MicroRNA Biogenesis Machinery Is Dysregulated in the Endometrium of Infertile Women Suggesting a Role in Receptivity and Infertility

MicroRNAs (miRs) regulate endometrial function and their dysregulation could underlie unexplained infertility in women. Ribonucleases including DICER and DROSHA, and the proteins, ARGONAUTE 1 (AGO 1) and 2 (AGO 2) regulate the biogenesis/maturation of miRs. We aimed to elucidate the expression and localization of miR biogenesis machinery components during the human menstrual cycle and compare their levels in endometrium from women with normal fertility and primary unexplained infertility. miR biogenesis components were measured by quantitative-RT-PCR and immunohistochemistry. In the endometrium of women with normal fertility, DROSHA immunolocalized maximally to the epithelium during the early and mid-secretory phases compared with the proliferative and late-secretory phases. Stromal DICER immunostaining intensity was higher in the late-secretory phase compared with all other phases in fertile women. DROSHA mRNA was reduced in the mid-secretory-infertile whole endometrial tissue (has all cells of the tissue), and primary epithelial and stromal cells while no differences were found in DICER, AGO1, and AGO2 mRNA. In the luminal epithelium, DROSHA staining intensity was reduced in early and mid-secretory-infertile while DICER staining was reduced in the early secretory-infertile compared with their respective fertile groups. DICER and DROSHA were dynamically regulated across the menstrual cycle and reduced levels during receptivity phase could underlie implantation failure/infertility.

The influence of migraine and female hormones on capsaicin-induced dermal blood flow

Background Migraine is much more common in females than in males, and occurrence is associated with changes in female sex hormones. Calcitonin gene-related peptide (CGRP) plays a key role in migraine, and variations in female sex hormones may affect CGRP sensitivity and/or production. Objectives Investigate repeatability, gender differences, influence of the menstrual cycle and of migraine on CGRP-dependent changes in dermal blood flow (DBF). Methods CGRP-dependent increases in DBF were assessed using laser Doppler perfusion imaging after topical application of 300 or 1000 µg capsaicin on the forearm of healthy subjects and migraine patients. Results In healthy males, DBF response did not vary over time and was comparable with DBF in male migraineurs. In healthy females, capsaicin-induced DBF responses to both doses of capsaicin were higher during menstruation compared to the late-secretory phase (p < 0.05); this menstrual cycle dependence was absent in female migraine patients. Compared to healthy subjects, female migraineurs displayed a higher DBF response both during menstruation and during the late-secretory phase (p < 0.05). Conclusions An increased capsaicin-induced, CGRP-mediated DBF response was observed during menstruation in healthy women, but in female migraine patients this increased response was not affected by the menstrual cycle.

Nuclear factor κB pathway and interleukin-6 are affected in eutopic endometrium of women with endometriosis

In order to investigate the role of the nuclear factor κB (NFKB) pathway on gene expression in the eutopic endometrium in endometriosis, and in particular of interleukin-6 (IL6), we evaluated RELA, IκB kinase (CHUK), NFKBIA and IL6 expressions and NFKB DNA binding in eutopic endometrium from women with endometriosis. Eutopic endometrium was obtained from 37 women with endometriosis and 42 fertile women during laparoscopy. We analysed RELA, CHUK, NFKBIA and IL6 mRNA levels (RT-PCR); RELA, CHUK and NFKBIA proteins and p-NFKBIA/NFKBIA ratio (western blot); and NFKB binding (DNA shift assay) and IL6 concentration (ELISA) in endometrial explants. Our results indicate that mRNA and cytoplasmic proteins of RELA and CHUK exhibit constant levels in normal endometrium during the menstrual cycle. A dramatic increase (P<0.05) in NFKBIA mRNA expression, RELA nuclear presence and the mRNA and the protein of IL6 during late secretory phase was also observed in this tissue. By contrast, in eutopic endometrium from endometriosis patients, a decrease (P<0.05) in IL6 mRNA and protein (61%), NFKBIA mRNA (46%), p-NFKBIA/NFKBIA ratio (42%), RELA nuclear stromal (68%) and CHUK (48%) proteins were found exclusively during the late secretory phase compared with normal endometrium. In conclusion, the canonical activation of NFKB pathway is deregulated and may have reduced transcriptional function affecting NFKBIA and IL6 expression, genes related local proinflammatory processes. These molecular alterations observed during the late secretory phase in eutopic endometrium from endometriosis patients constitute a NFKB system dysfunction, suggesting that NFKB could be an important factor in endometriosis aetiology.

