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A major cause of infertility in normal and assisted reproduction cycles is failure of the endometrium to undergo appropriate changes during the secretory phase of the menstrual cycle as it acquires receptivity for an implanting blastocyst. Current dogma states that loss of epithelial polarity in the luminal epithelial cells, the point of first contact between maternal endometrium and blastocyst, may facilitate embryo implantation. Loss of polarity is likely an important change during the secretory phase to overcome mutual repulsion between otherwise polarized epithelial surfaces. Although "plasma membrane transformation" describes morphological/molecular alterations associated with loss of polarity, direct measures of polarity have not been investigated. Transepithelial resistance, a proxy measure of polarity, was downregulated in endometrial epithelial (ECC-1) cells by combined estrogen/progestin, mimicking the hormonal milieu of the secretory phase. Examination of defined polarity markers within human endometrium throughout the menstrual cycle identified downregulation of atypical protein kinase C, Stardust, Crumbs, and Scribble within the luminal-epithelial layer, with upregulation of Scribble within the stromal compartment as the menstrual cycle progressed from the estrogen-dominated proliferative to progesterone-dominated secretory phase. Epithelial (ECC-1) Scribble expression was downregulated in vitro by combined estrogen/progestin and estrogen/progestin/human chorionic gonadotropin treatment, whereas knockdown of Scribble in these cells enhanced "embryo" (trophectodermal spheroid) adhesion. In contrast, Scribble was upregulated within decidualized primary human endometrial stromal cells, with decidualization downregulated upon Scribble knockdown. These data highlight an important contribution of polarity modulation within the human endometrium, likely important for receptivity. Clinical investigations examining how polarity may be modulated in the infertile endometrium may facilitate fertility.

Abnormal uterine bleeding can severely affect the quality of life for women. After menstruation, the endometrium must adequately repair to limit and stop bleeding. Abnormal uterine bleeding may result from incorrect or inadequate endometrial repair after menstruation. Previous studies have shown an important contribution of activin to skin wound healing, with severely delayed wound repair observed in animals transgenically induced to overexpress activin’s natural inhibitor, follistatin. Activin subunits have also been identified within human endometrium; however, their role in endometrial repair is unknown. We assessed the contribution of activin to endometrial repair after menses using a human in vitro cell wounding method and our well-characterized mouse model of endometrial breakdown and repair applied to mice overexpressing follistatin. Endometrial repair after menses is initiated with reepithelialization of the uterine surface. To mimic this repair, we utilized a human endometrial epithelial cell line (ECC-1) and demonstrated significant stimulation of wound closure after activin A administration, and attenuation of this response by addition of follistatin. Immunolocalization of activin subunits, βA and βB, in control endometrium from the mouse model demonstrated specific epithelial and stromal localization and some leukocyte staining (βA) around sites of endometrial repair, suggestive of a role for activin in this process. Follistatin-overexpressing animals had significantly higher circulating follistatin levels than wild-type littermates. There was a significant delay in endometrial repair after breakdown in follistatin transgenic animals compared with control animals. This study demonstrates for the first time a functional role for activin in endometrial repair after menses.

The current dogma surrounding endometrial regeneration after menses includes a critical need for estrogen-primed proliferation. Although some evidence suggests that estrogen may not be required for the initial reepithelialization of the uterine surface, it is widely believed that it is essential for successful stromal renewal. This study aimed to identify proliferating cell types during endometrial repair and to examine whether estrogen is required for successful repair using a previously developed mouse model. In the model, decidualization is artificially induced, and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and by 48 h has usually undergone complete repair. Although the mice are ovariectomized, restoration of both the stromal and epithelial components proceeds rapidly after breakdown and results in what appears to be a normal endometrium. However, potential estrogenic influences from extraovarian sources (particularly the diet and fat) remain. In this study, complete removal of extraovarian estrogen was achieved by maintenance of animals on a soy-free diet and administration of aromatase inhibitor letrozole. No significant differences in uterine weight or estrogen-responsive genes lactoferrin and progesterone receptor were observed compared with control ovariectomized but otherwise untreated mice, whereas significantly higher measurements were obtained from an estrogen-added group. Importantly, no significant difference in the rate of endometrial repair was observed in the complete absence of estrogen, demonstrating that estrogen is not essential for complete endometrial restoration in this model.

