Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer.

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.

The C Protein Is Recruited to Measles Virus Ribonucleocapsids by the Phosphoprotein.

Measles virus (MeV), like all viruses of the order Mononegavirales, utilizes a complex consisting of genomic RNA, nucleoprotein, the RNA-dependent RNA polymerase, and a polymerase cofactor, the phosphoprotein (P), for transcription and replication. We previously showed that a recombinant MeV that does not express another viral protein, C, has severe transcription and replication deficiencies, including a steeper transcription gradient than the parental virus and generation of defective interfering RNA. This virus is attenuated in vitro and in vivo However, how the C protein operates and whether it is a component of the replication complex remained unclear. Here, we show that C associates with the ribonucleocapsid and forms a complex that can be purified by immunoprecipitation or ultracentrifugation. In the presence of detergent, the C protein is retained on purified ribonucleocapsids less efficiently than the P protein and the polymerase. The C protein is recruited to the ribonucleocapsid through its interaction with the P protein, as shown by immunofluorescence microscopy of cells expressing different combinations of viral proteins and by split luciferase complementation assays. Forty amino-terminal C protein residues are dispensable for the interaction with P, and the carboxyl-terminal half of P is sufficient for the interaction with C. Thus, the C protein, rather than being an "accessory" protein as qualified in textbooks so far, is a ribonucleocapsid-associated protein that interacts with P, thereby increasing replication accuracy and processivity of the polymerase complex.IMPORTANCE Replication of negative-strand RNA viruses relies on two components: a helical ribonucleocapsid and an RNA-dependent RNA polymerase composed of a catalytic subunit, the L protein, and a cofactor, the P protein. We show that the measles virus (MeV) C protein is an additional component of the replication complex. We provide evidence that the C protein is recruited to the ribonucleocapsid by the P protein and map the interacting segments of both C and P proteins. We conclude that the primary function of MeV C is to improve polymerase processivity and accuracy, rather than uniquely to antagonize the type I interferon response. Since most viruses of the Paramyxoviridae family express C proteins, their primary function may be conserved.

Sexual Health Education in Obstetrics and Gynecology (Ob-Gyn) Residencies-A Resident Physician Survey.

BACKGROUND: Many women will experience a sexual health concern and present to their Obstetrics and Gynecology (Ob-Gyn) care provider, yet a large portion of graduating Ob-Gyn resident physicians in the United States may not feel comfortable helping patients with some sexual health issues. AIM: To perform a cross-sectional study of U.S. Ob-Gyn resident physicians that assesses sexual health education didactic sessions and comfort level with sexual health clinical vignettes. METHODS: A 32-item anonymous survey was sent to all 4,065 Ob-Gyn residents on June 7, 2016. Respondents voluntarily completed the survey electronically. OUTCOMES: The primary outcome measures are the comfort level of Ob-Gyn resident physicians in taking a sexual history and providing counseling to patients represented in clinical vignettes, which were based on sexual health learning objectives from the Council on Resident Education in Obstetrics and Gynecology (CREOG). RESULTS: Of the 4,065 eligible U.S. examinees, 968 (23.8%) agreed to participate in the study, and 802 (19.7%) completed the survey and were included in the final analysis. Nearly two-thirds of the residents indicated that sexual health training was a priority in residency. However, more than half were not able to describe the disorders of sexual function or list common medications that effect sexual function. When posed with clinical vignettes, residents felt very comfortable obtaining a sexual history (98.5%) and providing counseling (97.0%) for a 16-year-old seeking contraception, yet fewer felt very comfortable obtaining a history and providing counseling for a 26-year-old who is a refugee from Somalia (77.2% and 73.8%). Smaller cohorts felt prepared to take a sexual history and provide counseling for a 17-year-old who discloses that she is a victim of sex trafficking (61.2% and 57.7%), and for a 58-year-old transgender patient planning hormone therapy and surgery (49.9% and 37.9%). In logistic regression analysis, the factors that were influential in an Ob-Gyn resident physician's program to prepare them to describe the disorders of sexual function were post-graduate year (OR 1.387, 95% CI 1.189, 1.618; P = .0001), those that rated the importance of a sexual health curriculum highly (OR 0.701, 95% CI 0.569, 0.864; P = .0012), and a greater number of didactic sessions on sexual health in the residency curriculum (OR 0.685, 95% CI 0.626, 0.750; P < .0001). CONCLUSION: These findings highlight strengths in the comfort of Ob-Gyn resident physicians about sexual health and illustrate areas of opportunity to engage resident learners by improving the sexual health curriculum. Worly B, Manriquez M, Stagg A, et al. Sexual Health Education in Obstetrics and Gynecology (Ob-Gyn) Residencies-A Resident Physician Survey. J Sex Med 2021;18:1042-1052.

