Multiplexed serum biomarkers to discriminate nonviable and ectopic pregnancy.

OBJECTIVE: To evaluate combinations of candidate biomarkers to develop a multiplexed prediction model for identifying the viability and location of an early pregnancy. In this study, we assessed 24 biomarkers with multiple machine learning-based methodologies to assess if multiplexed biomarkers may improve the diagnosis of normal and abnormal early pregnancies. DESIGN: A nested case-control design evaluated the predictive ability and discrimination of biomarkers in patients at risk of early pregnancy failure in the first trimester to classify viability and location. SETTING: Three university hospitals. PATIENTS: A total of 218 individuals with pain and/or bleeding in early pregnancy: 75 had an ongoing intrauterine gestation; 68 had ectopic pregnancies (EPs); and 75 had miscarriages. INTERVENTIONS: Serum levels of 24 biomarkers were assessed in the same patients. Multiple machine learning-based methodologies to evaluate combinations of these top candidates to develop a multiplexed prediction model for the identification of a nonviable pregnancy (ongoing intrauterine pregnancy vs. miscarriage or EP) and an EP (EP vs. ongoing intrauterine pregnancy or miscarriage). MAIN OUTCOME MEASURES: The predicted classification using each model was compared with the actual diagnosis, and sensitivity, specificity, positive predictive value, negative predictive value, conclusive classification, and accuracy were calculated. RESULTS: Models using classification regression tree analysis using 3 (pregnancy-specific beta-1-glycoprotein 3 [PSG3], chorionic gonadotropin-alpha subunit, and pregnancy-associated plasma protein-A) biomarkers were able to predict a maximum sensitivity of 93.3% and a maximum specificity of 98.6%. The model with the highest accuracy was 97.4% (with 70.2% receiving classification). Models using an overlapping group of 3 (soluble fms-like tyrosine kinase-1, PSG3, and tissue factor pathway inhibitor 2) biomarkers achieved a maximum sensitivity of 98.5% and a maximum specificity of 95.3%. The model with the highest accuracy was 94.4% (with 65.6% receiving classification). When the models were used simultaneously, the conclusive classification increased to 72.7% with an accuracy of 95.9%. The predictive ability of the biomarkers in the random forest produced similar test characteristics when using 11 predictive biomarkers. CONCLUSION: We have demonstrated a pool of biomarkers from divergent biological pathways that can be used to classify individuals with potential early pregnancy loss. The biomarkers choriogonadotropin alpha, pregnancy-associated plasma protein-A, and PSG3 can be used to predict viability, and soluble fms-like tyrosine kinase-1, tissue factor pathway inhibitor 2, and PSG3 can be used to predict pregnancy location.

Evaluation of novel biomarkers for early pregnancy outcome prediction†.

OBJECTIVE: To assess performance and discriminatory capacity of commercially available enzyme-linked immunosorbent assays of biomarkers for predicting first trimester pregnancy outcome in a multi-center cohort. DESIGN: In a case-control study at three academic centers of women with pain and bleeding in early pregnancy, enzyme-linked immunosorbent assays of biomarkers were screened for assay performance. Performance was assessed via functional sensitivity, assay reportable range, recovery/linearity, and intra-assay precision (%Coefficient of Variation). Top candidates were analyzed for discriminatory capacity for viability and location among 210 women with tubal ectopic pregnancy, viable intrauterine pregnancy, or miscarriage. Assay discrimination was assessed by visual plots, area under the curve with 95% confidence intervals, and measures of central tendency with two-sample t-tests. RESULTS: Of 25 biomarkers evaluated, 22 demonstrated good or acceptable assay performance. Transgelin-2, oviductal glycoprotein, and integrin-linked kinase were rejected due to poor performance. The best biomarkers for discrimination of pregnancy location were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 1, insulin-like growth factor binding protein 1, kisspeptin (KISS1), pregnancy-specific beta-1-glycoprotein 3, and beta parvin (PARVB). The best biomarkers for discrimination of pregnancy viability were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 3, EH domain-containing protein 3, KISS1, WAP four-disulfide core domain protein 2 (HE4), quiescin sulfhydryl oxidase 2, and pregnancy-specific beta-1-glycoprotein 1. CONCLUSION: Performance of commercially available enzyme-linked immunosorbent assays was acceptable for a panel of novel biomarkers to predict early pregnancy outcome. Of these, six and seven candidates demonstrated good discriminatory capacity of pregnancy location and viability, respectively, when validated in a distinct external population. Four markers demonstrated good discrimination for both location and viability.

