 4-OHE1/4-OMeE1) and progesterone in daily urine at patients with fibrous and cystous mastopathy in the post-menopausal period.
 Materials and methods. The analysis of metabolites of estrogen (16a-OHE1, 2-OHE2, 2-OHE1, 2-OMEE1, 4-OMEE2, 4-OHE1, ratios 2-OHE1/16a-OHE1, 2-OHE1/2-OMeE1 and 4-OHE1/4-OMeE1) and progesterone in daily urine with use of solid-phase immunofermental set (IFA) of Amerscham International on the analyzer of Amerkard by means of standard sets is carried out. Statistical 10.0 and Microsoft Excel spreadsheets were used in statistical material processing.
 Results. It was found that taking 7-HMR (60 mg/day, 1 month) contributes to normalizing estrogen metabolism (decreasing estradiol, estrone, estriol, 16a-OHE1) and improves the clinical symptoms of mastopathy.
 Conclusions. In patients with FCM, 7-HMR contributes to a decrease in hyperestrogenia-related tumor risks by lowering estrogen metabolites levels.

Plasma leptin level mirrors metabolome alterations in young adults.

Leptin is known to regulate pathways of energy metabolism, reproduction, and control appetite. Whether plasma leptin levels reflect changes in metabolites of these pathways is unknown. We aimed to find whether there is an association between leptin levels and levels of metabolites of energy and hormone metabolism. We performed an untargeted metabolomics analysis of plasma from 110 healthy adults (men: women = 1:1; aged 18-40years), using liquid chromatography-tandem mass spectrometry. Blood samples were collected from all the study subjects in the fasting state. Clinical features and markers of obesity and Type 2 diabetes mellitus (T2DM) were assessed in all. The association between levels of metabolites and clinical and biochemical parameters was identified using the multivariable-adjusted linear regression model and PLS-DA analysis. The leptin level was found to have a significant association with a substantial number of metabolites in women and men. Leptin level was positively associated with glycocholic acid and arachidic acid, metabolites related to energy metabolisms, pregnanediol-3-glucuronide, a metabolite of progesterone metabolism, and quercetin 3'-sulfate, a diet-derived metabolite. Leptin level was negatively associated with ponasteroside A and barringtogenol C levels. Leptin level was positively correlated with adiponectin and negatively with total calorie intake and levels of triglyceride and very-low-density lipoprotein. Leptin levels were associated with lipid and sex hormone metabolism in women, while metabolites involved in amino acid metabolism were correlated to leptin in men. Our study indicates that leptin level reflects metabolome alterations and hence could be a useful marker to detect early changes in energy and hormone metabolisms.

Alterations in endogenous progesterone metabolism associated with spontaneous very preterm delivery

Alterations in endogenous progesterone metabolism associated with spontaneous very preterm delivery

Characteristics of lipid droplets and the expression of proteins involved in lipolysis in the murine cervix during mid-pregnancy.

Lipid droplets (LDs) are reservoirs of arachidonoyl lipids for prostaglandin (PG) E2 synthesis, and progesterone can stimulate PGE2 synthesis; however, the relationship between progesterone and LD metabolism in the murine cervix remains unclear. In the present study we examined LD distribution and changes in the expression of proteins involved in lipolysis and autophagy in the murine cervix during pregnancy, and compared the findings with those in dioestrous mice. During mid-pregnancy, LDs were predominantly distributed in the cervical epithelium. Electron microscopy revealed the transfer of numerous LDs from the basal to apical region in the luminal epithelium, marked catabolism of LDs, an elevated number of LDs and autophagosomes and a higher LD:mitochondrion size ratio in murine cervical epithelial cells (P<0.05). In addition, immunohistochemical and western blotting analyses showed significantly higher cAMP-dependent protein kinase, adipose triglyceride lipase and hormone-sensitive lipase expression, and a higher light chain 3 (LC3) II:LC3I ratio in the stroma and smooth muscles and, particularly, in murine cervical epithelial cells, during mid-pregnancy than late dioestrus. In conclusion, these results suggest that the enhanced lipolysis of LDs and autophagy in murine cervical tissues were closely related to pregnancy and were possibly controlled by progesterone because LD catabolism may be necessary for energy provision and PGE2 synthesis to maintain a closed pregnant cervix.

