Progesterone Withdrawal Research Articles

Research on the Expression of HOXA10 in Placental Villi during the Onset of Labor

Objective To investigate the difference of the expression of HOXA10 gene in placental villi before and after the onset of labor. Methods Immunohistochemistry was used to detect the location of HOXA10 in placental villi. The expression of HOXA10 mRNA and protein were measured by RT-PCR and Western blotting. Results Immunohistochemistry showed that HOXA10 was only weakly expressed in placental villi of the natural labor group. However, it was strongly expressed in placental villi of the surgical labor group. RT-PCR and Western blotting showed that the expression of HOXA10 mRNA and protein were significantly lower in the natural labor group than in the surgical labor group (P Conclusions The lower expression of HOXA10 during the onset of labor may be one of the major factors in initiating the normal labor. And to some extent, the lower expression of HOXA10 gene in the natural labor group may be related to “functional progesterone withdrawal”.

Gene regulation of UDP-galactose synthesis and transport: potential rate-limiting processes in initiation of milk production in humans

Lactose synthesis is believed to be rate limiting for milk production. However, understanding the molecular events controlling lactose synthesis in humans is still rudimentary. We have utilized our established model of the RNA isolated from breast milk fat globule from seven healthy, exclusively breastfeeding women from 6 h to 42 days following delivery to determine the temporal coordination of changes in gene expression in the carbohydrate metabolic processes emphasizing the lactose synthesis pathway in human mammary epithelial cell. We showed that milk lactose concentrations increased from 75 to 200 mM from 6 to 96 h. Milk progesterone concentrations fell by 65% at 24 h and were undetectable by day 3. Milk prolactin peaked at 36 h and then declined progressively afterward. In concordance with lactose synthesis, gene expression of galactose kinase 2, UDP-glucose pyrophosphorylase 2 (UGP2), and phosphoglucomutase 1 increased 18-, 10-, and threefold, respectively, between 6 and 72 h. Between 6 and 96 h, gene expression of UDP-galactose transporter 2 (SLC35A2) increased threefold, whereas glucose transporter 1 was unchanged. Gene expression of lactose synthase no. 3 increased 1.7-fold by 96 h, whereas α-lactalbumin did not change over the entire study duration. Gene expression of prolactin receptor (PRLR) and its downstream signal transducer and activator of transcription complex 5 (STAT5) were increased 10- and 2.5-fold, respectively, by 72 h. In summary, lactose synthesis paralleled the induction of gene expression of proteins involved in UDP-galactose synthesis and transport, suggesting that they are potentially rate limiting in lactose synthesis and thus milk production. Progesterone withdrawal may be the signal that triggers PRLR signaling via STAT5, which may in turn induce UGP2 and SLC35A2 expression.

Transforming growth factor β1 (TGFβ1) and progesterone regulate matrix metalloproteinases (MMP) in human endometrial stromal cells.

Menstruation is preceded by progesterone withdrawal and endometrial matrix remodeling predominantly through induction of matrix metalloproteinases (MMP) and recruitment of invading neutrophils. Using endometrial tissues from women during various phases of the menstrual cycle, we found that MMP2, MMP9, and MMP11 were up-regulated in the late secretory phase/premenstrual phase. Because TGFβ-responsive genes were also up-regulated in endometrium during this time, we tested the hypothesis that TGFβ1 and progesterone regulate expression of MMP in human endometrial stromal cells (HESC). Treatment of HESC with TGFβ1 resulted in marked increases in MMP2 and MMP11 mRNA and pro- and active MMP2 activity. Progesterone inhibited TGFβ1-induced stimulation of MMP2 and MMP11 through its nuclear hormone receptors. Interestingly, TGFβ1 also decreased progesterone receptor (PR)-A and PR-B in HESC with a more pronounced effect on PR-A. These data support the hypothesis that TGFβ1 has endogenous anti-progestational effects in HESC and that the opposing effects of progesterone and TGFβ1 are important in regulation of matrix integrity in human endometrium.

