Does human endometrial LGR5 gene expression suggest the existence of another hormonally regulated epithelial stem cell niche?

STUDY QUESTION: Is human endometrial leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) gene expression limited to the postulated epithelial stem cell niche, stratum basalis glands, and is it hormonally regulated? SUMMARY ANSWER: LGR5 expressing cells are not limited to the postulated stem cell niche but LGR5 expression is hormonally regulated. WHAT IS KNOWN ALREADY: The human endometrium is a highly regenerative tissue; however, endometrial epithelial stem cell markers are yet to be confirmed. LGR5 is a marker of stem cells in various epithelia. STUDY DESIGN, SIZE, DURATION: The study was conducted at a University Research Institute. Endometrial samples from 50 healthy women undergoing benign gynaecological surgery with no endometrial pathology at the Liverpool Women's hospital were included and analysed in the following six sub-categories; proliferative, secretory phases of menstrual cycle, postmenopausal, those using oral and local progestagens and samples for in vitro explant culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we used the gold standard method, in situ hybridisation (ISH) along with qPCR and a systems biology approach to study the location of LGR5 gene expression in full thickness human endometrium and Fallopian tubes. The progesterone regulation of endometrial LGR5 was examined in vivo and in short-term cultured endometrial tissue explants in vitro. LGR5 expression was correlated with epithelial proliferation (Ki67), and expression of previously reported epithelia progenitor markers (SOX9 and SSEA-1) immunohistochemistry (IHC). MAIN RESULTS AND THE ROLE OF CHANCE: LGR5 gene expression was significantly higher in the endometrial luminal epithelium than in all other epithelial compartments in the healthy human endometrium, including the endometrial stratum basalis (P < 0.05). The strongest SSEA-1 and SOX9 staining was observed in the stratum basalis glands, but the general trend of SOX9 and SSEA-1 expression followed the same cyclical pattern of expression as LGR5. Stratum functionalis epithelial Ki67-LI and LGR5 expression levels correlated significantly (r = 0.74, P = 0.01), however, they did not correlate in luminal and stratum basalis epithelium (r = 0.5 and 0.13, respectively). Endometrial LGR5 demonstrates a dynamic spatiotemporal expression pattern, suggesting hormonal regulation. Oral and local progestogens significantly reduced endometrial LGR5 mRNA levels compared with women not on hormonal treatment (P < 0.01). Our data were in agreement with in silico analysis of published endometrial microarrays. LARGE SCALE DATA: We did not generate our own large scale data but interrogated publically available large scale data sets. LIMITATIONS, REASONS FOR CAUTION: In the absence of reliable antibodies for human LGR5 protein and validated lineage markers for the various epithelial populations that potentially exist within the endometrium, our study does not formally characterise or examine the functional ability of the resident LGR5+ cells as multipotent. WIDER IMPLICATIONS OF THE FINDINGS: These data will facilitate future lineage tracing studies in the human endometrial epithelium; to identify the location of stem cells and further complement the in vitro functional studies, to confirm if the LGR5 expressing epithelial cells indeed represent the epithelial stem cell population. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Wellbeing of Women project grant (RTF510) and Cancer Research UK (A14895). None of the authors have any conflicts of interest to disclose.

Human endometrial epithelial telomerase is important for epithelial proliferation and glandular formation with potential implications in endometriosis.

