United States

BackgroundDisorders/Differences of sex development (DSD) may be associated with adverse psychosocial and psychosexual outcomes in adults. However, there is a paucity of information on health-related quality of life (QoL) outcomes in parents and children with DSD and a lack of instruments available for evaluating these outcomes. Recently, this has led to the development of Parent Self-Report and Proxy-Report QoL questionnaires (QoL-DSD), validated measures for parents of young children with DSD, comprising 63 items within 13 domains and 25 items within 5 domains, respectively.ObjectiveWhile retaining the original domain structure of the QoL-DSD questionnaires, we aimed to develop short forms of the QoL-DSD, optimizing their use in routine clinic settings.MethodsShort forms of the DSD-QoL Parent Self-Report (QoL-DSD Short PSR) and Parent Proxy-Report (QoL-DSD Short PPR) questionnaires were developed following exploratory factor analysis with maximum likelihood and varimax rotation, using previous QoL-DSD data from 132 parents. Long and resulting short form questionnaires were completed online by 18 parents of children with DSD, under 7 years of age, attending endocrine and urology clinics at one tertiary hospital in Scotland.ResultsItem selection for the short forms — QoL-DSD Short PSR and QoL-DSD Short PPR — based on item factor loadings of &gt;0.8, produced questionnaires containing 16 and 7 items, respectively. Eighteen parents completed both long and short forms of the Parent Self-Report for children aged &lt;7 years, and a subset of these (n=13) also completed long and short forms of the Parent Proxy-Report for children aged 2 to 7 years. Of the 18 target children, all were boys with a median age of 3.6 years (range 0.4, 6.6) and 10 (59%) had proximal hypospadias. Overall, agreement was achieved between the short and long questionnaires in 9 out of 12 (75%) domains on the Parent Self-Report and 4 out of 5 (80%) domains on the Parent Proxy-Report. Correlations between total scores on the short and long forms were 0.508 to 0.897 in 9 of 12 domains in the QoL-DSD Short PSR. Parental feedback (n=18) regarding the acceptability of the short versus long forms was evaluated using a 5-point Likert score: 83% (versus 66%) of parents agreed the length of time (less than 3 minutes) taken to complete short forms was acceptable, 39% preferred the short forms compared with 11% whom preferred the longer version, 45% (versus 22%) stated a preference to complete the short forms should they be implemented routinely at clinic visits in the future.Conclusions Short forms of the QoL-DSD for parents of young children with DSD may be more acceptable for use in a routine outpatient setting to evaluate psychosocial distress experienced by young children with DSD and their caregivers. Further psychometric validation in a larger cohort is warranted.

ENDO 2022

Development and Validation of a Short Version of the Quality of Life DSD Questionnaire (QoL-DSD) for Parents of Young Children with Disorders/Differences of Sex Development

**Program**
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over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
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more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

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These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
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**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

