Progesterone withdrawal up-regulates vascular endothelial growth factor receptor type 2 in the superficial zone stroma of the human and macaque endometrium: potential relevance to menstruation

Several reports indicate that vascular endothelial growth factor (VEGF) expression is increased in endometrial glands and stroma during the menstrual phase in the human endometrium. Here we report that VEGF receptor type 2 (KDR), normally expressed only in the vascular endothelium, was dramatically up-regulated in the stromal cells of the superficial endometrial zones during the premenstrual phase in both human and macaque endometrium. This increase was detectable by Northern analysis, in situ hybridization, and immunocytochemistry and was cell specific, zone specific, cycle phase specific, and VEGF receptor type specific. That is, it only occurred during the premenstrual/menstrual phase, did not occur in glandular epithelium, endothelium, or stromal cells of the deepest endometrial zones, and was not observed for VEGF receptor type 1. The upregulation of stromal KDR was induced by progesterone (P) withdrawal in both women and macaques, and adding back P 24 h after P withdrawal in macaques blocked stromal, but not vascular, endothelial KDR expression. Promatrix metalloproteinase-1 (MMP-1) was coordinately up-regulated in the same stromal cell population by P withdrawal. Because of reports that VEGF can enhance MMP expression, we hypothesize that VEGF-KDR interactions may influence MMP expression in the superficial zones of the primate endometrium during the premenstrual phase, and that these interactions play a role in the induction of menstruation.     

Coexpression of fractalkine and its receptor in normal human endometrium and in endometrium from users of progestin-only contraception supports a role for fractalkine in leukocyte recruitment and endometrial remodeling

Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyte subpopulations enter the uterus are currently unknown. Endometrial leukocytes have no genomic progesterone receptors; thus, we hypothesized that leukocyte migration is induced indirectly by progesterone-regulated chemokines. Fractalkine (CX3CL1), a chemotactic membrane-bound adhesion factor, and its receptor (CX3CR1) were assessed by immunohistochemistry in endometrial samples across the menstrual cycle, in early pregnancy, and in women using progestin-only contraceptives. Fractalkine was localized predominantly to glandular epithelial and decidualized stromal cells, with the highest staining intensity in the secretory phase and early pregnancy. It was also detected in subpopulations of endometrial leukocytes (macrophages and uterine NK cells), with maximal numbers during the proliferative phase and early pregnancy. CX3CR1 was similarly colocalized to the glandular epithelium and decidualized stromal cells, with the highest expression in the secretory phase. CX3CR1-positive leukocytes (macrophages and neutrophils) were in greatest abundance during the menstrual phase. In the endometrium of women using progestin-only contraceptives, immunoreactive fractalkine was markedly reduced in the glandular epithelium, but was increased in decidualized stroma and infiltrating leukocytes. These findings support a number of roles for fractalkine in the endometrium, in the secretory phase, in early pregnancy, and when influenced by progestin-only contraceptives.     

Expression of vascular endothelial growth/permeability factor by endometrial glandular epithelial and stromal cells in baboons during the menstrual cycle and after ovariectomy

