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The anti-cancer properties of plasma-treated solutions (PTS) and their interaction with drugs are one of the most popular topics in modern plasma medicine. Our research involved comparing the effects of four physiological saline solutions (0.9% NaCl, Ringer's solution, Hank's Balanced Salt Solution, Hank's Balanced Salt Solution with amino acids added in concentrations observed in the human blood) treated with cold atmospheric plasma and studying the combined cytotoxic effect of PTS with doxorubicin and medroxyprogesterone acetate (MPA). Analysis of the effect of the studied agents on the formation of radicals in the incubation medium, the vitality of K562 myeloid leukaemia cells, and the processes of autophagy and apoptosis in them revealed two key findings. The first is that when using PTS and doxorubicin-containing PTS, autophagy is the predominant process in cancer cells. The second is that combining PTS with MPA enhances apoptotic processes. It was hypothesised that while autophagy is stimulated by the accumulation of reactive oxygen species in the cell, apoptosis is stimulated through specific cell progesterone receptors.

Sickle cell disease (SCD) is associated with altered plasma and erythrocyte lipid profiles. In a previous study, SCD mice with deficiency of proprotein convertase subtilisin/kexin type 9 (PCSK9) were observed to have more severe anemia and increased sickling compared to control SCD mice. Although PCSK9 affects circulating low density lipoprotein (LDL) by regulation of the LDL receptor, the effect of PCSK9 on anemia was independent of LDL receptor expression. In the current study, erythrocyte metabolomics were performed and revealed altered erythrocyte lipid species between SCD mice with and without PCSK9. Of particular interest, the late endosome-specific lipid bis(mono)acylglycerol phosphate (BMP) 44:12 was markedly decreased in erythrocytes from SCD mice deficient in PCSK9 mice relative to control SCD mice. Incubation of sickle erythrocytes with a neutralizing antibody to BMP increased erythrocyte sickling in vitro. In vitro treatment of SCD erythrocytes with amiodarone (1.5 μM) or medroxyprogesterone (6.75 μM), two pharmacologic compounds known to increase BMP, resulted in reduced erythrocyte sickling. Treatment of SCD mice with amiodarone (10 mg/kg) for 2 weeks resulted in increased BMP, improvement in anemia with reduced reticulocytosis, and decreased ex vivo sickling. In conclusion, severity of anemia in SCD is improved with amiodarone treatment, an effect which may be mediated through increased erythrocyte BMP., (© 2022. The Author(s).)

Objective: To compare the clinical outcomes of dydrogesterone (DYG) and medroxyprogesterone (MPA) in the progestin-primed ovarian stimulation (PPOS) protocol for patients with poor ovarian response (POR)., Patients and Methods: This was a retrospective cohort study. Women with POR who underwent IVF/ICSI at the Reproductive Center of Third Affiliated Hospital of Zhengzhou University between January 2020 and January 2021 were included. The primary outcome measure of our study was the number of oocytes retrieved. The secondary outcome measures in the present study were the number of 2PN, number of available embryos, oocyte retrieval rate, fertilization rate, viable embryo rate per oocyte retrieved, cancellation rate and pregnancy outcomes of the first embryo transfer cycle, including the biochemical pregnancy, clinical pregnancy and miscarriage rates., Results: In total, 118 women underwent hMG +DYG protocols, and 692 women who underwent hMG +MPA met the Bologna criteria for POR. After baseline characteristics were balanced using the PSM model, 118 hMG +DYG protocols were matched to 118 hMG +MPA protocols, and the baseline characteristics were comparable between the two groups. The numbers of oocytes retrieved, 2PN, and available embryos and the oocyte retrieval rate, fertilization rate, viable embryo rate per oocyte retrieved and cancellation rate of the hMG+DYG and hMG+MPA protocols were comparable. Altogether, 66 women in the hMG+DYG group and 87 women in the hMG+MPA group underwent first embryo transfers. In the hMG+DYG group, 81.8% (54/66) of the patients underwent cleavage embryo transfers; similarly, 79.3% (69/87) of patients in the hMG+MPA group had cleavage embryo transfers (P=0.70).The biochemical pregnancy rate of the hMG+DYG group was 42.4%, and this was comparable to the rate in the hMG+DYG group, at 34.5% (P=0.32). The clinical pregnancy rates were similar between the two groups (36.4% vs. 31.0%, P=0.49), and there was no significant difference in the rate of miscarriage between the two groups (12.5% vs. 29.6%, P=0.14)., Conclusion: For women with POR, the clinical outcome of the hMG + DYG group was similar to that of the hMG + MPA group, indicating that both combinations can be useful options for PPOS protocols., 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 Zhang, Du, Li, Liu, Ren, Zhang and Guan.)

Endocrine therapy is a promising treatment for endometrial cancer (EC) that preserves fertility, however, progesterone-resistance is currently the major challenges. The Cancer Genome Atlas (TCGA) database analysis showed that CNR1 was closely have a negative correlation with overall survival (OS) and relapse-free survival (RFS) in endometrial cancer. To explore the role of CNR1 in progesterone resistance and possible molecular regulation mechanism, we established stable progesterone-resistant cell lines (IshikawaPR) via progesterone tolerance of ordinary cancer cells (Ishikawa). The difference of CNR1 level in two cell lines was assessed by MTT, RT-PCR, Western blot, immunofluorescence. Then, lentiviruses constructed CNR1-knockdown with GV248 as the tool vector were used to transfect IshikwaPR cells, and the changes of biological behavior and progesterone sensitivity was verified respectively through plate cloning experiment, EdU assay, flow cytometry cycle analysis, transwell, Scratch test, etc. We founded after CNR1 was knocked down, the proliferative activity and ability to migrate of IshikawaPR cells decreased, progesterone-response sensitivity could be improved. Moreover, knockdown of CNR1 can also down-regulate ERK and NFκ B expression and activation. Furthermore, subcutaneous xenograft in nude mice was tested similarly in vivo. The above datas suggest that targeting CNR1 may reverse the progesterone resistance in endometrial cancer and may coordinate the role of ERK pathway activation., 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 © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The fungal transformations of medroxyrogesterone (1) were investigated for the first time using Cunninghamella elegans, Trichothecium roseum, and Mucor plumbeus. The metabolites obtained are as following: 6β, 20-dihydroxymedroxyprogesterone (2), 12β-hydroxymedroxyprogesterone (3), 6β, 11β-dihydroxymedroxyprogesterone (4), 16β-hydroxymedroxyprogesterone (5), 11α, 17-dihydroxy-6α-methylpregn-4-ene-3, 20-dione (6), 11-oxo-medroxyprogesterone (7), 6α-methyl-17α-hydroxypregn-1,4-diene-3,20-dione (8), and 6β-hydroxymedroxyprogesterone (9), 15β-hydroxymedroxyprogesterone (10), 6α-methyl-17α, 11β-dihydroxy-5α-pregnan-3, 20-dione (11), 11β-hydroxymedroxyprogesterone (12), and 11α, 20-dihydroxymedroxyprogesterone (13). Among all the microbial transformed products, the newly isolated biotransformed product 13 showed the most potent activity against proliferation of SH-SY5Y cells. Compounds 12, 5, 6, 9, 11, and 3 (in descending order of activity) also showed some extent of activity against SH-SY5Y tumour cell line. The never been reported biotransformed product, 2, showed the most potent inhibitory activity against acetylcholinesterase. Molecular modelling studies were carried out to understand the observed experimental activities, and also to obtain more information on the binding mode and the interactions between the biotransformed products, and enzyme., (Copyright © 2020. Published by Elsevier Inc.)