Validation of a multiple marker test for early pregnancy outcome prediction.

PURPOSE: To validate the use of a multiple biomarker test panel for predicting first trimester pregnancy outcome in a multi-center cohort. METHODS: A case-control study of women presenting with pain and bleeding in early pregnancy at 5-10 weeks gestational age was performed at three academic centers. Sera from women with ectopic pregnancy (EP), viable intrauterine pregnancy (IUP), and miscarriage (SAB) were analyzed via immunoassay for Activin A (AA), Progesterone (P4), A Disintegrin And Metalloprotease-12 (ADAM12), pregnancy-associated plasma protein A (PAPP-A), glycodelin (Glyc), and human chorionic gonadotropin (hCG). Biomarkers were assessed for reproducibility using medians, ranges, standard deviations, and area under receiver-operating characteristic curve (AUC) and accuracy in early pregnancy outcome classification compared to a previous derivation population. RESULTS: In 192 pregnancies, the biomarkers demonstrated good reproducibility with similar medians, ranges, and AUCs when compared to the derivation population except glycodelin. Pregnancy location was conclusively classified in 53% (n = 94) of the whole study sample with 78% accuracy. Pregnancy viability was conclusively classified in 58% (n = 112) of the new sample with 89% accuracy. Results were similar with subsequent model revisions where glycodelin was excluded and in the subgroups of subjects with a hCG below 2000 mIU/mL and a gestational age less than 6 weeks. CONCLUSION: The use of a panel of biomarkers to maximize test accuracy of a prediction of pregnancy location and prediction of pregnancy viability was reproducible and validated in an external population from which it was derived, but clinical utility is limited based on the test characteristics obtained.

Willingness of Women with Endometriosis Planning to Undergo IVF to Participate in a Randomized Clinical Trial and the Effects of the COVID-19 Pandemic on Potential Participation.

The Pre-IVF Treatment with a GnRH Antagonist in Women with Endometriosis (PREGnant) Trial (clinicaltrials.gov no. NCT04173169) was designed to test the hypothesis that 60-day pre-treatment with an oral GnRH antagonist in women with documented endometriosis and planning an IVF cycle will result in a superior live birth rate to placebo. Eight hundred fourteen women are required from 4 national sites. To determine the feasibility of using an electronic medical record (EMR)-based strategy to recruit 204 participants at the Colorado site, we conducted a survey of women within the UCHealth system. Eligible women, identified using relevant ICD-10 codes, were invited to complete a 6-question survey to assess planned utilization of IVF, potential interest in participation, and whether delays in treatment due to COVID-19 would influence their decision to participate. Of 6354 age-eligible women with an endometriosis diagnosis, 421 had a concurrent infertility diagnosis. After eliminating duplicates, 212 were emailed a survey; 76 (36%) responded, 6 of whom reported no endometriosis diagnosis. Of the remaining 70, 29 (41%) were planning fertility treatment; only 19 planned IVF. All 19 expressed interest in participation. COVID-19 delays in treatment were not considered as a factor affecting participation by 8/19; the remaining 11 felt that it would "somewhat" affect their decision. None reported that they would not consider participation because of COVID-19. EMR-based recruitment for an endometriosis clinical trial is feasible although the overall yield of participants is low. Delays in treatment due to COVID-19 did not appear to overly influence potential recruitment.

Novel ploidy analysis in ectopic pregnancy.