Vascular endothelial growth/permeability factor (VEG/PF) has a crucial role in angiogenesis, and neovascularization is essential in preparing the uterine endometrium for implantation. However, the regulation of VEG/PF synthesis by particular cell types of the endometrium during the human menstrual cycle is not well understood. Therefore, in the present study the baboon was used as a nonhuman primate to determine the role of the ovary in vivo in endometrial VEG/PF expression. VEG/PF mRNA levels were quantified by competitive RT-PCR in whole uterine endometrium and in glandular epithelial and stromal cells isolated from the endometrium by laser capture microdissection of baboons during the normal menstrual cycle and after ovariectomy, which decreased serum estradiol and progesterone to undetectable levels. Mean (+/-SE) levels (attomoles per micrograms of total RNA) of the 323-bp VEG/PF mRNA product, which reflected collective expression of all VEG/PF isoforms, in whole endometrium were 785 and 727 +/- 158 during the mid and late follicular phases, respectively, and 1108 +/- 320 during the midcycle surge in serum estradiol. VEG/PF mRNA levels then declined briefly before increasing to 1029 +/- 365 attomoles/ micro g RNA during the late luteal phase of the menstrual cycle. VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) were similar in glandular epithelial (2.27 +/- 1.11) and stromal (2.54 +/- 0.70) cells at the midcycle estradiol peak and the midluteal phase of the menstrual cycle (2.34 +/- 1.30 and 1.49 +/- 0.53, respectively). Immunocytochemical expression of VEG/PF protein was abundant in glandular and luminal epithelium, stroma, and vascular endothelium. Endometrial vessel density and percent vascularized area, determined by morphometric image analysis, were similar during the various stages of the baboon menstrual cycle. After ovariectomy, VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) in the endometrial glands (0.52 +/- 0.21) and stroma (0.22 +/- 0.11) were decreased to values that were approximately 20% and 10% (P < 0.05), respectively, of those in intact baboons during the midcycle estrogen surge. Moreover, there was relatively little VEG/PF protein immunostaining in the endometrial glands, stroma, and vascular endothelium after ovariectomy. In summary, VEG/PF mRNA and protein expression in glandular epithelial and stromal cells were markedly suppressed after ovariectomy, indicating that synthesis of this angiogenic factor in these endometrial cells is dependent upon a product(s) secreted by the ovary. Moreover, endometrial VEG/PF expression remained relatively constant and thus was available as a component of the angiogenic system throughout the menstrual cycle, presumably to progressively promote vascular reconstruction of the endometrium.     

Stromal-epithelial interaction mediates steroidal regulation of metalloproteinase expression in human endometrium.

The hallmark of the menstrual cycle is extensive steroid-dependent tissue turnover. Estrogen mediates endometrial cell growth and structural remodeling, whereas progesterone suppresses estrogen-dependent proliferation and promotes cellular differentiation. In nonfertile cycles, tissue degradation and menstruation occur as a consequence of steroidal deprivation as the ovarian corpus luteum fails. Stromal-epithelial interactions are recognized as a necessary component in mediating steroid-induced endometrial turnover. Specific mRNAs for metalloproteinases of the stromelysin family are expressed during endometrial growth and menstrual breakdown but are absent in the progestin-dominated secretory phase. This expression pattern suggests involvement of stromelysins in remodeling the extracellular matrix of the endometrium during tissue growth and breakdown and implicates progesterone in the suppression of these enzymes. We examined the regulation of endometrial stromelysins in explant cultures and found no acute effect of estradiol on their expression, whereas progesterone was a potent inhibitor of stromelysin expression. Progesterone also suppressed stromelysin expression in cultures of isolated stromal cells, but epithelial cells were progesterone insensitive. Coculture of recombined stromal and epithelial cells restored steroidal suppression of the epithelial-specific metalloproteinase. Our data confirm that progesterone inhibits endometrial stromelysins and further demonstrate the necessity for a stromal-derived factor(s) as a mediator of steroid suppression of an epithelial metalloproteinase.     

The glucocorticoid receptor is specifically expressed in the stromal compartment of the human endometrium

The human endometrium is a classical target tissue for steroid hormones. While the expression pattern and functional roles of both the estrogen receptor (ER) and the progesterone receptor (PR) are well defined, expression of the glucocorticoid receptor (GR) in this tissue has not been described so far. In the present study, we used immunohistochemistry to analyze the expression of GR in the normal human endometrium throughout the menstrual cycle. The expression of GR was compared to that of ER and PR, which were analyzed in parallel. We show that GR is expressed in the human endometrium with a pattern that markedly differs from the expression patterns of ER and PR. ER and PR are expressed in the nuclei of endometrial glands, whereas GR is completely absent from these structures. However, GR is strongly expressed in the stromal compartment of the endometrium throughout the cycle. Both stromal fibroblasts and lymphocytes are GR-positive. In addition GR expression is also observed in the endothelium of small endometrial blood vessels, which are ER- and PR-negative. Western blot analysis performed on endometrial tumor cell lines of glandular (HEC-1B) and mesodermal (SKUT-1B) origin, respectively, showed GR expression only in the latter. In summary, we demonstrate that GR is expressed in fibroblasts, lymphocytes and endothelial cells of the human endometrial stroma, while it is absent from the glandular compartment. The specific expression pattern of GR within the human endometrium points to a possible functional role of glucocorticoids in the process of decidualization which occurs primarily in the stromal compartment.     

