Sarah J. Holdsworth-Carson

Pre-labour Fetal Membranes Overlying the Cervix Display Alterations in Inflammation and NF-κB 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.

Clonality of smooth muscle and fibroblast cell populations isolated from human fibroid and myometrial tissues

Uterine fibroids are conventionally defined as clonally derived benign tumours from the proliferation of a single smooth muscle cell (SMC). We have previously identified fibroblast-like cells in fibroids, the presence of which raises the question as to whether all cells within the fibroid have the same clonal origin. The first aim of this study was to develop a fluorescence-activated cell sorting (FACS)-based method to isolate different cell types from human myometrium and fibroid tissues. Secondly, we aimed to use X chromosome inactivation analysis to determine the clonality of cell subpopulations isolated from myometrial and fibroid tissues. Human myometrium and fibroid tissues were collected from women undergoing hysterectomy. Immunohistochemistry (IHC) and flow cytometry confirmed that in addition to SMCs, fibroblasts constitute a significant proportion of cells in human myometrium and fibroid tissues. FACS based on CD90 and ALDH1 reliably separated cells into three myometrial and four fibroid subpopulations: SMCs, vascular smooth muscle cells and two fibroblast subsets. Clonality was first determined by X chromosome inactivation using the classic DNA methylation-sensitive HUMARA assay. Data from this assay were highly variable, with only a quarter of samples meeting the definition of clonal fibroid and non-clonal myometrium. However, using an RNA-based X chromosome inactivation HUMARA assay, we were able to demonstrate clonality of all cellular constituents of most fibroids. Our results confirm that most fibroids are derived from a single cell, and for the first time demonstrates that these clonal cells differentiate into fibroblast and SMC subpopulation as the fibroid grows.

Proteomic Analysis of Human Cervico-Vaginal Fluid Displays Differential Protein Expression in Association with Labor Onset at Term

Human labor is characterized by dramatic physiological and structural alterations of the cervix and overlying fetal membranes, leading to myometrial activation and delivery. To investigate the potential mechanism of these changes, we performed 2D PAGE proteomic analysis on serial cervico-vaginal fluid samples obtained from women during late pregnancy and spontaneous labor. We identified 9 protein spots that were significantly altered ( p < 0.05) in association with spontaneous term labor. Eight protein spots were definitively characterized by electrospray ion-trap mass spectrometry yielding 7 different proteins: cystatin-A, interleukin-1 receptor antagonist, glutathione S-transferase P, peroxiredoxin-2, thioredoxin, copper-zinc superoxide dismutase, and epidermal fatty-acid binding protein. These proteins are involved in protease inhibition, anti-inflammatory cytokine activity, and oxidative stress defense. These findings may provide an insight into the biochemical processes and timing associated with extracellular matrix remodelling of the cervix, supracervical fetal membranes, and myometrial activation in association with spontaneous term labor. Application of these findings may lead to development of predictive biomarkers of labor onset.

Preterm and infection-driven preterm labor: the role of peroxisome proliferator-activated receptors and retinoid X receptor

Approximately 8% of births are complicated by preterm delivery. To improve neonatal outcomes, a greater understanding of the mechanisms surrounding preterm parturition is required. Peroxisome proliferator-activated receptors (PPARs) have been implicated in the regulation of labor at term where they exhibit anti-inflammatory properties. Thus, we hypothesize that dysregulation of PPAR expression and activity may be associated with preterm labor and infection-associated preterm labor. The aim of this study was to compare the expression and activity of PPARs and the expression of retinoid X-receptor alpha (RXRA) in gestational tissues from term and preterm deliveries, and from infection-associated preterm deliveries. Quantitative RT-PCR, western blotting and activity ELISA were used to study expression and DNA binding profiles. Compared with term, preterm parturition was associated with an increased expression of PPAR delta (PPARD; mRNA and protein), PPAR gamma (PPARG; protein) and RXRA (protein) in the placenta and PPARD (mRNA and protein) and RXRA (mRNA) in the choriodecidua. There was, however, no change in preterm PPAR DNA binding activity compared with term. Preterm chorioamnionitis (CAM) demonstrated protein degradation in the choriodecidua and was associated with a decline in the mRNA expression of PPAR alpha (PPARA) and RXRA compared with uninfected preterm cases. PPAR DNA binding activity increased in the placenta (PPARD and PPARG) and decreased in the amnion (PPARA and PPARG) in association with preterm CAM. In conclusion, idiopathic preterm deliveries were associated with an increase in PPAR:RXR expression and preterm CAM was associated with a decrease in PPAR:RXR expression and tissue-specific alterations in transcriptional activity. The reasons for such dysregulation remain to be determined; however, the data are consistent with the hypothesis that PPARs may play a role in preterm labor and infection-complicated preterm deliveries.