OBJECTIVE: To study whether a single-nucleotide polymorphism (SNP) array could be used to test tissue from ectopic pregnancy to distinguish whether ectopic pregnancies were aneuploid. DESIGN: Case series report. SETTING: Academic medical center. PATIENTS: One hundred seventy-eight women who underwent surgery for ectopic pregnancy at Northwestern Memorial Hospital between 2015 and 2018 were eligible for participation; written consent was obtained from 33 patients. Eight subjects had sufficient DNA samples and were included in the analysis. Maternal and paternal DNA samples were self-collected by buccal swab. Archived paraffin tissue containing chorionic villi from each surgically removed ectopic specimen was analyzed using SNP microarray technology to determine chromosome number and evaluate for maternal cell contamination. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Prevalence of aneuploidy in ectopic pregnancy specimens as well as success of SNP array technology in formalin-fixed and paraffin-embedded specimens. RESULTS: Subjects had a mean (±SD) age of 33.4 ± 5.4 years, body mass index of 23.4 ± 5.7 kg/m2, 3.3 ± 1.8 prior pregnancies, and 1.5 ± 1.4 live births. Genetic testing revealed that all eight tested samples were euploid, 6 female and 2 male (two arr(1-22)x2, (X,Y)x1 and 6 arr(1-22, X)x2); maternal cell contamination was ruled out in all cases. CONCLUSIONS: This study showed proof of concept for the use of routinely stored formalin-fixed, paraffin-embedded tissue blocks with DNA extraction for SNP array to detect ploidy status of ectopic pregnancy. Although all tested samples were euploid, further research is needed to gain a definitive answer to this question and better understand the mechanism that leads to ectopic implantation.

Can embryo morphokinetic parameters predict euploid pregnancy loss?

OBJECTIVE: To use time-lapse imaging to compare embryo morphokinetic parameters between embryos resulting in euploid pregnancy loss and euploid embryos resulting in live birth. DESIGN: Retrospective cohort study. SETTING: Single academic fertility center. PATIENT(S): All euploid single embryo transfers between October 2015 and January 2018. INTERVENTION(S): Collection and analysis of baseline characteristics, cycle parameters, and outcomes. MAIN OUTCOME MEASURE(S): Embryo morphokinetic measurements assessed with time-lapse imaging for time to syngamy (TPNf), time to two cells, time to three cells, time to four cells, time to eight cells, time to morula, and time to blastocyst. RESULT(S): The study included 192 euploid single-embryo transfers. Of these, the pregnancy rate was 78% (150 of 193) and the live-birth rate was 63% (121 of 193). There were 43 transfers that did not result in pregnancy, 15 biochemical pregnancy losses, 13 clinical losses, and 121 live births. There was no statistically significant difference in age, body mass index, or number of oocytes retrieved between the groups. Unadjusted and adjusted models revealed no differences in the morphokinetics of embryos resulting in euploid miscarriage compared with those resulting in live birth. CONCLUSION(S): Embryos that resulted in a euploid miscarriage did not display evidence of abnormal morphokinetics on time-lapse imaging. Euploid pregnancy loss is likely multifactorial, including both embryo and endometrial factors. Further research is needed to identify factors that can predict and prevent euploid loss.

Embryo transfer training in fellowship: national and institutional data.

OBJECTIVE: To evaluate current national practices in embryo transfer (ET) training in United States reproductive endocrinology and infertility (REI) fellowship programs and live birth rates after ET performed by fellows versus attending physicians. DESIGN: Cross-sectional survey of U.S. fellowship program directors and fellows in 2019 and retrospective cohort study of IVF cycle outcomes after ET performed by fellows versus attending physicians. SETTING: Not applicable. PATIENT(S): Fellowship program directors and fellows completed a survey. Embryo transfers from 2015-2018 were analyzed. INTERVENTION(S): A survey assessed experiences with ET training. Cycle outcomes were analyzed. MAIN OUTCOME MEASURE(S): Proportion of fellows performing ET during training, and live birth rate following fellow and faculty ETs. RESULT(S): Anonymous surveys were sent to 51 REI fellowship program directors and 142 fellows. Twenty-one percent (15/73) reported that no ETs were performed by fellows. Forty-four percent of third-year fellows had performed fewer than ten ETs during fellowship training. Retrospective review of 940 blastocyst ETs revealed no difference in live birth rates between fellows and attending physicians: 51.6% (131/254) versus 49.4% (339/686), respectively. CONCLUSION(S): This study revealed striking differences between fellowship programs regarding the adequacy of ET training; nearly one-half of third-year fellows had performed fewer than ten ETs. With appropriate supervision, there is no difference in live birth rate between ETs performed by fellows and attending physicians. Efforts should be made to address barriers and set minimums for the number of transfers performed during fellowship.

