Genome-wide association study meta-analysis of dizygotic twinning illuminates genetic regulation of female fecundity.

STUDY QUESTION: Which genetic factors regulate female propensity for giving birth to spontaneous dizygotic (DZ) twins? SUMMARY ANSWER: We identified four new loci, GNRH1, FSHR, ZFPM1, and IPO8, in addition to previously identified loci, FSHB and SMAD3. WHAT IS KNOWN ALREADY: The propensity to give birth to DZ twins runs in families. Earlier, we reported that FSHB and SMAD3 as associated with DZ twinning and female fertility measures. STUDY DESIGN, SIZE, DURATION: We conducted a genome-wide association meta-analysis (GWAMA) of mothers of spontaneous dizygotic (DZ) twins (8265 cases, 264 567 controls) and of independent DZ twin offspring (26 252 cases, 417 433 controls). PARTICIPANTS/MATERIALS, SETTING, METHODS: Over 700 000 mothers of DZ twins, twin individuals and singletons from large cohorts in Australia/New Zealand, Europe, and the USA were carefully screened to exclude twins born after use of ARTs. Genetic association analyses by cohort were followed by meta-analysis, phenome wide association studies (PheWAS), in silico and in vivo annotations, and Zebrafish functional validation. MAIN RESULTS AND THE ROLE OF CHANCE: This study enlarges the sample size considerably from previous efforts, finding four genome-wide significant loci, including two novel signals and a further two novel genes that are implicated by gene level enrichment analyses. The novel loci, GNRH1 and FSHR, have well-established roles in female reproduction whereas ZFPM1 and IPO8 have not previously been implicated in female fertility. We found significant genetic correlations with multiple aspects of female reproduction and body size as well as evidence for significant selection against DZ twinning during human evolution. The 26 top single nucleotide polymorphisms (SNPs) from our GWAMA in European-origin participants weakly predicted the crude twinning rates in 47 non-European populations (r = 0.23 between risk score and population prevalence, s.e. 0.11, 1-tail P = 0.058) indicating that genome-wide association studies (GWAS) are needed in African and Asian populations to explore the causes of their respectively high and low DZ twinning rates. In vivo functional tests in zebrafish for IPO8 validated its essential role in female, but not male, fertility. In most regions, risk SNPs linked to known expression quantitative trait loci (eQTLs). Top SNPs were associated with in vivo reproductive hormone levels with the top pathways including hormone ligand binding receptors and the ovulation cycle. LARGE SCALE DATA: The full DZT GWAS summary statistics will made available after publication through the GWAS catalog (https://www.ebi.ac.uk/gwas/). LIMITATIONS, REASONS FOR CAUTION: Our study only included European ancestry cohorts. Inclusion of data from Africa (with the highest twining rate) and Asia (with the lowest rate) would illuminate further the biology of twinning and female fertility. WIDER IMPLICATIONS OF THE FINDINGS: About one in 40 babies born in the world is a twin and there is much speculation on why twinning runs in families. We hope our results will inform investigations of ovarian response in new and existing ARTs and the causes of female infertility. STUDY FUNDING/COMPETING INTEREST(S): Support for the Netherlands Twin Register came from the Netherlands Organization for Scientific Research (NWO) and The Netherlands Organization for Health Research and Development (ZonMW) grants, 904-61-193, 480-04-004, 400-05-717, Addiction-31160008, 911-09-032, Biobanking and Biomolecular Resources Research Infrastructure (BBMRI.NL, 184.021.007), Royal Netherlands Academy of Science Professor Award (PAH/6635) to DIB, European Research Council (ERC-230374), Rutgers University Cell and DNA Repository (NIMH U24 MH068457-06), the Avera Institute, Sioux Falls, South Dakota (USA) and the National Institutes of Health (NIH R01 HD042157-01A1) and the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health and Grand Opportunity grants 1RC2 MH089951. The QIMR Berghofer Medical Research Institute (QIMR) study was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485, 552498, 1050208, 1075175). L.Y. is funded by Australian Research Council (Grant number DE200100425). The Minnesota Center for Twin and Family Research (MCTFR) was supported in part by USPHS Grants from the National Institute on Alcohol Abuse and Alcoholism (AA09367 and AA11886) and the National Institute on Drug Abuse (DA05147, DA13240, and DA024417). The Women's Genome Health Study (WGHS) was funded by the National Heart, Lung, and Blood Institute (HL043851 and HL080467) and the National Cancer Institute (CA047988 and UM1CA182913), with support for genotyping provided by Amgen. Data collection in the Finnish Twin Registry has been supported by the Wellcome Trust Sanger Institute, the Broad Institute, ENGAGE-European Network for Genetic and Genomic Epidemiology, FP7-HEALTH-F4-2007, grant agreement number 201413, National Institute of Alcohol Abuse and Alcoholism (grants AA-12502, AA-00145, AA-09203, AA15416, and K02AA018755) and the Academy of Finland (grants 100499, 205585, 118555, 141054, 264146, 308248, 312073 and 336823 to J. Kaprio). TwinsUK is funded by the Wellcome Trust, Medical Research Council, Versus Arthritis, European Union Horizon 2020, Chronic Disease Research Foundation (CDRF), Zoe Ltd and the National Institute for Health Research (NIHR) Clinical Research Network (CRN) and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London. For NESDA, funding was obtained from the Netherlands Organization for Scientific Research (Geestkracht program grant 10000-1002), the Center for Medical Systems Biology (CSMB, NVVO Genomics), Biobanking and Biomolecular Resources Research Infrastructure (BBMRI-NL), VU University's Institutes for Health and Care Research (EMGO+) and Neuroscience Campus Amsterdam, University Medical Center Groningen, Leiden University Medical Center, National Institutes of Health (NIH, ROI D0042157-01A, MH081802, Grand Opportunity grants 1 RC2 Ml-1089951 and IRC2 MH089995). Part of the genotyping and analyses were funded by the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health. Computing was supported by BiG Grid, the Dutch e-Science Grid, which is financially supported by NWO. Work in the Del Bene lab was supported by the Programme Investissements d'Avenir IHU FOReSIGHT (ANR-18-IAHU-01). C.R. was supported by an EU Horizon 2020 Marie Sklodowska-Curie Action fellowship (H2020-MSCA-IF-2014 #661527). H.S. and K.S. are employees of deCODE Genetics/Amgen. The other authors declare no competing financial interests. TRIAL REGISTRATION NUMBER: N/A.