BackgroundDisorders/Differences of sex development (DSD) may be associated with adverse psychosocial and psychosexual outcomes in adults. However, there is a paucity of information on health-related quality of life (QoL) outcomes in parents and children with DSD and a lack of instruments available for evaluating these outcomes. Recently, this has led to the development of Parent Self-Report and Proxy-Report QoL questionnaires (QoL-DSD), validated measures for parents of young children with DSD, comprising 63 items within 13 domains and 25 items within 5 domains, respectively.ObjectiveWhile retaining the original domain structure of the QoL-DSD questionnaires, we aimed to develop short forms of the QoL-DSD, optimizing their use in routine clinic settings.MethodsShort forms of the DSD-QoL Parent Self-Report (QoL-DSD Short PSR) and Parent Proxy-Report (QoL-DSD Short PPR) questionnaires were developed following exploratory factor analysis with maximum likelihood and varimax rotation, using previous QoL-DSD data from 132 parents. Long and resulting short form questionnaires were completed online by 18 parents of children with DSD, under 7 years of age, attending endocrine and urology clinics at one tertiary hospital in Scotland.ResultsItem selection for the short forms — QoL-DSD Short PSR and QoL-DSD Short PPR — based on item factor loadings of >0.8, produced questionnaires containing 16 and 7 items, respectively. Eighteen parents completed both long and short forms of the Parent Self-Report for children aged <7 years, and a subset of these (n=13) also completed long and short forms of the Parent Proxy-Report for children aged 2 to 7 years. Of the 18 target children, all were boys with a median age of 3.6 years (range 0.4, 6.6) and 10 (59%) had proximal hypospadias. Overall, agreement was achieved between the short and long questionnaires in 9 out of 12 (75%) domains on the Parent Self-Report and 4 out of 5 (80%) domains on the Parent Proxy-Report. Correlations between total scores on the short and long forms were 0.508 to 0.897 in 9 of 12 domains in the QoL-DSD Short PSR. Parental feedback (n=18) regarding the acceptability of the short versus long forms was evaluated using a 5-point Likert score: 83% (versus 66%) of parents agreed the length of time (less than 3 minutes) taken to complete short forms was acceptable, 39% preferred the short forms compared with 11% whom preferred the longer version, 45% (versus 22%) stated a preference to complete the short forms should they be implemented routinely at clinic visits in the future.Conclusions Short forms of the QoL-DSD for parents of young children with DSD may be more acceptable for use in a routine outpatient setting to evaluate psychosocial distress experienced by young children with DSD and their caregivers. Further psychometric validation in a larger cohort is warranted.

technical paper

Background: Hypospadias is a common human congenital anomaly occurring in ~1: 200 to 1: 300 live male births in the USA, with an unexplained doubling in incidence over previous decades. Historically, 46XY Differences of Sex Development (DSD) patients, with severe hypospadias have lacked a genetic diagnosis.  With the advent of commercially available DSD genomic panels, this is now changing (GeneDX. Neonatal 46 2021, In-vitae 2021).  The goal of this study is to review the anatomical characteristics of severe hypospadias that can accurately define patients that have 46XY DSD and to determine the practical utility of performing a genomic workup in these rare 46XY DSD patients with severe hypospadias.Patients: 14 patients seen in disorders of sexual development clinic between 2018 – 2020 with severe hypospadias defined by an ectopic urethral meatus typically in the scrotum or perineum, an abnormally small glans penis size (<14 mm at maximal width in the first year of life), severe curvature and associated foreskin fusion or penoscrotal transposition were enrolled in the study.Methods: Patients who met the criteria for 46XY disorder of sexual development and severe hypospadias were offered a genomic work-up in consultation with pediatric genetics. Patients were identified through chart review. Data extracted included karyotype, hypospadias phenotype including stretched penile length at diagnosis, age at genetic diagnosis/genomic workup, gene defect/ pathologic variant, gender identity, and clinical course.Main Outcome Measurements: The main outcome measured was the presence of a genetic mutation that can be linked to the disorder of sexual development. Secondary outcomes measured included gender identity, clinical course, and histological findings if a biopsy was obtained.Results: Of the 14 patients that underwent genomic analysis, 8 had documented pathologic variants known to cause hypospadias. Pathologic genetic variants include defects in the AR (3), SRD5A2 (1), NR5A1 (2), WT1 (1), and ARTX (1).  Five had no evidence of pathologic variants based on the Invitae and GeneDx commercial 46XY DSD panel (see Table 2 for genes tested).  One had a genetic variant of unknown significance, FREM2. The patient with WT1 was consequently found to have a Wilms tumor and the patients with SRD5A2 are now undergoing adrenal insufficiency surveillance after their genetic testing.Conclusions: Patients with 46XY DSD and severe hypospadias can benefit from genetic evaluation as their underlying disorders may unveil mutations that could have potentially life-altering consequences and change surveillance and monitoring.