Attenuation of 5α-reductase-mediated progesterone metabolism promotes differentiation of human endometrial stromal cells for the establishment of pregnancy

Attenuation of 5α-reductase-mediated progesterone metabolism promotes differentiation of human endometrial stromal cells for the establishment of pregnancy

Hepatic mRNA expression of enzymes associated with progesterone metabolism and its impact on ovarian and endocrine responses in Nelore (Bos indicus) and Holstein (Bos taurus) heifers with differing feed intakes

The aim of this study was to evaluate circulating progesterone concentration (P4), LH pulsatility and ovarian follicular dynamics in Nelore (B. indicus) and Holstein (B. taurus) heifers under high (HDMI) and low (LDMI) dry matter/energy intakes. In addition, the effects of dry matter/energy intake and breed on hepatic expression of six genes associated with P4 metabolism (AKR1C4, AKR1D1, CYP3A4, CYP2C19, SRD5A1, and SRD5A3) was evaluated. Heifers received an intravaginal P4 device (1 g), 2 mg of estradiol benzoate (EB) i.m. and 500 μg of PGF2α at the begging of the synchronization protocol (D0). Eight days later, the P4 device was removed and all heifers received 1 mg of EB 24h later. Regardless of dry matter/energy intake, the number of recruited follicles was greater in Nelore than in Holstein heifers. In contrast, the maximum diameter of the dominant follicle was greater in Holstein than in Nelore heifers. Circulating P4 concentrations were greater in Nelore than in Holstein from D2 to D9, and in heifers receiving LDMI than those receiving HDMI from D1 to D8 of hormonal protocol. In addition, Holstein heifers had greater LH pulsatility and area under the curve of LH peaks compared to Nelore heifers. However, no effects were observed for LH values between feed intake levels. Interestingly, Holstein heifers had higher expression of SRD5A1, AKR1C4, AKR1D1 than Nelore heifers; whereas, for Nelore heifers, only the expression of CYP3A4 was higher compared to Holstein heifers. In conclusion, there are important differences in the follicular dynamics, circulating P4 and LH pulsatility concentrations that need to be considered during synchronization protocols for Nelore and Holstein breeds. More importantly, these differences appear to be at least partially modulated by the level of feed intake and the contrasting enzyme system in the liver involved with P4 metabolism between these cattle breeds.

Approche GABAergique de la dépression du post-partum : une revue critique translationnelle