Genomic characterisation of the overlap of endometriosis with 76 comorbidities identifies pleiotropic and causal mechanisms underlying disease risk.

Comorbid conditions can be driven by underlying pleiotropic and causal mechanisms that can provide insights into shared molecular and biological processes contributing to disease risk. Endometriosis is a chronic condition affecting one in nine women of reproductive age and poses many challenges including lengthy diagnostic delays and limited treatment efficacy owing to poor understanding of disease aetiology. To shed light on the underlying biological mechanisms and to identify potential risk factors, we examine the epidemiological and genomic relationship between endometriosis and its comorbidities. In the UK Biobank 292 ICD10 codes were epidemiologically correlated with endometriosis diagnosis, including gynaecological, immune, infection, pain, psychiatric, cancer, gastrointestinal, urinary, bone and cardiovascular traits. A subset of the identified comorbidities (n = 76) underwent follow-up genetic analysis. Whilst Mendelian randomisation suggested causality was not responsible for most comorbid relationships, 22 traits were genetically correlated with endometriosis, including pain, gynaecological and gastrointestinal traits, suggestive of a shared genetic background. Pleiotropic genetic variants and genes were identified using gene-based and colocalisation analysis. Shared genetic risk factors and potential target genes suggest a diverse collection of biological systems are involved in these comorbid relationships including coagulation factors, development of the female reproductive tract and cell proliferation. These findings highlight the diversity of traits with epidemiological and genomic overlap with endometriosis and implicate a key role for pleiotropy in the comorbid relationships.

Polygenic risk score phenome-wide association study reveals an association between endometriosis and testosterone.

BACKGROUND: Endometriosis affects 1 in 9 women, yet it is poorly understood with long diagnostic delays, invasive diagnoses, and poor treatment outcomes. Characterised by the presence of endometrial-like tissue outside of the uterus, its main symptoms are pain and infertility. Endometriosis often co-occurs with other conditions, which may provide insights into the origins of endometriosis. METHODS: Here a polygenic risk score phenome-wide association study of endometriosis was conducted in the UK Biobank to investigate the pleiotropic effects of a genetic liability to endometriosis. The relationship between the polygenic risk score for endometriosis and health conditions, blood and urine biomarkers and reproductive factors were investigated separately in females, males and females without an endometriosis diagnosis. The relationship between endometriosis and the blood and urine biomarkers was further investigated using genetic correlation and Mendelian randomisation approaches to identify causal relationships. RESULTS: Multiple health conditions, blood and urine biomarkers and reproductive factors were associated with genetic liability to endometriosis in each group, indicating many endometriosis comorbidities are not dependent on the physical manifestation of endometriosis. Differences in the associated traits between males and females highlighted the importance of sex-specific pathways in the overlap of endometriosis with many other traits. Notably, an association of genetic liability to endometriosis with lower testosterone levels was identified. Follow-up analysis utilising Mendelian randomisation approaches suggested lower testosterone may be causal for both endometriosis and clear cell ovarian cancer. CONCLUSIONS: This study highlights the diversity of the pleiotropic effects of genetic risk to endometriosis irrespective of a diagnosis of endometriosis. A key finding was the identification of a causal effect of the genetic liability to lower testosterone on endometriosis using Mendelian randomisation.

Gene expression profiles separate endometriosis lesion subtypes and indicate a sensitivity of endometrioma to estrogen suppressive treatments through elevated ESR2 expression.