CONTEXT: Irregular bleeding affects many users of combined menopausal hormone therapy (HT) and commonly leads to invasive and expensive investigations to exclude underlying malignancy. In most cases no abnormality is found. OBJECTIVE: The main objective of this study was to explore the role of uterine natural killer (uNK) cells and their regulatory cytokine IL-15 in irregular bleeding in HT users. DESIGN: This was a prospective observational study conducted between 2002 and 2004. SETTING: The study was conducted in a tertiary referral menopause clinic at King Edward Memorial Hospital, Western Australia. PATIENTS: Patients included 117 postmenopausal women taking combined HT. INTERVENTIONS: Outpatient endometrial biopsies were taken during and outside bleeding episodes. MAIN OUTCOME MEASURES: The relationship between endometrial uNK cells (CD56+) and bleeding patterns was measured. We also addressed the impact of HT exposure on uNK cell populations, the relationship between endometrial IL-15 expression and uNK cell populations, and killer Ig like receptor genotype in subjects with irregular bleeding. RESULTS: Endometrial CD56+ uNK cells were significantly increased in biopsies obtained during bleeding episodes (P < 0.001), compared with HT users with no bleeding. The highest level of IL-15 expression was also seen in biopsies taken during bleeding. No clear relationship between killer Ig like receptor genotype and bleeding on HT was observed. CONCLUSIONS: Little is known about the mechanisms underlying irregular bleeding in HT users. This is the first report of uNK cells and their association with regulating cytokines in postmenopausal endometrium and demonstrates a possible mechanism by which HT may induce irregular bleeding.

Macrophage inhibitory cytokine-1 (MIC-1) is a recently described divergent member of the transforming growth factor-β superfamily. MIC-1 transcription up-regulation is associated with macrophage activation, and this observation led to its cloning. Northern blots indicate that MIC-1 is also present in human placenta. A sensitive sandwich enzyme-linked immunosorbent assay for the quantification of MIC-1 was developed and used to examine the role of this cytokine in pregnancy. High levels of MIC-1 are present in the sera of pregnant women. The level rises substantially with progress of gestation. MIC-1 can also be detected, in large amounts, in amniotic fluid and placental extracts. In addition, the BeWo placental trophoblastic cell line was found to constitutively express the MIC-1 transcript and secrete large amounts of MIC-1. These findings suggest that the placental trophoblast is a major source of the MIC-1 present in maternal serum and amniotic fluid. We suggest that MIC-1 may promote fetal survival by suppressing the production of maternally derived proinflammatory cytokines within the uterus.

The 11β-hydroxysteroid dehydrogenase type II enzyme (11βHSD2) is a potent inactivator of glucocorticoids and is present in high amounts in the placental syncytiotrophoblast and sodium-transporting epithelia. Placental 11βHSD2 is thought to protect the fetus from high circulating levels of maternal glucocorticoids, whereas the renal enzyme is important in conferring aldosterone specificity on the mineralocorticoid receptor. An isoform of 11βHSD (11βHSD1) is also present in a wide range of tissues, but usually acts as an oxoreductase, converting the biologically inactive cortisone to cortisol. In the present study we have used an immunopurified antibody to the carboxy-terminus of human 11βHSD2 (HUH23) to demonstrate localization of the enzyme in luminal and glandular epithelia of human endometrium. In some specimens staining was uniformly distributed, but in others there was clear evidence of heterogeneity both between and within epithelia. Although 11βHSD2 was found mainly in the cytoplasm, some cells show...

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are implicated in normal menstruation, but the mechanism of their regulation is not yet clear. Human endometrial stromal cell cultures were established to mimic the events of the late luteal phase of the menstrual cycle: after 6 days of culture with estradiol 17beta (10 nmol/L) and progestin (P, 100 nmol/L), half the cells were subjected to P withdrawal, and medium was harvested on day 10. Decidualization of the cells was verified by PRL immunohistochemistry. Latent MMP-1, -2, -3, and -9 were detected by zymography and quantitated by densitometry, and production of all enzymes was increased on withdrawal of P. This increase was confirmed by enzyme-linked immunosorbent assay for MMP-1. TIMP-1, -2, and -3 also were produced by the cells, with a mean ratio of 3.9:1:1.2, respectively. There was no effect of P withdrawal on either the amount of each TIMP or their relative concentrations. Expression of the messenger RNA for TIMP-1 or TIMP-2 also was not changed by P withdrawal. Thus, withdrawal of P alters the ratio of MMPs to TIMPs in this model in favor of MMPs and, hence, of tissue degradation. However, the focal nature of menstruation-associated MMP activity suggests that P withdrawal is unlikely to be the only factor responsible for in vivo induction of MMPs at menstruation.

