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.

Top 10 priorities for future infertility research: an international consensus development study.

STUDY QUESTION: Can the priorities for future research in infertility be identified? SUMMARY ANSWER: The top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care for people with fertility problems were identified. WHAT IS KNOWN ALREADY: Many fundamental questions regarding the prevention, management, and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems. STUDY DESIGN, SIZE, DURATION: Potential research questions were collated from an initial international survey, a systematic review of clinical practice guidelines, and Cochrane systematic reviews. A rationalized list of confirmed research uncertainties was prioritized in an interim international survey. Prioritized research uncertainties were discussed during a consensus development meeting. Using a formal consensus development method, the modified nominal group technique, diverse stakeholders identified the top 10 research priorities for each of the categories male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, people with fertility problems, and others (healthcare funders, healthcare providers, healthcare regulators, research funding bodies and researchers) were brought together in an open and transparent process using formal consensus methods advocated by the James Lind Alliance. MAIN RESULTS AND THE ROLE OF CHANCE: The initial survey was completed by 388 participants from 40 countries, and 423 potential research questions were submitted. Fourteen clinical practice guidelines and 162 Cochrane systematic reviews identified a further 236 potential research questions. A rationalized list of 231 confirmed research uncertainties were entered into an interim prioritization survey completed by 317 respondents from 43 countries. The top 10 research priorities for each of the four categories male infertility, female and unexplained infertility (including age-related infertility, ovarian cysts, uterine cavity abnormalities, and tubal factor infertility), medically assisted reproduction (including ovarian stimulation, IUI, and IVF), and ethics, access, and organization of care, were identified during a consensus development meeting involving 41 participants from 11 countries. These research priorities were diverse and seek answers to questions regarding prevention, treatment, and the longer-term impact of infertility. They highlight the importance of pursuing research which has often been overlooked, including addressing the emotional and psychological impact of infertility, improving access to fertility treatment, particularly in lower resource settings, and securing appropriate regulation. Addressing these priorities will require diverse research methodologies, including laboratory-based science, qualitative and quantitative research, and population science. LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods, which have inherent limitations, including the representativeness of the participant sample, methodological decisions informed by professional judgement, and arbitrary consensus definitions. WIDER IMPLICATIONS OF THE FINDINGS: We anticipate that identified research priorities, developed to specifically highlight the most pressing clinical needs as perceived by healthcare professionals, people with fertility problems, and others, will help research funding organizations and researchers to develop their future research agenda. STUDY FUNDING/ COMPETING INTEREST(S): The study was funded by the Auckland Medical Research Foundation, Catalyst Fund, Royal Society of New Zealand, and Maurice and Phyllis Paykel Trust. Geoffrey Adamson reports research sponsorship from Abbott, personal fees from Abbott and LabCorp, a financial interest in Advanced Reproductive Care, committee membership of the FIGO Committee on Reproductive Medicine, International Committee for Monitoring Assisted Reproductive Technologies, International Federation of Fertility Societies, and World Endometriosis Research Foundation, and research sponsorship of the International Committee for Monitoring Assisted Reproductive Technologies from Abbott and Ferring. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and editor for the Cochrane Gynaecology and Fertility Group. Hans Evers reports being the Editor Emeritus of Human Reproduction. Andrew Horne reports research sponsorship from the Chief Scientist's Office, Ferring, Medical Research Council, National Institute for Health Research, and Wellbeing of Women and consultancy fees from Abbvie, Ferring, Nordic Pharma, and Roche Diagnostics. M. Louise Hull reports grants from Merck, grants from Myovant, grants from Bayer, outside the submitted work and ownership in Embrace Fertility, a private fertility company. Neil Johnson reports research sponsorship from Abb-Vie and Myovant Sciences and consultancy fees from Guerbet, Myovant Sciences, Roche Diagnostics, and Vifor Pharma. José Knijnenburg reports research sponsorship from Ferring and Theramex. Richard Legro reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. Ben Mol reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. Ernest Ng reports research sponsorship from Merck. Craig Niederberger reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and retains a financial interest in NexHand. Jane Stewart reports being employed by a National Health Service fertility clinic, consultancy fees from Merck for educational events, sponsorship to attend a fertility conference from Ferring, and being a clinical subeditor of Human Fertility. Annika Strandell reports consultancy fees from Guerbet. Jack Wilkinson reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. Andy Vail reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and of the journal Reproduction. His employing institution has received payment from HFEA for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. Lan Vuong reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the present work. All authors have completed the disclosure form. TRIAL REGISTRATION NUMBER: Not applicable.

Developing a core outcome set for future infertility research: an international consensus development study.