Utility of Genomic work-up for 46XY Disorder of Sex Development Patients with Severe Hypospadias

Study Objective:This case series was designed to describe cases of polycythemia among transgender male adolescent patients undergoing treatment with testosterone.Methods:Retrospective data was collected for our Pediatric Gender Clinic between Jan 2014 and June 2021. Variables assessed included demographic and laboratory data, presence of comorbidities, testosterone dose and level at the time of polycythemia, concurrent treatment with other hormonal treatments, and duration of therapy. Polycythemia in children was defined as hematocrit (HCT) +2 SD above mean for age and gender. Male reference range was used for HCT after 3 months of treatment.Results:Seven of 131 patients were identified with polycythemia (5.3%). The mean age was 17.9 ± 3.7 (SD) years at the time of polycythemia and 6 of 7 patients were overweight or obese. All patients were receiving testosterone by injection. The mean testosterone dose was 51.4 ± 13.3 (SD) mg/m²/2wks or 92.9 mg/2wk ±22.1 (SD). All patients developed polycythemia developed at doses below 75 mg/m²/2wks. The mean testosterone level at the time of polycythemia was 588 ± 315.3. (SD) ng/dL and the mean length of treatment before polycythemia occurred was 18.6 ± 6.2 (SD) months; the shortest duration of treatment was 10 months. Two patients have received concurrent treatment with progestin therapy. Obstructive sleep apnea (OSA) was identified in 1 patient.Conclusion:Polycythemia occurred in 5.3% of our transgender male patients receiving testosterone therapy. This is a known complication of testosterone therapy that may appear with a relatively low dose and after a relatively short duration of treatment. Patient 1Patient 2Patient 3Patient 4*Patient 5Patient 6Patient 7Age at time of polycythemia, years19.116.616.721.816.917.616.6BMI at time of polycythemia, kg/m²25.239.634.130.949.619.426.7Smoking/vaping NNNVapingNNNComorbidities: Asthma, OSANAsthma, mild OSAAsthmaNNNNAdditional hormonal therapyNMedroxyprogesterone acetate injectionEtonogestrel implantNNNNHCT level at baseline, %46.544.143.050.444.245.841.1HCT level at the time of polycythemia, %
55.050.150.053.050.550.650.8Testosterone level at the time of polycythemia, ng/dL3997033455254821253416Length of Tx with testosterone, months10141426182523Dose of testosterone, mg/2wk801008060130100100Dose of testosterone, mg/m²/2wk47.369.741.231.249.664.556.5Table 1. Characteristics of patients with polycythemia.* Patient was taking an injectable dose of testosterone for 6-8 months prior to the first visit to our clinic.

Case Series of Transmasculine Adolescents Who Developed Polycythemia on Gender Affirming Hormone Therapy.

Objective: GnRH agonists (GnRHa), used to prevent development of undesired secondary sex characteristics for gender diverse youth (GDY), may impede pubertal bone mineral accrual increasing the risk for osteopenia/osteoporosis during adulthood. Based on findings of low bone mineral density (BMD) scores (relative to natal sex) prior to GnRHa [1], we hypothesized BMD might be associated with body mass index (BMI) in GDY.Method: To test our hypothesis, we reviewed medical records for 27 GDY; 16 were designated female at birth (DFAB) and 11 were designated male at birth (DMAB). Age at presentation, gender identity, ages at initiation of GnRHa and gender affirming hormone treatment (GAHT), duration of GnRHa treatment prior to GAHT initiation, auxologic parameters including BMI, and lumbar spine BMD obtained from GE lunar IDXa dual-energy X-ray absorptiometry (DEXA) were extracted. Lumbar spine BMD was used for statistical analysis. Statistical analysis was performed with Microsoft Excel Analysis ToolPak. A linear regression model was used to determine significance of BMI and GnRHa as predictors of BMD Z-score using a significance level of 0.05. Legal gender marker was used to determine BMD Z-scores.Results: BMI ranged from 16.93 kg/m² to 35.79 kg/m² (5th to the 99th percentile). DEXA scan results were available for 27 GDY with age range 10-25 years; mean age (±SD) was 17±3 years. BMD of the lumbar spine was obtained from 0-61 months (mean 30 ± 21 months) following initiation of GnRHa. Length of GnRHa treatment prior to GAHT initiation ranged from 0-53 months (mean 14 ± 12 months).Individuals were at different stages of the gender affirmation process including 1) No medical treatments, 2) GnRH agonist only, 3) GAHT only, and 4) combined GnRH agonist and GAHT. Using a linear regression model, length of GnRHa therapy and length of GnRHa without GAHT were not significantly associated with lumbar spine BMD in our sample (p = 0.44 and 0.29, respectively). Using a linear regression model, BMI was a significant positive predictor of higher lumbar bone mineral density (p = 0.03).Conclusion: Sex hormone exposure during adolescence and young adulthood is essential for bone health. While GnRH agonists lower endogenous sex hormone exposure, in our sample, lower BMD was not associated with total duration of GnRHa therapy or duration of GnRHa treatment prior to GAHT. Rather BMD was positively associated with BMI regardless of gender affirming medical therapies. Limitations include small sample size, retrospective design, and the use of legal gender marker for normative values. The next step will be larger, prospective studies to identify predictors of low BMD in GD youth. This information will guide future management regarding BMD monitoring.References: 1. Lee JY,et al., J Endocr Soc. 2020;4(9): bvaa065.

