Nicola Tempest

Theories on the Pathogenesis of Endometriosis

Endometriosis is a common, chronic inflammatory disease defined by the presence of extrauterine endometrial tissue. The aetiology of endometriosis is complex and multifactorial, where several not fully confirmed theories describe its pathogenesis. This review examines existing theories on the initiation and propagation of different types of endometriotic lesions, as well as critically appraises the myriad of biologically relevant evidence that support or oppose each of the proposed theories. The current literature suggests that stem cells, dysfunctional immune response, genetic predisposition, and aberrant peritoneal environment may all be involved in the establishment and propagation of endometriotic lesions. An orchestrated scientific and clinical effort is needed to consider all factors involved in the pathogenesis of this multifaceted disease and to propose novel therapeutic targets to reach effective treatments for this distressing condition.

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.

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.

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 β-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 β-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 β-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 β-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 Governments 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.

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.

Hormones and endometrial carcinogenesis.

Abstract Endometrial cancer (EC) is the commonest gynaecological cancer in the Western World with an alarmingly increasing incidence related to longevity and obesity. Ovarian hormones regulate normal human endometrial cell proliferation, regeneration and function therefore are implicated in endometrial carcinogenesis directly or via influencing other hormones and metabolic pathways. Although the role of unopposed oestrogen in the pathogenesis of EC has received considerable attention, the emerging role of other hormones in this process, such as androgens and gonadotropin-releasing hormones (GnRH) is less well recognised. This review aims to consolidate the current knowledge of the involvement of the three main endogenous ovarian hormones (oestrogens, progesterone and androgens) as well as the other hormones in endometrial carcinogenesis, to identify important avenues for future research.

Does advanced operative obstetrics still have a place in contemporary practice

Purpose of review This article reviews recent significant contributions to the literature concerning advanced operative obstetric procedures used for rotational vaginal deliveries and their alternative, primary caesarean section. Recent findings Rising caesarean section rates are a global concern. Caesarean section in the second stage of labour is associated with high rates of maternal and fetal morbidity. Rotational vaginal deliveries may reduce the caesarean section rate without additional adverse effects on maternal and fetal outcomes. A recent national trainees’ survey highlighted that training in the management of operative birth in the second stage of labour, especially when there is malposition of the fetal head, is a priority. Summary There is a need for evidence-based guidelines, including standardized documentation of these advanced procedures. Training strategies for junior practitioners to acquire these skills and for experienced practitioners to maintain and disseminate their skills should be prioritized. The safety of rotational delivery methods versus primary caesarean section is likely to prove difficult to assess directly, in the context of a randomized controlled trial, but may be approximated via a national prospective audit.

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

Abstract 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.

Neonatal and maternal outcomes of successful manual rotation to correct malposition of the fetal head; A retrospective and prospective observational study

Objective To evaluate the neonatal and maternal outcomes associated with successful operative vaginal births assisted by manual rotation. Design Prospective and retrospective observational study. Setting Delivery suite in a tertiary referral teaching hospital in England. Population A cohort of 2,426 consecutive operative births, in the second stage of labour, complicated with malposition of the fetal head during 2006–2013. Methods Outcomes of all births successfully assisted by manual rotation followed by direct traction instruments were compared with other methods of operative birth for fetal malposition in the second stage of labour (rotational ventouse, Kielland forceps and caesarean section). Main outcome measures Associated neonatal outcomes (admission to the special care baby unit, low cord pH, low Apgar and shoulder dystocia) and maternal outcomes (massive obstetric haemorrhage (blood loss of >1500ml) and obstetric anal sphincter injury). Results Births successfully assisted with manual rotation followed by direct traction instruments, resulted in 10% (36/346) of the babies being admitted to the Special Care Baby Unit, 4.9% (17/349) shoulder dystocia, 2% (7/349) massive obstetric haemorrhage and 1.7% (6/349) obstetric anal sphincter injury, similar to other methods of rotational births. Conclusions Adverse neonatal and maternal outcomes associated with successful manual rotations followed by direct traction instruments were comparable to traditional methods of operative births. There is an urgent need to standardise the practice (guidance, training) and documentation of manual rotation followed by direct traction instrumental deliveries that will enable assessment of its efficacy and the absolute safety in achieving a vaginal birth.

Authors' response to: The role of rotational forceps

Sir, I read with interest the article by Tempest et al. on a re-evaluation of the role of rotational forceps. Their elegant study clearly shows that assisted vaginal birth by Kielland forceps is an effective and safe means to achieving a vaginal delivery when malposition complicates the second stage of labour, if undertaken by experienced hands. Aye, there’s the rub. Experienced hands. Experienced hands are an increasingly rare commodity. I reviewed the maternity theatre register of a district general hospital where I once worked in 1993: middle-grade cover was provided by two doctors in training and one non-career post. In the space of 1 year, the two doctors in training each performed approximately 160 caesarean sections. I reviewed the maternity theatre register of the same hospital in 2011. The delivery numbers and caesarean section rate were broadly unchanged. But the staffing level was vastly different. In order to comply with the European Working Time Directive (EWTD), middle-grade cover was provided by seven doctors in training. In addition, some resident cover was also provided by consultants. In 2011, on average, trainees were performing 50 caesarean sections per annum. This demonstrates the massive reduction in practical experience that doctors in training gain, which has come with no corresponding increase in the time spent in training. Because of this reduction in practical experience and a lack of experienced hands, I believe it is unlikely that we will witness a return to the increased use of rotational forceps – at least in the foreseeable future. This study was run between 2006 and 2010. The full impact of the EWTD only came into force in 2009. There is well-documented evidence on the negative effect of the EWTD on the practical skills gained by doctors in training as well as the intensity with which practical skills are learned. And as a result, I feel trainees are less likely to opt for using an instrument that they may feel inadequately trained to use. And because of the perceived risks (real or otherwise) associated with the use of rotational forceps, local guidelines may well be designed to discourage their use. It is unfortunate that a safe and effective alternative to caesarean section for malposition complicating the second stage of labour is unlikely to be widely adopted because of a lack of experienced hands to teach the technique. Caesarean section in the second stage of labour is fraught with difficulties and dangers. It may be that the driving force for the re-introduction of Kielland forceps will be when we fully recognise the harm caused by caesarean sections performed at full cervical dilatation for malposition. Particularly so if these caesarean sections are undertaken by obstetricians who have had significantly less experience than trainees once had.&

Endometrial Stem Cell Markers: Current Concepts and Unresolved Questions

The human endometrium is a highly regenerative organ undergoing over 400 cycles of shedding and regeneration over a woman’s lifetime. Menstrual shedding and the subsequent repair of the functional layer of the endometrium is a process unique to humans and higher-order primates. This massive regenerative capacity is thought to have a stem cell basis, with human endometrial stromal stem cells having already been extensively studied. Studies on endometrial epithelial stem cells are sparse, and the current belief is that the endometrial epithelial stem cells reside in the terminal ends of the basalis glands at the endometrial/myometrial interface. Since almost all endometrial pathologies are thought to originate from aberrations in stem cells that regularly regenerate the functionalis layer, expansion of our current understanding of stem cells is necessary in order for curative treatment strategies to be developed. This review critically appraises the postulated markers in order to identify endometrial stem cells. It also examines the current evidence supporting the existence of epithelial stem cells in the human endometrium that are likely to be involved both in glandular regeneration and in the pathogenesis of endometrial proliferative diseases such as endometriosis and endometrial cancer.