Identification of genes with differential regulation in primate endometrium during the proliferative and secretory phases of the cycle

To study gene expression in the endometrium at different stages of the menstrual cycle, differential mRNA display and reverse Northern analysis were performed on uterine tissues from cynomolgus monkeys. Eutopic endometrial RNA was prepared from uteri of animals that were either ovariectomized and supplemented with hormones, or were not ovariectomized but were subjected to surgically induced endometriosis. A number of genes were identified whose levels fluctuated between the proliferative and secretory phases of the cycle. Expression of four genes thus identified was further examined by in situ hybridization to normal human endometrial biopsies. Iodothyronine deiodinase was uniformly expressed at all stages of the human cycle that were studied: proliferative, secretory, and menstrual. Fibulin 1, osteopontin, and cathepsin H exhibited complex temporal and spatial regulation. Fibulin 1 was expressed in glandular epithelia during the menstrual phase, but expression switched to the stroma during the secretory phase. During the menstrual phase, osteopontin was expressed at high levels in glandular epithelia and in isolated stromal cells that may be of immune origin. Secretory phase expression of osteopontin was confined to a sub-population of epithelial cells. Cathepsin H was expressed in proliferative and menstrual phase endometrium, but expression disappeared in the secretory phase. Messenger RNA for fibulin 1, osteopontin, and iodothyronine deiodinase was detected in an endometriosis sample. Our data support functional roles for fibulin 1, osteopontin, cathepsin H and thyroid hormone in endometrium.     

Endometrial regeneration and endometrial stem/progenitor cells

The functional layer of the human endometrium is a highly regenerative tissue undergoing monthly cycles of growth, differentiation and shedding during a woman??s reproductive years. Fluctuating levels of circulating estrogen and progesterone orchestrate this dramatic remodeling of human endometrium. The thin inactive endometrium of postmenopausal women which resembles the permanent basal layer of cycling endometrium retains the capacity to respond to exogenous sex steroid hormones to regenerate into a thick functional endometrium capable of supporting pregnancy. Endometrial regeneration also follows parturition and endometrial resection. In non menstruating rodents, endometrial epithelium undergoes rounds of proliferation and apoptosis during estrus cycles. The recent identification of adult stem cells in both human and mouse endometrium suggests that epithelial progenitor cells and the mesenchymal stem/stromal cells have key roles in the cyclical regeneration of endometrial epithelium and stroma. This review will summarize the evidence for endometrial stem/progenitor cells, examine their role in mouse models of endometrial epithelial repair and estrogen-induced endometrial regeneration, and also describe the generation of endometrial-like epithelium from human embryonic stem cells. With markers now available for identifying endometrial mesenchymal stem/stromal cells, their possible role in gynecological diseases associated with abnormal endometrial proliferation and their potential application in cell-based therapies to regenerate reproductive and other tissues will be discussed.     