This study was conducted in ewes to assess effects of human chorionic gonadotropin (hCG) administration after imposing an estrous induction treatment regimen. Ewes (n = 115) were treated with a 60 mg medroxyprogesterone-intravaginal-sponge for 6 d plus 200 IU of equine chorionic gonadotropin (eCG) im and 37.5 μg d-cloprostenol im 36 h before sponge removal (Day 0). After natural mating, ewes having at least one corpus luteum (CL; n = 108) were administered either 1 mL of saline (G-Control; n = 53) or 300 IU of hCG (G-hCG; n = 55) on Day 7.5 after sponge removal (Day 0). Ovarian ultrasonography and blood collection were performed on Days 7.5, 13.5, 17.5, 21.5, and 30.5. Accessory CL (aCL) were observed in 81.5 % (G-hCG) and 0.0 % (G-Control) of ewes (P = 0.0001). Diameter, area, and volume of luteal tissue were greater (P < 0.05) in G-hCG from Day 13.5 to 30.5. Progesterone (P 4 ) concentrations were greater (P < 0.05) on Days 13.5, 17.5, 21.5 and 30.5 for ewes of the G-hCG group. Pregnancy percentage was similar (P = 0.25) between groups [47.1 % (G-control) compared with 60.0 % (G-hCG)], although total number of lambs produced by estrous synchronized ewes was greater (P = 0.005) in ewes of the G-hCG group (90.9 % compared with 66.0 %). In conclusion, hCG administration 7.5 days after sponge removal from Morada Nova ewes during the non-breeding season is an effective treatment to induce aCL formation, improve luteal tissue biometry and P4 concentrations, and to enhance the total number of lambs born., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The effect of short-term administration of medroxyprogesterone acetate (MPA) or natural progesterone (P 4 ) during ovarian stimulation with FSH on oocyte recovery was investigated in Santa Inês ewes. Ewes were treated with an intravaginal sponge containing MPA for 6 d; GnRH was applied 36 h after sponge removal and FSH was given in 3 injections (40, 24, and 16 mg, respectively) every 12 h after (D0, approximate ovulation time). At the first FSH dose, the ewes received either a new MPA sponge (n = 10) or a controlled device for internal release impregnated with P 4 (n = 10) or did not receive any device (n = 10). Ovarian dynamics were assessed every 12 h by transrectal ultrasonography from D-3 to D2. Oocytes were recovered by laparoscopic ovum pick-up (LOPU) on D2 and graded by morphologic quality. The number of small, medium, and large follicles at D0 and D2 (ultrasound examinations), number of both follicles aspirated and oocytes recovered at LOPU, recovery rate, and oocyte grade did not differ (P > 0.05) among treatments. Thus, the short-term use of MPA or P 4 during ovarian stimulation did not affect the first-wave follicle population or morphologic quality of oocytes. We would suggest that, in this protocol, the use of exogenous progestin is unnecessary., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Turner Syndrome (TS) is associated with an increased risk of cardiovascular and metabolic complications. Furthermore, TS women need hormone replacement therapy (HRT), of which progestins can influence body weight. We aimed to analyze the metabolic and weight profile in a cohort of 111 TS women. They started receiving estrogen at 15.8 (±3.6) years old, with no change in hypertension, dysglycemia, and dyslipidemia incidence but with a tendency to increase overweight (p = 0.054). As the first used type of progestin, most had received cycles of 10 days per month of medroxyprogesterone (MPA) or levonorgestrel (LNG), then shifted to micronized progesterone (MP), which has currently become the most used one. By multiple linear regression analysis, we found that the prolonged use of MPA, LNG, or MP showed no metabolic change except for weight gain. The percentage of annual BMI increment was positive for all progestins used in TS women (MPA 2.2 ± 2.2; LNG 0.2 ± 1.2; and MP 2.2 ± 2.6 kg/m2), but LNG seemed to best prevent on weight gain over time (p < 0.05). In conclusion, metabolic comorbidities are prevalent in TS even before the HRT regimen, and LNG performed better on less weight gain than MPA and MP in our cohort of the TS population.

Objective: Supraphysiologic estradiol (E 2 ) levels associated with controlled ovarian hyperstimulation in high in vitro fertilization (IVF) responders may alter implantation and placentation and increase the risk of preeclampsia. Our hypothesis is that elevated E 2 levels in vitro significantly alter endometrial decidualization, sFlt1, and HOXA10 expression., Methods: Human endometrial stromal cells were treated with a decidualization cocktail of medroxyprogesterone, cyclic adenosine monophosphate, and 3 concentrations of E 2 10 nM (standard), 100 nM (intermediate), or 1000 nM E 2 (high). Effects on sFlt1, prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP-1), vascular endothelial growth factor (VEGF), and HOXA10 were studied., Results: Prolactin, IGFBP-1, and VEGF significantly increased at all 3 E 2 concentrations. While IGFBP-1 and VEGF did not change with increasing E 2 , PRL was less with high E 2 (6.0 ng/mL ± 1.4 standard error of the mean) compared to standard (21.4 ± 3.2) and intermediate (19.8 ± 3.8). sFlt1 decrease was similar at all E 2 concentrations. HOXA10 was lower at standard (10%) and intermediate (30%) as expected, but did not change with high E 2 ., Conclusions: Supraphysiologic E 2 levels associated with high IVF responders that exceed in vivo levels may impair in vitro endometrial decidualization. Although PRL did increase with high E 2 , the levels were, however, attenuated and 3.4-fold lower than standard and intermediate E 2 . sFlt1 was decreased under all 3 conditions with no differences between concentrations. Reduced HOXA10 was not observed with high E 2 . These findings suggest that elevated E 2 levels in vitro may alter endometrial decidualization and subsequently affect implantation and placentation.

Objective: Risk of high grade gliomas is lower in young females and its incidence enhances after menopause suggesting likely protective roles of female hormones. Hormone replacement therapy (HRT) was widely employed to treat osteoporosis and some epidemiological studies showed that HRT regimes including progesterone analogs such as medroxyprogesterone acetate (MPA) decreased risk of glial tumors. Tibolone is a unique progesterone analog employed in HRT with tissue specific estrogenic effects and stimulates gene expressions very similar to those induced by MPA. Tibolone's pro-estrogenic effects occur particularly in bone and brain and both MPA and tibolone inhibit AKR1C enzymes, which involve in temozolomide chemoresistance. Hence, we aimed to investigate interactions between MPA, tibolone and temozolomide in modification of glioma cell growth and fine structure., Patients and Methods: For our studies, we have particularly chosen C6 rat glioma cell line due to several reasons: i) We previously showed that MPA reduced growth and induced procarbazine-sensitization in C6 cells; ii) temozolomide has a triazene-type molecular structure like procarbazine; iii) other groups previously showed that C6 glioma cell line is more resistant to temozolomide than human glioma cells; hence it may provide a native model of chemoresistance. Monolayer plating efficacy, soft agar colony growth, 3D-spheroid S-phase (as determined by BrdU-labeling) and electron microscopical analyses were performed to assess mutual interactions between MPA, tibolone and temozolomide., Results: MPA inhibited clonogenic growth of C6 glioma and this effect is augmented by both tibolone and temozolomide. MPA and tibolone inhibited DNA synthesis in C6 glioma spheroids to similar levels which can be achieved with temozolomide. Electron microscopical analyses revealed synergisms between MPA, tibolone and temozolomide involved mitochondrial proliferation, condensation, mitophagy and autophagy., Conclusions: MPA and tibolone shall be studied in further experimental models of glioblastoma in vitro and in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: A new strategy for menopausal hormone therapy replaces medroxyprogesterone with the selective estrogen receptor modulator bazedoxifene. While the agonist or antagonist activity of bazedoxifene has been examined in other tissues, the current study explored the impact of bazedoxifene on resistance artery reactivity. We hypothesized that bazedoxifene may induce greater vasoprotective effects than estradiol due to enhanced activation of the G-protein-coupled estrogen receptor., Methods: We measured the vasodilation of mesenteric resistance arteries from adult male and female wild-type and G-protein-coupled estrogen receptor knockout mice (n = 58) in response to increasing concentrations of bazedoxifene, medroxyprogesterone, and estradiol, and also the impact of these compounds on the responses to phenylephrine and sodium nitroprusside., Results: Bazedoxifene-induced vasorelaxation was greater than estradiol and blunted phenylephrine-induced contraction-an effect not observed with estradiol. Neither estradiol nor bazedoxifene altered relaxation to sodium nitroprusside. The combination of bazedoxifene + estradiol promoted greater vasodilation than medroxyprogesterone + estradiol, and opposed phenylephrine-induced contraction, whereas medroxyprogesterone + estradiol failed to attenuate this response. Both bazedoxifene + estradiol and medroxyprogesterone + estradiol enhanced sodium nitroprusside-induced relaxation in females. Vascular responses were similar in both sexes in wild-type and G-protein-coupled estrogen receptor knockout mice., Conclusion: Bazedoxifene and bazedoxifene + estradiol relaxed mesenteric arteries and opposed vasoconstriction to a greater degree than estradiol or medroxyprogesterone + estradiol. These effects were independent of sex and G-protein-coupled estrogen receptor expression. We conclude that bazedoxifene may provide vascular benefits over estrogen alone or estrogen plus progestogen combinations in postmenopausal women.

Several tissue clearing methods have been developed for three-dimensional imaging of thick specimens. Here, we applied CUBIC and ScaleS approaches to whole-mounted vaginal wall to reveal spatial distribution of γδ T lymphocytes, the key cells engaged in the epithelial homeostasis control and immune surveillance. Both methods rendered the tissue transparent and enabled detection of the green fluorescent protein (GFP)-expressing γδ T cells in vaginal samples of Tcrd-H2BeGFP transgenic mice. Upon additional immunolabeling, however, only CUBIC preserved the GFP signal and allowed for cell localization assessment during the estrous cycle. Using a combination of single- and two-photon microscopy, we found that during the diestrus phase the number of γδ T cells in the vaginal wall increased compared to estrus, while the proportion of cells residing in epithelium and stroma remained constant, irrespective of the cycle phase, and was close to 3:1, respectively. Moreover, the distance from epithelial γδ T cells to laminin-positive basal membrane and collagen-rich stroma also increased in diestrus in spite of thinning of epithelium upon shedding cornified cells. Our data indicate that γδ T cells sense sex hormone fluxes which influence their number and position them closer to the vaginal lumen in the diestrus phase., (© 2018 Australasian Society for Immunology Inc.)

