A molecular staging model for accurately dating the endometrial biopsy.

Natural variability in menstrual cycle length, coupled with rapid changes in endometrial gene expression, makes it difficult to accurately define and compare different stages of the endometrial cycle. Here we develop and validate a method for precisely determining endometrial cycle stage based on global gene expression. Our 'molecular staging model' reveals significant and remarkably synchronised daily changes in expression for over 3400 endometrial genes throughout the cycle, with the most dramatic changes occurring during the secretory phase. Our study significantly extends existing data on the endometrial transcriptome, and for the first time enables identification of differentially expressed endometrial genes with increasing age and different ethnicities. It also allows reinterpretation of all endometrial RNA-seq and array data that has been published to date. Our molecular staging model will significantly advance understanding of endometrial-related disorders that affect nearly all women at some stage of their lives, such as heavy menstrual bleeding, endometriosis, adenomyosis, and recurrent implantation failure.

Genetic risk factors for endometriosis near estrogen receptor 1 and coexpression of genes in this region in endometrium.

The etiology and pathogenesis of endometriosis are complex with both genetic and environmental factors contributing to disease risk. Genome-wide association studies (GWAS) have identified multiple signals in the estrogen receptor 1 (ESR1) region associated with endometriosis and other reproductive traits and diseases. In addition, candidate gene association studies identified signals in the ESR1 region associated with endometriosis risk suggesting genetic regulation of genes in this region may be important for reproductive health. This study aimed to investigate hormonal and genetic regulation of genes in the ESR1 region in human endometrium. Changes in serum oestradiol and progesterone concentrations and expression of hormone receptors ESR1 and progesterone receptor (PGR) were assessed in endometrial samples from 135 women collected at various stages of the menstrual cycle. Correlation between hormone concentrations, receptor expression and expression of genes in the ESR1 locus was investigated. The effect of endometriosis risk variants on expression of genes in the region was analyzed to identify gene targets. Hormone concentrations and receptor expression varied significantly across the menstrual cycle. Expression of genes in the ESR1 region correlated with progesterone concentration; however, they were more strongly correlated with expression of ESR1 and PGR suggesting coregulation of genes. There was no evidence that endometriosis risk variants directly regulated expression of genes in the region. Limited sample size and cellular heterogeneity in endometrial tissue may impact the ability to detect significant genetic effects on gene expression. Effects of these variants should be validated in a larger dataset and in relevant individual cell types.

Superficial peritoneal endometriotic lesions are histologically diverse and rarely demonstrate menstrual cycle synchronicity with matched eutopic endometrium.

STUDY QUESTION: Do menstrual cycle-dependent changes occur in the histological appearance of superficial peritoneal endometriotic lesions, and are they equivalent to those observed in the eutopic endometrium? SUMMARY ANSWER: Only a small subset of superficial peritoneal endometriotic lesions exhibits some histological features in phase with menstrual cycle-related changes observed in eutopic endometrium. WHAT IS KNOWN ALREADY: Endometriotic lesions are frequently described as implants that follow menstrual cycle-related changes in morphology, as per the eutopic endometrium. This concept has been widely accepted despite the lack of conclusive published evidence. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study of 42 patients, from across the menstrual cycle, with surgically and histologically confirmed endometriosis. Patients were a subset selected from a larger endometriosis study being conducted at the Royal Women's Hospital, Melbourne since 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological features of epithelium, stroma and gland morphology were examined in haematoxylin and eosin stained sections of superficial peritoneal endometriotic lesions and matched eutopic endometrium (menstrual: n = 4, proliferative: n = 11, secretory: n = 17, hormone-treated: n = 10). At least two biopsies (average = 4, range = 2-8 biopsies) and a matched endometrial sample were analysed for each patient and results were presented per endometriotic gland profile (n = 1051). Data were analysed using mixed effects logistic regression to account for multiple patients and multiple endometriotic biopsies, each with multiple endometriotic gland profiles. This model also enabled analysis of endometriotic lesions versus eutopic endometrium. MAIN RESULTS AND THE ROLE OF CHANCE: There was considerable inter- and intra-patient variability in the morphology of superficial peritoneal endometriotic lesions. Menstrual cycle-associated changes were only observed for some features in a subset of endometriotic gland profiles. The proportion of endometriotic gland profiles with epithelial mitoses significantly increased in the proliferative phase (18% of gland profiles) relative to the menstrual phase (0% of endometriotic gland profiles) (odds ratios (OR) 9.30; 95% confidence intervals (CI) = 3.71-23.32; P < 0.001). Fewer blood-filled gland lumens were observed in the secretory phase (45% of endometriotic gland profiles) compared to the menstrual phase (67% of endometriotic gland profiles) (OR, 0.30; 95% CI = 0.11-0.79; P = 0.015). The features of the eutopic endometrium analysed in this study did not reflect the results in matched endometriotic lesions (P > 0.05). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This study focused on features observed in sections of superficial peritoneal lesions and these may differ from features of deep infiltrating endometriosis or ovarian endometriomas. Cycle phases were limited to menstrual, proliferative and secretory phases to allow appropriate statistical modelling. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights heterogeneity in the histological characteristics of superficial peritoneal lesions. It challenges the assumption that lesion morphology consistently reflects menstrual cycle-associated changes. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported in part by National Health and Medical Research Council (NHMRC) project grants GNT1012245, GNT1105321 and GNT1026033 (P.A.W.R., J.E.G. and S.J.H.-C.). There are no competing interests.