Body Mass Index (BMI) as a Predictor of Bone Mineral Density (BMD) in Gender Diverse Youth

Background: Studies have shown transgender and non-binary individuals encounter discrimination and harassment in medical settings leading to delays in seeking care and worse physical and mental health outcomes. Organizations, including the American Academy of Pediatrics, published recommendations for medical centers to foster respectful and inclusive care through systems, environmental, and educational changes. However, it is unclear whether health systems are making these changes and what the impact may be for transgender and non-binary youth.
Objective: To explore perspectives of transgender and non-binary patients regarding the current state of gender inclusive care at our medical center and identify areas for improvement.
Design/Methods: A qualitative study was conducted at a large academic center. Participants aged 13-21 years were recruited from the Center for Gender Affirming Care to participate in a semi-structured interview and demographics survey regarding their experiences across various clinical settings within the medical center. Interviews were audio-recorded and transcribed verbatim. Survey results were analyzed using descriptive statistics. Two independent coders reviewed the transcripts, developed a preliminary coding scheme, and reached consensus on any coding discrepancies. ATLAS.ti® was used for thematic analysis.
Results: Thirty participants completed an interview and survey. Mean age of participants was 17 years, with 30% identifying as female/trans female, 47% male/trans male, and 23% identifying as nonbinary or gender fluid. Several key themes emerged. Overall participants are satisfied with their care and attributed improvements to societal progress and systems/environmental changes. Parent advocacy and diversity among staff promoted positive and affirming experiences. However, participants identified personnel, systems, and environmental areas needing improvement including: 1) Provider training on use of pronouns, gender affirming terminology, and exam techniques, 2) Reliable and accurate display of chosen name and pronouns in the electronic medical record (EMR) and patient labels, and standardized check-in processes to prevent misnaming/misgendering, 3) Desire for more LGBTQ-inclusive décor and resources.
Conclusions: While national recommendations were published more than 5 years ago, it is clear from our participants that not all recommendations have been implemented consistently across all clinical settings. Eliciting feedback from patients may provide tangible ways to improve care for this population.

Designing a Gender Affirming Hospital: Patient Perspectives on Gender Inclusive Care

