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).     

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.     

Expression of progesterone receptor isoforms in corpora lutea of human subjects: correlation with serum oestrogen and progesterone concentrations

To understand the biological significance of progesterone receptor forms A (PR-A) and B (PR-B) in human corpus luteum, the expression of mRNA and serum steroid hormone concentrations were determined simultaneously in the luteal stages. The expression of PR-A mRNA predominated over PR-B mRNA in all samples analysed. Total PR (PR-AB) and PR-B mRNA concentrations at the late secretory phase were significantly (P < 0.01) lower than those at the early and mid secretory phases of the menstrual cycle. The ratio of PR-B to PR-AB mRNA concentration showed no significant change during the secretory phase. In the early and mid secretory phases, there was a negative correlation between PR-B mRNA concentration and serum progesterone concentration, and between the ratio of PR-B to PR-AB mRNA concentrations and serum progesterone concentration (P < 0.01). These findings suggest that human corpus luteum might intracellularly synthesize PR-A and PR-B, and thus be involved in the steroid functional regulation of the corpus luteum, especially at the early and mid secretory phases, and that progesterone might regulate the synthesis of PR-A and PR-B.      

5 Beta-dihydroprogesterone and steroid 5 beta-reductase decrease in association with human parturition at term

The role of progesterone withdrawal in human parturition continues to provoke controversy. One possible mechanism by which functional progesterone withdrawal may be achieved is by a decrease in the circulating concentration of its bioactive metabolites. The progesterone metabolite 5beta-dihydroprogesterone (5betaDHP) has been shown to be a potent tocolytic in vitro. We quantified plasma concentrations of 5betaDHP in association with the onset of spontaneous labour in women at term and steroid 5beta-reductase mRNA expression in placenta, myometrium, chorion and amnion in relation to parturition, using real time RT-PCR. Serial blood samples were obtained from patients late in pregnancy, before term labour, during term labour and within the first 24 h postpartum. Following organic solvent extraction, steroids including 5betaDHP were separated by high-performance liquid chromatography (HPLC) and then quantified by radioimmunoassay (RIA). 5betaDHP concentration decreased two-fold (P = 0.00001, n = 25) from 0.317 +/- 0.039 nmol/ml to 0.178 +/- 0.017 nmol/ml in association with active labour. Tissue 5beta-reductase mRNA-relative abundance was determined in placenta, myometrium, chorion and amnion obtained from labouring and non-labouring women. In placenta and myometrium, relative expression decreased significantly in association with labour, by about two-fold and 10-fold, respectively. These data are consistent with a possible role for 5betaDHP in the onset of spontaneous human labour. Further studies exploring this hitherto unrecognized endocrinological pathway are indicated.     

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.     

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.     

Differential expression of the metalloproteinase MMP3 and the alpha5 integrin subunit in human myometrium at labour

Extensive tissue remodelling occurs in the human myometrium before, during and after parturition. The aim of this study was to investigate the expression of two tissue remodelling molecules, matrix metalloproteinase 3 (MMP3) and alpha5 integrin (ITGA5) subunit in human myometrium, during pregnancy and labour. mRNA and protein were isolated from human pregnant labouring and non-labouring myometrial tissue, and also from human primary uterine smooth muscle cells (SMCs). Semi-quantitative RT-PCR, real-time fluorescence RT-PCR and western blotting were subsequently performed to determine the expression levels of MMP3 and ITGA5 in the myometrial tissues during pregnancy and labour, and in the primary uterine SMCs. The expression of MMP3 and ITGA5 mRNA and protein are reported for the first time during pregnancy and labour in human myometrium. Furthermore, a significant increase in expression of MMP3 mRNA (41-fold, P = 0.001), and a significant decrease in ITGA5 mRNA expression (4-fold, P < 0.001) at labour, were observed. Protein expression of these two molecules was also altered at labour, pro-MMP3 protein expression significantly increased while ITGA5 protein expression decreased, with labour onset. Expression of these molecules was also observed in primary cultured human uterine SMCs. The differential expression of these two tissue remodelling molecules at labour and their detection in uterine SMCs highlights their potential importance in myometrial function during pregnancy, labour and post-partum.     

