Immune-Metabolic Interactions and T Cell Tolerance in Pregnancy.

Pregnancy depends on a state of maternal immune tolerance mediated by CD4+ regulatory T (Treg) cells. Uterine Treg cells release anti-inflammatory factors, inhibit effector immunity, and support adaptation of the uterine vasculature to facilitate placental development. Insufficient Treg cells or inadequate functional competence is implicated in infertility and recurrent miscarriage, as well as pregnancy complications preeclampsia, fetal growth restriction, and preterm birth, which stem from placental insufficiency. In this review we address an emerging area of interest in pregnancy immunology-the significance of metabolic status in regulating the Treg cell expansion required for maternal-fetal tolerance. We describe how hyperglycemia and insulin resistance affect T cell responses to suppress generation of Treg cells, summarize data that implicate a role for altered glucose metabolism in impaired maternal-fetal tolerance, and explore the prospect of targeting dysregulated metabolism to rebalance the adaptive immune response in women experiencing reproductive disorders.

Patient experiences of being advised by a healthcare professional to get pregnant to manage or treat endometriosis: a cross-sectional study.

BACKGROUND: There is a lack of evidence that pregnancy reduces endometriotic lesions or symptoms, however studies indicate that people with endometriosis are commonly advised to get pregnant to manage or treat endometriosis. This study sought to examine the impact of this advice on patients with endometriosis when the advice was provided by healthcare professionals. METHODS: The Endometriosis Patient Experience Survey was a self-reported, community-based, cross-sectional online survey of people who had been medically diagnosed with endometriosis. Descriptive statistics were used to analyse the quantitative survey data and thematic analysis was undertaken for the qualitative survey data. RESULTS: 1892 participants had received the advice to get pregnant or have a baby to manage or treat their endometriosis, with 89.4% of participants receiving this advice from healthcare professionals. In exploring the qualitative data, seven themes were contextualised relating to the impact of this advice in terms of health literacy, accepting the advice, rejecting the advice, major life decisions, healthcare interactions, mental health and relationships. CONCLUSIONS: This study demonstrates profound and often negative patient impacts of the advice from healthcare professionals to get pregnant to manage or treat endometriosis. Impacts ranged from planning for pregnancy, hastening the making of major life decisions, eroding trust with healthcare professionals, worsening mental health and straining relationships. Providing evidence-based information on the treatment and management of endometriosis is essential. Pregnancy or having a baby should not be suggested as a treatment for endometriosis and the provision of this advice by healthcare professionals can have negative impacts on those who receive it.

Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation.

STUDY QUESTION: How is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human? SUMMARY ANSWER: Podocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state. WHAT IS KNOWN ALREADY: The human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized. STUDY DESIGN, SIZE, DURATION: This study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates' in vivo expression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity using in vitro human embryo attachment and invasion models. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined the in vivo expression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined using in vitro attachment (n = 3-5) and invasion models (n = 4-6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann-Whitney U-test and ANOVA analyses established statistical significance at *P ≤ 0.05 and **P ≤ 0.01. MAIN RESULTS AND THE ROLE OF CHANCE: PCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCX in vivo for receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation. LIMITATIONS, REASONS FOR CAUTION: Primary HEECs isolated from the human endometrial tissue contained a mixture of luminal and glandular epithelial cells, as further purification into subtypes was not possible due to the lack of specific markers. Future study would need to investigate how progesterone differentially regulates PCX in endometrial epithelial subtypes. In addition, this study used primary human trophoblast spheroids as human embryo mimics and Ishikawa as endometrial epithelial cells in functional models, future studies with human blastocysts and primary epithelial cells would further validate the findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study add important new knowledge to the understanding of human endometrial remodeling for receptivity. The identification of PCX as a negative regulator of epithelial receptivity and the knowledge that its specific downregulation in the luminal epithelium coincides with receptivity development may provide new avenues to assess endometrial receptivity and individualize endometrial preparation protocols in assisted reproductive technology (ART). The study also discovered PCX as progesterone target in HEECs, identifying a potentially useful functional biomarker to monitor progesterone action, such as in the optimization of progesterone type/dose/route of administration for luteal support. STUDY FUNDING/COMPETING INTEREST(S): Study funding was obtained from ESHRE, Monash IVF and NHMRC. LR reports potential conflict of interests (received grants from Ferring Australia; personal fees from Monash IVF Group and Ferring Australia; and non-financial support from Merck Serono, MSD, and Guerbet outside the submitted work. LR is also a minority shareholder and the Group Medical Director for Monash IVF Group, a provider of fertility preservation services). The remaining authors have no potential conflict of interest to declare. TRIAL REGISTRATION NUMBER: NA.

The BlastGen study: a randomized controlled trial of blastocyst media supplemented with granulocyte-macrophage colony-stimulating factor.

RESEARCH QUESTION: Does Embryogen®/BlastGen™ culture medium improve live birth rates compared with standard culture medium for women undergoing IVF and intracytoplasmic sperm injection (ICSI) with poor prognosis. DESIGN: Randomized clinical trial. A total of 100 couples undergoing IVF/ICSI were randomly allocated to having their inseminated oocytes incubated in either Embryogen®/BlastGen™ sequential culture media or standard Cleavage/Blastocyst sequential culture media for 5 days (ClinicalTrials.gov Identifier: NCT02305420). RESULTS: No statistically significant difference in live birth rate was found between the control group and the Embryogen®/BlastGen™ group (17 [34%] versus 11 [22%], respectively) (OR 0.55; 95% CI 0.22 to 1.32; P = 0.18). After adjustment for maternal age, body mass index and fertilization procedure, the blastulation rate reduced (40.6 ± 26.5 versus 24.6 ± 26.7; RR 0.70, CI 0.52 to 0.95; P < 0.05), and grade of the embryo transferred (OR 0.35, CI 0.16 to 0.77; P < 0.01) when Embryogen®/BlastGen™ medium was used. CONCLUSION: A significant reduction in day-5 embryo outcome parameters was found using Embryogen®/BlastGen™ compared with standard medium, and insufficient evidence of a difference in pregnancy outcomes. Taking into consideration the small samples size, study limitations and strict inclusion criteria of this single-centre study, further research is needed to determine the efficacy of Embryogen®/BlastGen™ medium in couples undergoing IVF/ICSI.