Generation of immortalized human endometrial stromal cell lines with different endometriosis risk genotypes.

Endometriotic lesions are composed in part of endometrial-like stromal cells, however, there is a shortage of immortalized human endometrial stromal cultures available for research. As genetic factors play a role in endometriosis risk, it is important that genotype is also incorporated into analysis of pathological mechanisms. Human telomerase reverse transcriptase (hTERT) immortalization (using Lenti-hTERT-green fluorescent protein virus) took place following genotype selection; 13 patients homozygous for either the risk or non-risk 'other' allele for one or more important endometriosis risk single nucleotide polymorphism on chromosome 1p36.12 (rs3820282, rs56318008, rs55938609, rs12037376, rs7521902 or rs12061255). Short tandem repeat DNA profiling validated that donor tissue matched that of the immortalized cell lines and confirmed that cultures were genetically novel. Expression of morphological markers (vimentin and cytokeratin) and key genes of interest (telomerase, estrogen and progesterone receptors and LINC00339) were examined and functional assays for cell proliferation, steroid hormone and inflammatory responses were performed for 7/13 cultures. All endometrial stromal cell lines maintained their fibroblast-like morphology (vimentin-positive) and homozygous endometriosis-risk genotype following introduction of hTERT. Furthermore, the new stromal cultures demonstrated positive and diverse responses to hormones (proliferation and decidualisation changes) and inflammation (dose-dependent response), while maintaining hormone receptor expression. In conclusion, we successfully developed a range of human endometrial stromal cell lines that carry important endometriosis-risk alleles. The wider implications of this approach go beyond advancing endometriosis research; these cell lines will be valuable tools for multiple endometrial pathologies offering a level of genetic and phenotypic diversity not previously available.

Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus.

Female mice lacking the follistatin gene but expressing a human follistatin-315 transgene (tghFST315) have reproductive abnormalities (reduced follicles, no corpora lutea and ovarian-uterine inflammation). We hypothesised that the absence of follistatin-288 causes the abnormal reproductive tract via both developmental abnormalities and abnormal ovarian activity. We characterised the morphology of oviducts and uteri in wild type (WT), tghFST315 and follistatin-knockout mice expressing human follistatin-288 (tghFST288). The oviducts and uteri were examined in postnatal Day-0 and adult mice (WT and tghFST315 only) using histology and immunohistochemistry. Adult WT and tghFST315 mice were ovariectomised and treated with vehicle, oestradiol-17ß (100ng injection, dissection 24h later) or progesterone (1mg×three daily injections, dissection 24h later). No differences were observed in the oviducts or uteri at birth, but abnormalities developed by adulthood. Oviducts of tghFST315 mice failed to coil, the myometrium was disorganised, endometrial gland number was reduced and oviducts and uteri contained abundant leukocytes. After ovariectomy, tghFST315 mice had altered uterine cell proliferation, and inflammation was maintained and exacerbated by oestrogen. These studies show that follistatin is crucial to postnatal oviductal-uterine development and function. Further studies differentiating the role of ovarian versus oviductal-uterine follistatin in reproductive tract function at different developmental stages are warranted.

Peroxisome proliferator-activated receptors are altered in pathologies of the human placenta: gestational diabetes mellitus, intrauterine growth restriction and preeclampsia.