Reduction of progesterone receptor expression in human cumulus cells at the time of oocyte collection during IVF is associated with good embryo quality

BACKGROUND: It has been reported that the progesterone receptor (PR) level is transiently increased within the follicle by LH stimulation and controls cumulus cells in follicles and oocyte maturation. The purpose of this study was to predict developmental competence of human oocytes during IVF via analysis of PR in cumulus cells surrounding mature oocytes. METHODS: Prior to oocyte retrieval, the follicular diameter was measured and follicular fluid was collected from each mature follicle. Cumulus cells were manually separated from the oocyte-cumulus complex under a microscope. PR and PR mRNA were assessed by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR) measurement in human cumulus cells. RESULTS: Immunoreactive PR-A was mainly localized in the cytoplasm and PR-B was localized in the nuclei. There was no significant relationship between PR expression and follicular diameter, follicular fluid concentration of steroids, or LH. There was no significant relationship between expression of PRs and fertilization or cleavage rate. However, PR expression was lower in the good morphology group (blastomeres > or =7 cells with fragmentation > or =5% on day 3) when compared to the other groups (P<0.05). CONCLUSIONS: These results suggest that follicular LH or steroids do not affect PR expression, and full reduction of total PR expression on cumulus cells at the time of oocyte collection is associated with good morphology in human oocytes.     

Labour is associated with increased expression of type-IIA secretory phospholipase A2 but not type-IV cytosolic phospholipase A2 in human myometrium

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the type-IV cytosolic phospholipase A2 (cPLA2-IV) and the type IIA secretory phospholipase A2 (sPLA2-IIA) in myometrium in association with labour onset at term and preterm deliveries. These enzymes are important for the release of the prostaglandin precursor, arachidonic acid, from phospholipid membrane stores. RT-PCR was used to determine differences in gene expression between non-labour and labour groups. Expression of sPLA2-IIA in human myometrium was significantly increased with pregnancy, and with labour, both at term and preterm. Expression of cPLA2-IV in myometrium was not significantly altered with respect to pregnancy or labour. Immunohistochemical analysis demonstrated differences in the spatial localization of cPLA2-IV and sPLA2-IIA protein in upper and lower segment myometrium. cPLA2-IV was predominantly in vascular endothelial cells, while sPLA2-IIA was observed in vascular, endothelial and smooth muscle cells. In addition, sPLA2-IIA showed a distinct nuclear or perinuclear localization in myometrial smooth muscle cells of the lower segment. We postulate that the increased expression of sPLA2-IIA rather than cPLA2-IV in the myometrium may play a role in the onset and/or maintenance of human parturition.     

Induction of c-Jun mRNA without changes of estrogen and progesterone receptor expression in myometrium during human labor

To elucidate the endocrine mechanism of human parturition, the expression of c-Jun and c-Fos mRNA were examined in relation to estrogen receptor (ER) and progesterone receptor (PR) in human myometrium. c-Jun mRNA was detected in all myometrial tissues (n=5) during labor but not before labor (n=5) and in oxytocin-resistant postterm pregnancy (n=3). c-Fos mRNA was detected in only one myometrial tissue from a woman in labor. The distribution and intensity of immunostaining for ER and PR were semiquantitatively scored. During the late pregnancies, no significant difference was seen in the receptor scores for myometrial ER and PR between the patients who experienced labor and those who did not. Receptor scores for ER and PR were significantly lower in postterm pregnancy than in late pregnancy, regardless of the labor status. These data suggest that there are no changes in ER and PR in human myometrium during parturition. On the other hand, postterm pregnancy is associated with low ER and PR. c-Jun, induced during labor without changes in ER and PR, may play a role as a signaling mechanism in human myometrium.     

Ovarian steroids reduce apoptosis induced by trophic insufficiency in nerve growth factor-differentiated PC12 cells and axotomized rat facial motoneurons

Previous studies have demonstrated that ovarian steroids exert neuroprotective effects in a variety of in vitro and in vivo systems. The mechanisms underlying these effects remain poorly understood. In the present study, the neuroprotective effects of estradiol (E(2)) and progesterone (P) were examined in two models of apoptosis induced by growth factor insufficiency: partially nerve growth factor (NGF)-differentiated PC12 cells, after serum and NGF withdrawal; and axotomized immature rat facial motor motoneurons. E(2) and P both increased the survival of trophically withdrawn NGF-differentiated PC12 cells, at physiologically relevant concentrations. However, neither steroid had a significant effect on the survival of PC12 cells that had not been NGF treated. Exposure to NGF had no effect on the expression of estrogen receptor (ER)beta, but markedly increased the levels of ERalpha and altered the expression of the progesterone receptor (PR) from predominantly PR-B in NGF naive cells, to predominantly PR-A after NGF. The survival promoting effects of E(2) and P were blocked by the specific steroid receptor antagonists Faslodex (ICI 182780) and onapristone (ZK98299), respectively. Inhibitors of RNA (actinomycin D) or protein (cycloheximide) synthesis also abrogated the protective effects of both steroids. In immature rats, E(2) and P both significantly increased the numbers of surviving facial motor neurons at 21 days after axotomy. These data demonstrate significant protective effects of E(2) and P in two well-characterized models of apoptosis induced by trophic withdrawal and suggest that, at least in PC12 cells, the effects of the steroids are mediated via interaction with nuclear steroid receptor systems. The lack of steroid responsiveness in NGF-naive PC12 cells despite the presence of abundant ERbeta and PR-B are consistent with the view that ERalpha and PR-A may be particularly important as mediators of the neuroprotective effects of their corresponding hormonal ligands.     