STUDY QUESTION: Can a core outcome set to standardize outcome selection, collection, and reporting across future infertility research be developed? SUMMARY ANSWER: A minimum data set, known as a core outcome set, has been developed for randomized controlled trials (RCT) and systematic reviews evaluating potential treatments for infertility. WHAT IS KNOWN ALREADY: Complex issues, including a failure to consider the perspectives of people with fertility problems when selecting outcomes, variations in outcome definitions, and the selective reporting of outcomes on the basis of statistical analysis, make the results of infertility research difficult to interpret. STUDY DESIGN, SIZE, DURATION: A three-round Delphi survey (372 participants from 41 countries) and consensus development workshop (30 participants from 27 countries). PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, researchers, and people with fertility problems were brought together in an open and transparent process using formal consensus science methods. MAIN RESULTS AND THE ROLE OF CHANCE: The core outcome set consists of: viable intrauterine pregnancy confirmed by ultrasound (accounting for singleton, twin, and higher multiple pregnancy); pregnancy loss (accounting for ectopic pregnancy, miscarriage, stillbirth, and termination of pregnancy); live birth; gestational age at delivery; birthweight; neonatal mortality; and major congenital anomaly. Time to pregnancy leading to live birth should be reported when applicable. LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods which have inherent limitations, including the representativeness of the participant sample, Delphi survey attrition, and an arbitrary consensus threshold. WIDER IMPLICATIONS OF THE FINDINGS: Embedding the core outcome set within RCTs and systematic reviews should ensure the comprehensive selection, collection, and reporting of core outcomes. Research funding bodies, the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) statement, and over 80 specialty journals, including the Cochrane Gynaecology and Fertility Group, Ferility and Sterility, and Human Reproduction, have committed to implementing this core outcome set. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Catalyst Fund, Royal Society of New Zealand, Auckland Medical Research Fund, and Maurice and Phyllis Paykel Trust. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and an editor of the Cochrane Gynaecology and Fertility group. Hans Evers reports being the Editor Emeritus of Human Reproduction. José Knijnenburg reports research sponsorship from Ferring and Theramex. Richard Legro reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. Ben Mol reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. Craig Niederberger reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and retains a financial interest in NexHand. Annika Strandell reports consultancy fees from Guerbet. Ernest Ng reports research sponsorship from Merck. Lan Vuong reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the work presented. All authors have completed the disclosure form. TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials Initiative: 1023.

Standardizing definitions and reporting guidelines for the infertility core outcome set: an international consensus development study.

STUDY QUESTION: Can consensus definitions for the core outcome set for infertility be identified in order to recommend a standardized approach to reporting? SUMMARY ANSWER: Consensus definitions for individual core outcomes, contextual statements, and a standardized reporting table have been developed. WHAT IS KNOWN ALREADY: Different definitions exist for individual core outcomes for infertility. This variation increases the opportunities for researchers to engage with selective outcome reporting, which undermines secondary research and compromises clinical practice guideline development. STUDY DESIGN, SIZE, DURATION: Potential definitions were identified by a systematic review of definition development initiatives and clinical practice guidelines and by reviewing Cochrane Gynaecology and Fertility Group guidelines. These definitions were discussed in a face-to-face consensus development meeting, which agreed consensus definitions. A standardized approach to reporting was also developed as part of the process. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, researchers, and people with fertility problems were brought together in an open and transparent process using formal consensus development methods. MAIN RESULTS AND THE ROLE OF CHANCE: Forty-four potential definitions were inventoried across four definition development initiatives, including the Harbin Consensus Conference Workshop Group and International Committee for Monitoring Assisted Reproductive Technologies, 12 clinical practice guidelines, and Cochrane Gynaecology and Fertility Group guidelines. Twenty-seven participants, from 11 countries, contributed to the consensus development meeting. Consensus definitions were successfully developed for all core outcomes. Specific recommendations were made to improve reporting. LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods, which have inherent limitations. There was limited representation from low- and middle-income countries. WIDER IMPLICATIONS OF THE FINDINGS: A minimum data set should assist researchers in populating protocols, case report forms, and other data collection tools. The generic reporting table should provide clear guidance to researchers and improve the reporting of their results within journal publications and conference presentations. Research funding bodies, the Standard Protocol Items: Recommendations for Interventional Trials statement, and over 80 specialty journals have committed to implementing this core outcome set. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Catalyst Fund, Royal Society of New Zealand, Auckland Medical Research Fund, and Maurice and Phyllis Paykel Trust. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and an editor of the Cochrane Gynaecology and Fertility group. Hans Evers reports being the Editor Emeritus of Human Reproduction. Richard Legro reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. Ben Mol reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. Craig Niederberger reports being the Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and a financial interest in NexHand. Ernest Ng reports research sponsorship from Merck. Annika Strandell reports consultancy fees from Guerbet. Jack Wilkinson reports being a statistical editor for the Cochrane Gynaecology and Fertility group. Andy Vail reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and of the journal Reproduction. His employing institution has received payment from HFEA for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. Lan Vuong reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the work presented. All authors have completed the disclosure form. TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials Initiative: 1023.

Standardizing definitions and reporting guidelines for the infertility core outcome set: an international consensus development study† ‡.