Diagnosis and Management of Infertility: A Review.

IMPORTANCE: In the US, approximately 12.7% of reproductive age women seek treatment for infertility each year. This review summarizes current evidence regarding diagnosis and treatment of infertility. OBSERVATIONS: Infertility is defined as the failure to achieve pregnancy after 12 months of regular unprotected sexual intercourse. Approximately 85% of infertile couples have an identifiable cause. The most common causes of infertility are ovulatory dysfunction, male factor infertility, and tubal disease. The remaining 15% of infertile couples have "unexplained infertility." Lifestyle and environmental factors, such as smoking and obesity, can adversely affect fertility. Ovulatory disorders account for approximately 25% of infertility diagnoses; 70% of women with anovulation have polycystic ovary syndrome. Infertility can also be a marker of an underlying chronic disease associated with infertility. Clomiphene citrate, aromatase inhibitors such as letrozole, and gonadotropins are used to induce ovulation or for ovarian stimulation during in vitro fertilization (IVF) cycles. Adverse effects of gonadotropins include multiple pregnancy (up to 36% of cycles, depending on specific therapy) and ovarian hyperstimulation syndrome (1%-5% of cycles), consisting of ascites, electrolyte imbalance, and hypercoagulability. For individuals presenting with anovulation, ovulation induction with timed intercourse is often the appropriate initial treatment choice. For couples with unexplained infertility, endometriosis, or mild male factor infertility, an initial 3 to 4 cycles of ovarian stimulation may be pursued; IVF should be considered if these approaches do not result in pregnancy. Because female fecundity declines with age, this factor should guide decision-making. Immediate IVF may be considered as a first-line treatment strategy in women older than 38 to 40 years. IVF is also indicated in cases of severe male factor infertility or untreated bilateral tubal factor. CONCLUSIONS AND RELEVANCE: Approximately 1 in 8 women aged 15 to 49 years receive infertility services. Although success rates vary by age and diagnosis, accurate diagnosis and effective therapy along with shared decision-making can facilitate achievement of fertility goals in many couples treated for infertility.

First PGT-A using human in vivo blastocysts recovered by uterine lavage: comparison with matched IVF embryo controls†.