BACKGROUND: Endometriosis is a common, gynaecological disease characterised by the presence of endometrial-like cells growing outside the uterus. Lesions appear at multiple locations, present with variation in appearance, size and depth of invasion. Despite hormones being the recommended first-line treatment, their efficacy, success and side effects vary widely amongst study populations. Current, hormonal medication for endometriosis is designed to suppress systemic oestrogen. Whether these hormones can influence the lesions themselves is not yet clear. Evidence of hormone receptor expression in endometriotic lesions and their ability to respond is conflicting. A variation in their expression, activation of transcriptional co-regulators and the potential to respond may contribute to their variation in patient outcomes. Identifying patients who would benefit from hormonal treatments remain an important goal in endometriosis research. METHODS: Using gene expression data from endometriosis lesions including endometrioma (OMA, n = 28), superficial peritoneal lesions (SUP, n = 72) and deeply infiltrating lesions (DIE, n = 78), we performed principal component analysis, differential gene expression and gene correlation analyses to assess the impact of menstrual stage, lesion subtype and hormonal treatment on the gene expression. RESULTS: The gene expression profiles did not vary based on menstrual stage, but could distinguish lesion subtypes with OMA significantly differentiating from both SUP and DIE. Additionally, the effect of oestrogen suppression medication altered the gene expression profile in OMA, while such effect was not observed in SUP or DIE. Analysis of the target receptors for hormonal medication indicated ESR2 was differentially expressed in OMA and that genes that correlated with ESR2 varied significantly between medicated and non-medicated OMA samples. CONCLUSIONS: Our results demonstrate of the different lesion types OMA present with strongest response to hormonal treatment directly through ESR2. The data suggests that there may be the potential to target treatment options to individual patients based on pre-surgical diagnoses.

A Common Genetic Factor Underlies Genetic Risk for Gynaecological and Reproductive Disorders and Is Correlated with Risk to Depression.

INTRODUCTION: Sex steroid hormone fluctuations may underlie both reproductive disorders and sex differences in lifetime depression prevalence. Previous studies report high comorbidity among reproductive disorders and between reproductive disorders and depression. This study sought to assess the multivariate genetic architecture of reproductive disorders and their loading onto a common genetic factor and investigated whether this latent factor shares a common genetic architecture with female depression, including perinatal depression (PND). METHOD: Using UK Biobank and FinnGen data, genome-wide association meta-analyses were conducted for nine reproductive disorders, and genetic correlation between disorders was estimated. Genomic Structural Equation Modelling identified a latent genetic factor underlying disorders, accounting for their significant genetic correlations. SNPs significantly associated with both latent factor and depression were identified. RESULTS: Excellent model fit existed between a latent factor underlying five reproductive disorders (χ2 (5) = 6.4; AIC = 26.4; CFI = 1.00; SRMR = 0.03) with high standardised loadings for menorrhagia (0.96, SE = 0.05); ovarian cysts (0.94, SE = 0.05); endometriosis (0.83, SE = 0.05); menopausal symptoms (0.77, SE = 0.10); and uterine fibroids (0.65, SE = 0.05). This latent factor was genetically correlated with PND (rG = 0.37, SE = 0.15, p = 1.4e-03), depression in females only (rG = 0.48, SE = 0.06, p = 7.2e-11), and depression in both males and females (MD) (rG = 0.35, SE = 0.03, p = 1.8e-30), with its top locus associated with FSHB/ARL14EP (rs11031006; p = 9.1e-33). SNPs intronic to ESR1, significantly associated with the latent factor, were also associated with PND, female depression, and MD. CONCLUSION: A common genetic factor, correlated with depression, underlies risk of reproductive disorders, with implications for aetiology and treatment. Genetic variation in ESR1 is associated with reproductive disorders and depression, highlighting the importance of oestrogen signalling for both reproductive and mental health.

Associations between endometriosis and common symptoms: findings from the Australian Longitudinal Study on Women's Health.