Immunization of ewes against the N-terminal peptide of inhibin alpha 43 (alpha N) reduces fertility; this is thought to be due to impaired oocyte release at ovulation. This study further investigates the effect of alpha N immunoneutralization on the ovulatory process. Light microscopy was used to examine the effects of alpha N immunization of the tissue-remodeling process during ovulation and formation of the corpus luteum (CL) structure. Changes in follicular levels of matrix metalloproteinase-2 (MMP-2) with approaching ovulation were also investigated in normal and alpha N-immunized ewes. Differences in structure of 2-day-old CL were observed between control and alpha N-immunized ewes. Control CL had confluent luteal tissue throughout the internal structure and invaginations of theca and vasculature were common and penetrated deep into the luteal tissue. Immunized ewe CL had large fluid-filled antra, giving them a cystic appearance; luteal tissue remained a thin 10- to 15-cell layer lining the wall surrounding the antrum. Infolding of the surrounding tissue was incomplete, and thecal/vascular invaginations were rare and failed to penetrate into the luteal tissue. Morphologically normal rupture stigma were seen at the apex of both control and alpha N-immunized CL. Gelatin-digesting activity in follicular fluid collected 0, 12, and 24 h after hCG administration in control ewes increased significantly as the time of ovulation approached (827 +/- 182, 842 +/- 159, and 1230 +/- 89 mU/ml, respectively, in Exp 1; 743 +/- 32, 1182 +/- 98, and 1306 +/- 91 mU/ml at the same times in Exp 2). alpha N immunization reduced follicular gelatinase activity at each time in Exp 1 (533 +/- 132, 740 +/- 67, and 809 +/- 147 mU/ml) and Exp 2 (587 +/- 21, 768 +/- 27, and 891 +/- 53 mU/ml); the reduction was significant at 24 h in Exp 1 and at all times in Exp 2. Gelatin zymography of follicular fluid revealed bands of gelatinase of 72/67 kilodaltons, consistent with latent and active MMP-2. The area digested by both latent and active MMP-2 increased with approaching time of ovulation and was reduced by alpha N immunization. These data suggest that MMP-2 has a role in the tissue-remodeling processes of ovulation and CL formation in the ewe and that immunization against alpha N, which impairs fertility, effects the preovulatory cascade of intrafollicular proteolytic activity, reducing MMP-2 levels and disrupting normal CL formation.

This study identified and characterized endothelin (ET) produced by human endometrial epithelial cells cultured under serum-free conditions, compared the ET released by cells derived from proliferative and secretory phase endometrium, and examined the regulation of ET released by these cells. ET messenger RNA was detected in normal human endometrium with maximal expression in the mid-late secretory phase. Immunoreactive ET released into culture media by separated endometrial epithelial and stromal cells was almost entirely of epithelial cell origin, consistent with the previous immunohistochemical findings. This was identified as ET-1 by reverse phase high-pressure liquid chromatography, and the fractionated conditioned media exhibited bioactivity similar to that of standard ET-1. Mean ET production was greater from cells derived from proliferative phase endometrium cultured either in serum (P0.02) or serum-free conditions (P0.02). Fetal calf serum stimulated ET-1 production from epithelial cells in a dose-responsive manner. ET production was also stimulated by transforming growth factor-beta 1 (2, 510 ng/mL) and IL-1 alpha (10100 IU/mL) under serum-free conditions but always to a lesser extent than stimulation by serum. The production of ET in human endometrium underlines a potential role for ET in endometrial function.

Matrix metalloproteinases (MMPs) together degrade virtually all the components of the extracellular matrix and are likely to play a role in remodeling of endometrial tissue during the normal menstrual cycle. Primary cultures of human endometrial stromal cells secreted a number of MMPs. MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) were measured in culture medium by specific enzyme assays. Production of the enzymes did not correlate with the time of the menstrual cycle at which the tissue was collected. Identities of MMP-1 and MMP-3 were confirmed by Western blots, by comparison of mol wt with those of purified enzymes on casein zymography, and by inhibition of these activities with EDTA and 1,10-phenanthroline. Northern analysis demonstrated specific messenger ribonucleic acid for pro-MMP-1 and pro-MMP-3 in phorbol myristate acetate-stimulated stromal cells. Two gelatinases were detected by gelatin zymography: MMP-2 (gelatinase-A) was present in two forms (72 and 67 kilodaltons), and MMP-9 (gelatinase-B) was present as a homodimer with a mol wt of approximately 180 kilodaltons. MMP-9, but not MMP-2, secretion was stimulated by phorbol myristate acetate. All enzymes could be activated in vitro by (4-aminophenyl)mercuric acetate. Both interleukin-1 alpha and tumor necrosis factor-alpha stimulated the secretion of MMP-1, MMP-3, and MMP-9, but not MMP-2, from the cells in a concentration-dependent manner. MMP production by endometrial stromal cells has a potentially important role in the processes of menstruation and implantation.

A heterologous radioimmunoassay system, using an antiserum to human FSH in combination with radioiodinated ovine FSH was highly specific for FSH for a number of species, and was used to measure plasma FSH levels in sheep, rat and man in a range of physiological states. Within-assay coefficient of variation (CV) was