STUDY QUESTION: Can consensus definitions for the core outcome set for infertility be identified in order to recommend a standardized approach to reporting? SUMMARY ANSWER: Consensus definitions for individual core outcomes, contextual statements and a standardized reporting table have been developed. WHAT IS KNOWN ALREADY: Different definitions exist for individual core outcomes for infertility. This variation increases the opportunities for researchers to engage with selective outcome reporting, which undermines secondary research and compromises clinical practice guideline development. STUDY DESIGN, SIZE, DURATION: Potential definitions were identified by a systematic review of definition development initiatives and clinical practice guidelines and by reviewing Cochrane Gynaecology and Fertility Group guidelines. These definitions were discussed in a face-to-face consensus development meeting, which agreed consensus definitions. A standardized approach to reporting was also developed as part of the process. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, researchers and people with fertility problems were brought together in an open and transparent process using formal consensus development methods. MAIN RESULTS AND THE ROLE OF CHANCE: Forty-four potential definitions were inventoried across four definition development initiatives, including the Harbin Consensus Conference Workshop Group and International Committee for Monitoring Assisted Reproductive Technologies, 12 clinical practice guidelines and Cochrane Gynaecology and Fertility Group guidelines. Twenty-seven participants, from 11 countries, contributed to the consensus development meeting. Consensus definitions were successfully developed for all core outcomes. Specific recommendations were made to improve reporting. LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods, which have inherent limitations. There was limited representation from low- and middle-income countries. WIDER IMPLICATIONS OF THE FINDINGS: A minimum data set should assist researchers in populating protocols, case report forms and other data collection tools. The generic reporting table should provide clear guidance to researchers and improve the reporting of their results within journal publications and conference presentations. Research funding bodies, the Standard Protocol Items: Recommendations for Interventional Trials statement, and over 80 specialty journals have committed to implementing this core outcome set. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Catalyst Fund, Royal Society of New Zealand, Auckland Medical Research Fund and Maurice and Phyllis Paykel Trust. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and an editor of the Cochrane Gynaecology and Fertility Group. J.L.H.E. reports being the Editor Emeritus of Human Reproduction. R.S.L. reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. B.W.M. reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. C.N. reports being the Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and a financial interest in NexHand. E.H.Y.N. reports research sponsorship from Merck. A.S. reports consultancy fees from Guerbet. J.W. reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. A.V. reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and of the journal Reproduction. His employing institution has received payment from Human Fertilisation and Embryology Authority for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. N.L.V. reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the work presented. All authors have completed the disclosure form. TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials Initiative: 1023.

Top 10 priorities for future infertility research: an international consensus development study† ‡.

STUDY QUESTION: Can the priorities for future research in infertility be identified? SUMMARY ANSWER: The top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction and ethics, access and organization of care for people with fertility problems were identified. WHAT IS KNOWN ALREADY: Many fundamental questions regarding the prevention, management and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems. STUDY DESIGN, SIZE, DURATION: Potential research questions were collated from an initial international survey, a systematic review of clinical practice guidelines and Cochrane systematic reviews. A rationalized list of confirmed research uncertainties was prioritized in an interim international survey. Prioritized research uncertainties were discussed during a consensus development meeting. Using a formal consensus development method, the modified nominal group technique, diverse stakeholders identified the top 10 research priorities for each of the categories male infertility, female and unexplained infertility, medically assisted reproduction and ethics, access and organization of care. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, people with fertility problems and others (healthcare funders, healthcare providers, healthcare regulators, research funding bodies and researchers) were brought together in an open and transparent process using formal consensus methods advocated by the James Lind Alliance. MAIN RESULTS AND THE ROLE OF CHANCE: The initial survey was completed by 388 participants from 40 countries, and 423 potential research questions were submitted. Fourteen clinical practice guidelines and 162 Cochrane systematic reviews identified a further 236 potential research questions. A rationalized list of 231 confirmed research uncertainties was entered into an interim prioritization survey completed by 317 respondents from 43 countries. The top 10 research priorities for each of the four categories male infertility, female and unexplained infertility (including age-related infertility, ovarian cysts, uterine cavity abnormalities and tubal factor infertility), medically assisted reproduction (including ovarian stimulation, IUI and IVF) and ethics, access and organization of care were identified during a consensus development meeting involving 41 participants from 11 countries. These research priorities were diverse and seek answers to questions regarding prevention, treatment and the longer-term impact of infertility. They highlight the importance of pursuing research which has often been overlooked, including addressing the emotional and psychological impact of infertility, improving access to fertility treatment, particularly in lower resource settings and securing appropriate regulation. Addressing these priorities will require diverse research methodologies, including laboratory-based science, qualitative and quantitative research and population science. LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods, which have inherent limitations, including the representativeness of the participant sample, methodological decisions informed by professional judgment and arbitrary consensus definitions. WIDER IMPLICATIONS OF THE FINDINGS: We anticipate that identified research priorities, developed to specifically highlight the most pressing clinical needs as perceived by healthcare professionals, people with fertility problems and others, will help research funding organizations and researchers to develop their future research agenda. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Auckland Medical Research Foundation, Catalyst Fund, Royal Society of New Zealand and Maurice and Phyllis Paykel Trust. G.D.A. reports research sponsorship from Abbott, personal fees from Abbott and LabCorp, a financial interest in Advanced Reproductive Care, committee membership of the FIGO Committee on Reproductive Medicine, International Committee for Monitoring Assisted Reproductive Technologies, International Federation of Fertility Societies and World Endometriosis Research Foundation, and research sponsorship of the International Committee for Monitoring Assisted Reproductive Technologies from Abbott and Ferring. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and editor for the Cochrane Gynaecology and Fertility Group. J.L.H.E. reports being the Editor Emeritus of Human Reproduction. A.W.H. reports research sponsorship from the Chief Scientist's Office, Ferring, Medical Research Council, National Institute for Health Research and Wellbeing of Women and consultancy fees from AbbVie, Ferring, Nordic Pharma and Roche Diagnostics. M.L.H. reports grants from Merck, grants from Myovant, grants from Bayer, outside the submitted work and ownership in Embrace Fertility, a private fertility company. N.P.J. reports research sponsorship from AbbVie and Myovant Sciences and consultancy fees from Guerbet, Myovant Sciences, Roche Diagnostics and Vifor Pharma. J.M.L.K. reports research sponsorship from Ferring and Theramex. R.S.L. reports consultancy fees from AbbVie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. B.W.M. reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. E.H.Y.N. reports research sponsorship from Merck. C.N. reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring and retains a financial interest in NexHand. J.S. reports being employed by a National Health Service fertility clinic, consultancy fees from Merck for educational events, sponsorship to attend a fertility conference from Ferring and being a clinical subeditor of Human Fertility. A.S. reports consultancy fees from Guerbet. J.W. reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. A.V. reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and the journal Reproduction. His employing institution has received payment from Human Fertilisation and Embryology Authority for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. N.L.V. reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the present work. All authors have completed the disclosure form. TRIAL REGISTRATION NUMBER: N/A.