BACKGROUND: Common complications of pregnancy arise in part from dysfunctional placental development, and include gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE). Peroxisome proliferator-activated receptors (PPARs), and their partner retinoid X receptor a (RXRalpha), mediate trophoblast differentiation and thus may offer insight into the pathophysiology of these diseases. METHODS: Human placentae were obtained from women at term with GDM and were compared to uncomplicated term placentae. Placentae from women who delivered preterm with IUGR, PE or co-existing PE and IUGR were compared to matched controls. Quantitative RT-PCR and Western blotting were used to examine mRNA and protein expression of PPARalpha, PPARdelta, PPARgamma and RXRalpha. DNA binding activity of PPAR isoforms were measured in nuclear protein extracts. RESULTS: GDM was associated with significantly lower placental PPARgamma mRNA and protein, PPARalpha protein and RXRalpha protein expression, while PPAR DNA binding activity remained unchanged. Placentae from women with PE did not demonstrate any changes in mRNA or protein expression or PPAR DNA binding activity, while IUGR/PE placenta showed significant increases in PPARalpha protein, PPARgamma mRNA and protein and RXRalpha mRNA and protein expression. Significantly elevated protein expression of PPARalpha and RXRalpha were associated with IUGR placentae. IUGR and IUGR/PE placentae had significantly higher PPARgamma DNA binding activity compared to controls. CONCLUSIONS: The data presented herein suggest that PPARs may be involved in the pathophysiology of GDM, PE and IUGR.

Peroxisome proliferator-activated receptors and retinoid X receptor-alpha in term human gestational tissues: tissue specific and labour-associated changes.

Peroxisome proliferator-activated receptors (PPARs) and their transcriptional partner retinoid X receptor (RXR) are involved in transcriptionally regulating the events that contribute to the control of parturition in humans. Definitive data, however, are lacking with respect to PPAR and RXR expression and activation during term labour in human gestational tissues. The aim of this study, therefore, was to identify tissue and labour-associated changes of PPAR isoforms (alpha, delta and gamma) and RXRalpha in placenta, amnion and choriodecidua. Gestational tissues from term non-labouring women were used for immunohistochemistry localisation and confirmation studies of PPAR isoforms (alpha, delta and gamma) and RXRalpha. Human gestational tissues were then collected from term women not-in-labour (NIL) (elective Caesarean section), in-labour (IL) (emergency Caesarean section) and post-labour (PL) (normal vaginal delivery). Quantitative RT-PCR (qRT-PCR) and Western blotting were employed to study mRNA and protein expression profiles respectively. Significantly higher mRNA expression was observed in placental tissues taken from women in labour (PPARdelta, PPARgamma and RXRalpha). Elevated PPARdelta and RXRalpha mRNA expression in fetal membranes was also associated with being in labour. In contrast, PPARgamma mRNA in the amnion was decreased with term PL compared to NIL. In placenta, PPARalpha, PPARdelta and PPARgamma protein expression was significantly increased in the IL group compared to the NIL or PL group. There was no significant difference in PPAR or RXRalpha protein expression in both amnion and choriodecidua between the three labour groups. PPAR (alpha and gamma) transcription factor DNA binding activity was found to decline IL compared to NIL and PL in the placenta. PPARdelta DNA binding activity also decreased in the choriodecidua IL compared to PL. In amnion, PPARalpha DNA binding activity was found to be higher IL compared to NIL. In conclusion, term human labour is associated with changes in expression and activity of PPAR isoforms and its transcription partner, RXRalpha. This data is consistent with the hypothesis that PPAR:RXR are involved in regulating of the processes of human term parturition.

Pre-labour fetal membranes overlying the cervix display alterations in inflammation and NF-kappaB signalling pathways.

To generate new insights into the process of fetal membrane rupture, we have developed a technique whereby fetal membranes overlying the cervix (i.e. supracervical site, SCS) are tagged in women undergoing term elective Caesarean section. The specific aim is to determine the effect of supracervical apposition on the release of inflammatory mediators and NF-kappaB signalling in pre-labour fetal membranes. Fetal membranes were collected from both the SCS and from a distal site (DS). The level of NF-kappaB proteins and its transcriptional co-activator protein CBP and p300 was determined by Western blotting and/or immunohistochemistry (IHC), and cytokine and prostaglandin release was quantified by enzyme immunoassay. Tissues obtained before labour at term possess an area of the fetal membranes (i.e. supracervical site) that exhibit decreased release of IL-1beta, IL-6, IL-8, TNF-alpha and PGE(2). IHC revealed that NF-kappaB signalling proteins, CBP and p300 were significantly increased in SC fetal membranes compared to distal membranes. In summary, data from this study confirm that supracervical fetal membranes display altered structural and biochemical characteristics.