Seminal Plasma Promotes Lesion Development in a Xenograft Model of Endometriosis.

The factors that predispose one-tenth of reproductive-aged women to endometriosis are poorly understood. We determined that genetic deficiency in transforming growth factor ß1 impairs endometriosis-like lesion growth in mice. Given that seminal plasma is an abundant source of transforming growth factor ß, we evaluated the effect of exposure to seminal plasma on the growth of endometrial lesions. Human endometrial explants were exposed to seminal plasma or to control medium before transfer to Prkdc(scid)-mutant (severe combined immunodeficient) mice. Xenografts exposed to seminal plasma showed an eightfold increase in volume and a 4.3-fold increase in weight after 14 days. These increases were associated with increased proliferation of endometrial epithelial cells and enhanced survival and proliferation of human stromal cells compared with those in control lesions, in which human stromal cell persistence was negligible. Although the distribution of macrophages was altered, their number and activation status did not change in response to seminal plasma. Seminal plasma stimulated the production of a variety of cytokines in endometrial tissue, including growth-regulated oncogene, granulocyte macrophage colony-stimulating factor, and IL-1ß. These data suggest that seminal plasma enhances the formation of endometriosis-like lesion via a direct effect on endometrial cell survival and proliferation, rather than via macrophage-mediated mechanisms. These findings raise the possibility that endometrial exposure to seminal plasma could contribute to endometriotic disease progression in women.

Corticosteroid therapy in assisted reproduction - immune suppression is a faulty premise.

There is ongoing interest in immune-suppressant corticosteroid drugs such as prednisolone to treat infertility in women with repeated IVF failure and recurrent miscarriage. The rationale draws on the pervasive but flawed view that immune activation is inconsistent with normal pregnancy. This ignores clear evidence that controlled inflammation and activation of the immune response is essential for embryo implantation. Generally, the immune response actively promotes reproductive success - by facilitating endometrial receptivity and tolerance of the foreign embryo, and promoting vascular adaptation to support placental morphogenesis. The peri-conception immune response also establishes developmental trajectories that can impact on fetal growth and gestational age at birth. Here, we describe immune changes accompanying conception that could be impeded by inappropriate corticosteroid administration. While women with specific clinical conditions may benefit from the anti-inflammatory and immune-deviating actions of prednisolone and related drugs, it is incorrect to assume a 'one-size-fits-all' approach. Better diagnostics and more preclinical studies are essential to define patient groups, build evidence for efficacy and fine-tune treatments so as not to inhibit essential actions of immune cells. We argue that unless overt immune pathology is evident, utilization of corticosteroids is not warranted and may be harmful. In most women, perturbing immune adaptation at implantation is expected to adversely influence placental development and impair immune-mediated quality control mechanisms, potentially elevating risk of altered fetal growth and developmental programming, congenital anomalies and preterm birth.

Imaging modalities for the non-invasive diagnosis of endometriosis.