Objective: Glial tumor growth may accelerate during gestation, but epidemiological studies consistently demonstrated that parousity reduces life long risk of glial tumors. Pregnancy may also accelerate growth of medulloblastoma and meningioma, but parousity does not confer protection against these tumors. We were the first to show that medroxyprogesterone acetate (MPA) reduces rat C6 glioma growth in vitro. Now we aimed to determine the effects of MPA on human brain cancers (particularly glioblastoma) in vitro and C6 glioma in vivo., Patients and Methods: We evaluated the effects of MPA on: i) monolayer growth of human U87 and U251 glioblastoma, ii) 3D-spheroid growth and invasion of C6 rat glioma and human U251 glioma, iii) interactions with PI3-Kinase inhibitors and coxsackie-adenovirus receptor (CAR) in modifying 3D-spheroid invasion of glioma., Results: MPA at low doses (3.25-13 μM) insignificantly stimulated and at high doses (above 52 μM) strongly suppressed the growth of human U87 and U251 cells in vitro. MPA also binds to glucocorticoid receptors similar to dexamethasone (Dex) and unexpectedly, PI3-Kinase inhibitors at low doses suppressed anti-invasive efficacies of MPA and Dex. MPA exerted higher invasion-inhibitory effects on CAR-expressing human glioma cells. Lastly, MPA suppressed growth of C6 glioma implanted into rat brain., Conclusion: Progesterone analogues deserve to be studied in future experimental models of high grade glial brain tumors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Context: Glycogen synthesis is a critical metabolic function of the endometrium to prepare for successful implantation and sustain embryo development. Yet, regulation of endometrial carbohydrate metabolism is poorly characterized. Whereas glycogen synthesis is attributed to progesterone, we previously found that the metabolic B isoform of the insulin receptor is maximally expressed in secretory-phase endometrium, indicating a potential role of insulin in glucose metabolism., Objective: We sought to determine whether insulin or progesterone regulates glycogen synthesis in human endometrium., Design, Participants, Outcome Measurements: Endometrial epithelial cells were isolated from 28 healthy women and treated with insulin, medroxyprogesterone (MPA), or vehicle. Intracellular glycogen and the activation of key enzymes were quantified., Results: In epithelia, insulin induced a 4.4-fold increase in glycogen, whereas MPA did not alter glycogen content. Insulin inactivated glycogen synthase (GS) kinase 3α/β (GSK3α/β), relieving inhibition of GS. In a regulatory mechanism, distinct from liver and muscle, insulin also increased GS by 3.7-fold through increased GS 2 (GYS2) gene expression., Conclusions: We demonstrate that insulin, not progesterone, directly regulates glycogen synthesis through canonical acute inactivation of GSK3α/β and noncanonical stimulation of GYS2 transcription. Persistently elevated GS enables endometrium to synthesize glycogen constitutively, independent of short-term nutrient flux, during implantation and early pregnancy. This suggests that insulin plays a key, physiological role in endometrial glucose metabolism and underlines the need to delineate the effect of maternal obesity and hyperinsulinemia on fertility and fetal development.

Background: Plasma aldosterone/renin ratio (ARR) is the most popular screening test for primary aldosteronism (PA). Because both estrogen and progesterone (including in oral contraceptive agents) affect aldosterone and renin levels, we studied the effects of combined hormonal replacement therapy (HRT) on ARR; renin was measured as both direct renin concentration (DRC) and plasma renin activity (PRA)., Methods: Fifteen normotensive, healthy postmenopausal women underwent measurement (seated, midmorning) of plasma aldosterone, DRC, PRA, electrolytes, and creatinine and urinary aldosterone, cortisol, electrolytes, and creatinine at baseline and after 2 weeks and 6 weeks of treatment with combined HRT (conjugated estrogens 0.625 mg and medroxyprogesterone 2.5 mg daily)., Results: Combined HRT was associated with statistically significant increases in aldosterone [median (range): baseline, 150 (85 to 600); 2 weeks, 230 (129 to 790); 6 weeks, 434 (200 to 1200) pmol/L; P < 0.001 (Friedman test)] and PRA [2.3 (1.2 to 4.3), 3.8 (1.4 to 7.0), 5.1 (1.4 to 10.8) ng/mL/h, respectively; P < 0.001] but decreases in DRC [21 (10 to 31), 21 (10 to 39), and 14 (8.0 to 30) mU/L, respectively; P < 0.01], leading to increases in ARR calculated by DRC [7.8 (3.6 to 34.8), 11.4 (5.4 to 48.5), and 30.4 (10.5 to 90.2), respectively; P < 0.001]. The ARR calculated by DRC exceeded the cutoff value (70) in three patients after 6 weeks. There were no significant changes in ARR calculated by PRA [79 (26 to 184), 91 (23 to 166), and 88 (50 to 230), respectively; P = 0.282], plasma electrolytes and creatinine, or any urinary measurements., Conclusion: The combined oral HRT used in this study is capable of significantly increasing ARR with a risk of false-positive results during screening for PA but only if DRC (and not PRA) is used to calculate the ratio., (Copyright © 2017 Endocrine Society)

Acute myeloid leukaemia (AML) is a life threatening cancer for which there is an urgent clinical need for novel therapeutic approaches. A redeployed drug combination of bezafibrate and medroxyprogesterone acetate (BaP) has shown anti-leukaemic activity in vitro and in vivo. Elucidation of the BaP mechanism of action is required in order to understand how to maximise the clinical benefit. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Synchrotron radiation FTIR (S-FTIR) and Raman microspectroscopy are powerful complementary techniques which were employed to probe the biochemical composition of two AML cell lines in the presence and absence of BaP. Analysis was performed on single living cells along with dehydrated and fixed cells to provide a large and detailed data set. A consideration of the main spectral differences in conjunction with multivariate statistical analysis reveals a significant change to the cellular lipid composition with drug treatment; furthermore, this response is not caused by cell apoptosis. No change to the DNA of either cell line was observed suggesting this combination therapy primarily targets lipid biosynthesis or effects bioactive lipids that activate specific signalling pathways.

The objective was to investigate the effect of short-term (7 days) and long-term (14 days) progesterone-based estrus synchronization on number of follicles, progesterone concentrations, cumulus-oocyte complex (COC) gene expression, and embryonic development in goats. Nulliparous Thai-native goats (n = 45) were randomly assigned to one of two estrus synchronization treatments. Goats were treated with intravaginal sponges containing 60-mg medroxyprogesterone acetate (MAP; Synchrogest esponjas, Spain) during 7 or 14 days (short-term or long-term protocol, respectively). Multiple follicular development was induced by intramuscularly injections of 300-IU eCG in both groups (1 day before sponge withdrawal). An ovariectomy was performed at 24 hours after sponge removal to evaluate number of follicle and collect oocyte for IVF. Oocyte quality (healthy or nonhealthy) was determined by morphology of COCs before IVM. Recovery of COCs and total cellular RNA isolation were applied to determine apoptosis-related gene expression. After IVF, embryos were evaluated during the eight-day culture as numbers of cleaved oocyte, morula, and blastocyst embryo. Total numbers of follicles and oocytes were similar for both treatments. Plasma progesterone concentrations were not different during MAP insertion period (P > 0.05). However, goats that received the short-term protocol had a greater number of 4 to 6-mm follicle, healthy oocytes, cleaved oocytes, and morula embryos than goats that received the long-term protocol (P < 0.01). In addition, the expression of B-cell lymphoma 2 messenger RNA was greater (P < 0.05) in COCs derived from the 7 days MAP-treated when compared to the 14 days MAP-treated goats. These data highlight that the 7-day progestin-based treatment may contribute to quality of oocytes and embryonic development in goats., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in the regulation of cell proliferation and cancer progression. Increased expression of S1P receptors has been detected in advanced breast tumours with poor prognosis suggesting that S1P receptors might control tumour response to chemotherapy. However, it remains unclear how the levels of S1P receptor expression are influenced by chemotherapy agents. Western immunoblotting, PCR analysis and fluorescent microscopy techniques were used in this study to analyze expression patterns of S1P receptors 2 and 3 (S1P2/S1P3) in MCF-7 breast adenocarcinoma cells treated by Tamoxifen (TAM) and/or Medroxyprogesterone acetate (MPA). We found that TAM/MPA induce downregulation of S1P3 receptors, but stimulate expression of S1P2. According to cell viability and caspase activity analyses, as expected, TAM activated apoptosis. We also detected TAM/MPA-induced autophagy marked by formation of macroautophagosomes and increased level of Beclin 1. Combined application of TAM and MPA resulted in synergistic apoptosis- and autophagy-stimulating effects. Assessed by fluorescent microscopy with autophagosome marker LAMP-2, changes in S1P receptor expression coincided with activation of autophagy, suggestively, directing breast cancer cells towards death. Further studies are warranted to explore the utility of manipulation of S1P2 and S1P3 receptor expression as a novel treatment approach., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Aim of the Study: To compare effect of six month transdermal 17 β-estradiol supplementation with oral medroxyprogresterone acetate to oral simvastatin treatment on nitric oxide (NO), endothelin-1, β-thromboglobulin, vascular endothelial growth factor (VEGF) and von Willebrand factor (vWF) levels during standard exercise test in post menopausal women., Patients and Methods: 32 women were included to the study. Group 1 treated with 17β-estradiol combined with medroxyprogesterone. Group 2 treated with simvastatin, group 3 was the controls. VEGF plasma levels as well as basal and standard exercise test induced levels of vWF, NO, endothelin- 1, β-thromboglobulin were measured at the beginning of the study, at 3rd and 6th month of the study. During standard exercise test these parameters were measured three times: at the beginning, at peak exercise and at the 15th minute of recovery., Results: 17β-estradiol supplementation and simvastatin treatment reduced basal and exercise test induced endothelin-1 plasma level. 17β-estradiol supplementation gradually increased NO release, whereas simvastatin initially reduced and finally increased nitric oxide release. NO/ET-1 ratio was increased at peak exercise and recovery time in group 1 whereas only at peak exercise in group 2. Basal VEGF plasma level and β-thromboglobulin level at recovery time were reduced after 6 month of simvastatin therapy., Conclusion: Six months long oral simvastatin exerted beneficial influence on endothelial function equal to that of continuous transdermal 17β-estradiol supplementation combined with medroxyprogesterone acetate. Simvastatin only exerted benefical effect on platelet function. The protective effect of both therapies was more pronounced during exercise and recovery time.