IntroductionPrevalence of postpartum depression (PPD) ranges from 10 to 15 % of parturients. The impact of the PPD is major on the maternal bond and the health of both mother and child. Its physiopathological mechanisms appear to differ from other types of depression. Today, pharmacotherapy is based on nonspecific treatment, and recent therapeutic advances in this field require a comprehensive approach of the implication of the GABAergic system in the development of PPD. Neurosteroid levels during pregnancy and after parturition and the GABA-A-r modulation are thought to be involved in PPD. ObjectiveTo evaluate if the GABAergic approach is relevant in postpartum depression management. MethodsWe conducted a systematic review of literature based on the MEDLINE database with the following Medical Subject Headings (MeSH): “postpartum depression”, “GABA”, “ganaxolone”, “brexanolone”, “allopregnanolone”, prior to September 2019. We selected articles in English: preclinical and clinical studies, literature review, observational and therapeutic studies. ResultsPreclinical models (mouse and rat) show changes in GABAergic inhibition in the peripartum period and correlation between allopregnanolone and GABA-A-r plasticity. This plasticity in the peripartum period maintains levels of inhibition adapted despite increased neurosteroid levels. KO models for the GABA-A-r δ subunit develop depression and anxiety symptoms in the postpartum period, and a change in the expression of the gene coding for the GABA-R alpha-4 subunit was found. Artificial inhibition of progesterone metabolism during post-partum increased depression symptoms. GABAergic fluctuation seems to be interrelated with other systems such as those of oxytocins. A synthetic neurosteroid (SGE-516) was tested on mouse models of PPD, KO for δ-GABA-A-r or KCC2, and showed decreased depressive symptoms and better mothering. Clinical studies confirm neurosteroid fluctuation and changes in the GABAergic system during the peripartum period. Allopregnanolone is the neurosteroid the most studied in PPD, and it is elevated in the brain during the pregnancy. Studies disagree on the presence of significant differences in allopregnanolone plasma levels during pregnancy or postpartum between women with PPD or not. Women with a history of PPD have greater susceptibility to neurosteroid withdrawal. Imagery and genetical data also show a link between allopregnanolone and PPD. The GABA-A-r may not recover in time following a reduced number during pregnancy, and this mismatch between neurosteroid levels and their receptor may trigger PPD. Several randomized controlled trials investigated brexanolone administrated IV, a synthetic formulation of allopregnanolone, and demonstrated a rapid and well tolerated reduction in depressive symptoms. In March 2019 brexanolone obtained FDA approval in PPD indication under the name Zulresso. However, there are differences in the time of beginning of PPD, which could constitute different subgroups of this disease, and which physiopathology could respond to different mechanisms. Prenatal depression does not respond to a GABAergic approach, but women without any risk factor or previous mood disorder developing PPD in the weeks following childbirth could be particularly responsive to this kind of treatment. ConclusionDisability to modulate GABA-A-r expression during pregnancy and restore its previous state after parturition appears to trigger PPD. The GABAergic system is a promising pharmacotherapy target. From preclinical to clinical studies for about twenty years the GABAergic system has been incriminated and targeted in this challenging mental disease.

The allopregnanolone to progesterone ratio across the menstrual cycle and in menopause

The neuroactive steroid 3α-5α-tetrahydroprogesterone (allopregnanolone), a metabolite of progesterone, is a positive allosteric modulator of GABAA receptors, and low levels have been implicated in the etiology of mood disorders. However, it is not known whether metabolism of progesterone to allopregnanolone varies across the menstrual cycle or is low after menopause. We hypothesized that the allopregnanolone/progesterone ratio would decrease from the follicular to luteal phase. We also hypothesized that postmenopausal women would have lower levels of progesterone and allopregnanolone but similar allopregnanolone/progesterone ratios as premenopausal women in the follicular phase.Serum fasting allopregnanolone and progesterone levels were measured by gas chromatography-mass spectrometry in ten premenopausal women at the follicular, mid-cycle, and luteal phases of the menstrual cycle and in twenty-four postmenopausal women. Although allopregnanolone and progesterone levels increased from the follicular to luteal phase, the allopregnanolone/progesterone ratio decreased 8-fold [0.33 ± 0.08 (follicular) vs 0.16 ± 0.09 (mid-cycle) vs 0.04 ± 0.007 (luteal), p = 0.0003]. Mean allopregnanolone and progesterone levels were lower in postmenopausal than premenopausal women at all menstrual cycle phases (p < 0.01). The mean allopregnanolone/progesterone ratio was similar in postmenopausal and premenopausal women in the follicular phase (0.39 ± 0.08 vs 0.33 ± 0.08, p = 0.94) but was significantly lower at mid-cycle and in the luteal phase than in postmenopausal women (p < 0.01). In conclusion, the serum allopregnanolone/progesterone ratio decreases 8-fold from the follicular to luteal phase and is lower at mid-cycle and the luteal phase than in postmenopausal women. Whether these data have implications for luteal phase and other mood disorders merits further study.