Cancer therapies often cause serious side effects, affecting the quality of life for cancer survivors. The ovary can be affected by cancer therapies, causing premature ovarian insufficiency, leading to endocrine dysfunction and infertility. Thus, maintaining ovarian function against cancer treatment is an unmet need for female cancer patients. Cyclophosphamide (CPA), a common chemotherapeutic agent, is metabolized in vivo and forms DNA crosslinks, inducing apoptosis in rapidly proliferating tumor cells. However, the underlying mechanism of the CPA-induced oocyte death in ovarian reserve remains unclear. This study aims to investigate the mechanism of oocyte death in primordial follicles by generating oocyte-specific Abl1 and p63 knockout and Pik3ca* knockin mouse models using Gdf9-iCre+. Postnatal day 7 female mice were i.p. injected w/wo CPA. Notably, we found CPA drastically reduced the number of primordial follicles without increasing the number of growing follicles. The quantification of surviving follicles validated that 90% of the primordial follicles from oocyte-specific Abl1 knockout mice were lost following CPA treatment in vivo and in vitro. Concurrently, high expression of CHK2 was detected in the oocytes of the ovary cultured with CPA metabolite in vitro. This implied CHK2 rather than ABL is a possible tyrosine kinase for CPA-induced oocyte death. Most of all, p63 was a key player for CPA-induced oocyte death, supported by the result that p63 knockout oocytes were rescued after CPA treatment. To clarify whether the ovaries lose follicles via activation or apoptosis pathway, PIK3CA* mice were examined with CPA. As expected, primordial follicles and primordial follicles with activated oocytes in the ovary of PIK3CA* mice survived against CPA due to constitutive PI3K activity in them. This indicates activated follicles resist gonadotoxic reagents, although p63 is expressed inside their oocytes. Accordingly, the apoptosis markers, BAX and cleaved PARP were highly induced with CPA injection in a time-dependent manner in the ovaries from wild-type female mice but not from the p63 knockout. The double-strand break also occurred in the nucleus of oocytes post CPA administration as γH2AX was detected. Interestingly, OPA1, a protein required for mitochondrial fusion, was highly induced inside the oocyte cytoplasm of p63 knockout, while oocytes in wild-type mice time-dependently lost the OPA1 expression by CPA treatment. This indicates that oocytes without p63 in the nucleus survive and induce mitochondrial fusion to escape apoptosis by mitochondrial damages. In conclusion, CPA induces depletion of oocytes of primordial follicles through the CHK2-TAp63 apoptotic pathway.

Cyclophosphamide Depletes Primordial Follicles via the p63 Apoptotic Pathway

While androgens are traditionally associated with male physiology, recently it has become more apparent that balanced levels of androgens are crucial to maintaining female health and fertility as well. Specifically, androgens are necessary for granulosa cell proliferation and follicle survival and growth. Excess androgen levels in females are a hallmark in polycystic ovary syndrome (PCOS), whereas insufficient androgen signaling due to androgen receptor (AR) knockout leads to diminished ovarian reserve (DOR) – with both extremes resulting in anovulatory infertility. However, little is known about the factors regulating AR in granulosa cells. Our previous findings demonstrate that paxillin, a cytoplasmic adaptor protein, mediates AR signaling in prostate cancer cells. However, our current interest is whether this interaction is also found in granulosa cells. As such, our results show that paxillin indeed enhances AR protein expression in granulosa cells. We have also demonstrated through immunofluorescence and proximity ligation assays that paxillin colocalizes with AR, notably in membrane-bound complexes. Moreover, paxillin-null KGN and mouse granulosa cells express lower levels of AR, as demonstrated through Western blotting and immunofluorescence imaging. We then conducted mRNA and protein degradation experiments in paxillin-null KGN cells in order to determine the mechanism by which paxillin increases AR expression. These results suggest that paxillin acts by preventing AR mRNA and protein degradation, possibly by trafficking AR to focal adhesions, where it may be sequestered from degradation. Finally, to investigate the role of paxillin in female fertility, we developed a novel granulosa cell-specific paxillin knockout mouse model using an Amhr2-driven Cre/lox system. These knockout mice experience altered estrous cycles compared to controls, spending fewer days in estrus and more days in metestrus/diestrus. When bred continuously through 6 months of age, the paxillin KO mice produced significantly more litters compared to their littermate controls. Taken together, these results suggest that paxillin acts as a regulator of androgen-mediated female fertility in granulosa cells, and may serve as a potential target for therapeutics in fertility-related diseases such as PCOS and DOR.