BACKGROUND: Endometriosis has been linked to higher rates of a variety of symptoms; however, the findings from longitudinal studies are scarce and inconsistent. OBJECTIVE: This study aimed to examine the association between endometriosis and common symptoms in a prospective cohort study. STUDY DESIGN: This study included 7606 women born from 1973 to 1978 using data from the Australian Longitudinal Study on Women's Health that were collected every 3 years from 2009 to 2018. We identified women with endometriosis based on self-reported incidence from each survey and linked administrative health data. At each survey, women also completed a checklist on the presence of 24 symptoms. Generalized estimating equations for multinomial responses were used for analyses. RESULTS: Women with endometriosis had significantly more menstrual symptoms than those without endometriosis with an adjusted odds ratio (95% confidence interval) of 3.61 (3.11-4.19) for severe period pain, 2.40 (2.10-2.74) for heavy menstrual bleeding, 1.76 (1.52-2.03) for irregular bleeding, and 1.52 (1.32-1.76) for premenstrual tension. They also had higher odds of mental health problems with adjusted odds ratios of 1.67 (1.39-2.01) for depression and 1.59 (1.24-2.03) for anxiety and higher odds of allergies and nonspecific symptoms with adjusted odds of 1.62 (1.40-1.89) for allergies or hay fever or sinusitis, 1.79 (1.56-2.05) for severe tiredness, 1.56 (1.35-1.81) for sleep difficulty, and 1.77 (1.37-2.18) for palpitations. There was also a strong association with other forms of pain with an adjusted odds ratio of 1.76 (1.53-2.04) for backpain, 1.50 (1.29-1.74) for headaches or migraines, and 1.65 (1.41-1.93) for stiff or painful joints. Women with endometriosis also had increased odds of developing bowel and urinary symptoms with an adjusted odds ratio (95% confidence interval) of 1.67 (1.35-2.08) for constipation, 1.46 (1.12-1.90) for hemorrhoids or piles, 1.25 (1.03-1.52) for indigestion or heartburn, 2.80 (1.71-4.58) for urine burn or stings, and 1.37 (1.03-1.82) for vaginal discharge or irritation. The association between each symptom and endometriosis was similar whether endometriosis was surgically confirmed or clinically suspected. No association was found between endometriosis and the risk for skin problems, leaking urine, or breathing difficulty. CONCLUSION: This study suggests that women with endometriosis are more likely to report not only menstrual symptoms but are also at an increased risk for mental health problems, other pain symptoms, bowel and urinary symptoms, and nonspecific symptoms, such as severe tiredness and difficulty sleeping.

New concepts on the etiology of endometriosis.

Endometriosis is a serious, chronic disorder where endometrial tissue grows outside the uterus, causing severe pelvic pain and infertility. It affects 11% of women. Endometriosis is a multifactorial disorder of unclear etiology, although retrograde menstruation plays a major role. It has a genetic component with over 40 genetic risk factors mapped, although their mechanism of action is still emerging. New evidence suggests a role for retrograde menstruation of endometrial stem/progenitor cells, now that identifying markers of these cells are available. Recent lineage tracing and tissue clearing microscopy and 3D reconstruction has provided new understanding of endometrial glandular structure, particularly the horizontal orientation and interconnection of basalis glands. New sequencing technologies, particularly whole genome DNA sequencing are revealing somatic mutations, including in cancer driver genes, in normal and eutopic endometrium of patients with endometriosis, as well as ectopic endometriotic lesions. Methylome sequencing is offering insight into the regulation of genes and the role of the environmental factors. Single cell RNA sequencing reveals the transcriptome of individual endometrial cells, shedding new light on the diversity and range of cellular subpopulations of the major cell types present in the endometrium and in endometriotic lesions. New endometrial epithelial organoid cultures replicating glandular epithelium are providing tractable models for studying endometriosis. Organoids derived from menstrual fluid offer a non-invasive source of endometrial tissue and a new avenue for testing drugs and developing personalized medicine for treating endometriosis. These new approaches are rapidly advancing our understanding of endometriosis etiology.

Genetic analyses of gynecological disease identify genetic relationships between uterine fibroids and endometrial cancer, and a novel endometrial cancer genetic risk region at the WNT4 1p36.12 locus.

Endometriosis, polycystic ovary syndrome (PCOS) and uterine fibroids have been proposed as endometrial cancer risk factors; however, disentangling their relationships with endometrial cancer is complicated due to shared risk factors and comorbidities. Using genome-wide association study (GWAS) data, we explored the relationships between these non-cancerous gynecological diseases and endometrial cancer risk by assessing genetic correlation, causal relationships and shared risk loci. We found significant genetic correlation between endometrial cancer and PCOS, and uterine fibroids. Adjustment for genetically predicted body mass index (a risk factor for PCOS, uterine fibroids and endometrial cancer) substantially attenuated the genetic correlation between endometrial cancer and PCOS but did not affect the correlation with uterine fibroids. Mendelian randomization analyses suggested a causal relationship between only uterine fibroids and endometrial cancer. Gene-based analyses revealed risk regions shared between endometrial cancer and endometriosis, and uterine fibroids. Multi-trait GWAS analysis of endometrial cancer and the genetically correlated gynecological diseases identified a novel genome-wide significant endometrial cancer risk locus at 1p36.12, which replicated in an independent endometrial cancer dataset. Interrogation of functional genomic data at 1p36.12 revealed biologically relevant genes, including WNT4 which is necessary for the development of the female reproductive system. In summary, our study provides genetic evidence for a causal relationship between uterine fibroids and endometrial cancer. It further provides evidence that the comorbidity of endometrial cancer, PCOS and uterine fibroids may partly be due to shared genetic architecture. Notably, this shared architecture has revealed a novel genome-wide risk locus for endometrial cancer.