STUDY QUESTION: After controlled ovarian stimulation (COS) and IUI, is it clinically feasible to recover in vivo conceived and matured human blastocysts by uterine lavage from fertile women for preimplantation genetic testing for aneuploidy (PGT-A) and compare their PGT-A and Gardner scale morphology scores with paired blastocysts from IVF control cycles? SUMMARY ANSWER: In a consecutive series of 134 COS cycles using gonadotrophin stimulation followed by IUI, uterine lavage recovered 136 embryos in 42% (56/134) of study cycles, with comparable in vivo and in vitro euploidy rates but better morphology in in vivo embryos. WHAT IS KNOWN ALREADY: In vivo developed embryos studied in animal models possess different characteristics compared to in vitro developed embryos of similar species. Such comparative studies between in vivo and in vitro human embryos have not been reported owing to lack of a reliable method to recover human embryos. STUDY DESIGN, SIZE, DURATION: We performed a single-site, prospective controlled trial in women (n = 81) to evaluate the safety, efficacy and feasibility of a novel uterine lavage catheter and fluid recovery device. All lavages were performed in a private facility with a specialized fertility unit, from August 2017 to June 2018. Subjects were followed for 30 days post-lavage to monitor for clinical outcomes and delayed complications. In 20 lavage subjects, a single IVF cycle (control group) with the same ovarian stimulation protocol was performed for a comparison of in vivo to in vitro blastocysts. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Women were stimulated with gonadotrophins for COS. The ovulation trigger was given when there were at least two dominant follicles ≥18 mm, followed by IUI of sperm. Uterine lavage occurred 4-6 days after the IUI. A subset of 20 women had a lavage cycle procedure followed by an IVF cycle (control IVF group). Recovered embryos were characterized morphologically, underwent trophectoderm (TE) biopsy, vitrified and stored in liquid nitrogen. Biopsies were analyzed using the next-generation sequencing technique. After lavage, GnRH antagonist injections were administered to induce menstruation. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 134 lavage cycles were performed in 81 women. Uterine lavage recovered 136 embryos in 56 (42%) cycles. At the time of cryopreservation, there were 40 (30%) multi-cell embryos and 96 (70%) blastocysts. Blastocysts were of good quality, with 74% (70/95) being Gardener grade 3BB or higher grade. Lavage blastocysts had significantly higher morphology scores than the control IVF embryos as determined by chi-square analysis (P < 0.05). This is the first study to recover in vivo derived human blastocysts following ovarian stimulation for embryo genetic characterization. Recovered blastocysts showed rates of chromosome euploidy similar to the rates found in the control IVF embryos. In 11 cycles (8.2%), detectable levels of hCG were present 13 days after IUI, which regressed spontaneously in two cases and declined after an endometrial curettage in two cases. Persistent hCG levels were resolved after methotrexate in three cases and four cases received both curettage and methotrexate. LIMITATIONS, REASON FOR CAUTION: The first objective was to evaluate the feasibility of uterine lavage following ovarian stimulation to recover blastocysts for analysis, and that goal was achieved. However, the uterine lavage system was not completely optimized in our earlier experience to levels that were achieved late in the clinical study and will be expected in clinical service. The frequency of chromosome abnormalities of in vivo and IVF control embryos was similar, but this was a small-size study. However, compared to larger historical datasets of in vitro embryos, the in vivo genetic results are within the range of high-quality in vitro embryos. WIDER IMPLICATIONS OF THE FINDINGS: Uterine lavage offers a nonsurgical, minimally invasive strategy for recovery of embryos from fertile women who do not want or need IVF and who desire PGT, fertility preservation of embryos or reciprocal IVF for lesbian couples. From a research and potential clinical perspective, this technique provides a novel platform for the use of in vivo conceived human embryos as the ultimate benchmark standard for future and current ART methods. STUDY FUNDING/COMPETING INTEREST(S): Previvo Genetics, Inc., is the sole sponsor for the Punta Mita, Mexico, clinical study. S.M. performs consulting for CooperGenomics. J.E.B. and S.A.C. are co-inventors on issued patents and patents owned by Previvo and ownshares of Previvo. S.N. is a co-author on a non-provisional patent application owned by Previvo and holds stock options in Previvo. S.T.N. and M.J.A. report consulting fees from Previvo. S.T.N., S.M., M.V.S., M.J.A., C.N. and J.E.B. are members of the Previvo Scientific Advisory Board (SAB) and hold stock options in Previvo. J.E.B and S. M are members of the Previvo Board of Directors. A.N. and K.C. are employees of Previvo Genetics. L.V.M, T.M.M, J.L.R and S. S have no conflicts to disclose. TRIAL REGISTRATION NUMBER: Protocol Registration and Results System (PRS) Trial Registration Number and Name: Punta Mita Study TD-2104: Clinical Trials NCT03426007.

Smoking in infertile women with polycystic ovary syndrome: baseline validation of self-report and effects on phenotype.