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.

Endometriosis risk alleles at 1p36.12 act through inverse regulation of CDC42 and LINC00339

Genome-wide association studies (GWAS) have identified markers within the WNT4 region on chromosome 1p36.12 showing consistent and strong association with increasing endometriosis risk. Fine mapping using sequence and imputed genotype data has revealed strong candidates for the causal SNPs within these critical regions; however, the molecular pathogenesis of these SNPs is currently unknown. We used gene expression data collected from whole blood from 862 individuals and endometrial tissue from 136 individuals from independent populations of European descent to examine the mechanism underlying endometriosis susceptibility. Association mapping results from 7,090 individuals (2,594 cases and 4,496 controls) supported rs3820282 as the SNP with the strongest association for endometriosis risk (P = 1.84 × 10−5, OR = 1.244 (1.126-1.375)). SNP rs3820282 is a significant eQTL in whole blood decreasing expression of LINC00339 (also known as HSPC157) and increasing expression of CDC42 (P = 2.0 ×10−54 and 4.5x10−4 respectively). The largest effects were for two LINC00339 probes (P = 2.0 ×10−54; 1.0 × 10−34). The eQTL for LINC00339 was also observed in endometrial tissue (P = 2.4 ×10−8) with the same direction of effect for both whole blood and endometrial tissue. There was no evidence for eQTL effects for WNT4. Chromatin conformation capture provides evidence for risk SNPs interacting with the promoters of both LINC00339 and CDC4 and luciferase reporter assays suggest the risk SNP rs12038474 is located in a transcriptional silencer for CDC42 and the risk allele increases expression of CDC42. However, no effect of rs3820282 was observed in the LINC00339 expression in Ishikawa cells. Taken together, our results suggest that SNPs increasing endometriosis risk in this region act through CDC42, but further functional studies are required to rule out inverse regulation of both LINC00339 and CDC42.