An evolutionary test of the isoform switching hypothesis of functional progesterone withdrawal for parturition: humans have a weaker repressive effect of PR-A than mice

A decrease in maternal serum progesterone (P4) concentrations precedes the onset of labor in most placental mammals. Humans differ by maintaining high levels of P4 throughout birth. Parturition in humans probably includes mechanisms that undercut the pregnancy sustaining function of P4. One attractive hypothesis is the isoform switching hypothesis (ISH). ISH is supported by in vitro evidence that progesterone receptor isoform A (PR-A) inhibits PR-B and that the PR-A/PR-B ratio increases towards term. Here, we test the hypothesis that isoform switching is an adaptation to high levels of P4 at term, predicting that, in humans, PR-A mediated repression of PR-B is stronger than in mouse. We use reporter assays with human and mouse PRs to detect species differences in the repressive effects of PR-A. We found that human PR-B is less sensitive to repression by human PR-A than mouse PR-B, contrary to our prediction. The difference between human and mouse PR-B sensitivity is most pronounced at PR-A/PR-B ratios typical for the preterm myometrium. Our results are inconsistent with the ISH. We speculate that, instead, the lower sensitivity of human PR-B to PR-A may be relevant for the maintenance of pregnancy at high progesterone levels and increasing PR-A concentrations towards term.

Progesterone Receptor-A and -B Have Opposite Effects on Proinflammatory Gene Expression in Human Myometrial Cells: Implications for Progesterone Actions in Human Pregnancy and Parturition

Progesterone promotes uterine relaxation during pregnancy and its withdrawal induces labor. Progesterone withdrawal in human parturition is mediated in part by changes in the relative levels of the nuclear progesterone receptor isoforms, PR-A and PR-B, in myometrial cells. Parturition also involves myometrial inflammation; however, the functional link between nuclear PR-mediated progesterone actions and inflammation in human myometrial cells is unclear. Our objective was to determine how PR-A and PR-B regulate progesterone action in human myometrial cells and specifically the expression of genes encoding contraction-associated proteins and proinflammatory mediators. Effects of PR-A and PR-B on the capacity for progesterone to modulate gene expression was determined using an immortalized human myometrial cell line stably transfected with inducible PR-A and PR-B expression transgenes and conditioned to express various PR-A and PR-B levels. Gene expression was assessed by genome wide transcriptome analysis, quantitative RT-PCR and immunoblotting. PR-A and PR-B were each transcriptionally active in response to progesterone and affected the expression of distinct gene cohorts. The capacity for progesterone to affect gene expression was dependent on the PR-A to PR-B ratio. This was especially apparent for the expression of proinflammatory genes. Progesterone decreased proinflammatory gene expression when the PR-A to PR-B ratio favored PR-B and increased proinflammatory gene expression when the ratio favored PR-A. Progesterone via PR-B increased expression of inhibitor-κBα, a repressor of the nuclear factor-κB transcription factor, and inhibited basal and lipopolysaccharide-induced proinflammatory gene expression. Both of those PR-B-mediated effects were inhibited by PR-A. Our data suggest that during most of human pregnancy, when myometrial cells are PR-B dominant, progesterone promotes myometrial quiescence through PR-B-mediated antiinflammatory actions. At parturition, the rise in PR-A expression promotes labor by inhibiting the antiinflammatory actions of PR-B and stimulating proinflammatory gene expression in response to progesterone.