BACKGROUND: About 10% of women of reproductive age suffer from endometriosis. Endometriosis is a costly chronic disease that causes pelvic pain and subfertility. Laparoscopy, the gold standard diagnostic test for endometriosis, is expensive and carries surgical risks. Currently, no non-invasive tests that can be used to accurately diagnose endometriosis are available in clinical practice. This is the first review of diagnostic test accuracy of imaging tests for endometriosis that uses Cochrane methods to provide an update on the rapidly expanding literature in this field. OBJECTIVES: • To provide estimates of the diagnostic accuracy of imaging modalities for the diagnosis of pelvic endometriosis, ovarian endometriosis and deeply infiltrating endometriosis (DIE) versus surgical diagnosis as a reference standard.• To describe performance of imaging tests for mapping of deep endometriotic lesions in the pelvis at specific anatomical sites.Imaging tests were evaluated as replacement tests for diagnostic surgery and as triage tests that would assist decision making regarding diagnostic surgery for endometriosis. SEARCH METHODS: We searched the following databases to 20 April 2015: MEDLINE, CENTRAL, EMBASE, CINAHL, PsycINFO, Web of Science, LILACS, OAIster, TRIP, ClinicalTrials.gov, MEDION, DARE, and PubMed. Searches were not restricted to a particular study design or language nor to specific publication dates. The search strategy incorporated words in the title, abstracts, text words across the record and medical subject headings (MeSH). SELECTION CRITERIA: We considered published peer-reviewed cross-sectional studies and randomised controlled trials of any size that included prospectively recruited women of reproductive age suspected of having one or more of the following target conditions: endometrioma, pelvic endometriosis, DIE or endometriotic lesions at specific intrapelvic anatomical locations. We included studies that compared the diagnostic test accuracy of one or more imaging modalities versus findings of surgical visualisation of endometriotic lesions. DATA COLLECTION AND ANALYSIS: Two review authors independently collected and performed a quality assessment of data from each study. For each imaging test, data were classified as positive or negative for surgical detection of endometriosis, and sensitivity and specificity estimates were calculated. If two or more tests were evaluated in the same cohort, each was considered as a separate data set. We used the bivariate model to obtain pooled estimates of sensitivity and specificity when sufficient data sets were available. Predetermined criteria for a clinically useful imaging test to replace diagnostic surgery included sensitivity ≥ 94% and specificity ≥ 79%. Criteria for triage tests were set at sensitivity ≥ 95% and specificity ≥ 50%, ruling out the diagnosis with a negative result (SnNout test - if sensitivity is high, a negative test rules out pathology) or at sensitivity ≥ 50% with specificity ≥ 95%, ruling in the diagnosis with a positive result (SpPin test - if specificity is high, a positive test rules in pathology). MAIN RESULTS: We included 49 studies involving 4807 women: 13 studies evaluated pelvic endometriosis, 10 endometriomas and 15 DIE, and 33 studies addressed endometriosis at specific anatomical sites. Most studies were of poor methodological quality. The most studied modalities were transvaginal ultrasound (TVUS) and magnetic resonance imaging (MRI), with outcome measures commonly demonstrating diversity in diagnostic estimates; however, sources of heterogeneity could not be reliably determined. No imaging test met the criteria for a replacement or triage test for detecting pelvic endometriosis, albeit TVUS approached the criteria for a SpPin triage test. For endometrioma, TVUS (eight studies, 765 participants; sensitivity 0.93 (95% confidence interval (CI) 0.87, 0.99), specificity 0.96 (95% CI 0.92, 0.99)) qualified as a SpPin triage test and approached the criteria for a replacement and SnNout triage test, whereas MRI (three studies, 179 participants; sensitivity 0.95 (95% CI 0.90, 1.00), specificity 0.91 (95% CI 0.86, 0.97)) met the criteria for a replacement and SnNout triage test and approached the criteria for a SpPin test. For DIE, TVUS (nine studies, 12 data sets, 934 participants; sensitivity 0.79 (95% CI 0.69, 0.89) and specificity 0.94 (95% CI 0.88, 1.00)) approached the criteria for a SpPin triage test, and MRI (six studies, seven data sets, 266 participants; sensitivity 0.94 (95% CI 0.90, 0.97), specificity 0.77 (95% CI 0.44, 1.00)) approached the criteria for a replacement and SnNout triage test. Other imaging tests assessed in small individual studies could not be statistically evaluated.TVUS met the criteria for a SpPin triage test in mapping DIE to uterosacral ligaments, rectovaginal septum, vaginal wall, pouch of Douglas (POD) and rectosigmoid. MRI met the criteria for a SpPin triage test for POD and vaginal and rectosigmoid endometriosis. Transrectal ultrasonography (TRUS) might qualify as a SpPin triage test for rectosigmoid involvement but could not be adequately assessed for other anatomical sites because heterogeneous data were scant. Multi-detector computerised tomography enema (MDCT-e) displayed the highest diagnostic performance for rectosigmoid and other bowel endometriosis and met the criteria for both SpPin and SnNout triage tests, but studies were too few to provide meaningful results.Diagnostic accuracies were higher for TVUS with bowel preparation (TVUS-BP) and rectal water contrast (RWC-TVS) and for 3.0TMRI than for conventional methods, although the paucity of studies precluded statistical evaluation. AUTHORS' CONCLUSIONS: None of the evaluated imaging modalities were able to detect overall pelvic endometriosis with enough accuracy that they would be suggested to replace surgery. Specifically for endometrioma, TVUS qualified as a SpPin triage test. MRI displayed sufficient accuracy to suggest utility as a replacement test, but the data were too scant to permit meaningful conclusions. TVUS could be used clinically to identify additional anatomical sites of DIE compared with MRI, thus facilitating preoperative planning. Rectosigmoid endometriosis was the only site that could be accurately mapped by using TVUS, TRUS, MRI or MDCT-e. Studies evaluating recent advances in imaging modalities such as TVUS-BP, RWC-TVS, 3.0TMRI and MDCT-e were observed to have high diagnostic accuracies but were too few to allow prudent evaluation of their diagnostic role. In view of the low quality of most of the included studies, the findings of this review should be interpreted with caution. Future well-designed diagnostic studies undertaken to compare imaging tests for diagnostic test accuracy and costs are recommended.

Combination of the non-invasive tests for the diagnosis of endometriosis.