Objective: To explore the effects of Wnt5a and Wint7a on the differentiation of human embryonic stem cells (hESCs) into endometrium-like cells, and provide a basis for establishing endometrium-like cell models and a cell source for carrying out further endometrium-related experiments., Methods: The hESCs established by our center were differentiated into endometrium-like cells in 4 different media including Wnt5a (Group A), Wnt7a (Group B), secreted frizzled related protein (sFRP, an inhibitor of Wnt signal pathway, Group C) and medium alone (Group D). In the differentiated terminal cells, the expressions of cytokeratin (CK) and vimentin were detected with immunofluorescence, and the mRNA levels of CK18, epithelial cell adhesion molecule (EPCAM), estrogen receptor (ER) and progesterone receptor (PR) were determined with RT-PCR. At the same time, the differentiated terminal cells were incubated in medium containing medroxyprogesterone followed by determination of prolactin (PRL)., Results: RT-PCR indicated that mRNA levels of CK18, EPCAM, ER and PR were significantly higher in Group A (Wnt5a) than in other groups (all P<0.05), but were significantly lower in Group C (sFRP2) than in other groups (all P<0.05). The changing trend of PRL mRNA was consistent with that of above genes in the 4 groups. Immunofluorescence displayed that the expression of cytokeratin was the strongest in Group A (Wnt5a), and the weakest in Group C (sFRP2) among the 4 groups., Conclusion: Wnt5a has promotive effects on the differentiation of hESCs into endometrium-like cells, but Wnt7a has no marked effects.

Endometrial decidualization is highly important for successful construction and maintenance of embryo implantation and pregnancy. Lefty gene at different menstrual cycle phases has different expressions, indicating its regulatory significance. To study the mechanism of Lefty in decidualization, human endometrial stromal cells (hESCs) were cultured and induced with medroxyprogesterone acetate (MPA) and 8-bromoadenosine-cAMP (8-Br-cAMP) in vitro as a research model. Our results showed that Lefty1 overexpression inhibited MPA- and 8-Br-cAMP-induced hESC decidualization and significantly reduced the secretion of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP-1). With the inhibition of Lefty1 expression, hESC decidualization induced by MPA and 8-Br-cAMP became more remarkable, and the secretions of PRL and IGFBP-1 were higher too. Further tests indicated that during the process of decidualization, P57 expression increased, whereas cyclin D1 expression decreased. Although Lefty1 overexpression did not significantly change the expressions of P57 and cyclin D1, inhibition of Lefty1 expression resulted in more evident changes in P57 and cyclin D1 expressions. Meanwhile, cell cycle examination showed that Lefty1 overexpression reduced the cell cycle arrest at G1/S phase in the in vitro hESC decidualization model. Therefore, Lefty1 could regulate the cell cycle via modulating the expressions of P57 and cyclin D1 and then inhibit the decidualization in vitro., (Copyright © 2014 John Wiley & Sons, Ltd.)

Both natural oestrogens and progesterone influence synaptic plasticity and neurogenesis within the female hippocampus. However, less is known of the impact of synthetic hormones on hippocampal structure and function. There is some evidence that the administration of the synthetic progestin, medroxyprogesterone acetate (MPA) is not as beneficial as natural progesterone and can attenuate oestrogen-induced neuroprotection. Although the effects of oestradiol have been well studied, little is known about the effects of natural and synthetic progestins alone and in combination with oestradiol on adult neurogenesis in females. In the present study, we investigated the effects of chronic oestradiol, progesterone, MPA and the co-administration of each progestin with oestradiol on neurogenesis within the dentate gyrus of adult ovariectomised female rats. Twenty-four hours after a bromodeoxyuridine (BrdU; 200 mg/kg) injection, female rats were repeatedly administered either progesterone (1 or 4 mg), MPA (1 or 4 mg), oestradiol benzoate (EB), progesterone or MPA in combination with EB (10 μg), or vehicle for 21 days. Rats were perfused on day 22 and brain tissue was analysed for the number of BrdU-labelled and Ki67 (an endogenous marker of cell proliferation)-expressing cells. EB alone and MPA + EB significantly decreased neurogenesis and the number of surviving BrdU-labelled cells in the dorsal region of the dentate gyrus, independent of any effects on cell proliferation. Furthermore, MPA (1 and 4 mg) and MPA + EB treated animals had significantly lower adrenal/body mass ratios and reduced serum corticosterone (CORT) levels. By contrast, progesterone + EB treated animals had significantly higher adrenal/body mass ratios and 1 mg of progesterone, progesterone + EB, and EB significantly increased CORT levels. The results of the present study demonstrate that different progestins alone and in combination with oestradiol can differentially affect neurogenesis (via cell survival) and regulation of the hypothalamic-pituitary-adrenal axis. These findings have implications for women using hormone replacement therapies with MPA for both neuroprotection and stress-related disorders., (© 2014 British Society for Neuroendocrinology.)

Progesterone (P4) facilitates exploration, anxiety and social behaviors in estrogen (E2)-primed mice. Some of these effects may be due to actions of its 5α-reduced metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP). In order to address the role of P4 and its metabolite, 3α,5α-THP, a mouse model was utilized. We hypothesized that if P4's metabolism to 3α,5α-THP is essential to facilitate exploratory, anti-anxiety and social behaviors of mice, then wildtype, but not 5α-reductase knockout (5α-RKO), mice will have greater expression of these behaviors. Experiment 1: Mice were ovariectomized (ovx), E2-primed and administered P4 (0, 125, 250, or 500μg) subcutaneously and then tested 4h later in a battery of tasks: open field, elevated plus maze, and social interaction. Experiment 2: Ovx, E2-primed mice were administered P4 (4mg/kg), 3α,5α-THP (4mg/kg), medroxyprogesterone acetate (MPA, which does not convert to 3α,5α-THP; 4mg/kg), or vehicle subcutaneously and tested 4h later. There was a dose-dependent effect of P4 to wildtype, but not 5α-RKO, mice. Neither wildtype, nor 5α-RKO, mice had increased exploration, anti-anxiety or pro-social behavior with MPA administration. Progesterone only exerted effects on anti-anxiety behavior, and increased 3α,5α-THP in the prefrontal cortex and hippocampus, when administered to wildtype mice. 3α,5α-THP to both WT and 5α-RKO mice increased exploration, anti-anxiety and social interaction and 3α,5α-THP levels in the hippocampus and prefrontal cortex. Thus, metabolism of P4 by the 5α-reductase enzyme may be essential for enhancement of these behaviors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sex steroids are important regulators of neuronal cell morphology, and this is critical for gender differences in brain function and dysfunction. Neuronal morphology is controlled by multiprotein complexes including moesin (a member of the ezrin/radixin/moesin family), focal adhesion kinase (FAK), or the Wiskott-Aldrich syndrome protein-family verprolin homologous (WAVE1) protein, controlling dynamic remodeling of the cytoskeleton and cell membrane. We investigated the actions of natural progesterone (P) and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin remodeling, focal adhesion complex formation, and actin branching in rat cortical neurons. Treatment with P and, to a lesser extent, MPA, increases the number and density of dendritic spines. P increases the phosphorylation of moesin, FAK, and WAVE1, and their redistribution toward cell membrane sites where spines are formed. Signaling to moesin is achieved by PR via a Gα/Gβ-dependent signaling to the small GTPase Ras homolog gene family, member A and its related kinase, Rho-associated kinase-2. In parallel, WAVE1 recruitment is triggered by a Gαi/Gβ-dependent signaling of PR to c-Src, FAK, and Rac1 GTPase. Rac1 recruits cyclin-dependent kinase-5, which phosphorylates WAVE1. Silencing of moesin, FAK, or WAVE1 abrogates the increase in dendritic spines induced by progesterone. In all applications, MPA is found to act similar to P, albeit with a lower efficacy. In conclusion, our findings indicate that the control of actin polymerization and branching and focal adhesion complex formation via moesin, FAK, and WAVE1 is a key function of progesterone receptor in neurons, which may be relevant for the regulation of dendritic spine turnover and neuronal plasticity.