Successful pregnancy following a novel endometrial preparation in a PCOS patient undergoing IVM: a case report

In-vitro fertilization (IVF) relies heavily on controlled ovarian stimulation to increase the number of available oocytes and embryos, and therefore the pregnancy rate. However, the side effects, such as nausea, mood swings, menopausal symptoms, ovarian hyperstimulation syndrome (OHSS), and potential cancer risk of ovarian stimulation may not be avoided. Therefore, the recovery of immature oocytes followed by in-vitro maturation (IVM) and fertilization is an attractive alternative to avoid the side effects of using gonadotrophin stimulation. In IVM, immature oocytes are retrieved from unstimulated or minimally stimulated cycles, then cultured using IVM culture media for maturation in vitro [1]. In comparison with conventional IVF, IVM technology is generally associated with the absence of OHSS, lower cost, and fewer complications. However, the efficiency of existing IVM technique is suboptimal because embryos generated from IVM exhibit frequent cleavage blocks and low implantation rate [2]. Women with polycystic ovarian syndrome (PCOS) are considered as the target population for this approach. This group of patients is extremely sensitive to ovarian stimulation and have a high potential for developing of OHSS [3]. In recent years, many improvements have been made in clinical and laboratory aspects of IVM treatment which have resulted in better pregnancy outcomes [4]. It was shown that pretreatment with FSH in the early days of folliculogenesis phase may increase the number of retrieved immature oocytes and/or maturation potential [5, 6]. In addition, hCG priming promotes some GV oocytes to reach the MI stage and increases the maturation rate of immature oocytes in vitro setting. Despite increased pregnancy rates following IVM in recent years, implantation rates are still lower than conventional IVF cycles. One possible explanation may be related to insufficient endometrial preparation during IVM cycles before ET [4, 7]. Here, we aim to report a novel endometrial preparation method in a PCOS case undergoing IVM. Case report A 33 years old patient with history of primary infertility for 3 years was referred to our program for infertility treatment. She was diagnosed as PCOS according to Rotterdam criteria [8] with history of acne, hirsutism and oligomenorrhea. The clinical profile showed that she had BMI = 28, LH = 4, FSH = 1.5, LH/FSH > 2. The patient had history of both clomiphen failure and metformin administration for more than 6 months. She also underwent laparoscopy and ovarian cauterization approximately 1 year before. Infertility work up for the husband showed abnormal sperm analysis including a count of 28 millions/ml, <20% progressive motility and <14% normal morphology according to WHO criteria. A recent varicoceletomy did not improve semen quality for her husband. In her first treatment cycle and due to a high response to the antagonist protocol and to avoid the risk of severe OHSS, it was decided to cancel the cycle, and to continue the treatment using IVM techniques. After progesterone withdrawal bleeding, FSH priming was delayed until 13th day of the cycle when endometrial thickness reached 7 mm. At that time, FSH priming started, 75 IU/day of FSH (Serono, Aubnne, Switzerland) was injected for 3 days, followed by an injection of 10,000 IU hCG (Pregnyl, Organon, Netherlands). Follicular diameters were less than 10 mm at the time of hCG injection. On the day of hCG injection, both endometrial thickness and level of estradiol were measured. Oocytes were retrieved 36 h after hCG injection using a 19-gauge aspiration needle under transvaginal ultrasound guidance. A total of 10 immature oocytes were retrieved, including 3 at GV stage, 5 at MI stage, and 2 were degenerated. Immature oocytes were transferred to IVM culture media (SAGE Co., CT) for 28 h, when 5 oocytes reached MII stage. The rest of oocytes were arrested. The mature oocytes were microinjected with husbands’ sperm and normal fertilization was confirmed when two polar bodies with two pronuclei were observed 18–20 h after insemination. A total of 3 oocytes were fertilized and 3 four cells embryos (grade B) were transferred 2 days after microinjection using ET catheter (Labotect, Gottingen, Germany). Lutheal phase support was done with 100 mg progesterone in oil (Aburaihan Co, Tehran, Iran) injected daily. Estradiol valerate (Aburaihan C,Tehran, Iran) was taken orally at the dose of 6 mg/day from day of oocytes retrieval, and continued until confirmation of fetal heart by sonography. βhCG was measured 14 days after ET, which was reported positive (>180 IU/L). A healthy baby girl was born at 39 weeks of pregnancy by cesarean section. The baby weigh at the time of birth was 3,500 g with the apgar of 9/10.