STUDY QUESTION: Do women with polycystic ovary syndrome (PCOS) seeking fertility treatment report smoking accurately and does participation in infertility treatment alter smoking? SUMMARY ANSWER: Self-report of smoking in infertile women with PCOS is accurate (based on serum cotinine levels) and smoking is unlikely to change over time with infertility treatment. WHAT IS KNOWN ALREADY: Women with PCOS have high rates of smoking and it is associated with worse insulin resistance and metabolic dysfunction. STUDY DESIGN, SIZE, DURATION: Secondary study of smoking history from a large randomized controlled trial of infertility treatments in women with PCOS (N = 626) including a nested case-control study (N = 148) of serum cotinine levels within this cohort to validate self-report of smoking. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with PCOS, age 18-40, seeking fertility who participated in a multi-center clinical trial testing first-line ovulation induction agents conducted at academic health centers in the USA. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, self-report of smoking in the nested case-control study agreed well with smoking status as determined by measure of serum cotinine levels, at 90% or better for each of the groups at baseline (98% of never smokers had cotinine levels <15 ng/ml compared with 90% of past smokers and 6% of current smokers). There were minor changes in smoking status as determined by serum cotinine levels over time, with the greatest change found in the smoking groups (past or current smokers). In the larger cohort, hirsutism scores at baseline were lower in the never smokers compared with past smokers. Total testosterone levels at baseline were also lower in the never smokers compared with current smokers. At end of study follow-up insulin levels and homeostatic index of insulin resistance increased in the current smokers (P < 0.01 for both) compared with baseline and with non-smokers. The chance for ovulation was not associated with smoking status, but live birth rates were increased (non-significantly) in never or past smokers. LIMITATIONS, REASONS FOR CAUTION: The limitations include the selection bias involved in our nested case-control study, the possibility of misclassifying exposure to second hand smoke as smoking and our failure to capture self-reported changes in smoking status after enrollment in the trial. WIDER IMPLICATIONS OF THE FINDINGS: Because self-report of smoking is accurate, further testing of smoking status is not necessary in women with PCOS. Because smoking status is unlikely to change during infertility treatment, extra attention should be focused on smoking cessation in current or recent smokers who seek or who are receiving infertility treatment. STUDY FUNDING/COMPETING INTERESTS: Sponsored by the Eugene Kennedy Shriver National Institute of Child Health and Human Development of the U.S. National Institutes of Health. CLINICAL TRIAL REGISTRATION NUMBERS: ClinicalTrials.gov numbers, NCT00068861 and NCT00719186.

ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis.

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis.

The androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here, we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 expression is abnormally gained in prostate cancer cells and its functional inhibition impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites, which is greater than 65% of the AR tumor cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and instead contain the canonical palindromic motifs. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in the physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Concordantly, a dual NSD1/2 PROTAC degrader, called LLC0150, was preferentially cytotoxic in AR-dependent prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the AR neo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 as viable paralog co-targets in advanced prostate cancer.

Human Uterine Lavage: First Live Births from In Vivo Conceived Genetically Screened Blastocysts.

INTRODUCTION: We wish to report the first live births from genetically screened human euploid blastocysts obtained by uterine lavage. The embryos transferred to infertile women were previously obtained using a novel fully automated uterine lavage catheter and fluid recovery device developed for this indication. The objective of this portion of the research was to confirm embryo implantation and live births with these unique in vivo conceived blastocysts obtained by uterine lavage. METHODS: In vivo conceived embryos recovered by uterine lavage 5 days after intrauterine insemination were available for embryo donation. In vivo embryos were the result of prior controlled ovarian stimulation cycles in oocyte donors and intrauterine insemination with donor sperm. An observational case series of nine embryo transfer procedures was performed at an outpatient fertility center. One to two embryos were transferred to eight infertile women since one woman had two separate embryo transfer procedures. RESULTS: Nine embryo transfer procedures were performed with 14 blastocysts in eight women resulting in a blastocyst implantation rate of 36% (5/14) and live birth rate of 44% (4/9). Five infants have been born from the four delivered pregnancies with one set of twins. CONCLUSIONS: This is the first report of live births from genetically screened human euploid blastocysts obtained by uterine lavage. The nonsurgical uterine lavage office procedure represents the only current approach to obtain in vivo conceived embryos and can provide a benchmark for comparison to standard in vitro cultured blastocysts. Live births of in vivo conceived blastocysts represent the validation that the nonsurgical uterine lavage procedure allows simplified access to naturally conceived embryos without performing the surgical procedure of an oocyte aspiration. Owing to its simplicity, uterine lavage may be useful in screening embryos for preimplantation genetic testing for aneuploidy in fertile and infertile couples. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (Identifier NCT03426007).