Elucidating the role of long intergenic non-coding RNA 339 in human endometrium and endometriosis.

Endometriosis is a complex disease, influenced by genetic factors. Genetic markers associated with endometriosis exist at chromosome 1p36.12 and lead to altered expression of the long intergenic non-coding RNA 339 (LINC00339), however, the role of LINC00339 in endometriosis pathophysiology remains unknown. The aim of this work was to characterize the expression patterns of LINC00339 mRNA in endometrium and endometriotic lesions in situ and to determine the functional role of LINC00339 in human endometrium. We employed RNA-sequencing (RNA-seq), quantitative RT-PCR and in situ hybridization to investigate the abundance of LINC00339 transcripts in endometrium and endometrial cell lines and to describe the pattern and localization of LINC00339 expression in endometrium and endometriotic lesions. LINC00339 mRNA expression was manipulated (overexpressed and silenced) in endometrial stromal cell lines and RNA-seq data from overexpression models were analysed using online bioinformatics platforms (STRING and Ingenuity Pathway Analysis) to determine functional processes. We demonstrated the expression of LINC00339 in endometriotic lesions for the first time; we found LINC00339 expression was restricted to the lesion foci and absent in surrounding non-lesion tissue. Furthermore, manipulation of LINC00339 expression in endometrial stromal cell lines significantly impacted the expression of genes involved in immune defence pathways. These studies identify a novel mechanism for LINC00339 activity in endometrium and endometriosis, paving the way for future work, which is essential for understanding the pathogenesis of endometriosis.

Genetic Regulation of Physiological Reproductive Lifespan and Female Fertility.

There is substantial genetic variation for common traits associated with reproductive lifespan and for common diseases influencing female fertility. Progress in high-throughput sequencing and genome-wide association studies (GWAS) have transformed our understanding of common genetic risk factors for complex traits and diseases influencing reproductive lifespan and fertility. The data emerging from GWAS demonstrate the utility of genetics to explain epidemiological observations, revealing shared biological pathways linking puberty timing, fertility, reproductive ageing and health outcomes. The observations also identify unique genetic risk factors specific to different reproductive diseases impacting on female fertility. Sequencing in patients with primary ovarian insufficiency (POI) have identified mutations in a large number of genes while GWAS have revealed shared genetic risk factors for POI and ovarian ageing. Studies on age at menopause implicate DNA damage/repair genes with implications for follicle health and ageing. In addition to the discovery of individual genes and pathways, the increasingly powerful studies on common genetic risk factors help interpret the underlying relationships and direction of causation in the regulation of reproductive lifespan, fertility and related traits.

Tissue specific regulation of transcription in endometrium and association with disease.

STUDY QUESTION: Are genetic effects on endometrial gene expression tissue specific and/or associated with reproductive traits and diseases? SUMMARY ANSWER: Analyses of RNA-sequence data and individual genotype data from the endometrium identified novel and disease associated, genetic mechanisms regulating gene expression in the endometrium and showed evidence that these mechanisms are shared across biologically similar tissues. WHAT IS KNOWN ALREADY: The endometrium is a complex tissue vital for female reproduction and is a hypothesized source of cells initiating endometriosis. Understanding genetic regulation specific to, and shared between, tissue types can aid the identification of genes involved in complex genetic diseases. STUDY DESIGN, SIZE, DURATION: RNA-sequence and genotype data from 206 individuals was analysed and results were compared with large publicly available datasets. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA-sequencing and genotype data from 206 endometrial samples was used to identify the influence of genetic variants on gene expression, via expression quantitative trait loci (eQTL) analysis and to compare these endometrial eQTLs with those in other tissues. To investigate the association between endometrial gene expression regulation and reproductive traits and diseases, we conducted a tissue enrichment analysis, transcriptome-wide association study (TWAS) and summary data-based Mendelian randomisation (SMR) analyses. Transcriptomic data was used to test differential gene expression between women with and without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE: A tissue enrichment analysis with endometriosis genome-wide association study summary statistics showed that genes surrounding endometriosis risk loci were significantly enriched in reproductive tissues. A total of 444 sentinel cis-eQTLs (P < 2.57 × 10-9) and 30 trans-eQTLs (P < 4.65 × 10-13) were detected, including 327 novel cis-eQTLs in endometrium. A large proportion (85%) of endometrial eQTLs are present in other tissues. Genetic effects on endometrial gene expression were highly correlated with the genetic effects on reproductive (e.g. uterus, ovary) and digestive tissues (e.g. salivary gland, stomach), supporting a shared genetic regulation of gene expression in biologically similar tissues. The TWAS analysis indicated that gene expression at 39 loci is associated with endometriosis, including five known endometriosis risk loci. SMR analyses identified potential target genes pleiotropically or causally associated with reproductive traits and diseases including endometriosis. However, without taking account of genetic variants, a direct comparison between women with and without endometriosis showed no significant difference in endometrial gene expression. LARGE SCALE DATA: The eQTL dataset generated in this study is available at http://reproductivegenomics.com.au/shiny/endo_eqtl_rna/. Additional datasets supporting the conclusions of this article are included within the article and the supplementary information files, or are available on reasonable request. LIMITATIONS, REASONS FOR CAUTION: Data are derived from fresh tissue samples and expression levels are an average of expression from different cell types within the endometrium. Subtle cell-specifc expression changes may not be detected and differences in cell composition between samples and across the menstrual cycle will contribute to sample variability. Power to detect tissue specific eQTLs and differences between women with and without endometriosis was limited by the sample size in this study. The statistical approaches used in this study identify the likely gene targets for specific genetic risk factors, but not the functional mechanism by which changes in gene expression may influence disease risk. WIDER IMPLICATIONS OF THE FINDINGS: Our results identify novel genetic variants that regulate gene expression in endometrium and the majority of these are shared across tissues. This allows analysis with large publicly available datasets to identify targets for female reproductive traits and diseases. Much larger studies will be required to identify genetic regulation of gene expression that will be specific to endometrium. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Health and Medical Research Council (NHMRC) under project grants GNT1026033, GNT1049472, GNT1046880, GNT1050208, GNT1105321, GNT1083405 and GNT1107258. G.W.M is supported by a NHMRC Fellowship (GNT1078399). J.Y is supported by an ARC Fellowship (FT180100186). There are no competing interests.

