Term myometrium is characterized by increased activating epigenetic modifications at the progesterone receptor-A promoter

Term human myometrial expression of progesterone receptor (PR)-A is increased relative to PR-B, and as PR-A is a repressor of progesterone action mediated through PR-B, this increase may mediate the withdrawal of progesterone action and precipitate the onset of labour. PR-A and PR-B expression is regulated by two separate promoters of the PR gene. We hypothesized that epigenetic histone modifications at the two promoters contribute to the labour-associated regulation of PR-A and PR-B expression in term myometrium. PR total, PR-B and PR-A mRNA levels were determined using quantitative real-time PCR, and chromatin immunoprecipitation was used to determine the levels of activating and repressive histone modifications at the PR-A and PR-B promoters in human myometrial samples not in labour (n = 4) and in labour (n = 4). Chromatin extracts were immunoprecipitated with antibodies against activating (histone H3 and H4 acetylation and histone H3 lysine 4 trimethylation), and repressive (histone H3 lysine 9 trimethylation, histone H3 lysine 27 trimethylation and asymmetrical histone H3 arginine 2 dimethylation) histone modifications. PR-A mRNA levels increased during labour, while PR-B mRNA levels remained constant resulting in an increase of PR-A/PR-B mRNA ratio, as expected. Regardless of labour status, significantly higher levels of the activating histone modifications were found at the PR-A promoter compared with the PR-B promoter (P <0.001). H3K4me3 increased significantly at both promoters with labour onset (P =0.001). Low levels of the repressive histone modifications were also present at both promoters, with no labour-associated changes observed. Our data indicate that the PR-A promoter is epigenetically marked for activation in term myometrium more extensively than the PR-B promoter, and that labour is associated with an increase in H3K4me3 activating modification, consistent with the previously described increase in PR protein at this time.     

Progesterone receptor expression in human decidua and fetal membranes before and after contractions: possible mechanism for functional progesterone withdrawal

In humans, progesterone levels are sustained before the onset of labour. Therefore, the mechanism for parturition that has been proposed for humans is 'functional' progesterone withdrawal. Immunohistochemical staining for the progesterone receptor (PR) was positive in the decidua with a decline after contractions began. Western blot analysis revealed a number of PR isoforms expressed in the decidua, with the PR-B form being dominant. After contractions began, all PR isoforms decreased sharply. PR-B and PR-A decreased by 85.8% +/- 6.7 and 78.2% +/- 7.1, respectively (P < 0.001). Incubation of decidua with Prostaglandin F2alpha 1.0 microg/ml decreased the expression of all forms of PR isoforms. PR-B was reduced by 64% +/- 6.09 (P < 0.01); PR-A was reduced by 77% +/- 5.9 (P < 0.05), while PR-C was reduced by 80% +/- 7.24 (P < 0.05). Progesterone (80 microg/ml) increased the PR-B, PR-C the 45 and 36 kDa isoforms to 150% +/- 7.89, 210% +/- 12.4, 270% +/- 9.7 and 216% +/- 13.5, respectively (P < 0.05). In immunohistochemical studies, the PR was not identified in the amnion or in the chorion, regardless of the presence or absence of contractions. Western blot analysis demonstrated that PR-C (60 kDa) and the 36 kDa isoforms were dominant in the amnion. After contractions began, PR-A decreased significantly by 61.9% +/- 7.1 (P < 0.001).     