Effect of Folic Acid and Zinc Supplementation in Men on Semen Quality and Live Birth Among Couples Undergoing Infertility Treatment: A Randomized Clinical Trial.

Importance: Dietary supplements marketed for male fertility commonly contain folic acid and zinc based on limited prior evidence for improving semen quality. However, no large-scale trial has examined the efficacy of this therapy for improving semen quality or live birth. Objective: To determine the effect of daily folic acid and zinc supplementation on semen quality and live birth. Design, Setting, and Participants: The Folic Acid and Zinc Supplementation Trial was a multicenter randomized clinical trial. Couples (n = 2370; men aged ≥18 years and women aged 18-45 years) planning infertility treatment were enrolled at 4 US reproductive endocrinology and infertility care study centers between June 2013 and December 2017. The last 6-month study visit for semen collection occurred during August 2018, with chart abstraction of live birth and pregnancy information completed during April 2019. Interventions: Men were block randomized by study center and planned infertility treatment (in vitro fertilization, other treatment at a study site, and other treatment at an outside clinic) to receive either 5 mg of folic acid and 30 mg of elemental zinc (n = 1185) or placebo (n = 1185) daily for 6 months. Main Outcomes and Measures: The co-primary outcomes were live birth (resulting from pregnancies occurring within 9 months of randomization) and semen quality parameters (sperm concentration, motility, morphology, volume, DNA fragmentation, and total motile sperm count) at 6 months after randomization. Results: Among 2370 men who were randomized (mean age, 33 years), 1773 (75%) attended the final 6-month study visit. Live birth outcomes were available for all couples, and 1629 men (69%) had semen available for analysis at 6 months after randomization. Live birth was not significantly different between treatment groups (404 [34%] in the folic acid and zinc group and 416 [35%] in the placebo group; risk difference, -0.9% [95% CI, -4.7% to 2.8%]). Most of the semen quality parameters (sperm concentration, motility, morphology, volume, and total motile sperm count) were not significantly different between treatment groups at 6 months after randomization. A statistically significant increase in DNA fragmentation was observed with folic acid and zinc supplementation (mean of 29.7% for percentage of DNA fragmentation in the folic acid and zinc group and 27.2% in the placebo group; mean difference, 2.4% [95% CI, 0.5% to 4.4%]). Gastrointestinal symptoms were more common with folic acid and zinc supplementation compared with placebo (abdominal discomfort or pain: 66 [6%] vs 40 [3%], respectively; nausea: 50 [4%] vs 24 [2%]; and vomiting: 32 [3%] vs 17 [1%]). Conclusions and Relevance: Among a general population of couples seeking infertility treatment, the use of folic acid and zinc supplementation by male partners, compared with placebo, did not significantly improve semen quality or couples' live birth rates. These findings do not support the use of folic acid and zinc supplementation by male partners in the treatment of infertility. Trial Registration: ClinicalTrials.gov Identifier: NCT01857310.

Economic evaluation of highly purified human menotropin or recombinant follicle-stimulating hormone for controlled ovarian stimulation in high-responder patients: analysis of the Menopur in Gonadotropin-releasing Hormone Antagonist Single Embryo Transfer-High Responder (MEGASET-HR) trial.