Obesity does not alter endometrial gene expression in women with endometriosis.

RESEARCH QUESTION: Does obesity affect endometrial gene expression in women with endometriosis, specifically women with stage I disease? DESIGN: Differential gene expression analysis was conducted on endometrium from women with and without endometriosis (n = 169). Women were diagnosed after surgical visualization and staged according to the revised American Society for Reproductive Medicine (stage I-IV). Women were grouped by body mass index (BMI) (kg/m2) as underweight, normal, pre-obese or obese. After accounting for menstrual cycle stage, endometrial gene expression was analysed by BMI (continuous and grouped) in women with endometriosis, and in non-endometriosis controls. RESULTS: No significant interaction effect was found between BMI and endometriosis status on endometrial gene expression. We have previously reported that obese women with endometriosis have a reduced incidence of stage I disease; however, stratifying our analysis into stage I endometriosis versus combined II, III and IV endometriosis failed to reveal any differentially expressed endometrial genes between normal, pre-obese and obese patients. CONCLUSIONS: Despite obesity having deleterious effects on endometrial gene expression in other gynaecological pathologies, e.g. endometrial cancer and polycystic ovary syndrome, our results do not support an association between BMI and altered endometrial gene expression in women with or without endometriosis.

Should Genetics Now Be Considered the Pre-eminent Etiologic Factor in Endometriosis?

Endometriosis is a common, estrogen-dependent, inflammatory disorder characterized by the growth of endometrial-like tissue at extrauterine locations. Its pathogenesis and mechanisms underlying its pathophysiology are poorly understood, although genetic variation is strongly implicated in these processes. Genetic studies reveal that approximately 50% of risk for endometriosis is due to genetic factors and the other 50% likely owing to environmental factors. As with other complex diseases, genetic variants in the DNA sequence increasing endometriosis risk all have small effects, unlike most single-gene disorders. It is the combinations of these variants adding together that contribute to higher risks for individual women. In addition, recent data on disease lesions demonstrate a high frequency of somatic (likely acquired) mutations, some of which are present in the eutopic endometrium and specifically in the epithelial cell compartment, raising the possibility that abnormal epithelial progenitors in the eutopic endometrium give rise to ectopic disease. Discovery in this field is occurring at a rapid pace, and further definitions of genetic (germline) and environmental (somatic) contributions to the pathogenesis and pathophysiology of this disorder are anticipated soon. These discoveries are expected to increase diagnostic, therapeutic, and preventive strategies to minimize disease and its associated morbidities.

Genome-wide association studies of 74 plasma metabolites of German shepherd dogs reveal two metabolites associated with genes encoding their enzymes.