The overall goal of this research was to develop a procedure that would allow collection of naturally conceived human embryos and compare them to embryos that result from the standard process of in vitro fertilization (IVF). IVF is a procedure where eggs are surgically removed from the ovaries and fertilized with sperm in a laboratory. Embryos from IVF are cultured for 3­7 days before they are placed back into a woman's uterus to establish a pregnancy. Uterine lavage is a different procedure where the sperm fertilizes an egg in the normal process of conception and the uterus is rinsed with fluid to recover the embryo before implantation. The embryos reported in this study were the first to be obtained in over 30 years owing to many improvements in the overall uterine lavage procedure. Until our initial study findings reported in 2020, the vast majority of information on embryo development was based on embryos fertilized and cultured in a laboratory. Our prior report of embryos obtained by uterine lavage compared with IVF embryos from the same women demonstrated a significantly better appearance of the embryos recovered by lavage. This current report documents the first live births from these genetically screened naturally conceived human embryos. The live births provide evidence that uterine lavage allows ready access to normal embryos without performing the surgical procedure IVF. Owing to the simplicity of uterine lavage, the procedure may improve access to genetic testing of embryos before pregnancy.

The transcriptome of a human polar body accurately reflects its sibling oocyte.

Improved methods are needed to reliably and accurately evaluate oocyte quality prior to fertilization and transfer into the woman of human embryos created through in vitro fertilization (IVF). All oocytes that are retrieved and matured in culture are exposed to sperm with little in the way of evaluating the oocyte quality. Furthermore, embryos created through IVF are currently evaluated for developmental potential by morphology, a criterion lacking in quantitation and accuracy. With the recent successes in oocyte vitrification and storage, clear metrics are needed to determine oocyte quality prior to fertilizing. The first polar body (PB) is extruded from the oocyte before fertilization and can be biopsied without damaging the oocyte. Here, we tested the hypothesis that the PB transcriptome is representative of that of the oocyte. Polar body biopsy was performed on metaphase II (MII) oocytes followed by single-cell transcriptome analysis of the oocyte and its sibling PB. Over 12,700 unique mRNAs and miRNAs from the oocyte samples were compared with the 5,431 mRNAs recovered from the sibling PBs (5,256 shared mRNAs or 97%, including miRNAs). The results show that human PBs reflect the oocyte transcript profile and suggests that mRNA detection and quantification through high-throughput quantitative PCR could result in the first molecular diagnostic for gene expression in MII oocytes. This could allow for both oocyte ranking and embryo preferences in IVF applications.

High throughput, cell type-specific analysis of key proteins in human endometrial biopsies of women from fertile and infertile couples.

BACKGROUND: Although histological dating of endometrial biopsies provides little help for prediction or diagnosis of infertility, analysis of individual endometrial proteins, proteomic profiling and transcriptome analysis have suggested several biomarkers with altered expression arising from intrinsic abnormalities, inadequate stimulation by or in response to gonadal steroids or altered function due to systemic disorders. The objective of this study was to delineate the developmental dynamics of potentially important proteins in the secretory phase of the menstrual cycle, utilizing a collection of endometrial biopsies from women of fertile (n = 89) and infertile (n = 89) couples. METHODS AND RESULTS: Progesterone receptor-B (PGR-B), leukemia inhibitory factor, glycodelin/progestagen-associated endometrial protein (PAEP), homeobox A10, heparin-binding EGF-like growth factor, calcitonin and chemokine ligand 14 (CXCL14) were measured using a high-throughput, quantitative immunohistochemical method. Significant cyclic and tissue-specific regulation was documented for each protein, as well as their dysregulation in women of infertile couples. Infertile patients demonstrated a delay early in the secretory phase in the decline of PGR-B (P < 0.05) and premature mid-secretory increases in PAEP (P < 0.05) and CXCL14 (P < 0.05), suggesting that the implantation interval could be closing early. Correlation analysis identified potential interactions among certain proteins that were disrupted by infertility. CONCLUSIONS: This approach overcomes the limitations of a small sample number. Protein expression and localization provided important insights into the potential roles of these proteins in normal and pathological development of the endometrium that is not attainable from transcriptome analysis, establishing a basis for biomarker, diagnostic and targeted drug development for women with infertility.