STUDY QUESTION: How does regulation of telomerase activity (TA) in human endometrial epithelial cells (EEC) by ovarian hormones impact on telomere lengths (TL) and cell proliferation? SUMMARY ANSWER: Healthy endometrial epithelial cell proliferation is characterized by high TA and endometrial TL changes according to the ovarian hormone cycle, with shortest TL observed in the progesterone dominant mid-secretory phase, when TA is lowest, implicating progesterone in the negative regulation of TA and TL. WHAT IS KNOWN ALREADY: Critical shortening of telomeres may result in permanent cell cycle arrest while the enzyme telomerase maintains telomere length (TL) and replicative capacity of cells. Telomerase expression and activity change in the human endometrium with the ovarian hormone cycle, however the effect of this on endometrial TL and cell growth is not known. STUDY DESIGN, SIZE, DURATION: A prospective observational study, which included endometrial and blood samples collected from 196 women. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied endometrial samples from five different groups of women. Endometrial and matched blood TL and circulating steroid hormones were studied in samples collected from 85 women (Group 1). Fresh epithelial and stromal cell isolation and culture in vitro for TL and TA was done on endometrial biopsies collected from a further 74 healthy women not on hormonal therapy (Group 2) and from 5 women on medroxyprogesterone acetate (MPA) for contraception (Group 3). The epithelial TL and telomerase protein expression was examined in active, peritoneal, ectopic endometriotic and matched uterine (eutopic) endometrial samples collected from 10 women with endometriosis (Group 4); the in vivo effect of mifepristone on telomerase protein expression by immunohistochemistry (IHC) was examined in endometrium from 22 healthy women in mid-secretory phase before (n = 8), and after administering 200 mg mifepristone (n = 14) (Group 5). TA was measured by telomere repeat amplification protocol (TRAP) assay; TL by qPCR, and Q-FISH; cell proliferation was assessed by immunoblotting of histone H3 and 3D-culture to assess the ability of EECs to form spheroids; telomerase reverse transcriptase protein levels and Ki-67 (proliferative index) were assessed with IHC. MAIN RESULTS AND THE ROLE OF CHANCE: Endometrial TLs correlated negatively with serum progesterone levels (n = 58, r = -0.54) and were significantly longer than corresponding blood TLs (4893 ± 929 bp versus 3955 ± 557 bp, P = 0.002) suggesting a tissue-specific regulation. High TA and short TLs were observed in proliferating EECs in vivo and in vitro. During the progesterone dominant mid-secretory phase endometrial TL were significantly shorter compared with the proliferative phase (P = 0.0002). Progestagen treatment suppressed EEC TA in vivo and reduced endometrial TA in explant (P = 0.01) and in vitro cultures (P = 0.02) compared with untreated cells. Mifepristone (progesterone receptor antagonist) increased telomerase protein levels in vivo (P < 0.05). In 2D culture, Imetelstat inhibited EEC TA (P = 0.03), proliferation (P = 0.009) and in 3D culture disrupted endometrial glandular architecture (P = 0.03). LIMITATIONS, REASONS FOR CAUTION: The in vitro telomerase inhibition data were tested in a mono-cellular system for a short-term. Further confirmation of the results in an in vivo model is necessary. The women in group 2 included a high proportion of women although with a regular menstrual cycle, with an increased BMI (>25) therefore this may affect extrapolation of data to other groups. WIDER IMPLICATIONS OF THE FINDINGS: The observed effects of telomerase inhibition in vitro on epithelial cell proliferation, suggest that telomerase might be an attractive target in developing new therapies for proliferative disorders of the endometrium, such as endometriosis.

A re-evaluation of the role of rotational forceps: retrospective comparison of maternal and perinatal outcomes following different methods of birth for malposition in the second stage of labour.

OBJECTIVE: To compare the outcomes of operative cephalic births by Kielland forceps (KF), rotational ventouse (RV), or primary emergency caesarean section (pEMCS) for malposition in the second stage of labour in modern practise. DESIGN: Retrospective observational study. POPULATION: Data were included from 1291 consecutive full-term, singleton cephalic births between 2 November 2006 and 30 November 2010 with malposition of the fetal head during the second stage of labour leading to an attempt to deliver by KF, RV or pEMCS. METHODS: Maternal and neonatal outcomes of all KF births were compared with other methods of operative birth for malposition in the second stage of labour (RV or pEMCS). MAIN OUTCOME MEASURES: Achieving a vaginal birth was the primary outcome and fetal (admission to special care baby unit, low cord pH, low Apgar, shoulder dystocia, Erb's palsy) and maternal (massive obstetric haemorrhage-blood loss of >1500 ml, sphincter injury, length of stay in hospital) safety outcomes were also recorded. RESULTS: Women were more likely to need caesarean section if RV (22.4%) was selected to assist the birth rather than KF (3.7%; adjusted odds ratio 8.20; 95% confidence interval 4.54-14.79). Births by KF had a rate of adverse maternal and neonatal outcomes comparable to those by RV and pEMCS in the second stage for malposition. CONCLUSIONS: Our results suggest that, in experienced hands, assisted vaginal birth by KF is likely to be the most effective and safe method to prevent the ever rising rate of caesarean sections when malposition complicates the second stage of labour.