The benefits of mid-luteal addition of human chorionic gonadotrophin in in-vitro fertilization using a down-regulation protocol and luteal support with progesterone

Luteal support is essential in in-vitro fertilization (IVF)when long-acting gonadotrophin-releasing hormone agonist (GnRHa)is used. Because progesterone lacks luteotrophic stimulation,it seems to be the drug of choice in cases with an increasedrisk of ovarian hyperstimulation syndrome (OHSS). The aim ofthis study was to assess the beneficial effect of the mid-lutealaddition of human choriomc gonadotrophin (HCG) in IVF, usinga down-regulation protocol and luteal support with progesterone,in a prospective randomized study. The study included 170 IVFcycles down-regulated with long-acting GnRHa which were supportedwith 50 mg/day progesterone i.m. during the luteal phase. Patientswere evaluated in the mid-luteal period. Those without clinicalsigns of OHSS, oestradiol concentrations <1000 pg/ml andprogesterone concentrations <50 mg/ml were randomly allocatedto either the addition of 2500 IU HCG (HCG+ group) or no HCG(HCG– group). End luteal phase progesterone concentrationsamong non-pregnant patients were used to assess the contributionof exogenous progesterone and to categorize pregnancies accordingto their corpus luteum function. Similar low OHSS (2.7 and 1.8%)and pregnancy (30 and 29%) rates were observed in the HCG+ andHCG– groups respectively. Of the 26 pregnancies in theHCG+ cases, there was only one case with reduced corpus luteumfunction, compared with 12 of the 25 pregnancies among HCG–patients. Cases with reduced corpus luteum function requiredcontinuous progesterone support and presented lower HCG concentrationsand a higher rate of adverse pregnancy outcome. We concludethat mid-luteal HCG addition does not affect pregnancy rate,but in fact helps to preserve corpus luteum function and avoidsthe need for further supplementation during early pregnancy.     

Toxic effects of cypermethrin and methamidophos on bovine corpus luteal cells and progesterone production

This study was planned and executed with the aims to explore corpus luteal primary cell culture as an “animal alternate testing system” in toxicity studies and in vitro toxic effects of cypermethrin (CY) 90% (pyrethroid) and methamidophos (MTP) 73% (organophosphate) on morphology and progesterone secretory activity of bovine corpus luteal cells and tissue. For this purpose, primary cell cultures of bovine corpus luteum (CL) cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal calf serum (FCS). Toxicity evaluation were based on viable CL cell counts, morphological changes in CL cells, ability of CL cells to produce progesterone and histological changes in CL tissue at different hours post exposure to CY and MTP. The changes induced by both the insecticides were time and dose dependant. Viable cell counts and progesterone concentration decreased significantly (P<0.05) with the treatment of CY and MTP when compared to control. Corpus luteal cells exposed to CY showed more severe toxic effects as compared to MTP, though the difference was non-significant. Cellular or tissue alterations included degenerative changes in luteal cells, pleomorphic changes, nuclear degeneration and vacuolation, cell shrinkage and rupture, cloudy swelling and hydropic degeneration, less cytoplasmic granulation, cell elongation, hyalinization and cytoplasmic haziness and stripling and necrosis. It was concluded that both the insecticides induce toxic effects in terms of viable counts, morphological and histological changes and progesterone production of bovine CL cells. Cypermethrin exhibited more adverse toxic changes in viable cell counts, progesterone production and histological findings as compared to methamidophos.     

Antioxidant concentrations in serum, follicular fluid, and corpus luteum of cyclic buffalo cows

The present study was undertaken to evaluate antioxidant’s vitamins concentrations in serum, follicular fluid and corpus luteum of cyclic buffalo cows. A total of 34 clinically healthy buffalo cows (Bubalus bubalis), aged 8–10 years, were subjected to study. All animals were examined before slaughtering and the findings on the ovaries and the uterus were recorded. Blood samples and the whole genital tract of each animal were collected just after slaughtering. Antioxidants were measured in serum, CL and follicular fluid at different stage of the estrus cycle (proestrus n?=?8, estrus n?=?7, metestrus n?=?7, and diestrus n?=?12). The results revealed significant increases in serum α-tocopherol concentration during metestrus and diestrus stages (p?<?0.05). On the other hand, there was a significant decrease (p?<?0.05) in follicular ascorbic acid concentration at diestrus phase. Follicular β-carotene (p?<?0.01) showed a significant increase at the metestrus than at estrus and diestrus phases of the cycle. Follicular Vitamin A significantly increased (p?<?0.01) at proestrus phase. Although during metestrus there was significant decrease in the corpus hemorrhagicum weight (p?<?0.01), ascorbic acid concentration was significantly increased (p?<?0.05). In conclusion, the results of the present study suggested that serum α-tocopherol concentration increased during metestrus and diestrus stages, follicular vitamin A increased in proestrus phases when regeneration and steroideogenesis are required. Ascorbic acid increased in diestrus phase to help corpus luteum to function properly.     