Developing a core outcome set for future infertility research: an international consensus development study† ‡.

STUDY QUESTION: Can a core outcome set to standardize outcome selection, collection and reporting across future infertility research be developed? SUMMARY ANSWER: A minimum data set, known as a core outcome set, has been developed for randomized controlled trials (RCTs) and systematic reviews evaluating potential treatments for infertility. WHAT IS KNOWN ALREADY: Complex issues, including a failure to consider the perspectives of people with fertility problems when selecting outcomes, variations in outcome definitions and the selective reporting of outcomes on the basis of statistical analysis, make the results of infertility research difficult to interpret. STUDY DESIGN, SIZE, DURATION: A three-round Delphi survey (372 participants from 41 countries) and consensus development workshop (30 participants from 27 countries). PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, researchers and people with fertility problems were brought together in an open and transparent process using formal consensus science methods. MAIN RESULTS AND THE ROLE OF CHANCE: The core outcome set consists of: viable intrauterine pregnancy confirmed by ultrasound (accounting for singleton, twin and higher multiple pregnancy); pregnancy loss (accounting for ectopic pregnancy, miscarriage, stillbirth and termination of pregnancy); live birth; gestational age at delivery; birthweight; neonatal mortality; and major congenital anomaly. Time to pregnancy leading to live birth should be reported when applicable. LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods which have inherent limitations, including the representativeness of the participant sample, Delphi survey attrition and an arbitrary consensus threshold. WIDER IMPLICATIONS OF THE FINDINGS: Embedding the core outcome set within RCTs and systematic reviews should ensure the comprehensive selection, collection and reporting of core outcomes. Research funding bodies, the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) statement, and over 80 specialty journals, including the Cochrane Gynaecology and Fertility Group, Fertility and Sterility and Human Reproduction, have committed to implementing this core outcome set. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Catalyst Fund, Royal Society of New Zealand, Auckland Medical Research Fund and Maurice and Phyllis Paykel Trust. The funder had no role in the design and conduct of the study, the collection, management, analysis or interpretation of data, or manuscript preparation. B.W.J.M. is supported by a National Health and Medical Research Council Practitioner Fellowship (GNT1082548). S.B. was supported by University of Auckland Foundation Seelye Travelling Fellowship. S.B. reports being the Editor-in-Chief of Human Reproduction Open and an editor of the Cochrane Gynaecology and Fertility group. J.L.H.E. reports being the Editor Emeritus of Human Reproduction. J.M.L.K. reports research sponsorship from Ferring and Theramex. R.S.L. reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. B.W.J.M. reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. C.N. reports being the Co Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and retains a financial interest in NexHand. A.S. reports consultancy fees from Guerbet. E.H.Y.N. reports research sponsorship from Merck. N.L.V. reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the work presented. All authors have completed the disclosure form. TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials Initiative: 1023.

Effects of depot-medroxyprogesterone acetate on the immune microenvironment of the human cervix and endometrium: implications for HIV susceptibility.

Depot-medroxyprogesterone acetate is a commonly used injectable contraceptive that has been associated with an increased risk of HIV acquisition. This study compares effects of depot-medroxyprogesterone acetate on immune parameters from several upper reproductive tract compartments relevant to HIV-1 susceptibility in repetitive samples from 15 depot-medroxyprogesterone acetate users and 27 women not on hormonal contraceptives. Compared with samples from unexposed women in the mid-luteal phase, depot-medroxyprogesterone acetate use was associated with: increased endocervical concentrations of MCP1 and IFNalpha2; decreased endocervical concentrations of IL1beta and IL6; increased proportions of endometrial CD4+ and CD8+ cells expressing the activation marker HLADR; increased density of endometrial macrophages; and decreased density of endometrial regulatory T cells. Unlike previous reports with samples from the vagina, we did not observe increased expression of the HIV co-receptor CCR5 on CD4+ T cells in the endocervix or endometrium. Our results indicate important differences in anatomic compartments regarding mechanisms by which depot-medroxyprogesterone acetate could be associated with increased risk of HIV acquisition, including increased recruitment of macrophages to the endometrium, decreased levels of pro-inflammatory cytokines in the endocervix possibly leading to enhanced susceptibility to viral infection, and activation of endometrial T cells.

Endometrial stromal fibroblasts from women with polycystic ovary syndrome have impaired progesterone-mediated decidualization, aberrant cytokine profiles and promote enhanced immune cell migration in vitro.