Spatial and temporal expression of ligands for CXCR3 and CXCR4 in human endometrium

In this study, we investigated the expression of ligands for CXCR3 (Mig, IP-10, and I-TAC) and CXCR4 (SDF-1) in the human endometrium throughout the menstrual cycle. By immunohistochemistry, immunostaining for Mig and IP-10 was found in the surface epithelia, glandular epithelia, and stroma with some menstrual cycle-dependent fluctuation. By contrast, immunostaining for I-TAC or SDF-1 was not detected. ELISA demonstrated that the concentrations of Mig and IP-10 were higher in the secretory phase than in the proliferative phase, but I-TAC and SDF-1alpha was detected in only a few samples. Endometrial Mig and IP-10 concentrations strongly correlated with the number of endometrial natural killer cells. Progesterone significantly induced Mig secretion and tended to induce IP-10 secretion from the cultured endometrial stromal cells, whereas 17beta-estradiol had no significant effect. Neither I-TAC nor SDF-1alpha was detected in the supernatant of cultured endometrial stromal cells in the presence or absence of 17beta-estradiol or progesterone. The results suggest that Mig and IP-10 may be involved in the recruitment of natural killer cells or other phenomena in the human endometrium.     

Apoptosis, proliferation, and sex hormone receptors in superficial parts of human endometrium at the end of the secretory phase.

Apoptosis with one regulator, Bcl-2, and proliferation with the marker Ki-67 were studied in 75 endometrial biopsies representing superficial parts of endometrium from 35 regularly menstruating women premenstrually and menstrually. Hormonal withdrawal was studied in serum samples and potentiated in epithelium by the decreasing 17beta-estradiol and progesterone receptor scores 4 days premenstrually. The apoptotic index increased 2 days before the onset of menstruation and peaked on the second menstrual day. The high apoptotic index together with low proliferation in endometrial epithelium at the end of the menstrual cycle are similar to the involution process seen in other hormone-dependent organs. In stroma, the apoptotic index increased later, at the onset of menstruation, and the increase was lower than that in epithelium. The Ki-67 index increased during the last 3 days of the secretory phase, parallel with an increasing progesterone receptor score and decreasing Bcl-2 staining, and peaked at the onset of menstruation. The findings in stroma concur with high proliferation at the end of the menstrual cycle and high cell turnover during menstruation, suggesting the participation of stroma in the renewal process of endometrium.     

Changes in endometrial PTEN expression throughout the human menstrual cycle

Frequent mutation of the PTEN tumor suppressor gene in endometrial adenocarcinoma has led to the prediction that its product, a phosphatase that regulates the cell cycle, apoptosis, and possibly cell adhesion, is functionally active within normal endometrial tissues. We examined PTEN expression in normal human endometrium during response to changing physiological levels of steroid hormones. PTEN ribonucleic acid levels, assessed by RT-PCR, increase severalfold in secretory compared to proliferative endometrium. This suggested that progesterone, a known antineoplastic factor for endometrial adenocarcinoma, increases PTEN levels. Immunohistochemistry with an anti-PTEN monoclonal antibody displayed a complex pattern of coordinate stromal and epithelial expression. Early in the menstrual cycle under the dominant influence of estrogens, the proliferative endometrium shows ubiquitous cytoplasmic and nuclear PTEN expression. After 3-4 days of progesterone exposure, glandular epithelium of early secretory endometrium maintains cytoplasmic PTEN protein in an apical distribution offset by expanding PTEN-free basal secretory vacuoles. By the midsecretory phase, epithelial PTEN is exhausted, but increases dramatically in the cytoplasm of stromal cells undergoing decidual change. We conclude that stromal and epithelial compartments contribute to the hormone-driven changes in endometrial PTEN expression and infer that abnormal hormonal conditions may, in turn, disrupt normal patterns of PTEN expression in this tissue.     

Endometrial and myometrial expression of nitric oxide synthase isoforms in pre- and postmenopausal women.