OBJECTIVE: To determine the cost of achieving a live birth after first transfer using highly purified human menotropin (HP-hMG) or recombinant follicle-stimulating hormone (FSH) for controlled ovarian stimulation in predicted high-responder patients in the Menopur in Gonadotropin-releasing hormone Antagonist Single Embryo Transfer-High Responder (MEGASET-HR) trial. DESIGN: Cost minimization analysis of trial results. SETTING: Thirty-one fertility centers. PATIENTS: Six hundred and nineteen women with serum antimüllerian hormone ≥5 ng/mL. INTERVENTIONS: Controlled ovarian stimulation with HP-hMG or recombinant FSH in a gonadotropin-releasing hormone (GnRH) antagonist assisted reproduction cycle where fresh transfer of a single blastocyst was performed unless ovarian response was excessive whereupon all embryos were cryopreserved and patients could undergo subsequent frozen blastocyst transfer within 6 months of randomization. MAIN OUTCOME MEASURES: Mean cost of achieving live birth after first transfer (fresh or frozen). RESULTS: First-transfer efficacy, defined as live birth after first fresh or frozen transfer, was 54.5% for HP-hMG and 48.0% for recombinant FSH (difference 6.5%). Average cost to achieve a live birth after first transfer (fresh or frozen) was lower with HP-hMG compared with recombinant FSH. For fresh transfers, the cost was lower with HP-hMG compared with recombinant FSH. The average cost to achieve a live birth after first frozen transfer was also lower in patients treated with HP-hMG compared with recombinant FSH. CONCLUSIONS: Treatment of predicted high-responders with HP-hMG was associated with lower cost to achieve a live birth after first transfer compared with recombinant FSH. CLINICAL TRIAL REGISTRATION NUMBER: NCT02554279.

A Randomized Trial to Evaluate the Effects of Folic Acid and Zinc Supplementation on Male Fertility and Livebirth: Design and Baseline Characteristics.

The Folic Acid and Zinc Supplementation Trial (FAZST) was a multicenter, double-blind, block-randomized, placebo-controlled trial to determine whether folic acid and zinc supplementation in men improves semen quality and increases livebirth rate among couples seeking infertility treatment (2013-2017). Eligible men were aged 18 years or older with female partners aged 18-45 years, seeking infertility treatment. Men were randomized (1:1) to 5 mg folic acid and 30 mg elemental zinc daily or matching placebo for 6 months. Randomization was stratified by site and intended infertility treatment (in vitro fertilization (IVF), non-IVF/study site, and non-IVF/outside clinic). Follow-up of men continued for 6 months, and female partners were passively followed for a minimum of 9 months. Women who conceived were followed throughout pregnancy. Overall, 2,370 men were randomized during 2013-2017 (1,185 folic acid and zinc, 1,185 placebo); they had a mean age of 33 years and body mass index (weight (kg)/height (m)2) of 29.8. Most participants were white (82%), well educated (83% with some college), and employed (72%). Participant characteristics were balanced across intervention arms. Study visits were completed by 89%, 77%, and 75% of men at months 2, 4, and 6, respectively. Here we describe the study design, recruitment, data collection, lessons learned, and baseline participant characteristics.

PLIN2 Functions As a Novel Link Between Progesterone Signaling and Metabolism in Uterine Leiomyoma Cells.

CONTEXT: Uterine leiomyoma (fibroids) are the most common tumors in women. Recently, perilipin-2 (PLIN2) was identified as a critical target gene of the progesterone receptor; however, its function in the pathogenesis of fibroids is unknown. OBJECTIVE: To determine the function of PLIN2 in leiomyoma cells. DESIGN: Tissue and primary cells from leiomyoma and myometrium were analyzed. PLIN2 function in leiomyoma was assessed using small interfering RNA. RNA-sequencing was performed to identify genome-wide effects of PLIN2 depletion. Metabolic activity was measured using the Seahorse XF96 analyzer. Real-time quantitative PCR and immunoblotting were also performed. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: Forty-one premenopausal women undergoing surgery for fibroids. MAIN OUTCOME MEASURES: Gene expression, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and cell proliferation. RESULTS: PLIN2 gene expression was 2.4-fold lower in leiomyoma compared with adjacent myometrium, suggesting a link between PLIN2 deficiency and fibroids. A total of 3877 genes were differentially expressed after PLIN2 knockdown. Gene ontology analysis identified metabolism as the second-highest biological process affected by PLIN2 depletion. OCR (mitochondrial respiration) and ECAR (glycolysis) were significantly upregulated after PLIN2 knockdown; PLIN2-depleted cells had a greater basal metabolic activity and higher metabolic stress response. Cell proliferation was also significantly increased after PLIN2 knockdown. CONCLUSIONS: PLIN2 depletion increases mitochondrial respiration and glycolysis, suggesting that PLIN2 is a critical regulator of metabolic function in leiomyoma cells. PLIN2 deficiency also reprograms leiomyoma cells to a proproliferative phenotype. These findings introduce metabolomics as an area to explore to better understand leiomyoma tumorigenesis.