Changes in PGRMC1, a potential progesterone receptor, in human myometrium during pregnancy and labour at term and preterm

This study assesses the role of progesterone receptor membrane component 1 (PGRMC1) in actions of progesterone (P4) on human myometrium during pregnancy and labour. Myometrial tissues were obtained from non-pregnant patients during hysterectomy or pregnant women undergoing C-section at term and preterm, before and during labour. PGRMC1 expression in myometrial tissues and in a human myometrial cell line (HM9) was assessed by western blots and RT-PCR. The subcellular localization of PGRMC1 in HM9 was performed by immunofluorescence staining. Isometric contractions of myometrial tissues were obtained in response to P4 with and without addition of specific antibodies against PGRMC1. Endogenous and over-expressed PGRMC1 proteins are detected by western blots in myometrial tissues, HM9 and 293 cells, respectively. PGRMC1 is localized to the plasma membrane, cytoplasm and nuclear membranes. PGRMC1 is lower in myometrium of women at term either not in labour (P = 0.004) or in labour (P = 0.005) compared with tissues from women in preterm non-labour. PGRMC1 levels are also decreased (P = 0.02) in myometrial tissues from women during preterm labour compared with preterm non-labour. P4 rapidly inhibits contractions of myometrial tissues compared with control (P < 0.05) in vitro. Pretreatment of myometrial strips with PGRMC1 antibody, suppresses the P4-induced relaxation (P < 0.05). PGRMC1 may mediate the non-genomic action of P4 and the relaxation effect on human myometrium during pregnancy. A decrease in PGRMC1 during term or preterm labour might contribute to the 'functional withdrawal' of P4 action and shift the balance to a state of heightened uterine contractility.     

Nitric oxide synthase activity and localization do not change in uterus and placenta during human parturition

Animal studies have suggested that nitric oxide, a smooth muscle relaxant, is a fundamental mediator in the initiation of parturition. The purpose of this study was to test the hypothesis that the onset of human labour is associated with a reduction in the activity of the enzyme nitric oxide synthase (NOS), within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were collected during Caesarean section from 11 women before and 11 women after the onset of labour at term. Immunocytochemistry was used to localize each of the three isoforms of NOS (endothelial NOS, brain NOS, and inducible NOS) in each of these tissues and the intensity of staining was qualitatively assessed. NOS enzyme activity was determined in homogenates of frozen myometrium, placenta and fetal membranes (with attached decidua), by measuring conversion of radio-labelled L-arginine to L-citrulline. Each of the three isoforms of NOS was localized in each of the tissues. We found no difference in either the expression or enzyme activity of NOS in myometrium, placenta or fetal membranes before and during labour at term. These results suggest that, in contrast to animal studies, a decrease in NOS enzyme activity may not be involved in the onset of parturition at term in the human.      

Identification and localization of prostaglandin E2 receptors in upper and lower segment human myometrium during pregnancy

Prostaglandin E2 (PGE2) plays a key role in the maintenance of human pregnancy and labour onset. PGE2 can elicit diverse actions within the uterus depending on the PGE2 receptors (EP1, EP2, EP3 and EP4) expressed. By signalling through different intracellular pathways the EP receptors may inhibit or promote smooth muscle contractility. Nine different EP3 receptor splice variants have been identified with divergent signalling pathways. RT-PCR and immunohistochemistry were utilized to identify and localize EP receptor isoforms within the upper segment (US) and lower segment (LS) myometrium. EP1 was significantly increased in the LS myometrium with term labour. EP3 (and EP3 splice variants EP3I(1b), EP3II, EP3III and EP3IV) was down-regulated in pregnancy (US and/or LS) with a further decrease at term labour in the LS. Overall, expression of EP2 was significantly higher in the LS while EP3 was significantly higher in the US. No significant EP4 changes were observed. Consistent with the RT-PCR results, immunohistochemistry confirmed the presence and, interestingly, showed nuclear localization of EP receptors in the myometrium with higher EP1 expression and lower expression of EP3. The differential regulation of EP receptors within the myometrium indicates that they may play a role in controlling the onset and maintenance of human labour.     