Expression of nitric oxide synthase (NOS) protein was examined by Western immunoblot analysis and immunohistochemistry in the endometrium and myometrium of 19 premenopausal and 18 postmenopausal women undergoing hysterectomy for benign gynecological reasons. The predominant isoform of NOS in the human uterus was endothelial NOS (eNOS). Using immunohistochemistry, eNOS was localized predominantly to the glandular epithelium and endometrial microvascular endothelium. eNOS was scant and inconsistently detected in endometrial stromal cells. In the myometrium, eNOS was predominantly found in smooth muscle cells (myocytes) and was also detected in the microvascular endothelium. Neuronal NOS was not detectable by immunohistochemical techniques, and inducible NOS (iNOS) was only detectable in occasional specimens, although more often in secretory specimens. iNOS, when present, was predominantly found in glandular epithelium and occasional stromal cells. Myometrial iNOS was scant and not consistently detected. By Western immunoblot analysis, neuronal NOS or iNOS was not detected. We observed a unique menstrual cycle-dependent expression of eNOS that was different in the endometrium compared to the myometrium and was independent of uterine pathology. In the endometrium, there was 62% higher expression of eNOS during the secretory phase (P = 0.00085) compared to the proliferative phase, whereas in the myometrium, there was 74% greater expression of eNOS in the proliferative phase (P = 0.03) compared to the secretory phase. Within the secretory phase, maximal endometrial eNOS expression was found in the midportion, whereas in the myometrium, highest eNOS expression occurred during the late secretory phase. In postmenopausal women not treated with hormones, a significant reduction in endometrial and myometrial expression of eNOS occurred, which was reversed by continuous hormone replacement therapy. In summary, both endogenous ovarian steroids and exogenous sex hormones influence uterine eNOS expression. Our results suggest that estrogen may regulate myometrial eNOS, whereas progesterone or a combination of estrogen and progesterone may be more important in regulating endometrial eNOS, and NO may be a critical mediator of sex steroid actions in the human uterus.     

Regulation of matrix metalloproteinase-9/gelatinase B expression and activation by ovarian steroids and LEFTY-A/endometrial bleeding-associated factor in the human endometrium

Various matrix metalloproteinases (MMPs) participate in the menstrual breakdown of the human endometrium. MMP-9/gelatinase B is proposed as a major factor because it degrades many extracellular matrix constituents, including in the vasculature. Although globally under ovarian steroids control, endometrial MMP-9 seems expressed differently than other MMPs, and conflicting publications prevent a clear understanding of its regulation. We therefore quantified MMP-9 expression in the cycling human endometrium, defined its localization, and analyzed its regulation by estradiol and progesterone and by LEFTY-A/endometrial bleeding-associated factor in explant cultures. In fresh tissues, a major increase in MMP-9 mRNA expression occurred at menstruation, after a larger increase in LEFTY-A mRNA. MMP-9 was immunodetected in all cell types throughout the cycle, especially in foci of stromal cells during menstruation. MMP-9 synthesis by these cells was confirmed in cultured explants. In proliferative explants, ovarian steroids slightly decreased MMP-9 mRNA. They had no consistent effect on MMP-9 release in culture medium but strongly inhibited proMMP-9 activation. Addition of recombinant LEFTY-A to explants induced MMP-9 in most samples, a response prevented by ovarian steroids. We propose that endometrial MMP-9 activity is overall controlled by the ovarian steroids and locally adjusted through a network of modulators, including LEFTY-A.     

