Diagnostic utility of clinicodemographic, biochemical and metabolite variables to identify viable pregnancies in a symptomatic cohort during early gestation.

A significant number of pregnancies are lost in the first trimester and 1-2% are ectopic pregnancies (EPs). Early pregnancy loss in general can cause significant morbidity with bleeding or infection, while EPs are the leading cause of maternal mortality in the first trimester. Symptoms of pregnancy loss and EP are very similar (including pain and bleeding); however, these symptoms are also common in live normally sited pregnancies (LNSP). To date, no biomarkers have been identified to differentiate LNSP from pregnancies that will not progress beyond early gestation (non-viable or EPs), defined together as combined adverse outcomes (CAO). In this study, we present a novel machine learning pipeline to create prediction models that identify a composite biomarker to differentiate LNSP from CAO in symptomatic women. This prospective cohort study included 370 participants. A single blood sample was prospectively collected from participants on first emergency presentation prior to final clinical diagnosis of pregnancy outcome: LNSP, miscarriage, pregnancy of unknown location (PUL) or tubal EP (tEP). Miscarriage, PUL and tEP were grouped together into a CAO group. Human chorionic gonadotrophin ß (ß-hCG) and progesterone concentrations were measured in plasma. Serum samples were subjected to untargeted metabolomic profiling. The cohort was randomly split into train and validation data sets, with the train data set subjected to variable selection. Nine metabolite signals were identified as key discriminators of LNSP versus CAO. Random forest models were constructed using stable metabolite signals alone, or in combination with plasma hormone concentrations and demographic data. When comparing LNSP with CAO, a model with stable metabolite signals only demonstrated a modest predictive accuracy (0.68), which was comparable to a model of ß-hCG and progesterone (0.71). The best model for LNSP prediction comprised stable metabolite signals and hormone concentrations (accuracy = 0.79). In conclusion, serum metabolite levels and biochemical markers from a single blood sample possess modest predictive utility in differentiating LNSP from CAO pregnancies upon first presentation, which is improved by variable selection and combination using machine learning. A diagnostic test to confirm LNSP and thus exclude pregnancies affecting maternal morbidity and potentially life-threatening outcomes would be invaluable in emergency situations.

Habitual physical activity levels in women attending the one stop infertility clinic: A prospective cross-sectional observational study.

Optimisation of lifestyle factors such as smoking and alcohol are encouraged to improve fecundability rates in the fertility setting. Currently, routine fertility consultations do not involve counselling or imparting advice regarding habitual physical activity (PA) and/or structured exercise, despite data showing that vigorous PA can be associated with delayed time to pregnancy. Therefore, this study aimed to determine habitual PA in a sample of women attending the one stop infertility (OSI) clinic. 250 women attending a large tertiary level NHS fertility unit prospectively anonymously completed a questionnaire over a period of 9 months. Participant's (mean age 34±5years, mean BMI 29±7kg/m2) habitual PA levels varied from vigorous exercise on ≥5 days/week (8%, n=17), to no moderate or high intensity activities across the whole week (66%, n=29). The majority of women reported no structured exercise (72%, n=179). No association was identified between any domain of PA and BMI, age, alcohol units, regular periods, or time spent trying to conceive (P > 0.05). Participant's habitual PA levels varied widely and no association between any domain of PA and background of the women was identified. No existing evidence and/or guidelines to explicitly inform women attempting to conceive regarding recommended PA levels are available, despite PA being a modifiable, affordable, and feasible lifestyle choice with the possible potential to improve fertility. A large-scale, clinical trial assessing effects of PA on fecundability is warranted to gain insights into the potential of this lifestyle factor to improve fertility outcomes and to explore the underlying biological mechanisms involved.

Altered endometrial oestrogen-responsiveness and recurrent reproductive failure.