Hormonal and experiential predictors of infant survivorship and maternal behavior in a monogamous primate (Callicebus cupreus).

To better understand the roles that hormones and experience play in infant survival and maternal behavior in a biparental primate species, we analyzed urinary estrone (E(1)C) and pregnanediol glucuronide (PdG) from 24 socially housed titi monkey (Callicebus cupreus) females over 54 pregnancies (N = 1,430 samples). Pregnancies were categorized according to whether the infant survived (N = 35) or not (N = 19), and by maternal parity (primiparous: N = 9; multiparous: N = 45). Mothers of infants that survived had a significantly greater drop in PdG from the third trimester to the first week postpartum than mothers of infants that did not survive. Multiparous mothers had a greater increase in PdG from the first to the third trimester as well as greater increases in the E(1)C:PdG ratio from the first to the third trimester and from the third trimester to the first week postpartum. There were positive relationships between third trimester PdG and maternal carrying and nursing during the first week postpartum, and between maternal age and carrying during the infant's first month of life. There was a negative correlation between maternal age and PdG during the third trimester. These results suggest that elevated progesterone during late pregnancy followed by progesterone withdrawal immediately following parturition is associated with greater probability of infant survivorship and maternal behavior in this species, and that older females engage in more postpartum maternal care.

Role of T cells in ovariectomy induced bone loss—revisited

Role of T cells in ovariectomy induced bone loss—revisited

Cervical Remodeling/Ripening at Term and Preterm Delivery: The Same Mechanism Initiated by Different Mediators and Different Effector Cells

BackgroundPremature cervical remodeling/ripening is believed to contribute to preterm delivery (PTD), the leading cause of perinatal morbidity and mortality. Despite considerable research, the causes of term and PTD remain unclear, and there is no effective treatment for PTD. We previously demonstrated that complement activation plays a causative role in cervical remodeling that leads to PTD in mice.Methodology/Principal FindingsHere we found that complement activation is not required for the physiological process that leads to term delivery in mice. Neither increased C3 cervical deposition nor increased C3a and C5a serum levels were observed at term. In addition, macrophages infiltration was found in PTD in contrast to term delivery were no leukocytes were found. Despite the different role of complement and different cellular effector cells, PTD and term delivery share a common dowsntream pathway characterized by increased metalloproteinases (MMPs) release and increased collagen degradation. However, different sources of MMPs were identified. Macrophages are the source of MMPs in PTD while cervical fibroblasts and columnar epithelial cells synthesize MMPs at term delivery. A dramatic diminution in serum progesterone levels precedes parturition at term but not in PTD, suggesting that progesterone withdrawal initiates cervical remodeling at term. On the other hand, MMPs release in PTD is triggered by C5a.Conclusion and SignificanceIn conclusion, preterm and term cervical remodeling occur through the same mechanism but they are initiated by different mediators and effector cells. That complement activation is required for PTD but not for the physiological process that leads to term delivery, suggests that complement is a potential specific biomarker and selective target to prevent PTD and thus avert neonatal mortality and morbidity.

Tumor necrosis factor (TNF)-α upregulates progesterone receptor-A by activating the NF-κB signaling pathway in human decidua after labor onset