INTRODUCTION: German shepherd dogs (GSDs) are a popular breed affected by numerous disorders. Few studies have explored genetic variations that influence canine blood metabolite levels. OBJECTIVES: To investigate genetic variants affecting the natural metabolite variation in GSDs. METHODS: A total of 82 healthy GSDs were genotyped on the Illumina CanineHD Beadchip, assaying 173,650 markers. For each dog, 74 metabolites were measured through liquid and gas chromatography mass spectrometry (LC-MS and GC-MS) and were used as phenotypes for genome-wide association analyses (GWAS). Sliding window and homozygosity analyses were conducted to fine-map regions of interest, and to identify haplotypes and gene dosage effects. RESULTS: Summary statistics for 74 metabolites in this population of GSDs are reported. Forty-one metabolites had significant associations at a false discovery rate of 0.05. Two associations were located around genes which encode for enzymes for the relevant metabolites: 4-hydroxyproline was significantly associated to D-amino acid oxidase (DAO), and threonine to L-threonine 3-dehydrogenase (LOC477365). Three of the top ten haplotypes associated to 4-hydroxyproline included at least one SNP on DAO. These haplotypes occurred only in dogs with the highest 15 measurements of 4-hydroxyproline, ranging in frequency from 16.67 to 20%. None of the dogs were homozygous for these haplotypes. The top two haplotypes associated to threonine included SNPs on LOC477365 and were also overrepresented in dogs with the highest 15 measurements of threonine. These haplotypes occurred at a frequency of 90%, with 80% of these dogs homozygous for the haplotypes. In dogs with the lowest 15 measurements of threonine, the haplotypes occurred at a frequency of 26.67% and 0% homozygosity. CONCLUSION: DAO and LOC477365 were identified as candidate genes affecting the natural plasma concentration of 4-hydroxyproline and threonine, respectively. Further investigations are needed to validate the effects of the variants on these genes.

Risk of Iron Deficiency in Women With Endometriosis: A Population-Based Prospective Cohort Study.

BACKGROUND: Endometriosis may be linked to the risk of iron deficiency through chronic systemic inflammation or heavy menstrual bleeding. No longitudinal studies, however, have examined the relationship between endometriosis and the risk of iron deficiency. METHODS: This study included 3,294 participants born from 1973 to 1978 and followed as part of the Australian Longitudinal Study on Women's Health from 2000 to 2018. Participants with endometriosis were identified using self-reported longitudinal surveys linked to administrative health records. During each survey, participants were also asked to report the diagnosis of iron deficiency, and we validated diagnoses using an administrative health database. Generalized estimating equations for binary responses with an autoregressive correlation matrix were used to examine the association between endometriosis and the risk of iron deficiency over the seven time points. FINDINGS: We found that women with endometriosis had a significantly higher risk of iron deficiency than those without endometriosis after adjusting for sociodemographic, lifestyle, reproductive, and nutrition factors (adjusted odds ratio [aOR] = 1.46; 95% confidence interval [CI] [1.29, 1.66]; p < .0001). Women with a surgically confirmed diagnosis and those with clinically suspected endometriosis had a higher risk of iron deficiency (aOR = 1.38; 95% CI [1.17, 1.64] and aOR = 1.53; 95% CI [1.30, 1.81]), respectively. These associations, however, were slightly attenuated (by 8%) when adjusted for the presence of heavy menstrual bleeding. CONCLUSIONS: Women with endometriosis are at a higher risk of developing iron deficiency than those without endometriosis. The findings suggest that iron deficiency should be concomitantly addressed during initial diagnosis and successive management of endometriosis.

Insights from Mendelian randomization and genetic correlation analyses into the relationship between endometriosis and its comorbidities.

BACKGROUND: Endometriosis remains a poorly understood disease, despite its high prevalence and debilitating symptoms. The overlap in symptoms and the increased risk of multiple other traits in women with endometriosis is becoming increasingly apparent through epidemiological data. Genetic studies offer a method of investigating these comorbid relationships through the assessment of causal relationships with Mendelian randomization (MR), as well as identification of shared genetic variants and genes involved across traits. This has the capacity to identify risk factors for endometriosis as well as provide insight into the aetiology of disease. OBJECTIVE AND RATIONALE: We aim to review the current literature assessing the relationship between endometriosis and other traits using genomic data, primarily through the methods of MR and genetic correlation. We critically examine the limitations of these studies in accordance with the assumptions of the utilized methods. SEARCH METHODS: The PubMed database was used to search for peer-reviewed original research articles using the terms 'Mendelian randomization endometriosis' and '"genetic correlation" endometriosis'. Additionally, a Google Scholar search using the terms '"endometriosis" "mendelian randomization" "genetic correlation"' was performed. All relevant publications (n = 21) published up until 7 October 2022 were included in this review. Upon compilation of all traits with published MR and/or genetic correlation with endometriosis, additional epidemiological and genetic information on their comorbidity with endometriosis was sourced by searching for the trait in conjunction with 'endometriosis' on Google Scholar. OUTCOMES: The association between endometriosis and multiple pain, gynaecological, cancer, inflammatory, gastrointestinal, psychological, and anthropometric traits has been assessed using MR analysis and genetic correlation analysis. Genetic correlation analyses provide evidence that genetic factors contributing to endometriosis are shared with multiple traits: migraine, uterine fibroids, subtypes of ovarian cancer, melanoma, asthma, gastro-oesophageal reflux disease, gastritis/duodenitis, and depression, suggesting the involvement of multiple biological mechanisms in endometriosis. The assessment of causality with MR has revealed several potential causes (e.g. depression) and outcomes (e.g. ovarian cancer and uterine fibroids) of a genetic predisposition to endometriosis; however, interpretation of these results requires consideration of potential violations of the MR assumptions. WIDER IMPLICATIONS: Genomic studies have demonstrated that there is a molecular basis for the co-occurrence of endometriosis with other traits. Dissection of this overlap has identified shared genes and pathways, which provide insight into the biology of endometriosis. Thoughtful MR studies are necessary to ascertain causality of the comorbidities of endometriosis. Given the significant diagnostic delay of endometriosis of 7-11 years, determining risk factors is necessary to aid diagnosis and reduce the disease burden. Identification of traits for which endometriosis is a risk factor is important for holistic treatment and counselling of the patient. The use of genomic data to disentangle the overlap of endometriosis with other traits has provided insights into the aetiology of endometriosis.