Validation of a Single-Nucleotide Polymorphism-Based Non-Invasive Prenatal Test in Twin Gestations: Determination of Zygosity, Individual Fetal Sex, and Fetal Aneuploidy.

We analyzed maternal plasma cell-free DNA samples from twin pregnancies in a prospective blinded study to validate a single-nucleotide polymorphism (SNP)-based non-invasive prenatal test (NIPT) for zygosity, fetal sex, and aneuploidy. Zygosity was evaluated by looking for either one or two fetal genome complements, fetal sex was evaluated by evaluating Y-chromosome loci, and aneuploidy was assessed through SNP ratios. Zygosity was correctly predicted in 100% of cases (93/93; 95% confidence interval (CI) 96.1%-100%). Individual fetal sex for both twins was also called with 100% accuracy (102/102; 95% weighted CI 95.2%-100%). All cases with copy number truth were also correctly identified. The dizygotic aneuploidy sensitivity was 100% (10/10; 95% CI 69.2%-100%), and overall specificity was 100% (96/96; 95% weighted CI, 94.8%-100%). The mean fetal fraction (FF) of monozygotic twins (n = 43) was 13.0% (standard deviation (SD), 4.5%); for dizygotic twins (n = 79), the mean lower FF was 6.5% (SD, 3.1%) and the mean higher FF was 8.1% (SD, 3.5%). We conclude SNP-based NIPT for zygosity is of value when chorionicity is uncertain or anomalies are identified. Zygosity, fetal sex, and aneuploidy are complementary evaluations that can be carried out on the same specimen as early as 9 weeks' gestation.

Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis.

OBJECTIVE: To investigate the rate of sperm DNA fragmentation in male partners of women with recurrent pregnancy loss and fertile control women. DESIGN: Systematic review and meta-analysis. SETTING: Not applicable. PATIENT(S): A total of 579 male partners of women with recurrent pregnancy loss and 434 male partners fertile control women. INTERVENTION(S): Prospective studies were identified through a Pubmed search. Recurrent pregnancy loss was defined as two or more previous pregnancy losses. Fertile control women had a history of a live birth or ongoing pregnancy. MAIN OUTCOME MEASURE(S): The primary outcome was the rate of sperm DNA fragmentation. The summary measures were reported as mean difference with 95% confidence interval (CI). RESULT(S): Fifteen prospective studies were included in a qualitative review. Pooled data from 13 studies with sufficient data for meta-analysis suggest that male partners of women with a history of recurrent pregnancy loss have a significantly higher rate of sperm DNA fragmentation compared to the partners of fertile control women: mean difference 11.91, 95% CI 4.97-18.86. CONCLUSION(S): These findings support an association between sperm DNA fragmentation and recurrent pregnancy loss. However, given the significant heterogeneity between studies and lack of prospective pregnancy outcome data, further large prospective studies are needed.

30 years of data: impact of the United States in vitro fertilization data registry on advancing fertility care.

OBJECTIVE: To summarize and assess the impact of key research generated through the Society of Assisted Reproductive Technology (SART)-initiated United States IVF registry and annual reporting system. DESIGN: Review. SETTING: Eligible studies included those that analyzed data generated by the National IVF data collection program (through SART or Centers for Disease Control and Prevention). PATIENT(S): Not applicable. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Summarize and report outcomes of research using National IVF registry data. RESULT(S): The Society of Assisted Reproductive Technology was founded in 1985 and published the first annual US IVF data report 30 years ago in 1988 in Fertility and Sterility. In 1995, the Centers for Disease Control and Prevention subsequently began collecting data from IVF programs and published their first report in 1997. This annual National IVF data collection and reporting is a significant responsibility and effort for IVF programs. Using these data sources, 199 articles have been published by clinicians and researchers from across the country. This research has guided the development of evidence-based assisted reproductive technology (ART) practice guidelines during the past 30 years, which have ultimately led to improved quality and patient care. CONCLUSION(S): Since the first SART National IVF data report publication 30 years ago, SART has achieved its original goals of creating a national IVF registry that successfully assesses clinical effectiveness, quality of care, and safety.