To date, the relationship between inflammatory cytokines and progesterone receptors (PRs) has been little studied, although both mediate the mechanism of parturition in human deciduas. Thus, the aim of study was to investigate the role of an inflammatory cytokine, tumor necrosis factor (TNF)-α, in regulating progesterone withdrawal in decidua at human parturition. TNF-α levels and PR isoforms were compared in intrauterine deciduas from women who were in labor (IL, n = 10) or who were not in labor (NIL, n = 10). Nuclear factor-kappaB (NF-κB) signaling and PR status were analyzed in NIL deciduas after TNF-α stimulation. Immunohistochemistry, western blotting, ELISA and reverse transcription-polymerase chain reaction (RT-PCR) were used to localize and quantitate protein and mRNA expression. TNF-α immunostaining, protein levels, PR-A/PR-B ratio and COX-2 level were significantly higher in IL deciduas (all P < 0.05). NF-κB was activated by TNF-α after 24 h stimulation in a dose-dependent manner, and was significantly inactivated by the NF-κB inhibitor panepoxydone, which was associated with decreased PR-A and COX-2 expression ( P < 0.05) in not in labor deciduas. In conclusion, TNF-α may have an important role in regulating progesterone withdrawal in human decidua following labor onset.

Immune aspects and myometrial actions of progesterone and CRH in labor.

Progesterone and corticotropin-releasing hormone (CRH) have a critical role in pregnancy and labor, as changes related to these hormones are crucial for the transition from myometrial quiescence to contractility. The mechanisms related to their effect differ between humans and other species, thus, despite extensive research, many questions remain to be answered regarding their mediation in human labor. Immune responses to progesterone and CRH are important for labor. Progesterone acts as an immunomodulator which controls many immune actions during pregnancy, and its withdrawal releases the inhibitory action on inflammatory pathways. In humans, a “functional” progesterone withdrawal occurs with onset of labor through changes in progesterone metabolism, progesterone receptors, and other molecules that either facilitate or antagonize progesterone function. Placental CRH acts on the fetal pituitary-adrenal axis to stimulate adrenal production of androgens and cortisol and also acts directly on myometrial cells via its receptors. CRH also affects inflammatory signals and vice versa. Interactions between progesterone and CRH additionally occur during labor. We describe the role of these two hormones in human myometrium and their interactions with the immune system during labor.

Estrous behavior, luteinizing hormone and estradiol profiles of intact ewes treated with insulin or endotoxin

Acute insulin administration causes a disparity between the onset of estrous behavior and the LH surge in ovary-intact ewes. To examine the considerable variation in responses, in the present study we used a large number of animals to confirm findings with insulin, and examine whether endotoxin has the same effect. During the breeding season, follicular phases of intact ewes were synchronized with progesterone vaginal pessaries and received saline vehicle (n=22; controls), insulin (4IU/kg; n=21 ewes) or endotoxin (LPS; 100ng/kg; n=10) at 28h after progesterone withdrawal (time zero). In controls, the LH surge onset occurred at 36.5±5.7h and were first mounted by a ram at 38.2±1.8h, but there was a delay of 17.6h (P<0.001) and 7.2h (P<0.05), respectively, in half the insulin-treated animals (‘insulin-delayed’) but not in the other half; and a delay of 22.5h (P<0.001) and 20.7h (P<0.001), respectively, in all LPS-treated animals. Plasma estradiol concentrations decreased after both stressors, and remained low for a period of time equivalent to the LH surge delay (P<0.001; Rs-q=78%). Cortisol increased for 12h after treatment in both insulin subgroups and the LPS group (P<0.05); whereas progesterone increased in the insulin-delayed and LPS groups from 4.0±0.5ng/ml and 5.3±1.0ng/ml to a maximum of 5.7±0.3ng/ml and 8.8±1.6ng/ml, respectively (P<0.05 for both comparisons). Plasma triglycerides were measured to assess insulin resistance, but concentrations were similar before and after treatment (0.25±0.01mmol/l versus 0.21±0.01 and 0.25±0.01mmol/l versus 0.26±0.01mmol/l in the insulin-non delayed and insulin delayed subgroups, respectively). Therefore, we hypothesize that a) when an acute stressor is applied during the late follicular phase, the duration of the LH surge delay is related to the duration of estradiol signal disruption b) cortisol is not the key disruptor of the LH surge after insulin, c) insulin (but not LPS) can separate the onsets of LH surge and estrus by approximately 10h, providing a model to identify the specific neuronal systems that control behavior distinct from those initiating the GnRH surge.