Ovulation induction in women with polycystic ovary syndrome: an update.

Infertility is frequently caused by anovulation. The affected women present with irregular menstrual cycles and the most common diagnosis is polycystic ovary syndrome. Ovulation induction is commonly used to treat these women. Clomiphene citrate (a selective estrogen receptor modulator or SERM) remains the most used medication for treating this condition. Alternatives that have been used include other SERMs such as tamoxifen, aromatase inhibitors, insulin sensitizing agents, and ovarian drilling. Evidence for and against the effectiveness of these agents has fluctuated over the last decade. Controversies surrounding the use of ovulation induction such as development of functional cysts, high-order multiple births, and development of ovarian cancer have been further studied and some controversies have almost been laid to rest in the last decade.

Phthalates and bisphenol do not accumulate in human follicular fluid.

OBJECTIVE: To determine if phthalates and bisphenol A accumulate in human follicular fluid after brief exposure to medical plastics during an IVF cycle STUDY DESIGN: Prospective collection of follicular fluid from five infertile women undergoing oocyte retrieval at a University IVF laboratory and analysis of Phthalate & Bisphenol A levels. RESULTS: All phthalate levels were detected at levels less than 15 ng/mL and Bisphenol A levels were undetectable in all five samples. The concentrations of phthalates are 200-1000 fold less than the minimum levels reported to cause reproductive toxicity in vitro to cumulus-oocyte complexes of laboratory animals. CONCLUSIONS: In reproductive age women undergoing infertility treatments there is little transfer or accumulation of phthalates, phthalate metabolites or bisphenol A into the microenvironment of the human preovulatory oocyte and the levels are not clinically significant. Further investigation of phthalate and bisphenol A accumulation in vivo in human follicular fluid may not be productive.

Pre-IVF treatment with a GnRH antagonist in women with endometriosis (PREGNANT): study protocol for a prospective, double-blind, placebo-controlled trial.

INTRODUCTION: Infertility is a common complication of endometriosis. While in vitro fertilisation-embryo transfer (IVF) successfully treats endometriosis-associated infertility, there is some evidence that pregnancy rates may be diminished in women seeing fertility treatment for endometriosis-associated infertility compared with other etiologies of infertility. The use of gonadotropin releasing hormone (GnRH) agonist prior to IVF has been suggested to improve success, however studies have been small and rarely reported live birth rates. Recent approval of an oral GnRH antagonist for endometriosis provides a novel option for women with endometriosis who are undergoing IVF. There have been no studies on the efficacy of GnRH antagonists for the treatment of endometriosis-related infertility. METHODS AND ANALYSIS: This study is a multicentre, prospective, randomised, double-blind, placebo-controlled trial to study the efficacy of GnRH antagonist pretreatment for women with endometriosis who are undergoing IVF. A total of 814 patients with endometriosis undergoing fertility treatment will be enrolled and randomised 1:1 into two groups: elagolix 200 mg two times per day or placebo for 8 weeks, prior to undergoing IVF. All participants will then undergo IVF treatment per local protocols. The primary outcome is live birth. Secondary outcomes include oocyte number, fertilisation rate, embryo morphology and implantation rates, as well as rates of known endometriosis-related obstetrical outcomes (pregnancy-induced hypertension, antepartum haemorrhage, caesarean delivery and preterm birth). ETHICS AND DISSEMINATION: The PREGnant trial was approved by the Institutional Review Board at Johns Hopkins University. Results will be published in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04173169.

TCR signal strength defines distinct mechanisms of T cell dysfunction and cancer evasion.