BACKGROUND: About 10% of women of reproductive age suffer from endometriosis, a costly chronic disease causing pelvic pain and subfertility. Laparoscopy is the gold standard diagnostic test for endometriosis, but is expensive and carries surgical risks. Currently, there are no non-invasive tests available in clinical practice to accurately diagnose endometriosis. This review assessed the diagnostic accuracy of combinations of different non-invasive testing modalities for endometriosis and provided a summary of all the reviews in the non-invasive tests for endometriosis series. OBJECTIVES: To estimate the diagnostic accuracy of any combination of non-invasive tests for the diagnosis of pelvic endometriosis (peritoneal and/or ovarian or deep infiltrating) compared to surgical diagnosis as a reference standard. The combined tests were evaluated as replacement tests for diagnostic surgery and triage tests to assist decision-making to undertake diagnostic surgery for endometriosis. SEARCH METHODS: We did not restrict the searches to particular study designs, language or publication dates. We searched CENTRAL to July 2015, MEDLINE and EMBASE to May 2015, as well as the following databases to April 2015: CINAHL, PsycINFO, Web of Science, LILACS, OAIster, TRIP, ClinicalTrials.gov, DARE and PubMed. SELECTION CRITERIA: We considered published, peer-reviewed, randomised controlled or cross-sectional studies of any size, including prospectively collected samples from any population of women of reproductive age suspected of having one or more of the following target conditions: ovarian, peritoneal or deep infiltrating endometriosis (DIE). We included studies comparing the diagnostic test accuracy of a combination of several testing modalities with the findings of surgical visualisation of endometriotic lesions. DATA COLLECTION AND ANALYSIS: Three review authors independently collected and performed a quality assessment of the data from each study by using the QUADAS-2 tool. For each test, the data were classified as positive or negative for the surgical detection of endometriosis and sensitivity and specificity estimates were calculated. The bivariate model was planned to obtain pooled estimates of sensitivity and specificity whenever sufficient data were available. The predetermined criteria for a clinically useful test to replace diagnostic surgery were a sensitivity of 0.94 and a specificity of 0.79 to detect endometriosis. We set the criteria for triage tests at a sensitivity of 0.95 and above and a specificity of 0.50 and above, which 'rules out' the diagnosis with high accuracy if there is a negative test result (SnOUT test), or a sensitivity of 0.50 and above and a specificity of 0.95 and above, which 'rules in' the diagnosis with high accuracy if there is a positive result (SpIN test). MAIN RESULTS: Eleven eligible studies included 1339 participants. All the studies were of poor methodological quality. Seven studies evaluated pelvic endometriosis, one study considered DIE and/or ovarian endometrioma, two studies differentiated endometrioma from other ovarian cysts and one study addressed mapping DIE at specific anatomical sites. Fifteen different diagnostic combinations were assessed, including blood, urinary or endometrial biomarkers, transvaginal ultrasound (TVUS) and clinical history or examination. We did not pool estimates of sensitivity and specificity, as each study analysed independent combinations of the non-invasive tests.Tests that met the criteria for a replacement test were: a combination of serum IL-6 (cut-off >15.4 pg/ml) and endometrial PGP 9.5 for pelvic endometriosis (sensitivity 1.00 (95% confidence interval (CI) 0.91 to 1.00), specificity 0.93 (95% CI, 0.80, 0.98) and the combination of vaginal examination and transvaginal ultrasound (TVUS) for rectal endometriosis (sensitivity 0.96 (95% CI 0.86 to 0.99), specificity 0.98 (95% CI 0.94 to 1.00)). Tests that met the criteria for SpIN triage tests for pelvic endometriosis were: 1. a multiplication of urine vitamin-D-binding protein (VDBP) and serum CA-125 (cut-off >2755) (sensitivity 0.74 (95% CI 0.60 to 0.84), specificity 0.97 (95% CI 0.86 to 1.00)) and 2. a combination of history (length of menses), serum CA-125 (cut-off >35 U/ml) and endometrial leukocytes (sensitivity 0.61 (95% CI 0.54 to 0.69), specificity 0.95 (95% CI 0.91 to 0.98)). For endometrioma, the following combinations qualified as SpIN test: 1. TVUS and either serum CA-125 (cut-off ≥25 U/ml) or CA 19.9 (cut-off ≥12 U/ml) (sensitivity 0.79 (95% CI 0.64 to 0.91), specificity 0.97 (95% CI 0.91 to 1.00)); 2. TVUS and serum CA 19.9 (cut-off ≥12 U/ml) (sensitivity 0.54 (95% CI 0.37 to 0.70), specificity 0.97 (95% CI 0.91 to 1.0)); 3-4. TVUS and serum CA-125 (cut-off ≥20 U/ml or cut-off ≥25 U/ml) (sensitivity 0.69 (95% CI 0.49 to 0.85), specificity 0.96 (95% CI 0.88 to 0.99)); 5. TVUS and serum CA-125 (cut-off ≥35 U/ml) (sensitivity 0.52 (95% CI 0.33 to 0.71), specificity 0.97 (95% CI 0.90 to 1.00)). A combination of vaginal examination and TVUS reached the threshold for a SpIN test for obliterated pouch of Douglas (sensitivity 0.87 (95% CI 0.69 to 0.96), specificity 0.98 (95% CI 0.95 to 1.00)), vaginal wall endometriosis (sensitivity 0.82 (95% CI 0.60 to 0.95), specificity 0.99 (95% CI 0.97 to 1.0)) and rectovaginal septum endometriosis (sensitivity 0.88 (95% CI 0.47 to 1.00), specificity 0.99 (95% CI 0.96 to 1.00)).All the tests were evaluated in individual studies and displayed wide CIs. Due to the heterogeneity and high risk of bias of the included studies, the clinical utility of the studied combination diagnostic tests for endometriosis remains unclear. AUTHORS' CONCLUSIONS: None of the biomarkers evaluated in this review could be evaluated in a meaningful way and there was insufficient or poor-quality evidence. Laparoscopy remains the gold standard for the diagnosis of endometriosis and using any non-invasive tests should only be undertaken in a research setting.

Endometrial biomarkers for the non-invasive diagnosis of endometriosis.

BACKGROUND: About 10% of reproductive-aged women suffer from endometriosis, which is a costly, chronic disease that causes pelvic pain and subfertility. Laparoscopy is the gold standard diagnostic test for endometriosis, but it is expensive and carries surgical risks. Currently, there are no non-invasive tests available in clinical practice that accurately diagnose endometriosis. This is the first diagnostic test accuracy review of endometrial biomarkers for endometriosis that utilises Cochrane methodologies, providing an update on the rapidly expanding literature in this field. OBJECTIVES: To determine the diagnostic accuracy of the endometrial biomarkers for pelvic endometriosis, using a surgical diagnosis as the reference standard. We evaluated the tests as replacement tests for diagnostic surgery and as triage tests to inform decisions to undertake surgery for endometriosis. SEARCH METHODS: We did not restrict the searches to particular study designs, language or publication dates. To identify trials, we searched the following databases: CENTRAL (2015, July), MEDLINE (inception to May 2015), EMBASE (inception to May 2015), CINAHL (inception to April 2015), PsycINFO (inception to April 2015), Web of Science (inception to April 2015), LILACS (inception to April 2015), OAIster (inception to April 2015), TRIP (inception to April 2015) and ClinicalTrials.gov (inception to April 2015). We searched DARE and PubMed databases up to April 2015 to identify reviews and guidelines as sources of references to potentially relevant studies. We also performed searches for papers recently published and not yet indexed in the major databases. The search strategies incorporated words in the title, abstract, text words across the record and the medical subject headings (MeSH). SELECTION CRITERIA: We considered published peer-reviewed, randomised controlled or cross-sectional studies of any size that included prospectively collected samples from any population of reproductive-aged women suspected of having one or more of the following target conditions: ovarian, peritoneal or deep infiltrating endometriosis (DIE). DATA COLLECTION AND ANALYSIS: Two authors independently extracted data from each study and performed a quality assessment. For each endometrial diagnostic test, we classified the data as positive or negative for the surgical detection of endometriosis and calculated the estimates of sensitivity and specificity. We considered two or more tests evaluated in the same cohort as separate data sets. We used the bivariate model to obtain pooled estimates of sensitivity and specificity whenever sufficient data were available. The predetermined criteria for a clinically useful test to replace diagnostic surgery was one with a sensitivity of 94% and a specificity of 79%. The criteria for triage tests were set at sensitivity at or above 95% and specificity at or above 50%, which in case of negative results rules out the diagnosis (SnOUT test) or sensitivity at or above 50% with specificity at or above 95%, which in case of positive result rules in the diagnosis (SpIN test). MAIN RESULTS: We included 54 studies involving 2729 participants, most of which were of poor methodological quality. The studies evaluated endometrial biomarkers either in specific phases of the menstrual cycle or outside of it, and the studies tested the biomarkers either in menstrual fluid, in whole endometrial tissue or in separate endometrial components. Twenty-seven studies evaluated the diagnostic performance of 22 endometrial biomarkers for endometriosis. These were angiogenesis and growth factors (PROK-1), cell-adhesion molecules (integrins α3ß1, α4ß1, ß1 and α6), DNA-repair molecules (hTERT), endometrial and mitochondrial proteome, hormonal markers (CYP19, 17ßHSD2, ER-α, ER-ß), inflammatory markers (IL-1R2), myogenic markers (caldesmon, CALD-1), neural markers (PGP 9.5, VIP, CGRP, SP, NPY, NF) and tumour markers (CA-125). Most of these biomarkers were assessed in single studies, whilst only data for PGP 9.5 and CYP19 were available for meta-analysis. These two biomarkers demonstrated significant diversity for the diagnostic estimates between the studies; however, the data were too limited to reliably determine the sources of heterogeneity. The mean sensitivities and specificities of PGP 9.5 (7 studies, 361 women) were 0.96 (95% confidence interval (CI) 0.91 to 1.00) and 0.86 (95% CI 0.70 to 1.00), after excluding one outlier study, and for CYP19 (8 studies, 444 women), they were were 0.77 (95% CI 0.70 to 0.85) and 0.74 (95% CI 0.65 to 84), respectively. We could not statistically evaluate other biomarkers in a meaningful way. An additional 31 studies evaluated 77 biomarkers that showed no evidence of differences in expression levels between the groups of women with and without endometriosis. AUTHORS' CONCLUSIONS: We could not statistically evaluate most of the biomarkers assessed in this review in a meaningful way. In view of the low quality of most of the included studies, the findings of this review should be interpreted with caution. Although PGP 9.5 met the criteria for a replacement test, it demonstrated considerable inter study heterogeneity in diagnostic estimates, the source of which could not be determined. Several endometrial biomarkers, such as endometrial proteome, 17ßHSD2, IL-1R2, caldesmon and other neural markers (VIP, CGRP, SP, NPY and combination of VIP, PGP 9.5 and SP) showed promising evidence of diagnostic accuracy, but there was insufficient or poor quality evidence for any clinical recommendations. Laparoscopy remains the gold standard for the diagnosis of endometriosis, and using any non-invasive tests should only be undertaken in a research setting. We have also identified a number of biomarkers that demonstrated no diagnostic value for endometriosis. We recommend that researchers direct future studies towards biomarkers with high diagnostic potential in good quality diagnostic studies.