Endometrial vezatin and its association with endometriosis risk

STUDY QUESTION Do endometriosis risk-associated single nucleotide polymorphisms (SNPs) found at the 12q22 locus have effects on vezatin ( ITALIC! VEZT) expression? SUMMARY ANSWER The original genome-wide association study (GWAS) SNP (rs10859871), and other newly identified association signals, demonstrate strong evidence for ITALIC! cis-expression quantitative trait loci (eQTL) effects on ITALIC! VEZT expression. WHAT IS KNOWN ALREADY GWAS have identified several disease-risk loci (SNPs) associated with endometriosis. The SNP rs10859871 is located within the ITALIC! VEZT gene. ITALIC! VEZT expression is altered in the endometrium of endometriosis patients and is an excellent candidate for having a causal role in endometriosis. Most of the SNPs identified from GWAS are not located within the coding region of the genome. However, they are likely to have an effect on the regulation of gene expression. Genetic variants that affect levels of gene expression are called expression quantitative trait loci (eQTL). STUDY DESIGN, SIZE, DURATION Samples for genotyping and ITALIC! VEZT variant screening were drawn from women recruited for genetic studies in Australia/New Zealand and women undergoing surgery in a tertiary care centre. Coding variants for ITALIC! VEZT were screened in blood from 100 unrelated individuals (endometriosis-dense families) from the QIMR Berghofer Medical Research Institute dataset. SNPs at the 12q22 locus were imputed and reanalysed for their association with endometriosis. Reanalysis of endometriosis risk-association was performed on a final combined Australian dataset of 2594 cases and 4496 controls. Gene expression was performed on 136 endometrial samples. eQTL analysis in whole blood was performed on 862 individuals from the Brisbane Systems Genetics Study. Endometrial tissue-specific eQTL analysis was performed on 122 samples (eutopic endometrium) collected following laparoscopic surgery. VEZT protein expression studies employed ITALIC! n = 56 (western blotting) and ITALIC! n = 42 (immunohistochemistry) endometrial samples. PARTICIPANTS/MATERIALS, SETTING, METHODS The women recruited for this study provided blood and/or endometrial tissue samples in a hospital setting. Genomic DNA was screened for common and coding variants. SNPs of interest in the 12q22 region were genotyped using Agena MassARRAY technology or Taqman SNP genotyping assay. Gene expression profiles from RNA extracted from blood and endometrial tissue samples were generated using Illumina whole-genome expression chips (Human HT-12 v4.0). Whole protein extracted from endometrium was used for VEZT western blots, and paraffin sections of endometrium were employed for VEZT immunohistochemistry semi-quantitative analysis. MAIN RESULTS AND THE ROLE OF CHANCE A total of 11 coding variants of ITALIC! VEZT (including one novel variant) were identified from an endometriosis-dense cohort. Polymorphic coding and imputed SNPs were combined with previous GWAS data to reanalyse the endometriosis risk association of the 12q22 region. The disease association signal at 12q22 was due to coding variants in ITALIC! VEZT or ITALIC! FGD6 (FYVE, RhoGEF and PH domain-containing 6) and SNPs with the strongest signals were either intronic or intergenic. We found strong evidence for ITALIC! VEZT cis-eQTLs with the sentinel SNP (rs10859871) in blood and endometrium, where the endometriosis risk allele (C) was associated with an increase in ITALIC! VEZT expression. We could not demonstrate this genotype-specific effect on VEZT protein expression in endometrium. However, we did observe a menstrual cycle stage specific increase in VEZT protein expression in endometrial glands, specific to the secretory phase ( ITALIC! P = 2.0 × 10(-4)). LIMITATIONS, REASONS FOR CAUTION In comparison to the blood sample datasets, the study numbers of endometrial tissues were substantially reduced. Protein studies failed to complement RNA results, also likely a reflection of the low study numbers in these experiments. ITALIC! In silico prediction tools used in this investigation are typically based on cell lines different to our tissues of interest, thus any functional annotations drawn from these approaches should be considered carefully. Therefore, functional studies on VEZT and related pathway components are still warranted to unequivocally implicate a causal role for VEZT in endometriosis pathophysiology. WIDER IMPLICATIONS OF THE FINDINGS GWAS have proven to be very valuable tools for deciphering complex diseases. Endometriosis is a text-book example of a complex disease, involving genetic, lifestyle and environmental influences. Our focused investigation of the 12q22 region validates an association with increased endometriosis risk. Endometriosis risk SNPs (including rs10859871) located within this locus demonstrated evidence for ITALIC! cis-eQTLs on ITALIC! VEZT expression. By examining women who possess an enhanced genetic risk of developing endometriosis, we have identified an effect on ITALIC! VEZT expression and therefore a potential gene/gene pathway in endometriosis disease establishment and development. STUDY FUNDING/COMPETING INTERESTS Funding for this work was provided by NHMRC Project Grants GNT1012245, GNT1026033, GNT1049472 and GNT1046880. G.W.M. is supported by the NHMRC Fellowship scheme (GNT1078399). S.J.H.-C. is supported by the J.N. Peters Bequest Fellowship. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