Allopregnanolone and progesterone in estradiol treated severe postpartum depression and psychosis – Preliminary findings

BackgroundPostpartum affective disorders may be associated with dysregulation of gonadal steroids. We investigated peripheral levels of allopregnanolone and progesterone in a combined group of women with postpartum onset of severe depression and/or psychosis who, as previously reported, responded with rapid symptom remission during sublingual estradiol treatment. The aim was to assess differences in allopregnanolone and progesterone between patients and healthy controls at baseline, and hormonal changes during estradiol treatment and symptom remission in patients. MethodsAllopregnanolone and progesterone in serum were analyzed with radioimmunoassay before and four weeks after initiation of sublingual estradiol treatment in ten women with postpartum depression and four women with postpartum psychosis (ICD-10). Twenty-eight healthy postpartum controls were included for baseline comparison. ResultsAllopregnanolone declined significantly during estradiol treatment while there was a trend for lower baseline allopregnanolone levels in patients compared with healthy postpartum controls. The ratio between allopregnanolone and progesterone was significantly lower in patients compared with controls and it remained unchanged after clinical recovery. LimitationsThis study is a secondary analysis of two estradiol treatment studies based on availability of samples for the analysis of allopregnanolone. Healthy controls were assessed earlier after delivery. Data on potential confounders (somatic health, breastfeeding, other medication) were not available. ConclusionsThese preliminary findings suggest that clinical recovery of severe postpartum depression and psychosis during estradiol treatment does not seem to depend on increasing levels of allopregnanolone. Differences in progesterone metabolism may constitute a risk factor for severe postnatal affective dysregulation.

Metabolism of cholesterol and progesterone is differentially regulated in primary trophoblastic subtypes and might be disturbed in recurrent miscarriages

During pregnancy, extravillous trophoblasts (EVTs) invade the maternal decidua and remodel the local vasculature to establish blood supply for the growing fetus. Compromised EVT function has been linked to aberrant pregnancy associated with maternal and fetal morbidity and mortality. However, metabolic features of this invasive trophoblast subtype are largely unknown. Using primary human trophoblasts isolated from first trimester placental tissues, we show that cellular cholesterol homeostasis is differentially regulated in EVTs compared with villous cytotrophoblasts. Utilizing RNA-sequencing, gene set-enrichment analysis, and functional validation, we provide evidence that EVTs display increased levels of free and esterified cholesterol. Accordingly, EVTs are characterized by increased expression of the HDL-receptor, scavenger receptor class B type I, and reduced expression of the LXR and its target genes. We further reveal that EVTs express elevated levels of hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1) (a rate-limiting enzyme in progesterone synthesis) and are capable of secreting progesterone. Increasing cholesterol export by LXR activation reduced progesterone secretion in an ABCA1-dependent manner. Importantly, HSD3B1 expression was decreased in EVTs of idiopathic recurrent spontaneous abortions, pointing toward compromised progesterone metabolism in EVTs of early miscarriages. Here, we provide insights into the regulation of cholesterol and progesterone metabolism in trophoblastic subtypes and its putative relevance in human miscarriage.

Comparative analysis of steroids in cyclic and pregnant killer whales, beluga whales and bottlenose dolphins by liquid chromatography tandem mass spectrometry

There exists a surprising diversity in the physiology and endocrinology of pregnancy among mammals in both the source (luteal/placental) and metabolism of progesterone. To evaluate the possible diversity of steroid metabolism within toothed cetaceans, we investigated 5α-reduced progesterone metabolites and androgens in cyclic (luteal phase) and pregnant captive killer whales, belugas and bottlenose dolphins (n = 5/species) bled longitudinally in early, mid- and late pregnancy (0.16, 0.50 and 0.85 fractions of 535, 464 and 380 gestation days, respectively). Mid-luteal samples were also collected. Serum was analyzed by liquid chromatography tandem-mass spectrometry as previously validated for (among others) progesterone, 20αOH-progesterone (20αOHP), 5α-dihydroprogesterone (DHP), several additional 5α-reduced metabolites and androgens (dehydroepiandrosterone, androstenedione and testosterone). The predominant mid-luteal pregnanes were: progesterone, belugas; progesterone and 20αOHP, dolphins; allopregnanolone (3α-DHP) and progesterone, killer whales. Progesterone was 2-4-fold higher in early pregnancy than mid-luteal samples but decreased thereafter. The predominant metabolite, 3β,20α-dihydroprogesterone (3β,20α-DHP; 40–80 ng/ml) was higher in mid- and late-than early gestation in all 3 species. Concentrations of 20αOHP and 3β,20α-DHP were similar at mid-gestation but 20αOHP declined in late-gestation in killer whales, and 20αOHP was lower than 3β,20α-DHP in belugas and dolphins throughout gestation. Other 5α-reduced metabolites, DHP, 3α-DHP and 20α-DHP, were far lower throughout pregnancy (<10 ng/ml). DHP and 3α-DHP decreased from early to mid-gestation in belugas, but changed little in killer whales and dolphins. These data suggest that progesterone metabolism is relatively conserved among these cetacean species. As in equine pregnancies, 3β,20α-DHP is the major metabolite, increasing at the expense of progesterone as pregnancy progresses. Androstenedione and testosterone also increased detectably in mid- to late-gestation in these species. The tissue source remains unknown, but progesterone metabolism during gestation in these cetaceans is similar to horses and, together with androgens, may be reliable biomarkers of pregnancy.