Distribution of progesterone in the uterus,broad ligament,and uterine arteries of beef cows

Samples of uterine horn, broad ligament (mesosalpinx and mesometrium), and uterine artery of ten unilaterally ovulating cows, 12–15 days after mating, were examined for differences in concentration of progesterone. Tissues (mesosalpinx, branches of the uterine artery, anterior uterine horn) adjacent to the ovary bearing the corpus luteum contained significantly greater quantities of progesterone than more distal tissues (mesometrium, uterine artery, posterior uterine horn) on the same side and all tissues on the contralateral side. In tissues on the side ipsilateral to the corpus luteum, a gradient of progesterone existed. Differences in tissue concentrations of progesterone may be the cause of observed differences in uterine physiology in the cow and ewe.     

Differential effect of exogenous human chorionic gonadotrophin on progesterone production from normal or malfunctioning corpus luteum

To examine whether luteal phase defect is, in part, causally related to insufficient gonadotrophin stimulation, we compared the relation of the increment of serum progesterone concentrations in response to human chorionic gonadotrophin (HCG) with its basal level at mid-luteal phase. Thirty-eight naturally cycling infertile women aged between 27-41 years old were evaluated for hormonal responses to HCG injection at the mid- luteal phase. We measured luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol and progesterone concentrations, before and 1, 2 and 3 h after the administration of HCG (5000 IU, i.m.) 7 days after ovulation verified by ultrasonography. Eleven out of 38 women exhibited progesterone concentrations below 10 ng/ml (low progesterone group), and those remaining showed progesterone concentrations of > or = 10 ng/ml (normal progesterone group). The basal LH, FSH and oestradiol concentrations were essentially the same in both groups. Progesterone concentrations rose significantly 1 h after the injection and levelled off thereafter. The increment of progesterone concentrations at 1 h in the normal progesterone group was 5.7 ng/ml on the average, whereas that in low progesterone group was 1.1 ng/ml. Furthermore, the percentage increase in progesterone concentrations at 1 h in the normal progesterone group was significantly greater than that in the low progesterone group. Both groups equally exhibited significant but marginal increases in oestradiol concentrations 1 h after the injection. LH and FSH concentrations at 3 h decreased significantly in both groups. In summary, HCG readily stimulates progesterone production in normally functioning corpus luteum whereas its stimulatory effect is minimal on malfunctioning corpus luteum. This suggests that luteal phase defect is not caused by inadequate gonadotrophin stimulation and, therefore, does not benefit from HCG administration.      

Pregnancy: Function of the corpus luteum, the endometrium and the trophoblast after treatment of tubal pregnancy by prostaglandin F2{alpha}

The activity of the corpus luteum, the endometrium and the trophoblastwas studied after local medical treatment of 31 women with tubalpregnancy. We measured the serum concentration of progesterone,the secretory endometrial protein placental protein 14 (PP14),and human chorionic gonadotrophin (HCG) before and after treatmentby injection of prostaglandin F₂ into the site of the gestationand into the corpus luteum. There was no significant differencein the pre-treatment serum progesterone and serum PP14 concentrationsof 26 women who were treated successfully and of five women,who were operated on after failure of the treatment. After theprostaglandin treatment the serum progesterone and PP14 concentrationsdecreased simultaneously with the serum HCG concentration orremained at a low, constant concentration. We conclude thatmeasurement of serum progesterone and PP14 cannot be used forselection of patients for treatment by prostaglandin F₂ or formonitoring the effect of the treatment. The injection of prostaglandininto the ovary has either no effect on the activity of the corpusluteum or induces only a partial luteolysis.     