Molecular and Cellular Causes of Abnormal Uterine Bleeding of Endometrial Origin

Women today may reasonably anticipate in the order of some 400 menstrual cycles over their reproductive lifespan. The endometrium is thus subject to repeat cycles of shedding and repair and notably healing of the endometrium post menses is "scarless". The local molecular and cellular mechanisms involved in post menstrual resolution of the inflammatory events associated with menstruation and endometrial repair remain to be fully determined. Menstrual complaints are common. It is highly likely that unrestrained local inflammatory events and/ or deficient repair processes within the endometrium contribute to the women's experience of heavy menstrual bleeding (HMB). The management of women with HMB may need to utilize therapeutic approaches that optimize endometrial repair processes, post menses. These approaches may be necessary in addition to current therapies that hitherto have focused on limiting the local inflammation associated with menstruation. Research endeavors thus need to focus upon the molecular and cellular causes of problematic uterine bleeding. Herein the events associated with pre-menstrual progesterone withdrawal, limitation of blood loss, the expression of vasoactive mediators and factors that may modulate vascular morphology are described. Such lines of enquiry and knowledge will be essential if novel targets for treatment of menstrual bleeding complaints, such as HMB, are to be identified.

Transforming growth factor beta 1(TGFβ1) and progesterone differentially regulate matrix metalloproteinases (MMPs) in human normal endometrium and endometriosis

IL-1β, TNF-α and TGFβ1 are increased in peritoneal fluid samples from women with endometriosis. Invasion of ectopic endometrium into peritoneal mesothelium requires matrix metalloproteinases (MMPs) for tissue remodeling. Menstruation is preceded by progesterone withdrawal, collagen degradation and matrix remodeling predominantly through induction MMPs and recruitment of invading neutrophils. Thus, elucidation of the mechanism underlying regulation of MMP gene expression in the endometrium will be helpful for understanding normal menstruation but also treatment of endometriosis.

P.2.b.018 Progesterone withdrawal models of depression in rats: behavioural characterisation and efficacy of Lu AA21004 and fluoxetine

P.2.b.018 Progesterone withdrawal models of depression in rats: behavioural characterisation and efficacy of Lu AA21004 and fluoxetine

The regulation of vascular endothelial growth factor by hypoxia and prostaglandin F₂α during human endometrial repair.

Context:The human endometrium has an exceptional capacity for repeated repair after menses, but its regulation remains undefined. Premenstrually, progesterone levels fall and prostaglandin (PG) F2α synthesis increases, causing spiral arteriole constriction. We hypothesized that progesterone withdrawal, PGF2α, and hypoxia increase vascular endothelial growth factor (VEGF), an endometrial repair factor.Design and Results:Endometrial biopsies were collected (n = 47) with ethical approval and consent. VEGF mRNA, quantified by quantitative RT-PCR, was increased during menstruation (P < 0.01).VEGF protein was maximally secreted from proliferative endometrial explants. Treatment of an endometrial epithelial cell line and primary human endometrial stromal cells with 100 nm PGF2α or hypoxia (0.5% O2) resulted in significant increases in VEGF mRNA and protein. VEGF was maximal when cells were cotreated with PGF2α and hypoxia simultaneously (P < 0.05–0.001). Secretory-phase endometrial explants also showed an increase in VEGF with cotreatment (P < 0.05). However, proliferative-phase explants showed no increase in VEGF on treatment with PGF2α and/or hypoxia. Proliferative tissue was induced to increase VEGF mRNA expression when exposed to progesterone and its withdrawal in vitro but only in the presence of hypoxia and PG. Hypoxia-inducible factor-1α (HIF-1α) silencing with RNA interference suppressed hypoxia-induced VEGF expression in endometrial cells but did not alter PGF2α-induced VEGF expression.Conclusions:Endometrial VEGF is increased at the time of endometrial repair. Progesterone withdrawal, PGF2α, and hypoxia are necessary for this perimenstrual VEGF expression. Hypoxia acts via HIF-1α to increase VEGF, whereas PGF2α acts in a HIF-1α-independent manner. Hence, two pathways regulate the expression of VEGF during endometrial repair.