T cell receptor (TCR) signal strength is a key determinant of T cell responses. We developed a cancer mouse model in which tumor-specific CD8 T cells (TST cells) encounter tumor antigens with varying TCR signal strength. High-signal-strength interactions caused TST cells to up-regulate inhibitory receptors (IRs), lose effector function, and establish a dysfunction-associated molecular program. TST cells undergoing low-signal-strength interactions also up-regulated IRs, including PD1, but retained a cell-intrinsic functional state. Surprisingly, neither high- nor low-signal-strength interactions led to tumor control in vivo, revealing two distinct mechanisms by which PD1hi TST cells permit tumor escape; high signal strength drives dysfunction, while low signal strength results in functional inertness, where the signal strength is too low to mediate effective cancer cell killing by functional TST cells. CRISPR-Cas9-mediated fine-tuning of signal strength to an intermediate range improved anti-tumor activity in vivo. Our study defines the role of TCR signal strength in TST cell function, with important implications for T cell-based cancer immunotherapies.

Use of in-cycle antimüllerian hormone levels to predict cycle outcome.

OBJECTIVE: The goal of this work is to expand the usefulness of antimüllerian hormone (AMH) in predicting in vitro fertilization cycle outcome by demonstrating that AMH concentration obtained in an ongoing treatment cycle predicts both oocyte number and pregnancy. STUDY DESIGN: Serum samples were obtained from 190 in vitro fertilization patients at onset of follicle-stimulating hormone stimulation. These were analyzed retrospectively during a single cycle in which clinicians were blinded to the results. Our major outcome measures were the number of oocytes obtained and ongoing pregnancy. RESULTS: Patients with an initial AMH concentration of >3 ng/mL were found to produce a mean of 19.8 oocytes and had an ongoing pregnancy rate of 60.3%. In contrast, those with AMH values of ≤1 ng/mL yielded a mean of 6.2 oocytes and had an ongoing pregnancy rate of 23.4% (P < .0001 for both). CONCLUSION: Greater AMH serum concentration strongly predicts an increased number of oocytes and ongoing pregnancy (P ≤ .0001).

The evolutionarily conserved long non-coding RNA LINC00261 drives neuroendocrine prostate cancer proliferation and metastasis via distinct nuclear and cytoplasmic mechanisms.

Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor-matched patient-derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+ /PSA+ ) or NEPC (AR- /SYN+ /CHGA+ ) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229-fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC-3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR-8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD-driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261-CBX2-FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease.

Clomiphene, metformin, or both for infertility in the polycystic ovary syndrome.

BACKGROUND: The polycystic ovary syndrome is a common cause of infertility. Clomiphene and insulin sensitizers are used alone and in combination to induce ovulation, but it is unknown whether one approach is superior. METHODS: We randomly assigned 626 infertile women with the polycystic ovary syndrome to receive clomiphene citrate plus placebo, extended-release metformin plus placebo, or a combination of metformin and clomiphene for up to 6 months. Medication was discontinued when pregnancy was confirmed, and subjects were followed until delivery. RESULTS: The live-birth rate was 22.5% (47 of 209 subjects) in the clomiphene group, 7.2% (15 of 208) in the metformin group, and 26.8% (56 of 209) in the combination-therapy group (P<0.001 for metformin vs. both clomiphene and combination therapy; P=0.31 for clomiphene vs. combination therapy). Among pregnancies, the rate of multiple pregnancy was 6.0% in the clomiphene group, 0% in the metformin group, and 3.1% in the combination-therapy group. The rates of first-trimester pregnancy loss did not differ significantly among the groups. However, the conception rate among subjects who ovulated was significantly lower in the metformin group (21.7%) than in either the clomiphene group (39.5%, P=0.002) or the combination-therapy group (46.0%, P<0.001). With the exception of pregnancy complications, adverse-event rates were similar in all groups, though gastrointestinal side effects were more frequent, and vasomotor and ovulatory symptoms less frequent, in the metformin group than in the clomiphene group. CONCLUSIONS: Clomiphene is superior to metformin in achieving live birth in infertile women with the polycystic ovary syndrome, although multiple birth is a complication. (ClinicalTrials.gov number, NCT00068861 [ClinicalTrials.gov].).