Lack of stimulatory effect of dienogest on the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by endothelial cell as compared with other synthetic progestins

Objectives: Monocyte adhesion to endothelial cells is an important initial event at the onset of atherosclerosis. It is partially mediated by the expression of adhesion molecules on the endothelial cell surface. While estrogens inhibit the development of atherosclerosis, the effect of co-administered progestin remains controversial. We examined the effect of progestins on cytokine-stimulated human umbilical venous endothelial cell (HUVEC) expression of adhesion molecules. Methods: In HUVECs, mRNA expression of progesterone receptors (PRs) and androgen receptors (AR) was determined by RT-PCR. HUVECs were stimulated by interleukin-1β (IL-1β) for 24 h with or without various steroids, and then the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was semiquantified by ELISA. Results: In all preparations of HUVECs used in this study, RT-PCR confirmed mRNA expression of both isoforms of PR, PR-A and PR-B, as well as AR. Addition of progesterone (10⁻¹⁰–10⁻⁷ M) or dienogest (DNG) (10⁻¹⁰–10⁻⁸ M) did not affect IL-1β-stimulated ICAM-1 or VCAM-1 expression. In contrast, medroxyprogesterone acetate, norethindrone acetate and levonorgestrel (10⁻¹⁰–10⁻⁸ M) dose-dependently increased cell adhesion molecules. The progestin-induced increase was blocked by the concomitant addition of mifepristone, a PR antagonist, but not by hydroxyflutamide, an AR antagonist, indicating that the progestin stimulation was mediated predominantly via PR. Conclusions: These results suggest that DNG, unlike other synthetic progestins, lacks stimulation of cell adhesion molecules. For the prevention of atherosclerosis, estrogen in combination with DNG may be a suitable regimen in hormone replacement therapy in postmenopausal women.     

Changes in the expression of nitric oxide synthase in the human uterine cervix during pregnancy and parturition

Nitric oxide (NO) has been proposed as a mediator of cervical ripening. We investigated the expression, using Western blotting, and localization, using immunohistochemistry, of the nitric oxide synthase (NOS) enzymes, inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (bNOS) in the human cervix during pregnancy and parturition. Cervical biopsies were obtained from non-pregnant women, women in the first trimester of pregnancy, and pregnant women at term before and after the onset of labour. Each of the NOS isoforms was localized in the cervices of both non-pregnant and pregnant subjects using immunohistochemistry. iNOS expression was significantly greater in early pregnancy compared with the non-pregnant state (P: < 0.005). iNOS expression was up-regulated further in samples obtained in the third trimester compared with the first trimester. bNOS expression was greater in samples from the first trimester of pregnancy than in non-pregnant samples (P: < 0. 005), but showed no additional increase in late pregnancy or with the onset of labour. eNOS expression was increased in samples obtained in the third trimester both before (P: = 0.002) and after the onset of labour (P: < 0.002) when compared with non-pregnant samples. The increased expression of NOS isoforms in late pregnancy supports the hypothesis that NO is involved in the process of cervical ripening.     

Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process.

Inflammatory mediators in the cervix, placenta and fetal membranes play a crucial role in human parturition. The aim of this study was to determine whether the upper and lower segments of the myometrium are infiltrated by inflammatory cells during pregnancy and parturition. Myometrial biopsies were obtained from non-pregnant women, and pregnant women at term before and after the onset of spontaneous labour. Subpopulations of inflammatory cells were identified using immunocytochemistry. The intercellular adhesion molecules, 1 and 2, platelet endothelial cell adhesion molecule, vascular cell adhesion molecule and E-selectin were immunolocalized to investigate their involvement in leukocyte accumulation. Histological analysis demonstrated that inflammatory cells, predominantly neutrophils and macrophages, infiltrate human myometrium during spontaneous labour at term. The infiltrate is predominant in the lower uterine segment but is also present in the upper segment. Increased expression of E-selectin was found on the vascular endothelium of biopsies obtained during labour, suggesting a role for this molecule in the accumulation of leukocytes. These results suggest that inflammatory cell infiltration is part of the physiological mechanisms that occur in the myometrium during parturition. Further understanding of this process may suggest new strategies aimed at preventing preterm delivery.