Progesterone as a mediator of gonadotrophin action in the corpus luteum: beyond steroidogenesis

Studies using newer, potent GnRH antagonists and pure gonadotrophins have clarified the importance of: (i) the strength-duration of the midcycle surge of pituitary gonadotrophins (LH, FSH) in follicle rupture and conversion to the corpus luteum; (ii) the continued requirement for pituitary LH throughout development and the functional lifespan of the primate corpus luteum in the menstrual cycle; and (iii) the exponential secretion of chorionic gonadotrophin (CG) by the developing placenta to extend the functional lifespan of the primate corpus luteum in early pregnancy. Although studies continue to increase current understanding of the cellular and molecular actions of LH/CG to stimulate luteal steroidogenesis, knowledge of the processes whereby these gonadotrophins promote the development and maintenance of the functional corpus luteum remains limited. This review summarizes evidence that the primate ovulatory follicle and corpus luteum is a target for the primary steroid produced by luteinizing/luteal tissue (i.e. progesterone). With evidence for dynamic expression of genomic progesterone receptors (PRA/B), and possibly other progesterone-receptor systems, recent studies addressed the hypothesis that progesterone is a critical 'local luteotrophin' that promotes luteal development and sustains luteal structure-function during the menstrual cycle and early pregnancy. Specific progesterone actions to regulate tissue remodelling (via protease expression), health (anti-apoptotic effects) and sensitivity to other local factors (e.g. via estrogen receptor expression) are discussed. The collective data suggest that there are gonadotrophin-stimulated, progesterone-dependent processes that promote luteotrophic and suppress luteolytic pathways in the primate corpus luteum. However, further studies are needed to verify their role in normal ovarian function and relevance to possible ovarian defects in natural and assisted reproduction technique-related cycles.     

Progesterone receptor modulator CDB-2914 down-regulates vascular endothelial growth factor, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of graded concentrations (10(-8), 10(-7) and 10(-6) M) of progesterone receptor (PR) modulator CDB-2914 on the protein contents of PR, of vascular endothelial growth factor (VEGF), adrenomedullin (ADM) and their receptors in cultured human uterine leiomyoma and matching myometrial cells. METHODS: PR-A, PR-B, VEGF-A, VEGF-B, VEGF receptor (VEGFR)-1, VEGFR-2, ADM and ADM receptor (ADMR) contents were assessed by Western blot analysis. RESULTS: Treatment with 100 ng/ml progesterone increased VEGF-A, VEGF-B and ADM contents in cultured leiomyoma cells and normal myometrial cells. The concomitant treatment with 10(-6) M CDB-2914 significantly decreased the progesterone-induced VEGF-A, VEGF-B and ADM contents in cultured leiomyoma cells but not in normal myometrial cells. CDB-2914 treatment alone decreased VEGFR-1, VEGFR-2 and ADMR contents in cultured leiomyoma cells but not in normal myometrial cells. CDB-2914 treatment increased PR-A and decreased PR-B contents in cultured leiomyoma cells in a dose-dependent manner compared with untreated cultures, whereas no significant changes in PR isoform contents were observed in normal myometrial cells. CONCLUSIONS: These results suggest that CDB-2914 down-regulates VEGF, ADM and their receptor contents and modulates PR isoform contents in cultured leiomyoma cells in a cell-type-specific manner.     