The Essential Role of GATA6 in the Activation of Estrogen Synthesis in Endometriosis.

Endometriotic stromal cells synthesize estradiol via the steroidogenic pathway. Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is critical, but alone not sufficient, in activating this cascade that involves at least 5 genes. To evaluate whether another transcription factor is required for the activation of this pathway, we examined whether GATA Binding Protein 6 (GATA6) can transform a normal endometrial stromal cell (NoEM) into an endometriotic-like cell by conferring an estrogen-producing phenotype. We ectopically expressed GATA6 alone or with NR5A1 in NoEM or silenced these transcription factors in endometriotic stromal cells (OSIS) and assessed the messenger RNAs or proteins encoded by the genes in the steroidogenic cascade. Functionally, we assessed the effects of GATA6 expression or silencing on estradiol formation. In OSIS, GATA6 was necessary for catalyzing the conversion of progesterone to androstenedione (CYP17A1; P < .05). In NoEM, ectopic expression of GATA6 was essential for converting pregnenolone to estrogen (HSD3B2, CYP17A1, and CYP19A1; P < .05). However, simultaneous ectopic expression of both GATA6 and NR5A1 was required and sufficient to confer induction of all 5 genes and their encoded proteins that convert cholesterol to estrogen. Functionally, only simultaneous knockdown of GATA6 and NR5A1 blocked estradiol formation in OSIS ( P < .05). The presence of both transcription factors was required and sufficient to transform endometrial stromal cells into endometriotic-like cells that produced estradiol in large quantities ( P < .05). In summary, GATA6 alone is essential but not sufficient for estrogen formation in endometriosis. However, simultaneous addition of GATA6 and NR5A1 to an endometrial stromal cell is sufficient to transform it into an endometriotic-like cell, manifested by the activation of the estradiol biosynthetic cascade.

Optimal treatment for women with a persisting pregnancy of unknown location, a randomized controlled trial: The ACT-or-NOT trial.

OBJECTIVE: Pregnancy of unknown location (PUL) is not a diagnosis but a transient state used to classify a woman when she has a positive pregnancy test without definitive evidence of an intra-uterine or extra-uterine pregnancy on transvaginal ultrasonography. Management of a persisting PUL varies substantially, including expectant or active management. Active management can include uterine cavity evacuation or systemic administration of methotrexate. To date, no consensus has been reached on whether either management strategy is superior or non-inferior to the other. DESIGN: Randomized controlled trial. SETTING: Academic medical centers. PATIENTS: We plan to randomize 276 persisting PUL-diagnosed women who are 18 years or older from Reproductive Medicine Network clinics and additional interested sites, all patients will be followed for 2 years for fertility and patient satisfaction outcomes. INTERVENTIONS: Randomization will be 1:1:1 ratio between expectant management, uterine evacuation and empiric use of methotrexate. After randomization to initial management plan, all patients will be followed by their clinicians until resolution of the PUL. The clinician will determine whether there is a change in management, based on clinical symptoms, and/or serial human chorionic gonadotropin (hCG) concentrations and/or additional ultrasonography. MAIN OUTCOME: The primary outcome measure in each of the 3 treatment arms is the uneventful clinical resolution of a persistent PUL without change from the initial management strategy. Secondary outcome measures include: number of ruptured ectopic pregnancies, number and type of re-interventions (additional methotrexate injections or surgical procedures), treatment complications, adverse events, number of visits, time to resolution, patient satisfaction, and future fertility. CONCLUSION: This multicenter randomized controlled trial will provide guidance for evidence-based management for women who have persisting pregnancy of unknown location.