Blood biomarkers for the non-invasive diagnosis of endometriosis.

BACKGROUND: About 10% of reproductive-aged women suffer from endometriosis, a costly chronic disease causing pelvic pain and subfertility. Laparoscopy is the gold standard diagnostic test for endometriosis, but is expensive and carries surgical risks. Currently, there are no non-invasive or minimally invasive tests available in clinical practice to accurately diagnose endometriosis. Although other reviews have assessed the ability of blood tests to diagnose endometriosis, this is the first review to use Cochrane methods, providing an update on the rapidly expanding literature in this field. OBJECTIVES: To evaluate blood biomarkers as replacement tests for diagnostic surgery and as triage tests to inform decisions on surgery for endometriosis. Specific objectives include:1. To provide summary estimates of the diagnostic accuracy of blood biomarkers for the diagnosis of peritoneal, ovarian and deep infiltrating pelvic endometriosis, compared to surgical diagnosis as a reference standard.2. To assess the diagnostic utility of biomarkers that could differentiate ovarian endometrioma from other ovarian masses. SEARCH METHODS: We did not restrict the searches to particular study designs, language or publication dates. We searched CENTRAL to July 2015, MEDLINE and EMBASE to May 2015, as well as these databases to 20 April 2015: CINAHL, PsycINFO, Web of Science, LILACS, OAIster, TRIP, ClinicalTrials.gov, DARE and PubMed. SELECTION CRITERIA: We considered published, peer-reviewed, randomised controlled or cross-sectional studies of any size, including prospectively collected samples from any population of reproductive-aged women suspected of having one or more of the following target conditions: ovarian, peritoneal or deep infiltrating endometriosis (DIE). We included studies comparing the diagnostic test accuracy of one or more blood biomarkers with the findings of surgical visualisation of endometriotic lesions. DATA COLLECTION AND ANALYSIS: Two authors independently collected and performed a quality assessment of data from each study. For each diagnostic test, we classified the data as positive or negative for the surgical detection of endometriosis, and we calculated sensitivity and specificity estimates. We used the bivariate model to obtain pooled estimates of sensitivity and specificity whenever sufficient datasets were available. The predetermined criteria for a clinically useful blood test to replace diagnostic surgery were a sensitivity of 0.94 and a specificity of 0.79 to detect endometriosis. We set the criteria for triage tests at a sensitivity of ≥ 0.95 and a specificity of ≥ 0.50, which 'rules out' the diagnosis with high accuracy if there is a negative test result (SnOUT test), or a sensitivity of ≥ 0.50 and a specificity of ≥ 0.95, which 'rules in' the diagnosis with high accuracy if there is a positive result (SpIN test). MAIN RESULTS: We included 141 studies that involved 15,141 participants and evaluated 122 blood biomarkers. All the studies were of poor methodological quality. Studies evaluated the blood biomarkers either in a specific phase of the menstrual cycle or irrespective of the cycle phase, and they tested for them in serum, plasma or whole blood. Included women were a selected population with a high frequency of endometriosis (10% to 85%), in which surgery was indicated for endometriosis, infertility work-up or ovarian mass. Seventy studies evaluated the diagnostic performance of 47 blood biomarkers for endometriosis (44 single-marker tests and 30 combined tests of two to six blood biomarkers). These were angiogenesis/growth factors, apoptosis markers, cell adhesion molecules, high-throughput markers, hormonal markers, immune system/inflammatory markers, oxidative stress markers, microRNAs, tumour markers and other proteins. Most of these biomarkers were assessed in small individual studies, often using different cut-off thresholds, and we could only perform meta-analyses on the data sets for anti-endometrial antibodies, interleukin-6 (IL-6), cancer antigen-19.9 (CA-19.9) and CA-125. Diagnostic estimates varied significantly between studies for each of these biomarkers, and CA-125 was the only marker with sufficient data to reliably assess sources of heterogeneity.The mean sensitivities and specificities of anti-endometrial antibodies (4 studies, 759 women) were 0.81 (95% confidence interval (CI) 0.76 to 0.87) and 0.75 (95% CI 0.46 to 1.00). For IL-6, with a cut-off value of > 1.90 to 2.00 pg/ml (3 studies, 309 women), sensitivity was 0.63 (95% CI 0.52 to 0.75) and specificity was 0.69 (95% CI 0.57 to 0.82). For CA-19.9, with a cut-off value of > 37.0 IU/ml (3 studies, 330 women), sensitivity was 0.36 (95% CI 0.26 to 0.45) and specificity was 0.87 (95% CI 0.75 to 0.99).Studies assessed CA-125 at different thresholds, demonstrating the following mean sensitivities and specificities: for cut-off > 10.0 to 14.7 U/ml: 0.70 (95% CI 0.63 to 0.77) and 0.64 (95% CI 0.47 to 0.82); for cut-off > 16.0 to 17.6 U/ml: 0.56 (95% CI 0.24, 0.88) and 0.91 (95% CI 0.75, 1.00); for cut-off > 20.0 U/ml: 0.67 (95% CI 0.50 to 0.85) and 0.69 (95% CI 0.58 to 0.80); for cut-off > 25.0 to 26.0 U/ml: 0.73 (95% CI 0.67 to 0.79) and 0.70 (95% CI 0.63 to 0.77); for cut-off > 30.0 to 33.0 U/ml: 0.62 (95% CI 0.45 to 0.79) and 0.76 (95% CI 0.53 to 1.00); and for cut-off > 35.0 to 36.0 U/ml: 0.40 (95% CI 0.32 to 0.49) and 0.91 (95% CI 0.88 to 0.94).We could not statistically evaluate other biomarkers meaningfully, including biomarkers that were assessed for their ability to differentiate endometrioma from other benign ovarian cysts.Eighty-two studies evaluated 97 biomarkers that did not differentiate women with endometriosis from disease-free controls. Of these, 22 biomarkers demonstrated conflicting results, with some studies showing differential expression and others no evidence of a difference between the endometriosis and control groups. AUTHORS' CONCLUSIONS: Of the biomarkers that were subjected to meta-analysis, none consistently met the criteria for a replacement or triage diagnostic test. A subset of blood biomarkers could prove useful either for detecting pelvic endometriosis or for differentiating ovarian endometrioma from other benign ovarian masses, but there was insufficient evidence to draw meaningful conclusions. Overall, none of the biomarkers displayed enough accuracy to be used clinically outside a research setting. We also identified blood biomarkers that demonstrated no diagnostic value in endometriosis and recommend focusing research resources on evaluating other more clinically useful biomarkers.