Progesterone profiles of individual bitches may vary considerably both between and within individuals during pregnancy and non-pregnancy. Suspected luteal deficiency is commonly purported but is difficult to evaluate in clinical cases when progesterone is supplemented because this masks the underlying hormone changes. Therefore, in this study, suspected cases of luteal deficiency (six pregnancies from five bitches) were supplemented with oral medroxyprogesterone acetate (MPA), allowing measurement of endogenous progesterone using conventional assay. MPA (0.1 mg/kg) treatment commenced between days 30 and 36 after estimated ovulation and was continued for 18-28 days. Endogenous progesterone was measured throughout treatment, and blood was additionally analysed for prolactin (PRL) and relaxin (RLN) as well as MPA. The latter revealed delayed MPA clearance in two bitches, in which Caesarean operation had to be performed because of a low foetal heart rate. In two cases with confirmed basal concentrations of both P(4) and MPA at term, spontaneous parturition occurred. Low endogenous progesterone during pregnancy was not apparent in three bitches that had previously had a short inter-oestrous interval of which two had previously had confirmed short luteal phase. However, in the remaining two cases, there had been previous pregnancy failure, but in only one of these, a premature decrease in endogenous progesterone to <2 ng/ml was detected. The latter had also low concentrations of PRL and RLN. The results of this preliminary clinical study suggest that abnormal progesterone profiles in pregnancy may be uncommon in bitches even when there has been previously documented short inter-oestrous interval. However, luteal deficiency may be suspected in bitches with a history of repeated pregnancy failure or abortion. MPA supplementation appears to be efficacious for management of suspected luteal deficiency and verification of the ovarian dysfunction, but care should be taken regarding the timing of MPA withdrawal and prolongation of pregnancy because of delayed elimination of MPA from blood circulation., (© 2012 Blackwell Verlag GmbH.)

Aberrant nuclear factor-erythroid 2 (NRF2) expression correlates with tumor development. We investigated NRF2 expression in ovarian epithelial carcinoma (OEC), aiming to identify associations with clinicopathological factors, hormones and induced reactive oxygen species (ROS). Immunohistochemical staining for NRF2 expression was performed on 10 benign cystadenomas, 4 boderline tumors and 64 OECs. Western blotting was used to determine the effects of hormones on NRF2 expression in OEC. NRF2 protein was found to be overexpressed in OEC. Gonadotrophins and estrogen induced ROS production in OEC; these hormones also enhanced NRF2 expression. Therefore, aberrant expression may be induced by hormones through ROS signaling. Increased NRF2 expression may be a molecular event in OEC carcinogenesis.

Previous research from our laboratory in beef cattle suggests that the pre-ovulatory follicle size, maturity and subsequent susceptibility to gonadotropin are influenced by the length of progestagen treatment in artificial insemination programme in beef cows. To test this hypothesis, two experiments were conducted. In experiment 1, 35 anoestrous beef cows received an intravaginal sponge containing 200 mg of medroxyprogesterone acetate. The treatment lasted for 7 (n = 12), 8 (n = 11) or 9 (n = 12) days. Half of the animals in each group were injected with 0.7 mg of oestradiol benzoate (EB) at device removal (0 h) and the other half 24 h later. In experiment 2, 38 cycling beef cows were treated with the same protocols as in experiment 1. Ultrasound examinations were performed to determine the follicular diameter at device removal (dominant follicle), interval to ovulation and ovulatory follicle diameter. The dominant follicle of anoestrous cows with progestagen for 7 days (8.4 ± 1.6 mm) resulted smaller (p < 0.05) than the cows treated for 8 (10.5 ± 1.6 mm) and 9 days (10.6 ± 1.2 mm). However, regardless of the length of the treatments, ovulation time after device removal was longer (p < 0.05) when EB was injected 24 h after withdrawal than at 0 h in anoestrous cows (EB0 = 52.7 ± 4.0 h; EB24 = 70.8 ± 6.2 h) and in cyclic cows (EB0 = 50.0 ± 21.0 h; EB24 = 73.0 ± 20.0 h). In anoestrous cows, the treatment with progestagen for 9 days and EB at 24 h increased the diameter of the ovarian follicle (p = 0.033) but did not affect the diameter of the ovulatory follicle in cyclic cows. In conclusion, increasing the length of progestagen treatment for 8 or 9 days compared to 7 days increased the diameter of the dominant follicle, in anoestrous and cyclic beef cows. Oestradiol benzoate administered at device removal resulted in a shorter interval from device removal to ovulation compared with EB injection 24 h after the end of a progestagen treatment., (© 2011 Blackwell Verlag GmbH.)

In the present study, the effect of medroxyprogesterone (MPA) is evaluated for its effect on pentylenetetrazole (PTZ) kindling model of epileptogenesis in mice followed by evaluation on kindling-induced changes in cognitive and motor functions. To explore whether the effects are mediated via progesterone receptors, a selective antagonist of progesterone (mifepristone, MIF) was also taken. Kindling was induced by once every 2 days treatment with PTZ (25 mg/kg, i.p.) for 5 weeks. The seizure severity during induction of kindling and % incidence of animals kindled at the end of 5 weeks were recorded. The motor function was assessed using a grip strength meter, whereas spatial memory was assessed in a cross maze. MPA (5 and 10 mg/kg, i.p.) significantly reduced the seizure severity scores and produced a significant decrease in the incidence of animals kindled at the end of 5 weeks (P<0.01). A higher efficacy was observed against male mice as compared with females following MPA. MIF neither reduced nor delayed the development of PTZ-induced kindling in mice. Also, it couldn't reverse the antiepileptogenic effects of MPA. On grip strength test (GST) and spontaneous alternation behavior (SAB), a significant decline in GST and % alternation was observed in kindled mice which was reversed by pre-treatment with MPA. MIF, however, could reverse only the reduced % alternation and not grip strength (GS) in PTZ-kindled animals. The study shows that MPA has antiepileptogenic effects against development of PTZ-induced kindling in mice that may not be mediated via progesterone receptors., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Two experiments were designed to investigate the administration of intravaginal progesterone in protocols for oestrus and ovulation synchronization in beef heifers. In Experiment 1, cyclic Black Angus heifers (n = 20) received an Ovsynch protocol and were randomly assigned to receive (CIDR-Ovsynch) or not (Ovsynch) a progesterone device between Days 0 and 7. Treatment with a controlled internal drug release (CIDR) device significantly increased the size of the dominant follicle prior to ovulation (12.8 ± 0.4 CIDR-Ovsynch vs 11.4 ± 0.4 Ovsynch) (p < 0.02). Plasma progesterone concentrations throughout the experiment were affected by the interaction between group and day effects (p < 0.004). In Experiment 2, cyclic Polled Hereford heifers (n = 382) were randomly assigned to one of the six treatment groups (3 × 2 factorial design) to receive a CIDR, a used bovine intravaginal device (DIB), or a medroxiprogesterone acetate (MAP) sponge and GnRH analogues (lecirelin or buserelin). All heifers received oestradiol benzoate plus one of the devices on Day 0 and PGF on Day 7 pm (device withdrawal). Heifers were detected in oestrus 36 h after PGF and inseminated 8-12 h later, while the remainder received GnRH 48 h after PGF and were inseminated on Day 10 (60 h). The number of heifers detected in oestrus on Day 8 and conception rate to AI on Day 9 were higher (p < 0.01) in the used-DIB than in the CIDR or MAP groups, while the opposite occurred with the pregnancy rate to FTAI on Day 10 (p < 0.01). There was no effect of progesterone source, GnRH analogue or their interaction on overall pregnancy rates (64.9%). Progesterone treatment of heifers during an Ovsynch protocol resulted in a larger pre-ovulatory follicle in beef heifers. Progesterone content of intravaginal devices in synchronization protocols is important for the timing of AI, as the use of low-progesterone devices can shorten the interval to oestrus., (© 2011 Blackwell Verlag GmbH.)