Expression of Delta-like protein 4 in the human endometrium

Activation of Delta-Notch signaling pathway promotes the development of the vascular system in embryo, normal adult tissues, and cancerous lesions. Delta and Notch genes are known to be expressed in endothelial cells, and little is known of their expression beyond the vascular system. The purpose of this study was to investigate whether Delta gene would be expressed in cells of the uterine endometrium. In this study, we found that the human endometrial cells expressed one of the Delta ligands, Delta-like 4 protein (Dll4). Dll4 was expressed in human endometrium in a spatiotemporal fashion. Immunohistochemistry studies showed the cytoplasm as well as membrane staining with apical localization both in the luminal and glandular epithelium and moderate diffuse staining in the cytoplasm of the stromal cells. Western blot analysis showed that the size of the endometrial Dll4 was identical to that in the human umbilical endothelial cells. The expression of Dll4 mRNA in human endometrial cells was quantitatively determined by real-time PCR. Dll4 mRNA expressed in the glandular epithelium showed large variations, and it was significantly elevated in the mid and late proliferative and early secretory endometrium. Endometrial stromal cells contained less Dll4 mRNA and had no clear correlation with the menstrual cycle. The effect of hormones was studied in the primary culture of isolated glandular epithelial and stromal cells. In glandular cells, estradiol had little effect, and medroxyprogesterone acetate significantly reduced the mRNAs compared with that of control. Relaxin induced the Dll4 mRNA. In stromal cells, both estradiol and medroxyprogesterone acetate reduced the Dll4 mRNA. To our knowledge, this is the first report of the expression of Dll4 in the endometrium. We propose that endometrial Dll4 may enhance the development of the endometrial microvascular system and facilitate the implantation of blastocyst in a fertile cycle.     

Opposite regulation of transforming growth factors-beta2 and -beta3 expression in the human endometrium

TGF-betas have been reported to mediate the repression by progesterone of several matrix metalloproteinases in the human endometrium, thereby preventing menstrual breakdown. Because of conflicting reports on the expression profiles, source, and regulation of the TGF-beta system in this tissue, we investigated by real-time RT-PCR and ELISA the expression of the three TGF-betas (total and mature forms) and their two receptors throughout the menstrual cycle, and their regulation by ovarian steroids in cultured explants including their microdissected epithelial and stromal compartments. Regulation by cAMP and MAPK was further investigated. This comprehensive study on a large collection of endometrial samples evidenced a differential regulation of TGF-beta isoforms expression, both in vivo and in explant culture. In vivo, TGF-beta2 increased by about 5-fold at the mid-late secretory phase then declined after menstruation; TGF-beta3 increased at menstruation and remained high during the proliferative phase; TGF-beta1 was maximal at menstruation. In explants cultured without ovarian steroids both TGF-beta2 and -beta3 were preferentially expressed in the stroma. Ovarian steroids strongly repressed both TGF-beta2 and -beta3 in stroma but only TGF-beta2 in glands. cAMP prevented inhibition by ovarian steroids of TGF-beta2 but not -beta3. In presence of ovarian steroids, MAPK inhibitors (p38 and ERK pathways) stimulated TGF-beta3 but inhibited TGF-beta2 expression. In conclusion, TGF-beta2 and -beta3 are differentially expressed during the menstrual cycle and regulated by progesterone in epithelial vs stromal cells. The opposite regulation of TGF-beta2 and -beta3 by cAMP and MAPK could account for their distinct expression in vivo.     

Expression of macrophage inflammatory protein-1beta in human endometrium: its role in endometrial recruitment of natural killer cells

Human endometrium is infiltrated by natural killer (NK) cells throughout the menstrual cycle. The number of endometrial NK cells is low in the proliferative phase, but acutely increases after ovulation, and reaches a peak in the late secretory phase, suggesting that endometrium recruits these leukocytes selectively from circulating peripheral blood. We investigated the expression of macrophage inflammatory protein (MIP)-1beta, a potential chemoattractant for NK cells, in the endometrium. RT-PCR and ELISA revealed that MIP-1beta is expressed in the endometrium throughout the menstrual cycle at both the message and protein levels. MIP-1beta expression is stronger in the secretory phase endometrium than in the proliferative phase endometrium. Immunohistochemistry revealed that MIP-1beta is localized in the surface epithelial cells, glandular epithelial cells, and perivascular stromal cells throughout the menstrual cycle. Stromal cells in a wider perivascular area became immunoreactive in the secretory phase. There was a strong correlation between the endometrial MIP-1beta concentration and the number of endometrial NK cells. Progesterone significantly induced MIP-1beta secretion from cultured endometrial stromal cells, whereas 17beta-estradiol had a weak effect. These results suggest that endometrial MIP-1beta may be involved in the recruitment of NK cells from circulating peripheral blood.     