SSEA-1 isolates human endometrial basal glandular epithelial cells: phenotypic and functional characterization and implications in the pathogenesis of endometriosis.

STUDY QUESTION: Can the basal epithelial compartment of the human endometrium be defined by specific markers? SUMMARY ANSWER: Human endometrial epithelial cells from the basalis express nuclear SOX9 and the cell-surface marker SSEA-1, with some cells expressing nuclear ß-catenin. In vitro, primary endometrial epithelial cells enriched for SSEA-1+ show some features expected of the basalis epithelium. WHAT IS KNOWN ALREADY: The endometrial glands of the functionalis regenerate from the basalis gland stumps following menstruation. Endometriosis is thought to originate from abnormal dislocation of the basalis endometrium. In the highly regenerative intestinal epithelium, SOX9 and nuclear ß-catenin are more highly expressed in the intestinal crypt, the stem/progenitor cell region. STUDY DESIGN, SIZE, DURATION: A large prospective observational study analysing full-thickness human endometrial hysterectomy samples from 115 premenopausal women, 15 post-menopausal women and ectopic endometriotic lesions from 20 women with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Full-thickness endometrium from hysterectomy tissues was analysed by immunohistochemistry for SSEA-1, SOX9 and ß-catenin. Primary human endometrial epithelial cells from short-term cultures were sorted into SSEA1+/- fractions with a cell sorter or magnetic beads and analysed for markers of differentiation and pluripotency and telomere lengths (TLs) using qPCR, telomerase activity [telomere repeat amplification protocol (TRAP)] and growth in 3D culture. MAIN RESULTS AND THE ROLE OF CHANCE: Similar to the intestinal crypt epithelium, human endometrial basal glandular epithelial cells expressed nuclear SOX9 and contained a rare subpopulation of cells with nuclear ß-catenin suggestive of an activated Wnt pathway. The embryonic stem cell-surface marker, SSEA-1, also marked the human endometrial basal glandular epithelial cells, and isolated SSEA-1(+) epithelial cells grown in monolayer showed significantly higher expression of telomerase activity, longer mean TLs, lower expression of genes for steroid receptors and produced a significantly higher number of endometrial gland-like spheroids in 3D culture compared with SSEA-1(-) epithelial cells (P = 0.009). Cells in ectopic endometriosis lesions also expressed SSEA-1 and nuclear SOX9, suggesting that the basalis contributes to ectopic lesion formation in endometriosis following retrograde menstruation. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study with only short-term culture of the primary human epithelial cells in vitro. WIDER IMPLICATIONS OF THE FINDINGS: The surface marker SSEA1 enriches for an endometrial epithelial cell subpopulation from the basalis. Since the functional endometrium originates from these cells, it is now possible to study basalis epithelium for stem/progenitor cell activity to extend our current understanding of endometrial biology in health and diseases. STUDY FUNDING/COMPETING INTEREST(S): The work included in this manuscript was funded by Wellbeing of Women project grant RG1073 (D.K.H. and C.G.). We also acknowledge the support by National Health and Medical Research Council, RD Wright Career Development Award 465121 and Senior Research Fellowship 1042298, and the Victorian Government's Operation Infrastructure Support Program to C.G. and MRC G0601333 to T.V.Z. All authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.