Ovarian hormones modulate acquisition processes involved in classical conditioning. Although progesterone has been indirectly implicated, its role in classical conditioning of the eyeblink response has not been directly investigated. We assessed the effects of daily dosing of progesterone or medroxyprogesterone (MPA), a non-metabolized synthetic progestin, upon the acquisition of a classically conditioned eyeblink response in ovariectomized (OVX) female rats. Rats were dosed 4h prior to each training session with 0.1 or 1.5 mg/kg of either of these hormones or sesame oil. A delay conditioning paradigm was employed using a 500 ms conditioned stimulus coterminating with a 10 ms 10 V unconditioned stimulus. At the low dose, progesterone and MPA rats did differ from each other, with MPA-treated rats learning slower, but neither group differed from OVX-oil or Sham-oil controls. No group differences in acquisition were observed at the higher dose. During extinction trials, high-dose MPA-treatment and OVX-oil groups extinguished quicker than the high-dose progesterone-treated group. In addition, unconditional response (UR) amplitudes were lower in all OVX groups, regardless of hormone or oil treatment, compared to the sham-oil group. Since MPA did not affect extinction, it is likely the slower extinction in the progesterone-treated rats is due to a metabolite of progesterone. Corticosterone is discussed as a likely candidate for such a role. In addition, we found chronic absence of ovarian hormones decreased UR amplitudes, although differences in UR amplitudes were not associated with changes in the acquisition process. These results are discussed with respect to differences in the hormonal effects upon acquisition versus extinction processes and how these data may explain reports of learning differences in women based on oral contraceptive usage., (Published by Elsevier Inc.)

The importance of the transcription factor TWIST1 for uterine decidualization was examined in human uterine fibroblast (HUF) cells decidualized in vitro with medroxyprogesterone, estradiol (E2), and prostaglandin E2. TWIST1 mRNA levels increased by 6.0- to 6.8-fold during the first 1-2 d of decidualization and remained above predecidualization levels for up to 15 d. Pretreatment of HUF cells with a TWIST1 small interfering RNA (siRNA) for 3 d before the induction of decidualization resulted in less morphologic differentiation than HUF cells pretreated with a nonsilencing control RNA. In addition, the cells pretreated with TWIST1 siRNA expressed 75-95% less IGF binding protein 1, LEFTY2, fibromodulin, laminin, and several other mRNA during decidualization, including the mRNA for the transcription factors forkhead box protein O1 and v-ets-erythroblastosis virus E26, both of which were previously shown to be critical for the induction of decidualization. The HUF cells pretreated with the TWIST1 siRNA also underwent less apoptosis during decidualization than the control cells, as evidenced by a 20% decrease in DNA fragmentation (terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling assay) and a 43-48% decrease in caspase 3, BCL2-associated X protein, and TNF receptor superfamily member 6 mRNA levels. Although the knockdown of TWIST1 expression markedly attenuated the induction of decidualization, overexpression of TWIST1 alone was insufficient to induce the decidualization of HUF cells. Taken together, these findings strongly implicate an essential role for TWIST1 in the initiation of human decidualization and uterine stromal cell apoptosis that occurs upstream of the induction of forkhead box protein O1 and v-ets-erythroblastosis virus E26 mRNA.

Ciliary beat frequency (CBF) was measured in slice preparations of the Fallopian tube fimbria, using videomicroscopy with a high-speed (500 Hz) camera in guinea pigs that were treated with β-oestradiol benzoate (βE2B) and medroxy progesterone (mPRG). In non-ovulating guinea pigs at 4 weeks of age, the CBF of the fimbria was high (17.8 Hz). In sexually mature guinea pigs (12-16 weeks of age) with constant ovulation, the CBF varied from 12 Hz to 16 Hz. The in vivo administration of both ICI-182,780 (a blocker of βE2 receptors) and mifepristone (a blocker of PRG receptors) induced high CBF (17.4 Hz). The administration of βE2B at a low (3.2 mg/kg/day) or high (32 mg/kg/day) dose decreased the CBF to 14.5 Hz or 11 Hz, respectively. ICI-182,780 abolished the βE2B-induced changes in CBF and decreased CBF to 12 Hz. The administration of mPRG (6.4 mg/kg/day) decreased CBF to 12.5 Hz. Mifepristone abolished this mPRG-induced decrease in CBF and maintained the CBF at 15 Hz. However, administering both βE2B and mPRG increased CBF to 17.5 Hz, suggesting that βE2B inhibits mPRG actions and vice versa. To confirm the interactions between βE2B and mPRG, we administered both βE2B and mPRG to guinea pigs that were pretreated for 1.5 days with either mPRG (6.4 mg/kg/day) or βE2B (3.2 mg/kg/day). Prior treatment with βE2B or mPRG prevented the increase in CBF that was otherwise by βE2B plus mPRG, and maintained the CBF at 14.5 Hz or 13 Hz, respectively. The administration of βE2B plus mPRG still induced the expression of PRG receptors, indicating that the highest CBF is not the result of no expression of the receptors. In the beating cilia of the fimbria, the signals that are activated by the βE2 and PRG receptors are proposed to antagonize each other in regulating the frequency.

Tamoxifen, a protein kinase C (PKC) inhibitor and antiestrogenic drug, has clinical antimanic effects and blocks psychostimulant-induced hyperlocomotion. Medroxyprogesterone (MPA), which has antiestrogenic effects, also exerts some clinical benefits in female manic patients and partially blocks amphetamine-induced hyperlocomotion, indicating that the antiestrogenic effect of tamoxifen could contribute to its antimanic effect. The present study evaluated the effect of acute and chronic (21 day) treatment of two antiestrogenic drugs, MPA and clomiphene (an estrogenic receptor antagonist), on methylphenidate (MPH, 5.0mg/kg)-induced hyperlocomotion in mice, an animal model of mania. Acute and chronic tamoxifen administration was used as a positive control. Acute and chronic tamoxifen (1.0mg/kg) administration blocked MPH-induced hyperlocomotion. Acute and chronic MPA (acute: 3.0 or 6.0mg/kg; chronic: 3.0mg/kg) and clomiphene (acute: 1.5 or 3.0mg/kg; chronic: 1.5mg/kg) treatment did not alter MPH-induced hyperlocomotion. These results indicate that tamoxifen exerts antimanic-like effects, and reduced estrogenic activity does not have antimanic-like effects in this psychostimulant-induced hyperlocomotion model. Therefore, the antiestrogenic effect of tamoxifen likely does not contribute to its antimanic effect, which may instead be related to its effect on PKC activity. Therefore, PKC inhibition may be associated with the antimanic effect of mood stabilizers., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Progestins and antiprogestins are widely used therapeutic agents in humans. In many cases, these are indicated for the treatment of reproductive activities. However, progesterone has widespread physiological effects including a reduction of the response to stress. We have reported that 5 min of restraint reduced lordosis behavior of ovariectomized rats hormonally primed with estradiol benzoate. When ovariectomized rats received both estradiol benzoate and progesterone priming, restraint had minimal effects on lordosis. Progesterone influences behavior through classical intracellular progesterone receptor-mediated nuclear events as well as extranuclear events. How these multiple events contribute to the response to stress is unclear. The current project was designed to initiate examination of the mechanisms responsible for progesterone's ability to protect against the effects of the restraint. In the first experiment, ovariectomized rats, primed with 10 μg estradiol benzoate, received 500 μg progesterone 4 h, 1 h, or 30 min before restraint. When progesterone was injected 4h before restraint, progesterone eliminated the effects of restraint. In contrast, progesterone 30 min before restraint offered no protection. Effects of progesterone 1h before restraint were equivocal allowing the suggestion that less than 4h of progesterone priming might be sufficient. In the second experiment, the synthetic progestin, medroxyprogesterone, was shown to mimic effects of progesterone in preventing effects of restraint. Finally, the progesterone receptor antagonist, RU486, attenuated progesterone's protection against restraint. These findings offer evidence that ligand-activated progesterone receptor mechanisms contribute to the maintenance of lordosis behavior in the presence of mild stress., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The zona pellucida (ZP) is an external glycoprotein membrane of oocytes of mammals and embryos in the early stage of their development. ZP first appears in growing ovarian follicles as an extracellular substance between the oocyte and granular cells. The zona pellucid markedly affects the development and maturation of the oocyte. The morphology of the ZP-oocyte complex allows a more precise determination of the oocyte maturity. According to numerous experimental studies, ZP is essential for preimplantation embryonic development of humans and other mammals. It prevents dispersion of blastomeres and enhances their mutual interactions. ZP is a dynamic structure responsible for the provision of nutrients to early forms of oocytes in mammals. The aim of the present study was untrastructural evaluation of the ZP-oocyte contact during inhibited ovulation. Female white rats (Wistar strain) received a suspension of medroxyprogesterone acetate (MPA) in incremental intramuscular bolus doses of 3.7 mg (therapeutic dose), 7.4 mg and 11.1 mg. The animals were decapitated 5 days after the administration of MPA. Ovarian sections were evaluated under a transmission electron microscope (TEM) Zeiss EM 900. Morphometric analysis of ZP was conducted using the cell imaging system by Olympus. In females exposed to therapeutic doses of MPA, ZP showed the structure of granular-fibrous reticulum of a medium electron density with single cytoplasmic processes originating from the surrounding structures. The oocyte cell membrane generated single, delicate processes directed toward ZP. Microvilli of the oocyte were short and thin. In the group receiving 7.4 mg of MPA, ZP had the structure of a delicate, loose granular-fibrous reticulum, and the oocyte cell membrane generated single microvilli directed toward ZP. In both those groups, the close ZP-oocyte contact was observed. Otherwise, in the group exposed to the highest MPA doses (11.1 mg), thicker and more numerous oocyte microvilli were found, which did not penetrate ZP matrix. They were dense, irregularly separated contour, forming a barrier between ZP and oocyte. The present findings are likely to suggest that MPA has inhibiting effects on the synthesis of binding proteins and causes the loss of the oocyte contact with ZP.