STUDY QUESTION: Do endometrial stromal fibroblasts (eSF) in women with polycystic ovary syndrome (PCOS) (eSFpcos) exhibit altered estrogen and/or progesterone (P4) responses, which may explain some of the adverse reproductive outcomes and endometrial pathologies in these women? SUMMARY ANSWER: In vitro, eSF from women with PCOS exhibit an aberrant decidualization response and concomitant changes in pro-inflammatory cytokine, chemokine and matrix metalloproteinase (MMP) release and immune cell chemoattraction. In vivo these aberrations may result in suboptimal implantation and predisposition to endometrial cancer. WHAT IS KNOWN ALREADY: The endometrium in women with PCOS has several abnormalities including progesterone (P4) resistance at the gene expression level, likely contributing to subfertility, pregnancy complications and increased endometrial cancer risk in PCOS women. STUDY DESIGN, SIZE, DURATION: Prospective, university-based, case-control, in vitro study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cultures of eSFPCOS (n = 12, Rotterdam and NIH criteria) and eSFControl (Ctrl) (n = 6, regular cycle length, no signs of hyperandrogenism) were treated with vehicle, estradiol (E2, 10 nM) or E2P4 (10 nM/1 µM) for 14 days. Progesterone receptor (PGR) mRNA was assessed with quantitative real-time PCR (qRT-PCR) and eSF decidualization was confirmed by insulin-like growth factor-binding protein-1 (IGFBP-1) transcript and protein expression. Fractalkine (CX3CL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 6, 8 and 11, macrophage chemoattractant protein (MCP) 1 and 3, CCL5 (RANTES) and MMPs (MMP1, 2, 3, 7, 9, 10 and 12) were measured in conditioned media by Luminex multiplex assays, and chemotactic activity of the conditioned media was tested in a migration assay using CD14+ monocyte and CD4+ T-cell migration assay. Effects of IL-6 (0.02, 0.2, 2 or 20 ng/ml) or IL-8 (0.04, 0.4, 4, or 40 ng/ml) or combination (0.2 ng/ml IL-6 and 4.0 ng/ml IL-8) on 14-d decidualization were also tested. ANOVA with pre-planned contrasts was used for statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Hormonal challenge with E2P4 to induce decidualization revealed two distinct subsets of eSFPCOS. Eight eSFPCOS (dPCOS) and all eSFCtrl (dCtrl) cultures showed a normal decidualization response to E2P4 as determined by morphology and IGFBP-1 secretion. However, 4 eSFPCOS cultures showed blunted decidualization (ndPCOS) in morphological assessment and low IGFBP-1 levels even though all three groups exhibited normal estrogen-mediated increase in PGR expression. Interestingly dPCOS had decreased IL-6 and GM-SCF secretion compared with dCtrl, whereas the ndPCOS cultures showed increased IL-6 and 8, MCP1, RANTES and GM-CSF secretion at base-line and/or in response to E2 or E2P4 compared with dCtrl and/or dPCOS. Furthermore, even though PGR expression was similar in all three groups, P4 inhibition of MMP secretion was attenuated in ndPCOS resulting in higher MMP2 and 3 levels. The conditioned media from ndPCOS had increased chemoattractic activity compared with dCtrl and dPCOS media. Exogenously added IL-6 and/or 8 did not inhibit decidualization in eSFCtrl indicating that high levels of these cytokines in ndPCOS samples were not likely a cause for the aberrant decidualization. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study with a small sample size, utilizing stromal cell cultures from proliferative and secretory phase endometrium. The effect of PCOS on endometrial epithelium, another major histoarchitectural cell compartment of the endometrium, was not evaluated and should be considered in future studies. Furthermore, results obtained should also be confirmed in a larger data set and with mid/late secretory phase in vivo samples and models. WIDER IMPLICATIONS OF THE FINDINGS: The alterations seen in ndPCOS may contribute to endometrial dysfunction, subfertility and pregnancy complications in PCOS women. The results emphasize the importance of understanding immune responses related to the implantation process and normal endometrial homeostasis in women with PCOS. STUDY FUNDING/COMPETING INTERESTS: Sigrid Juselius Foundation, Academy of Finland, Finnish Medical Foundation, Orion-Farmos Research Foundation (to T.T.P.), the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) U54HD 055764-07 Specialized Cooperative Centers Program in Reproduction and Infertility Research (to L.C.G.), the NICHD the Ruth L. Kirschstein National Research Service Awards grant 1F32HD074423-03 (to J.C.C.). The authors have no competing interests.

Impact of surgical excision of lesions on pain in a rat model of endometriosis.

BACKGROUND: Chronic pain is the most common and disabling feature of endometriosis. Surgical excision of endometriosis lesions provides relief but pain relapse is common. Studies in a preclinical model of endometriosis might help to unravel the role of the ectopic lesions as the source of pain. Thus, we evaluated the impact of lesion excision on mechanical hyperalgesia in a preclinical model of endometriosis pain. METHODS: Endometriosis was induced by implanting autologous uterine tissue onto the gastrocnemius muscle. Surgical excision or aspiration drainage of the cystic lesion was performed at different times post-implant and mechanical nociceptive thresholds were assessed at the site of the lesion. RESULTS: Lesions at 2, 8 and 16 weeks post-implant produced mechanical hyperalgesia of similar magnitude (n = 6/group). Excision of lesions (n = 6/group) produced a longer inhibition, with a magnitude and time course depending upon the timing of excision. Excision at 2 and 8 weeks produced a rapid onset marked attenuation of hyperalgesia, which returned to pre-excision values by post-surgical week 3. In contrast, excision of the lesion at 16 weeks produced a peak of inhibition of hyperalgesia 2 weeks post-excision, but then the inhibition was sustained. Aspiration of fluid from cysts in the lesions briefly attenuated mechanical hyperalgesia (n = 6/group). CONCLUSIONS: In this preclinical model, we demonstrate that endometriosis pain is alleviated by surgical excision of the ectopic lesion or drainage of its cysts, providing support for the clinical observation that endometriosis pain is dependent upon the ongoing presence of the lesions.