Abstract: Recurrent reproductive failure (RRF) encompasses recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL). These highly prevalent, distressing conditions have many unanswered questions regarding aetiology and management. Oestrogen receptor beta (ERß) is the predominant oestrogen receptor expressed in the vascular endothelium of the endometrium during the window of implantation (WOI). The establishment of normal endometrial receptivity is integrally associated with progesterone receptor (PR). Therefore, we aimed to investigate whether women with RRF have clinical, type-specific endometrial aberrations of ERß, PR and Ki-67 expression during the WOI. Thirty-eight endometrial biopsies were collected; 29 RRF (10 RIF, 9 recurrent loss of early pregnancy (RLEP) and 10 recurrent fetal loss (RFL)) and 9 fertile controls (FC). Within RIF, RLEP and RFL groups, the perivascular compartment showed significantly lower levels of ERß vs FC (P = 0.02, P = 0.03 and P = 0.01, respectively). Vascular endothelium also displayed significantly lower levels of ERß within RIF and RFL cohorts vs FC (P = 0.03 and P = 0.003). The expression of Ki-67 was significantly lower within vascular endothelium of all RRF; RIF (P = 0.02), RLEP (P = 0.02) and RFL (P <0.01). PR was significantly reduced (P <0.001) in the perivascular area of women with RIF. These findings provide novel insights into biological correlates of clinical subtypes of RRF. The endometrium of women with RRF display significantly altered levels of ERß, PR and Ki-67 during the WOI, furthering our understanding of the defective endometrial phenotype of women suffering from RRF, with possible impaired glandular function, angiogenesis and decidualisation. Lay summary: Recurrent reproductive failure (RRF) refers to a group of devastating conditions with many unanswered questions regarding their causes and treatment options. The lining of the womb, the endometrium, is primed and suitable for successful embryo implantation for a short time during the menstrual cycle; the window of implantation (WOI). Oestrogen is a key hormone that plays an important role in regulating the endometrium and its effects are exerted via two oestrogen receptor subtypes. Oestrogen receptor beta (ERß) is the main oestrogen receptor present during the WOI. Progesterone receptor allows the other main hormone, progesterone, to influence the endometrial activity and Ki-67 reflects the proliferative activity of the cells within the endometrium. We investigated the expression of these markers in endometrial samples collected from women with RRF and proven fertility. We found that the endometrium of women with RRF has significantly lower levels of ERß and Ki-67 during the WOI, possibly leading to unsuccessful pregnancies.

Novel microarchitecture of human endometrial glands: implications in endometrial regeneration and pathologies.

BACKGROUND: Human endometrium remains a poorly understood tissue of the female reproductive tract. The superficial endometrial functionalis, the site of embryo implantation, is repeatedly shed with menstruation, and the stem cell-rich deeper basalis is postulated to be responsible for the regeneration of the functionalis. Two recent manuscripts have demonstrated the 3D architecture of endometrial glands. These manuscripts have challenged and replaced the prevailing concept that these glands end in blind pouches in the basalis layer that contain stem cells in crypts, as in the intestinal mucosa, providing a new paradigm for endometrial glandular anatomy. This necessitates re-evaluation of the available evidence on human endometrial regeneration in both health and disease in the context of this previously unknown endometrial glandular arrangement. OBJECTIVE AND RATIONALE: The aim of this review is to determine if the recently discovered glandular arrangement provides plausible explanations for previously unanswered questions related to human endometrial biology. Specifically, it will focus on re-appraising the theories related to endometrial regeneration, location of stem/progenitor cells and endometrial pathologies in the context of this recently unravelled endometrial glandular organization. SEARCH METHODS: An extensive literature search was conducted from inception to April 2021 using multiple databases, including PubMed/Web of Science/EMBASE/Scopus, to select studies using keywords applied to endometrial glandular anatomy and regeneration, and the references included in selected publications were also screened. All relevant publications were included. OUTCOMES: The human endometrial glands have a unique and complex architecture; branched basalis glands proceed in a horizontal course adjacent to the myometrium, as opposed to the non-branching, vertically coiled functionalis glands, which run parallel to each other as is observed in intestinal crypts. This complex network of mycelium-like, interconnected basalis glands is demonstrated to contain endometrial epithelial stem cells giving rise to single, non-branching functionalis glands. Several previous studies that have tried to confirm the existence of epithelial stem cells have used methodologies that prevent sampling of the stem cell-rich basalis. More recent findings have provided insight into the efficient regeneration of the human endometrium, which is preferentially evolved in humans and menstruating upper-order primates. WIDER IMPLICATIONS: The unique physiological organization of the human endometrial glandular element, its relevance to stem cell activity and scarless endometrial regeneration will inform reproductive biologists and clinicians to direct their future research to determine disease-specific alterations in glandular anatomy in a variety of endometrial pathological conditions.

Symptomatology and Serum Nuclear Magnetic Resonance Metabolomics; Do They Predict Endometriosis in Fertile Women Undergoing Laparoscopic Sterilisation? A Prospective Cross-sectional Study.