Distinct localisation of lipids in the ovarian follicular environment.

Obesity is associated with decreased pregnancy rates due, in part, to compromised oocyte quality. The aim of the present cross-sectional study of 84 women undergoing oocyte aspiration was to: (1) compare insulin, lipids and glucose in follicular fluid with serum; (2) determine whether increased body mass index (BMI) and waist circumference, hyperinsulinaemia, dyslipidaemia or metabolic syndrome altered follicular fluid metabolites; and (3) determine relative lipid content in oocytes to reveal any influence of these parameters on oocyte quality and IVF outcomes. Insulin, glucose, triglyceride and free fatty acids were lower in follicular fluid than blood and not strictly correlated between compartments. Insulin, glucose and triglyceride positively correlated with increasing BMI and waist circumference in blood and follicular fluid. Insulin increased in follicular fluid in association with metabolic syndrome. Free fatty acid composition analysis showed saturated fatty acids, particularly palmitic and stearic acid, to be more prevalent in follicular fluid than blood. There were no associations between follicular fluid metabolites or oocyte lipid content and clinical outcomes; however, oocyte immaturity correlated with follicular fluid glucose and fatty acid levels, as well as metabolic syndrome. The present study confirms that the human ovarian follicular environment surrounding the oocyte exhibits a unique metabolite profile compared with blood, with distinct localisation of lipids within follicular fluid and oocytes.

Urinary biomarkers for the non-invasive diagnosis of endometriosis.

BACKGROUND: About 10% of reproductive-aged women suffer from endometriosis which is a costly chronic disease that causes pelvic pain and subfertility. Laparoscopy is the 'gold standard' diagnostic test for endometriosis, but it is expensive and carries surgical risks. Currently, there are no simple non-invasive or minimally-invasive tests available in clinical practice that accurately diagnoses endometriosis. OBJECTIVES: 1. To provide summary estimates of the diagnostic accuracy of urinary biomarkers for the diagnosis of pelvic endometriosis compared to surgical diagnosis as a reference standard.2. To assess the diagnostic utility of biomarkers that could differentiate ovarian endometrioma from other ovarian masses.Urinary biomarkers were evaluated as replacement tests for surgical diagnosis and as triage tests to inform decisions to undertake surgery for endometriosis. SEARCH METHODS: The searches were not restricted to particular study design, language or publication dates. We searched the following databases to 20 April - 31 July 2015: CENTRAL, MEDLINE, EMBASE, CINAHL, PsycINFO, Web of Science, LILACS, OAIster, TRIP and ClinicalTrials.gov (trial register). MEDION, DARE, and PubMed were also searched to identify reviews and guidelines as reference sources of potentially relevant studies. Recently published papers not yet indexed in the major databases were also sought. The search strategy incorporated words in the title, abstract, text words across the record and the medical subject headings (MeSH) and was modified for each database. SELECTION CRITERIA: Published peer-reviewed, randomised controlled or cross-sectional studies of any size were considered, which included prospectively collected samples from any population of reproductive-aged women suspected of having one or more of the following target conditions: ovarian, peritoneal or deep infiltrating endometriosis (DIE). We included studies comparing the diagnostic test accuracy of one or more urinary biomarkers with surgical visualisation of endometriotic lesions. DATA COLLECTION AND ANALYSIS: Two authors independently collected and performed a quality assessment of the data from each study. For each diagnostic test, the data were classified as positive or negative for the surgical detection of endometriosis and sensitivity and specificity estimates were calculated. If two or more tests were evaluated in the same cohort, each was considered as a separate data set. The bivariate model was used to obtain pooled estimates of sensitivity and specificity whenever sufficient data sets were available. The predetermined criteria for a clinically useful urine test to replace diagnostic surgery was one with a sensitivity of 94% and a specificity of 79% to detect endometriosis. The criteria for triage tests were set at sensitivity of equal or greater than 95% and specificity of equal or greater than 50%, which in case of negative result rules out the diagnosis (SnOUT test) or sensitivity of equal or greater than 50% with specificity of equal or greater than 95%, which in case of positive result rules the diagnosis in (SpIN test). MAIN RESULTS: We included eight studies involving 646 participants, most of which were of poor methodological quality. The urinary biomarkers were evaluated either in a specific phase of menstrual cycle or irrespective of the cycle phase. Five studies evaluated the diagnostic performance of four urinary biomarkers for endometriosis, including three biomarkers distinguishing women with and without endometriosis (enolase 1 (NNE); vitamin D binding protein (VDBP); and urinary peptide profiling); and one biomarker (cytokeratin 19 (CK 19)) showing no significant difference between the two groups. All of these biomarkers were assessed in small individual studies and could not be statistically evaluated in a meaningful way. None of the biomarkers met the criteria for a replacement test or a triage test. Three studies evaluated three biomarkers that did not differentiate women with endometriosis from disease-free controls. AUTHORS' CONCLUSIONS: There was insufficient evidence to recommend any urinary biomarker for use as a replacement or triage test in clinical practice for the diagnosis of endometriosis. Several urinary biomarkers may have diagnostic potential, but require further evaluation before being introduced into routine clinical practice. Laparoscopy remains the gold standard for the diagnosis of endometriosis, and diagnosis of endometriosis using urinary biomarkers should only be undertaken in a research setting.