The ciliary beat frequency (CBF) of guinea-pig fimbria during the ovarian cycle was measured by video microscopy using a high-speed camera (500 Hz). In the follicular phase, with increasing concentrations of beta-oestradiol ([betaE(2)]) and a low concentration of progesterone ([PRG]), CBF increased from 13.5 to 16 Hz. In the ovulatory phase, with further increase of [betaE(2)], CBF decreased gradually from 16 to 13.5 Hz. In the early luteal phase, with low [PRG] and [betaE(2)], CBF increased to 17 Hz; however, in the middle luteal phase, with increasing [PRG], CBF decreased (12 Hz), and in the late luteal phase, with decreasing [PRG], CBF increased to 15 Hz. Then, in the resting phase, with low [betaE(2)] and [PRG], CBF decreased immediately to 14 Hz. The CBF of the fimbria was measured in guinea-pigs treated with beta-oestradiol benzoate (betaE(2)B) or medroxyprogesterone (mPRG). A low dose of betaE(2)B increased CBF to 14.5 Hz, whereas a high dose decreased it to 11 Hz. A betaE(2) receptor blocker, ICI-182,780, abolished the betaE(2)B-induced CBF changes and maintained CBF at 12.0 Hz. Medroxyprogesterone decreased CBF to 12.5 Hz, and mifepristone (a PRG receptor blocker) abolished the mPRG-induced CBF decrease and maintained CBF at 15 Hz. The addition of both blockers increased CBF to 18 Hz, suggesting that activation of betaE(2) or PRG receptors decreases the CBF of the fimbria. In conclusion, a moderate [betaE(2)] increase maintains a high CBF (15.5 Hz) in the follicular phase, and then further [betaE(2)] increase decreases CBF to 13.5 Hz in the ovulatory phase. In the early and late luteal phase, low [betaE(2)] and [PRG] increase CBF to 17 and 15 Hz, respectively, and in the middle luteal phase a high [PRG] decreases CBF (to 12 Hz). Thus, the CBF of the fimbria was controlled by signals via betaE(2) and PRG receptors in guinea-pigs.

Aims: To investigate the medium-term effects of estrogen plus progesterone therapy (EPT) on vascular reactivity, endothelial function and hemodynamic responses in 20 hypertensive postmenopausal women., Methods: This randomized, double-blind, cross-over, placebo-controlled study investigates the effect of 6 months of EPT (conjugated equine estrogen plus medroxyprogesterone). Blood pressure (office and ambulatory), heart rate and heart rate variability (HRV) were measured at baseline and following EPT/placebo treatment. In eight women, we used a wire-myograph to assess endothelial function and contractile response of subcutaneous arteries to transmural nerve stimulation (TNS) and exogenous noradrenaline., Results: EPT decreased vascular reactivity to cumulative TNS compared with baseline (p<0.01) and placebo (p<0.05). Moreover, EPT diminished sensitivity to exogenous noradrenaline (p<0.05). Although EPT reinforced response to acetylcholine, we observed no difference in maximal relaxation induced by substance P or acetylcholine. EPT did not affect ambulatory blood pressure, heart rate or HRV., Conclusions: Oral combined medium-term EPT reduces adrenergic reactivity in subcutaneous arteries from treated hypertensive postmenopausal women. EPT might act postjunctionally at the adrenergic vascular receptor level. In the present study, EPT neither reduces sympathetic activity nor increases vagal tone, and thus does not support an effect on the central hemodynamic system.

Introduction: Reduction of ovarian estrogen secretion at menopause increases net bone resorption and leads to bone loss. Isoflavones have been reported to protect bone from estrogen deficiency, but their modest effects on bone resorption have been difficult to measure with traditional analytical methods., Methods: In this randomized-order, crossover, blinded trial in 11 healthy postmenopausal women, we compared four commercial sources of isoflavones from soy cotyledon, soy germ, kudzu, and red clover and a positive control of oral 1 mg estradiol combined with 2.5 mg medroxyprogesterone or 5 mg/d oral risedronate (Actonel) for their antiresorptive effects on bone using novel (41)Ca methodology., Results: Risedronate and estrogen plus progesterone decreased net bone resorption measured by urinary (41)Ca by 22 and 24%, respectively (P < 0.0001). Despite serum isoflavone profiles indicating bioavailability of the phytoestrogens, only soy isoflavones from the cotyledon and germ significantly decreased net bone resorption by 9% (P = 0.0002) and 5% (P = 0.03), respectively. Calcium absorption and biochemical markers of bone turnover were not influenced by interventions., Conclusions: Dietary supplements containing genistein-like isoflavones demonstrated a significant but modest ability to suppress net bone resorption in postmenopausal women at the doses supplied in this study over a 50-d intervention period.

Background: Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination., Methods: Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR., Results: In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus., Conclusion: These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Endometrial hyperplasia is a precursor lesion of endometrial carcinoma. Clinical studies of endometrial hyperplasia have shown that levonorgestrel (LNG) is more therapeutically effective than medroxyprogesterone acetate (MPA). The present pharmacological in vitro study was performed to compare progestin effects on human endometrial cancer (Ishikawa) cells. Supraphysiological concentrations of progesterone (PG) and high concentrations of LNG and MPA were employed to determine the order of potency in reducing cell density. The order of potency was LNG>MPA>PG with respective 50% inhibitory concentrations (IC(50)) of 3.9+/-0.4, 30.4+/-3.4 and 45.3+/-2.7 microM. Mifepristone (MF) is a potent antiprogestin, but was unable to antagonize the PG-induced cell density reduction. For MF concentrations from 0.2 to 70 microM alone, a PG-mimetic effect was observed with an IC(50) value of 19.0+/-1.7 muM. When PG and MF were combined, a marked reinforcement of the effect was seen. These observations indicate that extranuclear initiated signaling pathways are involved in the reduction of endometrial cancer cells exposed to high concentrations of PG and MF.

Changes in tumor necrosis factor (TNF) production by lipopolysaccharide-stimulated peripheral blood mononuclear cells significantly correlated with serum P levels in postmenopausal women subjected to estrogen/medroxyprogesterone therapy. This may suggest that low physiologic or hormone therapy-related changes of serum P in healthy postmenopausal women may affect an ability of peripheral blood mononuclear leukocytes to produce TNF, thus having an impact on a variety of TNF-dependent physiologic and pathologic phenomena.

Objective: There is evidence that long-term hormone replacement therapy (HRT) is associated with an increased breast cancer risk. The aim of this study was to assess the effects of tibolone on estrogen and progesterone receptors in comparison to the effects of conventional HRT in the breast of surgically postmenopausal macaques., Method: Sixty macaques were bilaterally ovariectomized 3 months before hormonal treatment was initiated. The animals were randomized into four treatment groups, including tibolone (TIB), conjugated equine estrogens (CEE), conjugated equine estrogens+medroxyprogesterone acetate (CEE+MPA) and control animals (C). After 2 years treatment, breast tissues were collected, fixed and paraffin embedded. Immunohistochemistry assays with monoclonal antibodies for estrogen receptors (ERalpha and ERbeta) and progesterone receptors (PRA and PRB) were performed., Results: The expression of ERalpha was markedly decreased in the CEE+MPA group as compared to C and TIB groups. The TIB group was not different from the C and CEE groups. No significant differences were found for ERbeta immunostaining. The expression of PRA was strongly increased in the TIB group as compared to the C and CEE+MPA groups. Immunostaining of PRB was increased in the CEE and TIB treated animals as compared to both C and CEE+MPA groups., Conclusions: Tibolone increased the expression of both PRA and PRB, without affecting ERalpha and ERbeta expression in the macaque breast. These findings indicate that the effects of tibolone in breast tissue could be mediated via differential regulation of PRA and PRB isoforms and therefore distinct from those observed with conventional HRT.