PLIN2 Functions As a Novel Link Between Progesterone Signaling and Metabolism in Uterine Leiomyoma Cells.

Uterine leiomyoma (fibroids) are the most common tumors in women. Recently, perilipin-2 (PLIN2) was identified as a critical target gene of the progesterone receptor; however, its function in the pathogenesis of fibroids is unknown. To determine the function of PLIN2 in leiomyoma cells. Tissue and primary cells from leiomyoma and myometrium were analyzed. PLIN2 function in leiomyoma was assessed using small interfering RNA. RNA-sequencing was performed to identify genome-wide effects of PLIN2 depletion. Metabolic activity was measured using the Seahorse XF96 analyzer. Real-time quantitative PCR and immunoblotting were also performed. Laboratory. Forty-one premenopausal women undergoing surgery for fibroids. Gene expression, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and cell proliferation. PLIN2 gene expression was 2.4-fold lower in leiomyoma compared with adjacent myometrium, suggesting a link between PLIN2 deficiency and fibroids. A total of 3877 genes were differentially expressed after PLIN2 knockdown. Gene ontology analysis identified metabolism as the second-highest biological process affected by PLIN2 depletion. OCR (mitochondrial respiration) and ECAR (glycolysis) were significantly upregulated after PLIN2 knockdown; PLIN2-depleted cells had a greater basal metabolic activity and higher metabolic stress response. Cell proliferation was also significantly increased after PLIN2 knockdown. PLIN2 depletion increases mitochondrial respiration and glycolysis, suggesting that PLIN2 is a critical regulator of metabolic function in leiomyoma cells. PLIN2 deficiency also reprograms leiomyoma cells to a proproliferative phenotype. These findings introduce metabolomics as an area to explore to better understand leiomyoma tumorigenesis.

In ovo metabolism of progesterone to 5β-pregnanedione in chicken eggs: Implications for how yolk progesterone influences embryonic development

Progesterone has received substantial attention for the essential role it plays in establishing and maintaining pregnancy in placental vertebrates. Despite the prevalence of progesterone during development, relatively little is known about how embryos respond to progesterone. This is true of placental vertebrates as well as egg-laying vertebrates where levels of progesterone in the yolk tend to be higher than most other steroids in the yolk. Bird eggs provide an opportunity to investigate the effects of progesterone on embryonic development because progesterone can be easily manipulated without any confounding effects on maternal physiology. To understand how progesterone might influence embryonic development, it is important to characterize the metabolic fate of progesterone given its potential to be converted to a wide range of steroids. We investigated the metabolic fate of tritiated progesterone over the first four days of development using chicken eggs (Gallus gallus) and identified 5β-pregnanedione as the primary metabolite during this period. After only one day of development, 5β-pregnanedione could be detected within the yolk. Levels of 5β-pregnanedione in both the yolk and albumen tended to rise early in development but conjugated metabolites began to accumulate towards the end of our sampling period. Additionally, in vitro assays using embryo homogenates collected after 72 h of development demonstrated that embryos were capable of carrying out the conversion of progesterone to 5β-pregnanedione. Overall these results have important implications for deciphering the mechanisms through which yolk progesterone might influence embryonic development. Effects could arise via progesterone receptors or receptors capable of binding 5β-pregnanedione but we found no evidence that progesterone is serving as a precursor for androgen or estrogen production.