Ectopic endometrium-derived leptin produces estrogen-dependent chronic pain in a rat model of endometriosis.

Endometriosis pain is a very common and extremely disabling condition whose mechanism is still poorly understood. While increased levels of leptin have been reported in patients with endometriosis, their contribution to endometriosis pain has not been explored. Using a rodent model of endometriosis we provide evidence for an estrogen-dependent contribution of leptin in endometriosis-induced pain. Rats implanted with autologous uterine tissue onto the gastrocnemius muscle developed endometriosis-like lesions and local chronic pain. Compared to eutopic uterine tissue, leptin mRNA and protein were up-regulated in the endometriosis-like lesions. Intramuscular injection of recombinant leptin in naive rats produced dose-dependent local mechanical hyperalgesia and nociceptor sensitization to mechanical stimulation. Ovariectomy attenuated the mechanical hyperalgesia induced by recombinant leptin, in rats treated with vehicle compared to those treated with 17ß-estradiol replacement, at 1 and 24 h after leptin injection. Finally, intralesional injections of a pegylated leptin receptor (Ob-R) antagonist or of an inhibitor of Janus kinase2, which transduces the Ob-R signal, markedly attenuated pain in the endometriosis model. Taken together these data support the hypothesis that leptin, generated in ectopic endometrial lesions produces mechanical hyperalgesia by acting on nociceptors innervating the lesion. This sensitivity to leptin is dependent on estrogen levels. Thus, interventions targeting leptin signaling, especially in combination with interventions that lower estrogen levels, might be useful for the treatment of endometriosis pain.

The human oviduct transcriptome reveals an anti-inflammatory, anti-angiogenic, secretory and matrix-stable environment during embryo transit.

The human oviduct serves as a conduit for spermatozoa in the peri-ovulatory phase and nurtures and facilitates transport of the developing embryo for nidation during the luteal phase. Interactions between the embryo and oviductal epithelial surface proteins and secreted products during embryo transit are largely undefined. This study investigated gene expression in the human oviduct in the early luteal versus follicular phases to identify candidate genes and biomolecular processes that may participate in maturation and transport of the embryo as it traverses this tissue. Oviductal RNA was hybridized to oligonucleotide arrays and resulting data were analysed by bioinformatic approaches. There were 650 genes significantly down-regulated and 683 genes significantly up-regulated (P<0.05) in the luteal versus follicular phase. Quantitative real-time PCR, immunoblot analysis and immunohistochemistry confirmed selected gene expression and cellular protein localization. Down-regulated genes involved macrophage recruitment, immunomodulation and matrix-degeneration, and up-regulated genes involved anti-inflammatory, ion transport, anti-angiogenic and early pregnancy recognition. The oviduct displayed some similarities and differences in progesterone-regulated genes compared with the human endometrium. Together, these data suggest a unique hormonally regulated environment during embryo development, maturation and transport through human oviduct and some conservation of progesterone signalling in tissues of common embryological origin. The oviduct serves as a conduit for spermatozoa in the peri-ovulatory phase and it nurtures and facilitates transport of the developing embryo during the luteal phase of the menstrual cycle, although precise interactions between the embryo and oviductal epithelium and secreted products are largely undefined. Herein, we investigated gene expression in human oviduct to identify candidate genes and processes that may participate in maturation and transport of the embryo as it develops implantation competence. Total RNA from human ampullary oviducts in the early luteal versus follicular phases was isolated and hybridized to oligonucleotide arrays. The data, analysed by bioinformatic approaches, revealed that 650 genes were significantly down- and 683 genes were significantly up-regulated in the luteal phase. Quantitative real-time PCR, immunoblot analysis and immunohistochemistry confirmed selected gene expression and cellular protein localization. The data demonstrated down-regulation of genes involved in macrophage recruitment, immunomodulation and matrix degeneration and up-regulation of ion transport and secretions, as well as anti-angiogenic and early pregnancy recognition. Together, these data suggest a unique hormonally regulated environment during embryo development, maturation and transport through the human oviduct and provide insight into mechanisms influencing acquisition of implantation competence of the human embryo during its passage through the oviduct en route to the uterine endometrium.

Mesenchymal stem/progenitors and other endometrial cell types from women with polycystic ovary syndrome (PCOS) display inflammatory and oncogenic potential.