Endometriosis is a common, chronic inflammatory condition, thought to have a higher incidence in symptomatic women, yet, commonly associated symptoms do not always correlate with the presence or severity of disease and diagnosis requires surgery. We prospectively collected data and assessed symptomology and NMR spectroscopy-based metabolomics of 102 women undergoing laparoscopic sterilisation at a tertiary referral centre in a cross-sectional study. Twelve women were incidentally diagnosed with endometriosis (11.7%). According to the pre-operative questionnaire, presence and absence of many symptoms usually attributed to endometriosis were declared at similar frequencies in women with or without endometriosis. Women with endometriosis reported apparently more persistent heavy periods (50% vs 18.9%), prolonged periods (25% versus 7.8%) and problems conceiving (27.3% versus 9%) than those without endometriosis. NMR could not discern any distinguishable differences in the serum metabolome between those with and without endometriosis. Our paper highlights the complex symptomology experienced by women, regardless of a surgical diagnosis of endometriosis. Previous literature and the current study failed to identify clear, distinguishable symptoms or biomarkers pertinent to surgically confirmed endometriosis in the general population. Therefore, development of effective, non-invasive tests for identifying this heterogenous benign condition, endometriosis, is likely to be challenging.

Anterior Gradient Protein 3 and S100 Calcium-Binding Protein P Levels in Different Endometrial Epithelial Compartments May Play an Important Role in Recurrent Pregnancy Failure.

Recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) are distressing conditions without effective treatments. The luminal epithelium (LE) is integral in determining receptivity of the endometrium, whereas functionalis glands and stroma aid in nurturing early embryo development. Calcium signalling pathways are known to be of vital importance to embryo implantation and pregnancy establishment, and anterior gradient protein 3 (AGR3) and S100 calcium-binding protein P (S100P) are involved with these pathways. We initially examined 20 full-thickness endometrial biopsies from premenopausal women across the menstrual cycle to characterize levels of AGR3 protein in each endometrial sub-region at the cellular level. A further 53 endometrial pipelle biopsies collected in the window of implantation were subsequently assessed to determine differential endometrial AGR3 and S100P levels relevant to RIF (n = 13) and RPL (n = 10) in comparison with parous women (n = 30) using immunohistochemistry. Significantly higher AGR3 and S100P immunostaining was observed in ciliated cells of the LE of women with recurrent reproductive failure compared with parous women, suggesting aberrant subcellular location-associated pathophysiology for these conditions. The nuclear localisation of S100P may allow transcriptional regulatory function, which is necessary for implantation of a viable pregnancy. Further work is thus warranted to assess their utility as diagnostic/therapeutic targets.

Histological 3D reconstruction and in vivo lineage tracing of the human endometrium.

Regular menstrual shedding and repair of the endometrial functionalis is unique to humans and higher-order primates. The current consensus postulates endometrial glands to have a single-tubular architecture, where multi-potential stem cells reside in the blind-ending glandular-bases. Utilising fixed samples from patients, we have studied the three-dimensional (3D) micro-architecture of the human endometrium. We demonstrate that some non-branching, single, vertical functionalis glands originate from a complex horizontally interconnecting network of basalis glands. The existence of a multipotent endometrial epithelial stem cell capable of regenerating the entire complement of glandular lineages was demonstrated by in vivo lineage tracing, using naturally occurring somatic mitochondrial DNA mutations as clonal markers. Vertical tracking of mutated clones showed that at least one stem-cell population resides in the basalis glands. These novel findings provide insight into the efficient and scar-less regenerative potential of the human endometrium. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Endometriosis and the Fallopian Tubes: Theories of Origin and Clinical Implications.

Endometriosis is a common, oestrogen driven chronic condition, where endometrium-like epithelial and stromal cells exist in ectopic sites. At present, no curative treatments are available and the existing evidence for disease progression is conflicting. The pathogenesis is still unknown and evidently complex, as mechanisms of initiation may depend on the anatomical distribution of endometriotic lesions. However, amongst the numerous theories and plethora of mechanisms, contributions of the fallopian tubes (FT) to endometriosis are rarely discussed. The FT are implicated in all endometriosis associated symptomatology and clinical consequences; they may contribute to the origin of endometriotic tissue, determine the sites for ectopic lesion establishment and act as conduits for the spread of proinflammatory media. Here, we examine the available evidence for the contribution of the human FT to the origin, pathogenesis and symptoms/clinical consequences of endometriosis. We also examine the broader topic linking endometriosis and the FT epithelium to the genesis of ovarian epithelial cancers. Further studies elucidating the distinct functional and phenotypical characteristics of FT mucosa may allow the development of novel treatment strategies for endometriosis that are potentially curative.

Human Uterine Biopsy: Research Value and Common Pitfalls.

The human uterus consists of the inner endometrium, the myometrium, and the outer serosa. Knowledge of the function of the uterus in health and disease is relevant to reproduction, fertility, embryology, gynaecology, endocrinology, and oncology. Research performed on uterine biopsies is essential to further the current understanding of human uterine biology. This brief review explores the value of the uterine biopsy in gynaecological and human fertility research and explores the common problems encountered when analysing data generated from different types of uterine biopsies, with the aim of improving the quality, reproducibility, and clinical translatability of future research.

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.

Hormones and endometrial carcinogenesis.

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.