Liquiritin, as a Natural Inhibitor of AKR1C1, Could Interfere With the Progesterone Metabolism.

Low progesterone level is always linked with pre-term birth. Therefore, maintaining of progesterone level is vital during pregnancy. Aldo-keto reductase family one member C1 (AKR1C1) catalyzes the reduction of progesterone to its inactive form of 20-alpha-hydroxy-progesterone and thus limits the biological effect of progesterone. In our effort to identify the natural compound that would specifically inhibit AKR1C1, liquiritin was found to be a selective and potent inhibitor of AKR1C1. Kinetic analyses in the S-(+)-1,2,3,4-tetrahydro-1-naphthol (s-tetralol) catalyzed by AKR1C1 in the presence of the inhibitors suggest that liquiritin is a competitive inhibitor by targeting the residues Ala-27, Val-29, Ala-25, and Asn-56 of AKR1C1. In HEC-1-B cells, treatment with liquiritin results in 85.00% of reduction in progesterone metabolism, which is mediated by AKR1C1 enzymatic activity. Overall, our study not only identify liquiritin as an inhibitor against AKR1C1, but also reveal that liquiritin may be served as a potential intervention strategy for preventing pre-term birth caused by low progesterone level.

MON-449 Serum Neuroactive Steroid Levels in Postmenopausal Women with Treatment-Resistant Major Depressive Disorder

Background: Neuroactive steroids such as 3α-5α-tetrahydroprogesterone (allopregnanolone) and 5α-androstane-3α,17β-diol (3α-androstanediol) are modulators of traditional neurotransmitter receptors and have been implicated in the etiopathology of psychiatric disorders, including depression, but levels have not been assessed in women with treatment-resistant major depressive disorder (MDD). We hypothesized that allopregnanolone, 3α-androstanediol, and the ratio of these steroid levels to their precursors (progesterone and testosterone, respectively) would be lower in postmenopausal women with treatment-resistant MDD than non-depressed controls. Methods: Fasting serum neuroactive steroid levels measured by gas chromatography/mass spectrometry were compared in women with treatment-resistant MDD (MDD, n=12) [Montgomery-Asberg Depression Rating Scale (MADRS) >12 despite an adequately dosed antidepressant] and in healthy controls without depression (HC, n=28). All subjects were postmenopausal nonsmokers; none were receiving systemic estrogen. Results: Mean age and BMI did not differ between groups. In MDD, mean number of antidepressants per subject was 1.5±0.5 (SD), with 58% receiving selective serotonin reuptake inhibitors and 50% bupropion. Mean MADRS was 24.4±5.8 (moderate depression severity). In MDD vs HC, the mean allopregnanolone/progesterone ratio was lower (0.20±0.19 vs 0.47±0.46, p=0.03) and progesterone levels were higher (153±177 vs 57±50 pg/mL, p=0.04). There was no difference in mean allopregnanolone levels between groups. Compared with HC, MDD subjects had lower serum free testosterone (0.21±0.16 vs 0.38±0.18 ng/dL, p=0.006) and a trend toward lower total testosterone (13.3±5.9 vs 18.6±9.9 ng/dL, p=0.06). There was no difference in 3α-androstanediol levels or 3α-androstanediol/total testosterone ratio between groups. There was a trend toward a positive association between progesterone levels and depression severity (r=0.34, p=0.06) and an inverse association between the allopregnanolone/progesterone ratio and depression severity (r=-0.36, p=0.07). Lower free testosterone levels were associated with greater depression severity (r=-0.45, p=0.03). Conclusion: In postmenopausal women with treatment-resistant MDD, the allopregnanolone/progesterone ratio was lower and progesterone levels higher than in non-depressed controls. Allopregnanolone levels did not differ between groups. This may be due to reduced metabolism of progesterone to allopregnanolone and could have treatment implications. Additionally, testosterone levels were lower in depressed women, but there was no difference in 3α-androstanediol levels or the 3α-androstanediol/total testosterone ratio between depressed and non-depressed women, suggesting that testosterone may play a greater role in depression symptomatology than its metabolite.