Artificial intelligence as a teaching tool for gynaecological ultrasound: A systematic search and scoping review.

Purpose: The aim of this study was to investigate the current application of artificial intelligence (AI) tools in the teaching of ultrasound skills as they pertain to gynaecological ultrasound. Methods: A scoping review was performed. Eight databases (MEDLINE, EMBASE, EMCARE, CINAHL, Scopus, Web of Science, IEEE Xplore and ACM digital library) were searched in December 2022 using predefined keywords. All types of publications were eligible for inclusion so long as they reported the use of an AI tool, included reference to or discussion of teaching or the improvement of ultrasound skills and pertained to gynaecological ultrasound. Conference abstracts and non-English language papers which could not be adequately translated into English were excluded. Results: The initial database search returned 481 articles. After screening against our inclusion and exclusion criteria, two were deemed to meet the inclusion criteria. Neither of the articles included reported original research (one systematic review and one review article). Neither of the included articles explicitly provided details of specific tools developed for the teaching of ultrasound skills for gynaecological imaging but highlighted similar applications within the field of obstetrics which could potentially be expanded. Conclusion: Artificial intelligence can potentially assist in the training of sonographers and other ultrasound operators, including in the field of gynaecological ultrasound. This scoping review revealed however that to date, no original research has been published reporting the use or development of such a tool specifically for gynaecological ultrasound.

Enabling Health Information Recommendation Using Crowdsourced Refinement in Web-Based Health Information Applications: User-Centered Design Approach and EndoZone Informatics Case Study.

BACKGROUND: In the digital age, search engines and social media platforms are primary sources for health information, yet their commercial interests-focused algorithms often prioritize irrelevant content. Web-based health applications by reputable sources offer a solution to circumvent these biased algorithms. Despite this advantage, there remains a significant gap in research on the effective integration of content-ranking algorithms within these specialized health applications to ensure the delivery of personalized and relevant health information. OBJECTIVE: This study introduces a generic methodology designed to facilitate the development and implementation of health information recommendation features within web-based health applications. METHODS: We detail our proposed methodology, covering conceptual foundation and practical considerations through the stages of design, development, operation, review, and optimization in the software development life cycle. Using a case study, we demonstrate the practical application of the proposed methodology through the implementation of recommendation functionalities in the EndoZone platform, a platform dedicated to providing targeted health information on endometriosis. RESULTS: Application of the proposed methodology in the EndoZone platform led to the creation of a tailored health information recommendation system known as EndoZone Informatics. Feedback from EndoZone stakeholders as well as insights from the implementation process validate the methodology's utility in enabling advanced recommendation features in health information applications. Preliminary assessments indicate that the system successfully delivers personalized content, adeptly incorporates user feedback, and exhibits considerable flexibility in adjusting its recommendation logic. While certain project-specific design flaws were not caught in the initial stages, these issues were subsequently identified and rectified in the review and optimization stages. CONCLUSIONS: We propose a generic methodology to guide the design and implementation of health information recommendation functionality within web-based health information applications. By harnessing user characteristics and feedback for content ranking, this methodology enables the creation of personalized recommendations that align with individual user needs within trusted health applications. The successful application of our methodology in the development of EndoZone Informatics marks a significant progress toward personalized health information delivery at scale, tailored to the specific needs of users.

A disrupted FOXP3 transcriptional signature underpins systemic regulatory T cell insufficiency in early pregnancy failure.

Regulatory T (Treg) cell defects are implicated in disorders of embryo implantation and placental development, but the origins of Treg cell dysfunction are unknown. Here, we comprehensively analyzed the phenotypes and transcriptional profile of peripheral blood Treg cells in individuals with early pregnancy failure (EPF). Compared to fertile subjects, EPF subjects had 32% fewer total Treg cells and 54% fewer CD45RA+CCR7+ naive Treg cells among CD4+ T cells, an altered Treg cell phenotype with reduced transcription factor FOXP3 and suppressive marker CTLA4 expression, and lower Treg:Th1 and Treg:Th17 ratios. RNA sequencing demonstrated an aberrant gene expression profile, with upregulation of pro-inflammatory genes including CSF2, IL4, IL17A, IL21, and IFNG in EPF Treg cells. In silico analysis revealed 25% of the Treg cell dysregulated genes are targets of FOXP3. We conclude that EPF is associated with systemic Treg cell defects arising due to disrupted FOXP3 transcriptional control and loss of lineage fidelity.