Impact of endometriosis on women's health-related quality of life: A national prospective cohort study.

OBJECTIVE: To examine the association between endometriosis and women's health-related quality of life (HRQoL). STUDY DESIGN: This study included 3728 women born in 1973-78 using data from the Australian Longitudinal Study on Women's Health. Women with endometriosis were identified using self-reported longitudinal surveys linked to administrative health records. A mixed effect model with only random intercept and generalised estimating equations with binary logistic regressions were used to examine the association between endometriosis and health-related quality of life over eight time points. Each HRQoL scale was analysed in terms of binary outcomes by comparing women who had a lower HRQoL (scoring below the 25th percentile) with those who had a higher HRQoL (scoring above the 25th percentile). MAIN OUTCOME MEASURES: Women's HRQoL was assessed using the 36-item Short Form Survey every 3 years from 1996 to 2018. RESULTS: Endometriosis was associated with significantly worse reports of HRQoL over time. In the comparison against women without endometriosis, the following adjusted odds ratios (95 % confidence intervals) were calculated for women with endometriosis having worse scores on the eight domains of the Short Form Survey: physical functioning 1.33 (1.19, 1.50), role physical 1.57 (1.41, 1.74), bodily pain 1.65 (1.48, 1.82), general health 1.61 (1.42, 1.81), vitality 1.38 (1.23, 1.55), social functioning 1.38 (1.25, 1.53), role emotion 1.19 (1.06, 1.33), mental health 1.32 (1.18, 1.48). Women with endometriosis also had significantly lower physical health 1.68 (1.51, 1.88) and mental health components scores 1.28 (1.14, 1.44). CONCLUSIONS: Endometriosis is associated with worse physical, mental, and social functioning and well-being. Bodily pain was the most affected HRQoL domain.

Evidence of shared genetic factors in the etiology of gastrointestinal disorders and endometriosis and clinical implications for disease management.

In clinical practice, the co-existence of endometriosis and gastrointestinal symptoms is often observed. Using large-scale datasets, we report a genetic correlation between endometriosis and irritable bowel syndrome (IBS), peptic ulcer disease (PUD), gastro-esophageal reflux disease (GORD), and a combined GORD/PUD medicated (GPM) phenotype. Mendelian randomization analyses support a causal relationship between genetic predisposition to endometriosis and IBS and GPM. Identification of shared risk loci highlights biological pathways that may contribute to the pathogenesis of both diseases, including estrogen regulation and inflammation, and potential therapeutic drug targets (CCKBR; PDE4B). Higher use of IBS, GORD, and PUD medications in women with endometriosis and higher use of hormone therapies in women with IBS, GORD, and PUD, support the co-occurrence of these conditions and highlight the potential for drug repositioning and drug contraindications. Our results provide evidence of shared disease etiology and have important clinical implications for diagnostic and treatment decisions for both diseases.

Global Analysis of Transcription Start Sites and Enhancers in Endometrial Stromal Cells and Differences Associated with Endometriosis.

Identifying tissue-specific molecular signatures of active regulatory elements is critical to understanding gene regulatory mechanisms. In this study, transcription start sites (TSS) and enhancers were identified using Cap analysis of gene expression (CAGE) across endometrial stromal cell (ESC) samples obtained from women with (n = 4) and without endometriosis (n = 4). ESC TSSs and enhancers were compared to those reported in other tissue and cell types in FANTOM5 and were integrated with RNA-seq and ATAC-seq data from the same samples for regulatory activity and network analyses. CAGE tag count differences between women with and without endometriosis were statistically tested and tags within close proximity to genetic variants associated with endometriosis risk were identified. Over 90% of tag clusters mapping to promoters were observed in cells and tissues in FANTOM5. However, some potential cell-type-specific promoters and enhancers were also observed. Regions of open chromatin identified using ATAC-seq provided further evidence of the active transcriptional regions identified by CAGE. Despite the small sample number, there was evidence of differences associated with endometriosis at 210 consensus clusters, including IGFBP5, CALD1 and OXTR. ESC TSSs were also located within loci associated with endometriosis risk from genome-wide association studies. This study provides novel evidence of transcriptional differences in endometrial stromal cells associated with endometriosis and provides a valuable cell-type specific resource of active TSSs and enhancers in endometrial stromal cells.