The genetic regulation of transcription in human endometrial tissue

Study question Do genetic effects regulate gene expression in human endometrium? Summary answer This study demonstrated strong genetic effects on endometrial gene expression and some evidence for genetic regulation of gene expression in a menstrual cycle stage-specific manner. What is known already Genetic effects on expression levels for many genes are tissue specific. Endometrial gene expression varies across menstrual cycle stages and between individuals, but there are limited data on genetic control of expression in endometrium. Study design, size, duration We analysed genome-wide genotype and gene expression data to map cis expression quantitative trait loci (eQTL) in endometrium. Participants/materials, setting, methods We recruited 123 women of European ancestry. DNA samples from blood were genotyped on Illumina HumanCoreExome chips. Total RNA was extracted from endometrial tissues. Whole-transcriptome profiles were characterized using Illumina Human HT-12 v4.0 Expression Beadchips. We performed eQTL mapping with ~8 000 000 genotyped and imputed single nucleotide polymorphisms (SNPs) and 12 329 genes. Main results and the role of chance We identified a total of 18 595 cis SNP-probe associations at a study-wide level of significance (P < 1 × 10-7), which correspond to independent eQTLs for 198 unique genes. The eQTLs with the largest effect in endometrial tissue were rs4902335 for CHURC1 (P = 1.05 × 10-32) and rs147253019 for ZP3 (P = 8.22 × 10-30). We further performed a context-specific eQTL analysis to investigate if genetic effects on gene expression regulation act in a menstrual cycle-specific manner. Interestingly, five cis-eQTLs were identified with a significant stage-by-genotype interaction. The strongest stage interaction was the eQTL for C10ORF33 (PYROXD2) with SNP rs2296438 (P = 2.0 × 10-4), where we observe a 2-fold difference in the average expression levels of heterozygous samples depending on the stage of the menstrual cycle. Large scale data The summary eQTL results are publicly available to browse or download. Limitations, reasons for caution A limitation of the present study was the relatively modest sample size. It was not powered to identify trans-eQTLs and larger sample sizes will also be needed to provide better power to detect cis-eQTLs and cycle stage-specific effects, given the substantial changes in expression across the menstrual cycle for many genes. Wider implications of the findings Identification of endometrial eQTLs provides a platform for better understanding genetic effects on endometriosis risk and other endometrial-related pathologies. Study funding/competing interest(s) Funding for this work was provided by NHMRC Project Grants GNT1026033, GNT1049472, GNT1046880, GNT1050208, GNT1105321 and APP1083405. There are no competing interests.

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-17β (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.

Aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids

Uterine fibroids are the most common benign tumour afflicting women of reproductive age. Despite the large healthcare burden caused by fibroids, there is only limited understanding of the molecular mechanisms that drive fibroid pathophysiology. Although a large number of genes are differentially expressed in fibroids compared with myometrium, it is likely that most of these differences are a consequence of the fibroid presence and are not causal. The aim of this study was to investigate the expression and regulation of NR2F2 and CTNNB1 based on their potential causal role in uterine fibroid pathophysiology. We used real-time quantitative RT-PCR, western blotting and immunohistochemistry to describe the expression of NR2F2 and CTNNB1 in matched human uterine fibroid and myometrial tissues. Primary myometrial and fibroid smooth muscle cell cultures were treated with progesterone and/or retinoic acid (RA) and sonic hedgehog (SHH) conditioned media to investigate regulatory pathways for these proteins. We showed that NR2F2 and CTNNB1 are aberrantly expressed in fibroid tissue compared with matched myometrium, with strong blood vessel-specific localisation. Although the SHH pathway was shown to be active in myometrial and fibroid primary cultures, it did not regulate NR2F2 or CTNNB1 mRNA expression. However, progesterone and RA combined regulated NR2F2 mRNA, but not CTNNB1, in myometrial but not fibroid primary cultures. In conclusion, we demonstrate aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids compared with normal myometrium, consistent with the hypothesis that these factors may play a causal role uterine fibroid development.