Protein kinase C (PKC) is an important cellular target for mood stabilizers such as lithium and valproate, and tamoxifen, an antiestrogenic drug with PKC inhibition activity, also demonstrates an antimanic effect. Thus, the aim of the present study was to evaluate whether the antimanic effect of tamoxifen is mediated through the PKC inhibitory and/or the antiestrogenic action(s) of the drug. In the present study, the effects of tamoxifen, chelerythrine (a PKC inhibitor) and medroxyprogesterone (an antiestrogenic drug) were investigated in amphetamine-induced hyperlocomotion of mice, an animal model of a manic state. Lithium carbonate (100 and 150 mg/kg, i.p.), tamoxifen (1.0 mg/kg, i.p.) and chelerythrine (1 microg/site, i.c.v.) completely blocked the amphetamine-induced hyperlocomotion. However, while the intermediate medroxyprogesterone dose (3.0 mg/kg, i.p.) partially reduced the amphetamine-induced hyperlocomotion, lower (1.0 mg/g) and higher (6.0 mg/kg) doses produced no effect. Our results indicate a major role for PKC inhibition in the antimanic-like effect of tamoxifen, although its antiestrogenic action may also contribute to this effect.

Amado and Cavalheiro [Amado, D., Cavalheiro, E.A., 1998. Hormonal and gestational parameters in female rats submitted to the pilocarpine model of epilepsy. Epilepsy Res. 32, 266-274], studying the establishment of the pilocarpine epilepsy model in female rats observed that the estrous cycle was dramatically altered during the three periods of this experimental model. This work was delineated to study the function of sexual hormones in the development of the epilepsy model induced by pilocarpine in ovariectomized rats. Experimental groups were: (a) control animals during estrus phase of the estrous cycle (E) and ovariectomized female rats (OVX) treated with saline instead of pilocarpine in the same volume, (b) experimental animals, that developed status epilepticus (SE) and were studied during the chronic phase of this model: intact chronic rats (CHRON) and ovariectomized chronic rats (OVX+CHRON) and (c) ovariectomized chronic rats, that were submitted to hormonal replacement therapy treated with: medroxyprogesterone (OVX+CHRON+MPA); 17beta-estradiol (OVX+CHRON+E2), or both (OVX+CHRON+E2+MPA). All ovariectomized animals showed genital atrophy 4 days after the surgical procedure. Moreover, all animals that developed SE and survived showed spontaneous recurrent seizures during the chronic phase. Concerning to seizure frequency, animals receiving medroxyprogesterone associated with 17beta-estradiol showed decreased seizures' number. However, animals that received only medroxyprogesterone therapy also showed reduction in the number of seizures. In addition, hormonal treatment was also able to stabilize the mossy fibers sprouting process, showing the importance of these hormones in the development of the epilepsy in female rats.

Context: Endothelial dysfunction in resistance arteries after menopause is important for the development of high blood pressure and cardiovascular disease., Objectives: Our objectives were to study the effects of different hormone replacement therapies on the function and morphology of isolated resistance arteries, and to look for their mechanistic basis., Design and Setting: This was a randomized, placebo-controlled double-blind study in a University hospital, along with laboratory based studies., Patients and Interventions: We isolated resistance arteries in sc biopsies from 55 postmenopausal women before and after 3-month therapy with estradiol (E2), medroxyprogesterone acetate (MPA), E2 plus MPA, or placebo. In addition, we studied isolated human endothelial cells., Main Outcome Measures and Results: Artery flow-mediated dilatation was augmented after treatment with E2 or E2 plus MPA, whereas MPA or placebo had no effect. Pressure-induced myogenic tone was reduced by E2 plus MPA, whereas it was unchanged in the other groups. Scanning microscopy showed that E2 improved endothelial cell morphology and decreased signs of endothelial apoptosis, but the addition of MPA impaired these events. E2, MPA, or the combination all increased the expression and phosphorylation of the actin-binding protein, moesin and of the focal adhesion complex controller, focal adhesion kinase, and induced the rearrangement of cytoskeletal actin and vinculin fibers. All treatments promoted endothelial cell horizontal migration, with E2 inducing the strongest effect., Conclusions: This study suggests that hormone replacement therapy with estrogens or in combination with MPA may benefit the function of resistance arteries and may preserve the morphological integrity of endothelial cells by regulatory actions on the cytoskeleton.

Investigations of the modulation of prostaglandin F(2alpha) receptor (FP) expression in primary cultures of human uterine myocytes showed that FP mRNA expression was reduced by progesterone, unaltered by cAMP (8-bromo cAMP or forskolin), but increased by the PKA antagonist H89. Interleukin (IL)-1beta, tumour necrosis factor-alpha and oxytocin increased FP mRNA expression and IL-6 and prostaglandin E(2) reduced FP mRNA expression. The changes in FP protein levels were similar to the mRNA responses. We found that the IL-1beta-induced increase in FP expression was mediated at least in part via protein kinase C (PKC), but was independent of mitogen-activated protein kinase, phospholipase C and PI3 kinase. Since IL-1beta activates NFkappaB, AP-1 and C/EBP, we over-expressed these transcription factors alone and in combination and found that only NFkappaB alone increased FP mRNA expression. Finally, we found that the IL-1beta-induced increase in FP expression was unaffected by progesterone and/or cAMP, but was accentuated by H89. These data suggest that the pregnancy-induced down-regulation in myometrial FP expression is mediated by progesterone and cAMP and that the increase with labour is induced by inflammatory cytokine activation of PKC and NFkappaB.

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA.

Objective: To investigate the effect of nonhormonal contraception (NHC), combination oral contraception (COC), and depo-medroxyprogesterone acetate (DMPA) on lipids and blood pressure in women with recent gestational diabetes mellitus (GDM)., Research Design and Methods: An observational cohort of 972 nondiabetic, normotensive, postpartum Latino women who elected NHC (n = 448), COC (n = 430), or DMPA (n = 94) were followed for at least one subsequent metabolic evaluation on the same contraception. Baseline and follow-up measures included glucose tolerance testing, fasting serum LDL and HDL cholesterol, triglycerides, and systolic (SBP) and diastolic (DBP) blood pressure. Patterns of changes in lipids and blood pressure were evaluated by comparing slopes over follow-up time using random coefficient linear mixed-effects models., Results: Median follow-up times were 20, 12, and 11 months in the NHC, COC, and DMPA groups. The DMPA users gained significantly more weight (4.3 +/- 6.9 kg/year) compared with NHC and COC users (1.2 +/- 4.7 and 0.7 +/- 6.0 kg/year, respectively; P < 0.0001). Patterns of change in LDL cholesterol, triglycerides, and DBP were not significantly different among groups. HDL cholesterol change differed only between COC and NHC groups (adjusted slopes: 1.0 vs. -1.6 mg x dl(-1) x year(-1), respectively; P < 0.0001). SBP change differed only between COC and DMPA groups (adjusted slopes: 1.3 vs. -1.7 mmHg/year, respectively; P = 0.01)., Conclusions: These results, derived predominantly from the initial 1-2 years of treatment in Hispanic women, demonstrate that DMPA was associated with greater weight gain than NHCs or COCs. Other differences in blood pressure and lipid effects were very small. These findings should be taken into account when advising women with recent GDM about their contraceptive choices.

Objective: Recent studies have shown changes in coagulation factors, suggesting an increased risk of thrombotic events, in women on progestin-containing contraceptives. To investigate this, we studied the effects of injectable depomedroxyprogesterone acetate (DMPA), given as a contraceptive, on coagulation and inflammation markers., Design: Prospective nonrandomized study., Setting: Department of Obstetrics and Gynecology, University of Vermont College of Medicine., Patient(s): In this substudy of subjects undergoing a phase III trial comparing IM and SC DMPA, 14 healthy reproductive-age women were randomly assigned to receive either IM or SC DMPA every 3 months during a pharmaceutical trial., Intervention(s): All subjects had blood samples obtained at baseline and 6 and 12 months., Main Outcome Measure(s): D-Dimer, C-reactive protein (CRP), antithrombin (AT), factor VIIIc, activated partial thromboplastin time (aPTT), and aPTT plus activated protein C (APC) were analyzed on all samples., Result(s): D-Dimer concentration was significantly decreased at 6 and 12 months with injectable DMPA compared t degrees o baseline. There was also a modest decrease in aPTT. The CRP, AT, factor VIIIc, and aPTT + APC were not significantly altered by the use of DMPA., Conclusion(s): In this preliminary study the decline in D-dimer represents a potentially beneficial change in coagulation function following the use of DMPA given SC or IM. A slight decrease in aPTT may reflect a prothrombotic tendency with this contraceptive, but in contrast with previous studies examining the effect of oral contraceptive this injectable progestin contraceptive did not demonstrate the adverse effect on markers for thrombosis risk.