Effect of Progesterone Withdrawal on Hypothalamic Mechanisms Related to Prolactin Release in Late Pregnant Rats

Background/Aims: Progesterone (P₄) fall provoked by spontaneous or prostaglandin F2α (PGF2α)-induced luteolysis in late pregnant rats triggers a prolactin (PRL) surge 12–24 h later. Methods: To investigate the hypothalamic mechanism mediating this response, we determined expression of tyrosine hydroxylase (TH), PRL receptors (long form, PRLR_long), estrogen-α (ERα) and ERβ, P₄ (PR) A and B receptors, and STAT5a, STAT5b, suppressors of cytokine signaling 1 (SOCS1), SOCS3 and CIS at mRNA (by semiquantitative and real-time RT-PCR) and protein (by Western blot only for TH, ERα and PRs) levels, and dopamine and DOPAC (by high-performance liquid chromatography) contents in the mediobasal hypothalamus (MBH) 24 h after luteolysis induced by a PGF2α analogue (cloprostenol, 25 µg/rat s.c. at 8 and 12 h on day 19 of pregnancy). Results: PGF2α treatment decreased circulating P₄ and estradiol and increased PRL and the estradiol/P₄ ratio. MBH DOPAC and DOPAC/dopamine ratio fell, indicating decreased dopaminergic transmission. PRLR_long, PRB and ERα mRNA increased. ERα and PR proteins were not modified. However, TH protein and mRNA did not change. PRA, the small PR isoform, was much more abundant than PRB, the isoform considered to mediate P₄ genomic actions. STAT5a, SOCS1 and SOCS3 mRNA were also increased. Conclusion: The P₄ fall induced by PGF2α treatment induces PRL release through diminution in MBH dopaminergic transmission without change in TH expression. The increased PRLR along with elevated circulating PRL may be responsible for maintaining high TH expression through activation of short-loop feedback mechanisms, counteracting the effect of the fall in circulating P₄. In parallel, SOCS expression contributes to limit PRL signaling.

The expression and regulation of adrenomedullin in the human endometrium: a candidate for endometrial repair.

After menstruation, the endometrium has a remarkable capacity for repair, but the factors involved remain undefined. We hypothesize adrenomedullin (AM) plays a role in this process. Premenstrually progesterone levels decline, stimulating prostaglandin (PG) synthesis, vasoconstriction, and hypoxia. This study aimed to determine 1) AM expression throughout the menstrual (M) cycle and 2) its regulation by PG and hypoxia. Human endometrial biopsies (n = 51) were collected with ethical approval and consent. AM mRNA expression was examined by quantitative RT-PCR and was found to be selectively elevated in endometrium from the menstrual (M) phase (P < 0.001). AM immunohistochemical staining was maximal in M and proliferative (P) endometrium. Culture of secretory, but not P, explants with 100 nm PGF2α or hypoxia (0.5% O2) increased AM mRNA (P < 0.05). P explants were induced to increase AM expression using in vitro progesterone withdrawal but required the presence of hypoxia (P < 0.05). Short hairpin sequences against hypoxia-inducible factor-1α (HIF-1α) inhibited AM hypoxic up-regulation but did not alter PGF2α-induced expression. The AM receptor was immunolocalized to endothelial cells in both lymphatic and blood vessels. Conditioned medium from PGF2α-treated cells increased endothelial cell proliferation and branching (P < 0.05). This was abolished by AM receptor antagonists. In conclusion, AM is elevated at the time of endometrial repair and induces both angiogenesis and lymphangiogenesis by stimulating endothelial cell proliferation and tube formation. In the human endometrium, AM expression is up-regulated by two mechanisms: a HIF-1α-mediated hypoxic induction and a HIF-1α-independent PGF2α pathway. These physiological mechanisms may provide novel therapeutic targets for disorders such as heavy menstrual bleeding.