CONTEXT: Endometrium in polycystic ovary syndrome (PCOS) presents altered gene expression indicating progesterone resistance and predisposing to reduced endometrial receptivity and endometrial cancer. OBJECTIVE: We hypothesized that an altered endocrine/metabolic environment in PCOS may result in an endometrial "disease phenotype" affecting the gene expression of different endometrial cell populations, including stem cells and their differentiated progeny. DESIGN AND SETTING: This was a prospective study conducted at an academic medical center. PATIENTS AND MAIN OUTCOME MEASURES: Proliferative-phase endometrium was obtained from 6 overweight/obese PCOS (National Institutes of Health criteria) and 6 overweight/obese controls. Microarray analysis was performed on fluorescence-activated cell sorting-isolated endometrial epithelial cells (eEPs), endothelial cells, stromal fibroblasts (eSFs), and mesenchymal stem cells (eMSCs). Gene expression data were validated using microfluidic quantitative RT-PCR and immunohistochemistry. RESULTS: The comparison between eEP(PCOS) and eEP(Ctrl) showed dysregulation of inflammatory genes and genes with oncogenic potential (CCL2, IL-6, ORM1, TNAIFP6, SFRP4, SPARC). eSF(PCOS) and eSF(Ctrl) showed up-regulation of inflammatory genes (C4A/B, CCL2, ICAM1, TNFAIP3). Similarly, in eMSC(PCOS) vs eMSC(Ctrl), the most up-regulated genes were related to inflammation and cancer (IL-8, ICAM1, SPRR3, LCN2). Immunohistochemistry scoring showed increased expression of CCL2 in eEP(PCOS) and eSF(PCOS) compared with eEP(Ctrl) and eSF(Ctrl) and IL-6 in eEP(PCOS) compared with eEP(Ctrl). CONCLUSIONS: Isolated endometrial cell populations in women with PCOS showed altered gene expression revealing inflammation and prooncogenic changes, independent of body mass index, especially in eEP(PCOS) and eMSC(PCOS), compared with controls. The study reveals an endometrial disease phenotype in women with PCOS with potential negative effects on endometrial function and long-term health.

Ectopic uterine tissue as a chronic pain generator.

While chronic pain is a main symptom in endometriosis, the underlying mechanisms and effective therapy remain elusive. We developed an animal model enabling the exploration of ectopic endometrium as a source of endometriosis pain. Rats were surgically implanted with autologous uterus in the gastrocnemius muscle. Within two weeks, visual inspection revealed the presence of a reddish-brown fluid-filled cystic structure at the implant site. Histology demonstrated cystic glandular structures with stromal invasion of the muscle. Immunohistochemical studies of these lesions revealed the presence of markers for nociceptor nerve fibers and neuronal sprouting. Fourteen days after surgery rats exhibited persistent mechanical hyperalgesia at the site of the ectopic endometrial lesion. Intralesional, but not contralateral, injection of progesterone was dose-dependently antihyperalgesic. Systemic administration of leuprolide also produced antihyperalgesia. In vivo electrophysiological recordings from sensory neurons innervating the lesion revealed a significant increase in their response to sustained mechanical stimulation. These results are consistent with clinical and pathological findings observed in patients with endometriosis, compatible with the ectopic endometrium as a source of pain. This model of endometriosis allows mechanistic exploration at the lesion site facilitating our understanding of endometriosis pain.

Effect of bisphenol A on human endometrial stromal fibroblasts in vitro.

This study evaluated the effects of bisphenol A (BPA) on human endometrial stromal fibroblast (ESF) differentiation and expression of genes involved in oestrogen metabolism. Human ESF from eight hysterectomy specimens were cultured and treated with 5-100 µmol/l of BPA ± oestradiol or 8-br-cAMP for 48 h. mRNA expression was analysed by real-time reverse-transcription PCR. 8-br-cAMP-induced human ESF decidualization was confirmed by expression of insulin-like growth factor binding protein-1 (IGFBP1) and prolactin secretion. Short-term exposure (48 h) decreased human ESF proliferation (P<0.04) not due to apoptosis. High doses of BPA significantly induced IGFBP1 mRNA and protein, decreased P450scc mRNA, reversed the 8-br-cAMP-induced increase in HSD17B2 (oestradiol to oestrone conversion) in a dose-dependent manner and down-regulated HSD17B1 expression (oestrone to oestradiol conversion; P ≤ 0.03). 8-br-cAMP significantly potentiated this effect (P=0.028). BPA had no significant effect on aromatase and PPAR γ expression. The oestrogen-receptor antagonist ICI had no effect on gene expression in BPA-treated cells, and oestrogen receptor α, but not oestrogen receptor ß, was significantly down-regulated by high doses of BPA (P=0.028). BPA has an endocrine-disrupting effect on human ESF function and gene expression but the underlying mechanisms appear not to involve oestrogen-mediated pathways.

Unique transcriptome, pathways, and networks in the human endometrial fibroblast response to progesterone in endometriosis.

Eutopic endometrium in endometriosis has molecular evidence of resistance to progesterone (P(4)) and activation of the PKA pathway in the stromal compartment. To investigate global and temporal responses of eutopic endometrium to P(4), we compared early (6-h), intermediate (48-h), and late (14-Day) transcriptomes, signaling pathways, and networks of human endometrial stromal fibroblasts (hESF) from women with endometriosis (hESF(endo)) with hESF from women without endometriosis (hESF(nonendo)). Endometrial biopsy samples were obtained from subjects with and without mild peritoneal endometriosis (n = 4 per group), and hESF were isolated and treated with P(4) (1 µM) plus estradiol (E(2)) (10 nM), E(2) alone (10 nM), or vehicle for up to 14 days. Total RNA was subjected to microarray analysis using a Gene 1.0 ST (Affymetrix) platform and analyzed by using bioinformatic algorithms, and data were validated by quantitative real-time PCR and ELISA. Results revealed unique kinetic expression of specific genes and unique pathways, distinct biological and molecular processes, and signaling pathways and networks during the early, intermediate, and late responses to P(4) in both hESF(nonendo) and hESF(endo), although a blunted response to P(4) was observed in the latter. The normal response of hESF to P(4) involves a tightly regulated kinetic cascade involving key components in the P(4) receptor and MAPK signaling pathways that results in inhibition of E(2)-mediated proliferation and eventual differentiation to the decidual phenotype, but this was not established in the hESF(endo) early response to P(4). The abnormal response of this cell type to P(4) may contribute to compromised embryonic implantation and infertility in women with endometriosis.

