Abstract
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.
Objective
To calculate the cost‐effectiveness of implementing PlGF testing alongside a clinical management algorithm in maternity services in the UK, compared with current standard care.
Design
...Cost‐effectiveness analysis.
Setting
Eleven maternity units participating in the PARROT stepped‐wedge cluster‐randomised controlled trial.
Population
Women presenting with suspected pre‐eclampsia between 20+0 and 36+6 weeks’ gestation.
Methods
Monte Carlo simulation utilising resource use data and maternal adverse outcomes.
Main outcome measures
Cost per maternal adverse outcome prevented.
Results
Clinical care with PlGF testing costs less than current standard practice and resulted in fewer maternal adverse outcomes. There is a total cost‐saving of UK£149 per patient tested, when including the cost of the test. This represents a potential cost‐saving of UK£2,891,196 each year across the NHS in England.
Conclusions
Clinical care with PlGF testing is associated with the potential for cost‐savings per participant tested when compared with current practice via a reduction in outpatient attendances, and improves maternal outcomes. This economic analysis supports a role for implementation of PlGF testing in antenatal services for the assessment of women with suspected pre‐eclampsia.
Tweetable
Placental growth factor testing for suspected pre‐eclampsia is cost‐saving and improves maternal outcomes.
Tweetable
Placental growth factor testing for suspected pre‐eclampsia is cost‐saving and improves maternal outcomes.
Abstract
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.
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?
Only a ...small subset of superficial peritoneal endometriotic lesions exhibits some histological features in phase with menstrual cycle-related changes observed in eutopic endometrium.
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.
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.
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.
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).
Not applicable.
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.
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.
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.
Abstract
STUDY QUESTION
Do endometriosis risk-associated single nucleotide polymorphisms (SNPs) found at the 12q22 locus have effects on vezatin (VEZT) expression?
SUMMARY ANSWER
The original ...genome-wide association study (GWAS) SNP (rs10859871), and other newly identified association signals, demonstrate strong evidence for cis-expression quantitative trait loci (eQTL) effects on VEZT expression.
WHAT IS KNOWN ALREADY
GWAS have identified several disease-risk loci (SNPs) associated with endometriosis. The SNP rs10859871 is located within the VEZT gene. 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 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 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 n = 56 (western blotting) and 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 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 VEZT or FGD6 (FYVE, RhoGEF and PH domain-containing 6) and SNPs with the strongest signals were either intronic or intergenic. We found strong evidence for VEZT cis-eQTLs with the sentinel SNP (rs10859871) in blood and endometrium, where the endometriosis risk allele (C) was associated with an increase in 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 (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. 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 cis-eQTLs on VEZT expression. By examining women who possess an enhanced genetic risk of developing endometriosis, we have identified an effect on VEZT expression and therefore a potential gene/gene pathway in endometriosis disease establishment and development.
STUDY FUNDING/COMPETING INTEREST(S)
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.
Abstract
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.
Abstract
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.
BACKGROUND The mammalian placenta plays a central role in maternal tolerance of the semi-allogeneic fetus and fluid balance between the maternal and fetal compartments. The lymphatics play a role in ...both these function. The aim of this study was to describe the distribution of lymphatic vessels in human decidua, with particular focus on the lymphatics that surround remodelling spiral arteries during decidualization and trophoblast invasion. METHODS Placental bed and non-placental bed (decidua parietalis) biopsies were obtained from 41 women undergoing elective termination of pregnancy at 6–18 weeks gestational age as well as placental bed biopsies from 5 women undergoing elective Caesarean section at term. In addition to routine haematoxylin and eosin staining, double immunohistochemical labelling was performed on serial 3-µm sections to identify lymphatic vessels in conjunction with one of the following: blood vessels, smooth muscle, epithelial and trophoblast cells or proliferating cells. Representative photomicrographs of all sections were obtained from a total of 273 areas (46 samples, average 6 range 3–15 areas per sample). Descriptive findings of the organization of lymphatics in human placental bed and decidua parietalis were made from a total of 1638 images. RESULTS Lymphatic vessels positive for podoplanin were abundant in non-decidualized hypersecretory endometrium at all stages of gestation. By contrast, the decidua was nearly always devoid of lymphatics. In some samples, structures that appeared to be regressing lymphatics could be observed at the boundary between non-decidualized hypersecretory and decidualized endometrium. Lymphatic vessels were notably absent from the vicinity of spiral arteries that were surrounded by decidualized stromal cells. Lymphatic vessels in non-decidualized hypersecretory endometrium appeared larger and more elongated as gestation progressed. Proliferating lymphatic vascular endothelial cells were identified in both large vessels, and in streaks of D2-40 positive cells that could have been newly forming lymphatic vessels. Placental bed lymphatics exhibited limited and variable staining with LYVE-1 at all stages of pregnancy apart from term. CONCLUSIONS We have made novel observations on lymphatics in the placental bed and their relationship with other structures throughout pregnancy. Endometrial stromal cell decidualization results in a loss of lymphatics, with this phenomenon being particularly apparent around the spiral arteries.
Identifying suitable housekeeping genes for quantitative RT–PCR in the uterus is problematic, as this tissue undergoes significant structural and functional alterations during the oestrous cycle and ...pregnancy in response to circulating hormones. The suitability of 18S rRNA as a housekeeping gene in mouse uterus was investigated by introducing an ‘RNA spike’ standard into the reverse transcription reaction. 18S rRNA levels increased by Day 4 of pregnancy and after progesterone administration in ovariectomized mice. We conclude that 18S rRNA is not a suitable housekeeping gene for quantitative RT–PCR analysis in progesterone-responsive tissues, and the RNA spiking method provides a suitable alternative.
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