The production of leukaemia inhibitory factor by human endometrium: presence in uterine flushings and production by cells in culture

The concentration of leukaemia inhibitory factor (LIF) was measured in uterine flushings obtained from normal fertile women, from women with unexplained infertility and from women who suffered recurrent miscarriage. In normal fertile women, LIF was not detected in flushings obtained on days luteinizing hormone (LH)+0 to LH+6 of the cycle, but concentrations gradually increased from day LH+7 to a maximum at day LH+12. The amount of LIF in flushings obtained from women with unexplained infertility was significantly lower than in those from normal fertile women on day LH+10 (P < 0.05). The production of LIF by cultured human epithelial and stromal cells was also investigated. LIF was not detectable in the supernatants of cultured stromal cells. Basal LIF production by epithelial cells varied according to the stage in the cycle at which the biopsy was taken. Significantly more LIF was produced by epithelial cells from late proliferative and early secretory endometrium compared with amounts produced by cells from early proliferative (P < 0.001) and late secretory (P < 0.01) endometrium. High doses of progesterone and oestradiol caused a small decrease in epithelial cell LIF production: the combined effect of progesterone and oestradiol (P < 0.01) was greater than the effect of either steroid alone (P < 0.05). The results show, for the first time, the capability of human endometrium to produce LIF in vivo. The fact that maximum LIF concentrations are present at implantation and that decreased concentrations occur in women with unexplained infertility suggest the importance of this cytokine in embryo implantation.      

Possible involvement of very low density lipoproteins in steroidogenesis in the human ovary

To determine whether human luteal cells can utilize very low density lipoproteins (VLDL)-carried cholesterol for steroidogenesis, we investigated the expression of VLDL receptor mRNA in human ovarian tissues and progesterone production by human luteinized granulosa cells after the addition of VLDL. The production of progesterone in the presence of human chorionic gonadotrophin (HCG) was increased significantly (P < 0.05) by VLDL (2479 +/- 1477 ng/10(5) cells, mean +/- SD, n = 6) and low density lipoproteins (LDL) (2726 +/- 1287), in comparison with the level in the absence of these lipoproteins (1350 +/- 739). Northern blot analysis revealed that the levels of expression of VLDL and LDL receptor mRNA in granulosa cells were almost equal to those in whole ovarian tissue. VLDL receptor mRNA was abundant in granulosa cells of preovulatory follicles and cells of the corpus luteum. Preovulatory thecal cells and stromal cells expressed lower amounts of VLDL receptor mRNA than granulosa cells of preovulatory follicles and cells of the corpus luteum. From the present study, it might be suggested that VLDL is utilized for steroidogenesis in human luteinized granulosa cells.      

Nitric oxide induces apoptosis in the human corpus luteum in vitro

The present study aimed to investigate the role of nitric oxide (NO) in regression of the human corpus luteum. We therefore examined the effect of both NO and human chorionic gonadotrophin (HCG) on luteal cell apoptosis, and Bcl-2 production. The effect of NO on oestrogen production during corpus luteum regression was also studied. Slices from corpus luteum collected throughout the luteal phase were incubated for 4 h with the nitric oxide synthase (NOS) substrate, L-arginine (L-Arg, 1 mmol/l), the NOS inhibitor N-monomethyl-L-arginine (L-NMMA) (1 mmol/l), or with HCG (10 IU/ml). Oestradiol concentrations were determined by radioimmunoassay; Bcl-2 concentrations were measured by enzyme-linked immunosorbent assay; apoptosis was detected in-situ by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; and inducible nitric oxide synthase (iNOS) was assessed by immunohistochemistry. Consistent with our previous findings, L-Arg elicited an inhibitory action on the production of oestradiol (P< 0.05). The number of apoptotic cells increased (P<0.05) from early to late corpus luteum, as did the number of cells positive for the expression of iNOS. The percentage of apoptotic cells in mid and late luteal phase was increased by L-Arg (56% and 310% respectively; P <0.05), and decreased by L-NMMA and HCG. Although no changes were observed in Bcl-2 concentration during the corpus luteum life span, L-Arg inhibited, and HCG augmented, Bcl-2 production (P<0.05) from mid and late corpus luteum cells in vitro. In summary, these results suggest that the opposite actions of L-Arg and HCG on human corpus luteum viability may, in part, be mediated by changes in the level of the anti-apoptotic activities caused by oestradiol and Bcl-2 protein.     