Estrogen and progesterone regulate expression of the endothelins in the rhesus macaque endometrium.

Endothelins (EDNs) are thought to modulate endometrial blood flow during menses, stromal healing and endometrial growth during the proliferative phase. Our goal was to assess the effects of estrogen and progesterone on the EDN paracrine system in the endometrium of rhesus macaques. In this study, archived samples were used. These samples were collected from oophorectomized rhesus macaques that were treated sequentially with estradiol (E(2)) and then E(2) plus progesterone to create artificial menstrual cycles. Endometrium from animals in the menstrual, proliferative and secretory phases of the artificial cycle were analyzed by real-time PCR, in situ hybridization and immunocytochemistry to detect changes in EDN peptides (EDN1, EDN2, EDN3), EDN receptors (EDNRA, EDNRB), EDN-converting enzyme 1 (ECE1) and membrane metalloendopeptidase (MME)-an enzyme that degrades the EDNs. Compared with the late secretory phase, progesterone withdrawal at the end of the artificial menstrual cycle triggered an increase (P< 0.05) in EDN1, EDNRB and ECE1 in the upper functionalis zone during menses of the next cycle. Treatment with E(2) alone in the proliferative phase increased (P< 0.05) EDNRA transcript, which was confined predominantly to the stromal cells. E(2) plus progesterone in the artificial secretory phase suppressed (P< 0.05) the expression of EDN3 in the functionalis zone stroma and epithelia, tended (P= 0.08) to attenuate levels of epithelial EDN2 and markedly up-regulated (P< 0.05) the stromal expression of MME. Our results indicate that estrogen and progesterone regulate the EDN family during the menstrual cycle. The changes in the EDN paracrine system during the mid-secretory phase may indicate a role for EDN during embryo implantation.

Mechanisms of normal and abnormal endometrial bleeding

Expression of tissue factor (TF), the primary initiator of coagulation, is enhanced in decidualized human endometrial stromal cells (HESC) during the progesterone-dominated luteal phase. Progesterone also augments a second HESC hemostatic factor, plasminogen activator inhibitor-1 (PAI-1). In contrast, progestins inhibit HESC matrix metalloproteinase (MMP)-1, 3 and 9 expression to stabilize endometrial stromal and vascular extracellular matrix. Through these mechanisms decidualized endometrium is rendered both hemostatic and resistant to excess trophoblast invasion in the mid-luteal phase and throughout gestation to prevent hemorrhage and accreta. In non-fertile cycles, progesterone withdrawal results in decreased HESC TF and PAI-expression and increased MMP activity and inflammatory cytokine production promoting the controlled hemorrhage of menstruation and related tissue sloughing. In contrast to these well ordered biochemical processes, unpredictable endometrial bleeding associated with anovulation reflects absence of progestational effects on TF, PAI-1 and MMP activity as well as unrestrained angiogenesis rendering the endometrium non-hemostatic, proteolytic and highly vascular. Abnormal bleeding associated with long-term progestin-only contraceptives results not from impaired hemostasis but from unrestrained angiogenesis leading to large fragile endometrial vessels. This abnormal angiogenesis reflects progestational inhibition of endometrial blood flow promoting local hypoxia and generation of reactive oxygen species that increase production of angiogenic factors such as vascular endothelial growth factor (VEGF) in HESCs and Angiopoietin-2 (Ang-2) in endometrial endothelial cells while decreasing HESC expression of angiostatic, Ang-1. The resulting vessel fragility promotes bleeding. Aberrant angiogenesis also underlies abnormal bleeding associated with myomas and endometrial polyps however there are gaps in our understanding of this pathology.