MicroRNA expression profiling of eutopic secretory endometrium in women with versus without endometriosis.

Endometriosis is a common gynecologic disorder characterized by pain and infertility. In addition to estrogen dependence, progesterone resistance is an emerging feature of this disorder. Specifically, a delayed transition from the proliferative to secretory phase as evidenced by dysregulation of progesterone target genes and maintenance of a proliferative molecular fingerprint in the early secretory endometrium (ESE) has been reported. MicroRNAs (miRNAs) are small noncoding RNAs that collectively represent a novel class of regulators of gene expression. In an effort to investigate further the observed progesterone resistance in the ESE of women with endometriosis, we conducted array-based, global miRNA profiling. We report distinct miRNA expression profiles in the ESE of women with versus without endometriosis in a subset of samples previously used in global gene expression analysis. Specifically, the miR-9 and miR-34 miRNA families evidenced dysregulation. Integration of the miRNA and gene expression profiles provides unique insights into the molecular basis of this enigmatic disorder and, possibly, the regulation of the proliferative phenotype during the early secretory phase of the menstrual cycle in affected women.

Steroidogenic enzyme and key decidualization marker dysregulation in endometrial stromal cells from women with versus without endometriosis.

Identification of mechanisms underlying endometriosis pathogenesis will facilitate understanding and treatment of infertility and pain associated with this disorder. Herein, we investigated the expression of steroidogenic pathway enzymes and key decidualization biomarkers in endometrial tissue and in eutopic endometrial stromal fibroblasts (hESFs) from women with vs. those without endometriosis, and subsequently treated in vitro with 8-bromo-cAMP (8-Br-cAMP) or progesterone (P4). Real-time quantitative PCR, immunohistochemistry, ELISA, and radiometric aromatase activity assay were used. The results demonstrate significantly increased (14.5-fold; P=0.037) expression of aromatase in eutopic endometrium of women with disease. In 8-Br-cAMP-treated hESF from eutopic endometrium of women with endometriosis, the balance in estradiol (E2) and P4 biosynthetic and metabolizing enzymes is disturbed (decreased HSD3B1 and HSD17B2, and increased HSD17B1 and aromatase), with the equilibrium being shifted towards an E2-enriched milieu. However, hESF from the same group of women treated with P4 did not demonstrate such responsiveness. Lower expression of IGFBP1 and prolactin mRNA and protein was observed in hESF from women with vs. those without endometriosis in response to 8-Br-cAMP, but not P4, suggesting a blunted response of these decidual biomarkers to activation of the PKA pathway in eutopic endometrium in women with disease. The dichotomy of 8-Br-cAMP regulation of select steroidogenic enzymes leading to an enriched E2 milieu within the endometrium and a blunted response of decidual biomarkers to this decidualizing agent of hESF from women with endometriosis suggests resistance to full decidualization of the stromal fibroblasts and mechanisms underlying implantation failure and the pathophysiology of this disorder.

Expression of membrane progesterone receptors on human T lymphocytes and Jurkat cells and activation of G-proteins by progesterone.

Although there is significant evidence for progesterone's role as an immunomodulator, nuclear progesterone receptors have not been consistently identified in immune cells. Recently, three new putative membrane progesterone receptors (mPRs), mPRalpha, mPRbeta, and mPRgamma have been described. The objective of this study was to examine whether mPRs are expressed in peripheral blood leukocytes (PBLs) in women of reproductive age, and to further characterize them in T lymphocytes and immortalized T cells (Jurkat cells). Transcripts for mPRalpha and mPRbeta but not mPRgamma, were detected by RT-PCR in PBLs, T lymphocytes, and Jurkat cells. Western blot analysis showed the presence of the mPRalpha and mPRbeta proteins on cell membranes of T lymphocytes and Jurkat cells. Expression of the mPRalpha mRNA was upregulated in the luteal phase of the menstrual cycle in cluster of differentiation (CD)8+, but not in CD4+, T lymphocytes. Radioreceptor assays revealed specific [(3)H]progesterone binding to T- and Jurkat cell membranes (K(d) 4.25 nM) characteristic of steroid membrane receptors. Progesterone activated an inhibitory G-protein (G(i)), suggesting that mPRs are coupled to G(i) in Jurkat cells. These results suggest a potential novel mechanism for progesterone's immunoregulatory function through activation of mPRs.

Uterine receptivity to human embryonic implantation: histology, biomarkers, and transcriptomics.

Embryonic implantation is a dynamic process of paracrine interactions between the maternal compartment and the conceptus and involves a receptive endometrium and a developmentally competent blastocyst. Herein, we review histology, clinical approaches, and the promise of transcriptomics in elucidating mechanisms underlying implantation and development of biomarkers of uterine receptivity-with an eye to diagnose and treat implantation-based disorders of miscarriage, fetal growth restriction, pre-eclampsia, and infertility.