Effects of RU486 in the expression of progesterone receptor isoforms in the hypothalamus and the preoptic area of the rat during postpartum estrus

In several mammalian species females undergo postpartum estrus, a brief period of ovulation and sexual receptivity that in rats usually occurs during the first 24 h following parturition. The maximal lordotic expression occurs at 12 h after the initiation of parturition and depends on intracellular progesterone receptor (PR). We studied the regulation of PR expression by its antagonist, RU486 in the hypothalamus and the preoptic area of the rat during postpartum estrus by Western blot. Adult female rats were treated with RU486 (1.25 and 5 mg) 3 h after parturition, and Western blot was performed to assess the expression of PR-A and PR-B at 12 h postpartum. RU486 (1.25 and 5 mg) reduced the expression of PR-A (63% and 95%) and that of PR-B (75% and 99%), respectively in the preoptic area whereas it had no effects in the hypothalamus. These results suggest a differential regulation of PR expression in the rat brain during postpartum estrus.     

Source of circulating levels of inhibin A, pro alpha C-containing inhibins and activin A in early pregnancy

It is now established that the glycoprotein hormone inhibin is produced by primate granulosa cells, corpus luteum and trophoblast of human placenta. This study was designed to investigate the major source of inhibins and activin A in early pregnancy using a novel panel of assays with high specificity and sensitivity. A total of 12 women (aged 20-35 years) with singleton pregnancy undergoing first trimester (group 1: 6- 8; group 2: 8-10; group 3: 10-12 weeks of gestation) termination of pregnancy (TOP) was recruited for the study. Blood samples were taken before TOP, every 15 min for the first hour and hourly for the next 3 h after TOP (total of 4 h of measurements). Circulating concentrations of inhibin A, pro alpha C, activin A, human chorionic gonadotrophin (HCG), oestradiol and progesterone were higher in early pregnancy than at any stage of the menstrual cycle. Peripheral concentrations of inhibin A and activin A were significantly decreased within the first hour in all three groups and gradually decreased to even lower concentrations within the study period. Pro alpha C concentrations decreased within the first hour and then remained unaltered during the next 3 h. Similarly, HCG, oestradiol and progesterone concentrations in circulation decreased substantially within 4 h of TOP. Correlation analyses showed a significant positive correlation (P < 0.001) between inhibin A, activin A, HCG, and oestradiol concentrations throughout the study period. In summary, this study shows that the feto-placental unit is the major source of increased circulating concentrations of inhibin A in early pregnancy. Activin A is produced by the feto-placental unit and the corpus luteum. Pro alpha C-containing inhibins are mainly secreted by the corpus luteum in early pregnancy.      

Endocrine responses to gonadotrophins after LHRH agonist administration on cycle days 1-4: prevention of premature luteinization

A treatment regime comprising an intranasally administered luteinizinghormone-releasing hormone (LHRH) agonist analogue (buserelin)on cycle days 1–4, followed by gonadotrophin administration[follicle stimulating hormone (FSH)/human menopausal gonadotrophin(HMG)] resulted in identical oestradiol (E₂) responses comparedwith the reference method using clomiphene citrate (CC) andgonadotrophins. Immediately after analogue administration (day4), buserelin-treated women showed short-lived elevations inserum LH and progesterone concentrations, but in the later follicularphase, the serum LH concentration was lowered compared withthe controls. None of the women treated with analogue displayedelevated serum LH or progesterone concentrations at the timeof injection of human chorionic gonadotrophin. In the earlyluteal phase, these women had higher serum levels of progesteroneand higher progesterone to E2 ratios than the controls, butthe length of the luteal phase was slightly shortened. Hence,in hyperstimulated cycles, 4-day treatment with buserelin causedprofound endocrinological changes: namely, short-term rescueof the corpus luteum, prevention of an endogenous LH rise andpremature luteinization and increased progesterone productionin the early luteal phase