Vascular proliferation and vascular endothelial growth factor expression in the rhesus macaque endometrium

The relationship between vascular endothelial growth factor (VEGF) expression and the pattern of vascular proliferation in the rhesus macaque endometrium has not been studied. In this report, we used in situ hybridization to evaluate VEGF, VEGF receptor type 1 and VEGF receptor type 2 mRNA expression during hormonally regulated menstrual cycles in ovariectomized macaques. Proliferating endothelial cells were identified by a double immunocytochemistry procedure that detected Ki-67 antigen and von Willebrand factor in the same endothelial cells. One and 2 d after progesterone withdrawal (premenstrual), VEGF mRNA was up-regulated in the glands and stroma of the superficial endometrial zones, a finding that supports our previous suggestion that VEGF may play a role in the menstrual induction cascade. During the postmenstrual repair phase, the healing surface epithelium showed a further, dramatic increase in expression of VEGF mRNA, accompanied by strong increases in signals for VEGF receptor types 1 and 2 in multiple profiles of small blood vessels immediately below the surface epithelium. This finding implicates VEGF in the early angiogenic processes associated with endometrial healing and regeneration. Vascular endothelial proliferation persisted throughout the cycle in the upper endometrial zones and showed a dramatic estrogen- dependent peak during the midproliferative phase. This proliferative peak coincided with a peak in VEGF expression in the endometrial stroma. Endothelial proliferation was also significantly correlated with the degree of stromal VEGF expression during the proliferative and secretory stages of the cycle. These results implicate VEGF of stromal origin in endometrial vascular proliferation.     

Endothelin-1 gene expression and protein biosynthesis in human endometrium: potential modulator of endometrial blood flow.

We present evidence that endothelin-1 (ET-1) is produced by two distinct cell types (other than vascular endothelial cells) in human endometrial tissue. The supportive findings of this investigation are summarized as follows: 1) prepro-ET-1 mRNA is present in endometrial tissue and in separated endometrial stromal and glandular epithelial cells in culture; 2) immunoreactive ET is secreted into the medium of isolated endometrial stromal cells and glandular epithelium maintained in culture; and 3) the level of prepro-ET-1 mRNA in endometrial tissues obtained at the premenstrual-menstrual phase of the endometrial cycle is greater than that in tissues from the proliferative or early and midsecretory phases. We also found that transforming growth factor-beta and interleukin-1 alpha act to increase the levels of prepro-ET-1 mRNA in endometrial stroma cells in monolayer culture. We speculate that ET-1 derived from endometrial stromal cells may act on the adventitial surface of contiguous spiral arterioles of the endometrium to modulate endometrial blood flow.     

Elafin in human endometrium: an antiprotease and antimicrobial molecule expressed during menstruation

Elafin is an antiproteinase and antimicrobial molecule that is expressed at epithelial sites (for example, cervix). This study details the expression and regulation of elafin in the human endometrium. Elafin mRNA and protein expression were examined in endometrium throughout the menstrual cycle and in first-trimester decidua. Real-time quantitative PCR showed that expression of elafin mRNA peaked during menstruation. Elafin protein was localized to leukocytes scattered in the endometrial stroma during the late secretory and menstrual phases. Faint immunostaining was also present in glandular epithelium at these cycle stages. Immunofluorescent colocalization of elafin with neutrophil elastase confirmed that elafin was expressed by endometrial neutrophils around the time of menstruation. This is consistent with the expression profile observed from immunohistochemical studies. Primary endometrial epithelial cells were treated with proinflammatory molecules, and elafin mRNA was studied. A combination of the proinflammatory mediators, IL-1 beta and TNFalpha, increased elafin mRNA levels by 4.6-fold. These results show that endometrium expresses elafin in a menstruation-dependent manner. This is attributable to the presence of infiltrating leukocytes and increased inflammatory signaling. Elafin will regulate proteolytic enzymes during menstruation and will contribute to the innate defense against uterine infection.