Host-derived TGFB1 deficiency suppresses lesion development in a mouse model of endometriosis.

Transforming growth factor-ß1 (TGFB1) is a multifunctional cytokine that is abundant in both endometriotic lesions and the peritoneal fluid in women with endometriosis. However, the role of TGFB1 in the development of endometriosis is as yet undefined. In the present study, we investigated the physiologic function of TGFB1 in endometriotic lesion development, using Tgfb1-null mutant mice on a background of severe combined immunodeficiency. Xenotransplantation of human eutopic endometrial tissue resulted in development of endometriosis-like lesions in 63% of ovariectomized estrogen-supplemented Tgfb1-null mutant mice and in 68% of wild-type control mice. Median lesion weight was reduced by 11-fold in Tgfb1-null mice compared with wild-type control mice, and the fraction of glandular epithelium in lesions from Tgfb1-null mice was reduced by 32% compared with that in control mice. In lesions from Tgfb1-null mice, the relative abundance of both macrophages and α-smooth muscle actin-positive myofibroblasts was reduced by 66% and 47%, respectively. Deficiency of TGFB1 neither altered the percentage of proliferating cells in the epithelial or stromal compartments of the lesions nor affected blood vessel density or vessel size. Observation of this study indicates that host-derived TGFB1 deficiency suppresses endometriotic lesion development and provides proof of principle that targeting TGFB1 signaling pathways in cells that support the survival of ectopic endometrium may be an effective therapeutic approach in women with endometriosis.

Tissue and circulating microRNA influence reproductive function in endometrial disease.

microRNA (miRNA) have emerged as important epigenetic modulators of gene expression in diverse pathological and physiological processes. In the endometrium, miRNA appear to have a role in the dynamic changes associated with the menstrual cycle, in implantation and in the pathophysiology associated with reproductive disorders such as recurrent miscarriage and endometriosis. This review explores the role of miRNA in endometrial physiology and endometrial disorders of reproduction and also raises the prospect that circulating miRNA may modulate endometrial function or reflect disordered endometrial activity. The clinical potential to use miRNA in diagnostic tests of endometrial function or in the treatment of endometrial disorders will also be discussed.

A phase 1/2, open-label, parallel group study to evaluate the preliminary efficacy and usability DARE-HRT1 (80 µg estradiol/4 mg progesterone and 160 µg estradiol/8 mg progesterone intravaginal RinGSM) over 12 weeks in healthy postmenopausal women.

OBJECTIVES: The exploratory objectives of this study were to evaluate the usability and acceptability and to conduct a preliminary evaluation of the efficacy of DARE-HRT1. DARE-HRT1 is an intravaginal ring (IVR) that releases 17ß2-estradiol (E2) with progesterone (P4) over 28 days. It is the first combination E2 and P4 IVR being developed for the treatment of vasomotor symptoms (VMS) in healthy postmenopausal women with an intact uterus. METHODS: This was a randomized, open-label, 2-arm, parallel group study in 21 healthy postmenopausal women. Women were randomized (1:1) to either DARE-HRT1 IVR1 (E2 80 µg/d with P4 4 mg/d) or DARE-HRT1 IVR2 (E2 160 µg/d with P4 8 mg/d). They used the assigned IVR for three 28-day cycles, inserting a new IVR monthly. Preliminary genitourinary syndrome of menopause (GSM) treatment efficacy was estimated by measuring changes from baseline in vaginal pH, vaginal maturation index (VMI), and changes in the severity of GSM symptoms. Preliminary systemic VMS efficacy was measured by changes in responses to the Menopause-Specific Quality of Life (MENQOL) questionnaire. Acceptability was assessed by product experience surveys. RESULTS: Preliminary local GSM treatment efficacy was supported by significant decreases in vaginal pH and % parabasal cells, and significant increases in the overall VMI and % superficial cells for both IVR groups (all P values <0.01). Preliminary VMS efficacy was supported by significant decreases in all domains of the MENQOL questionnaire from baseline for both dosing groups (all P values <0.01). CONCLUSIONS: Data from this study support further development of DARE-HRT1 for the treatment of menopausal symptoms.

A phase 1/2, open-label, parallel group study to evaluate the safety and pharmacokinetics of DARE-HRT1 (80 µg estradiol/4 mg progesterone and 160 µg estradiol/8 mg progesterone intravaginal rings) over 12 weeks in healthy postmenopausal women.

OBJECTIVES: Primary objectives were to evaluate the safety and systemic pharmacokinetics (PK) of DARE-HRT1, an intravaginal ring (IVR), which releases 17ß2-Estradiol (E2) with progesterone (P4) for 28 days in healthy postmenopausal women. METHODS: This was a randomized, open-label, 2-arm, parallel group study in 21 healthy postmenopausal women with an intact uterus. Women were randomized (1:1) to either DARE-HRT1 IVR1 (E2 80 µg/d with P4 4 mg/d) or DARE-HRT1 IVR2 (E2 160 µg/d with P4 8 mg/d). They used the IVR for three 28-day cycles, inserting a new IVR monthly. Safety was measured by treatment emergent adverse events and changes in systemic laboratories and the endometrial bilayer width. Baseline adjusted plasma PK of E2, P4, and estrone (E1) was described. RESULTS: Both DARE-HRT1 IVR were safe. All treatment emergent adverse events were mild or moderate and were distributed similarly among IVR1 versus IVR2 users. Month 3 median maximum plasma ( Cmax ) P4 concentrations were 2.81 and 3.51 ng/mL and Cmax E2 was 42.95 and 77.27 pg/mL for IVR1 and IVR2 groups, respectively. Month 3 median steady state ( Css ) plasma P4 concentrations were 1.19 and 1.89 ng/mL, and Css E2 was 20.73 and 38.16 pg/mL for IVR1 and IVR2 users, respectively. CONCLUSIONS: Both DARE-HRT1 IVRs were safe and released E2 in systemic concentrations, which were in the low, normal premenopausal range. Systemic P4 concentrations predict endometrial protection. Data from this study support further development of DARE-HRT1 for the treatment of menopausal symptoms.