Opposite Regulation of Transforming Growth Factors-β2 and -β3 Expression in the Human Endometrium

TGF-βs 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-β system in this tissue, we investigated by real-time RT-PCR and ELISA the expression of the three TGF-βs (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-β isoforms expression, both in vivo and in explant culture. In vivo, TGF-β2 increased by about 5-fold at the mid-late secretory phase then declined after menstruation; TGF-β3 increased at menstruation and remained high during the proliferative phase; TGF-β1 was maximal at menstruation. In explants cultured without ovarian steroids both TGF-β2 and -β3 were preferentially expressed in the stroma. Ovarian steroids strongly repressed both TGF-β2 and -β3 in stroma but only TGF-β2 in glands. cAMP prevented inhibition by ovarian steroids of TGF-β2 but not -β3. In presence of ovarian steroids, MAPK inhibitors (p38 and ERK pathways) stimulated TGF-β3 but inhibited TGF-β2 expression. In conclusion, TGF-β2 and -β3 are differentially expressed during the menstrual cycle and regulated by progesterone in epithelial vs stromal cells. The opposite regulation of TGF-β2 and -β3 by cAMP and MAPK could account for their distinct expression in vivo.

241. Changes in the expression of Annexin IV mRNA and protein in human endometrium

In a previous study investigating global gene expression throughout the menstrual cycle,1 Annexin 4 (ANXIV) was identified as having significant cyclic changes in human endometrium. ANXIV belongs to a ubiquitous family of Ca2+-dependent phospholipid and membrane-binding proteins. The aims of this study were to investigate the cellular localization and regulation of ANXIV mRNA and temporal expression of ANXIV protein in human endometrium during the menstrual cycle. mRNA Expression: The menstrual cycle was divided into seven stages by histological evaluation. Curettings of endometrium were collected from 60 cycling women. For cellular localization, tissues from eight endometrial curettings were dissociated with collagenase into single cells, separated into epithelial and stromal cell fractions and snap frozen. Total RNA was extracted and ANXIV mRNA was quantified by real-time PCR. Immunohistochemistry: Full thickness endometrial tissue was collected from 50 reproductive age women undergoing hysterectomy. Tissue sections were formalin-fixed and paraffin-embedded. Goat polyclonal ANXIV antibody was used to localize ANXIV protein. ANXIV mRNA was significantly upregulated in the whole tissue during mid-late secretory phase of the cycle, and was predominantly expressed in epithelial cells. ANXIV protein was detected in the luminal and glandular epithelium in high levels throughout the menstrual cycle except in early secretory (ES) phase. The intensity of immunostaining was stronger in the glands of the basalis compared to functionalis in early proliferative phase, however, by the late secretory phase the functionalis glands showed higher expression levels. ANXIV mRNA data are consistent with a role for progesterone in upregulating the expression of ANXIV, although protein levels remain high through menstruation and into the proliferative phase. ANXIV can indirectly inhibit prostaglandin production, which is important for implantation. Hence the low levels of ANXIV protein at ES phase may relate to processes involved in implantation. (1)Ponnampalam et al. (2004). Mol. Hum. Reprod. 10(12), 879–893.

Polo-like kinase expression in normal human endometrium during the menstrual cycle

The enzyme, polo-like kinase (PLK), is a mammalian serine/threonine kinase involved in cell cycle regulation. A great deal of evidence regarding the role of PLK in the cell cycle has been obtained through studies of cultured cells, though little is known about its function or even expression in vivo. The endometrium undergoes rapid proliferation and differentiation under ovarian steroid hormone control during the 28-day cycle. Thus, normal endometrium provides an excellent model in which to study the hormone dependency of PLK expression. In the present study, we examined the features of PLK expression in 20 samples of normal human endometrium during the menstrual cycle. The expression of Ki-67 and proliferating cell nuclear antigen (PCNA) were also examined as markers of proliferation. Immunohistochemical studies showed that PLK staining was detected in the basement membrane of many endometrial glands, stromal cells, and some endothelial cells. The number of PLK-positive endometrial gland cells was significantly higher in the late proliferative phase (19.16% 4.98%) and the early secretory phase (19.28% 4.99%) than in the early proliferative phase (2.60% 2.33%) or the late secretory phase (5.76% 2.16%) (P<0.0001). PLK expression seemed to be correlated with the expression of Ki-67 and PCNA in many endometrial glands and stromal cells particularly in the late proliferative phase, reflecting a role of PLK in cellular proliferation. Nevertheless, in the early secretory phase, at which point the expression of Ki-67 and PCNA decreased in endometrial glands, PLK was strongly expressed. This finding suggests that PLK may have some post-mitotic functions in certain specialized cell types. Although the highest expression of PLK was observed in the late proliferative and the early secretory phases, the expression drastically decreased in the late secretory phase. These findings, taken together, indicate that the expression of PLK in normal endometrium fluctuates over the course of the menstrual cycle, suggesting in turn that PLK is associated with hormone-dependent cellular proliferation and that hormone functions may be involved in its regulation.