Paxillin and Androgen Receptor Interact to Modulate Female Fertility

Introduction: Estrogen is vital to human reproduction and acts primarily through two receptors: estrogen receptor alpha (ER𝛼, encoded by ESR1) and estrogen receptor beta (ERβ, encoded by ESR2). Surprisingly, very few human ESR1 pathogenic variants have been recognized, despite the existence of >1000 different androgen receptor pathogenic variants causing androgen insensitivity syndrome. Functional analysis of an ESR1 homozygous missense variant (p.Glu375His) in the ER𝛼 ligand-binding domain (LBD) showed a 240-fold decrease in estrogen response in a patient with complete estrogen insensitivity and abnormal pubertal development. We predict that less severe ESR1 variants will present with unexplained infertility. Previously, we performed whole exome sequencing on 200 females with unexplained infertility and identified 4 likely pathogenic ESR1 heterozygous missense variants confirmed by Sanger sequencing, including one variant (p.Thr313Met) in the LBD. In this study, we performed in vitro functional analysis to evaluate if the p.Thr313Met likely pathogenic variant found in a patient with unexplained infertility causes incomplete estrogen insensitivity.
Hypothesis: We hypothesize that the p.Thr313Met likely pathogenic variant in the ERa LBD will result in incomplete estrogen insensitivity.
Methods: The p.Thr313Met variant plasmid was constructed using site-directed mutagenesis (SDM), and confirmed by Sanger sequencing. An in vitro cell model was used to evaluate estrogenic activity. HepG2 cells were transiently transfected with WT ESR1, p.Thr313Met, or p.Glu375His plasmid, along with the Firefly estrogen response element (ERE) luciferase reporter (3X ERE TATA luc) and Renilla luciferase (pRL-TK luc) plasmids using the Effectene transfection protocol. Cells were treated with 17β-estradiol (0.01, 0.1, 1, 10, and 100 nM). A dual luciferase reporter assay was used to compare relative Firefly and Renilla luciferase activity between WT ESR1, p.Thr313Met, and p.Glu375His plasmids using a luminometer. Estrogen dose response curves were generated, and a two-way ANOVA with Tukey's test was performed to compare the relative luciferase activity across WT and variant plasmids using GraphPad Prism v.9. Differences were considered significant at p<0.05.
Results: Estrogen response curves were generated for WT, p.Thr313Met, and p.Glu375His plasmids. The p.Thr313Met variant showed lower overall activity with a right shifted dose response curve with a statistically significant decrease in relative luciferase activity in comparison to WT (p<0.05). The p.Thr313Met variant was more active at higher doses (1 and 10 nM) with a left shifted dose curve when compared to the p.Glu375His variant (p<0.05).
Conclusion: We have identified a human ESR1 variant that impairs estrogen signaling in vitro, suggesting incomplete estrogen insensitivity as a putative mechanism for unexplained infertility. The identification of ESR1 pathogenic variants and the evaluation of their functionality could provide improved diagnosis and genetic counseling for patients with unexplained infertility.

ESR1 Pathogenic Variant With Incomplete Estrogen Insensitivity

Although obesity is closely associated with reproductive dysfunction, many women with obesity remain eumenorrheic. The degree to which obesity affects menstrual cycle characteristics in this population is limited to endocrine assessments with few data available on how endocrine aberrations align with defects in antral follicle development.We have previously shown alterations in the distribution of recruitable (2-5mm) versus selectable (6-9mm) follicles in those with obesity that were associated with decreased gonadotropin and progesterone production despite normal menstrual cycle length. The objectives of the present study were to further contrast follicular growth and endocrine dynamics between obese and non-obese eumenorrheic women with the goal of determining any alterations in distinct events in folliculogenesis.
21 non-obese (total percent body fat <35%) and 21 obese (total percent body fat ≥35%) reproductive age females with regular menstrual cycles (21-35d) were evaluated by serial ovarian transvaginal ultrasonography and venipuncture every-other-day for one complete inter-ovulatory interval (IOI). The number and diameters of all follicles ≥2mm at each visit were documented offline using the Grid Method. Growth profiles of individual follicles which grew to ≥7mm were assessed using the Identity Method to characterize antral follicle recruitment, selection, and ovulation. Clinical, morphologic, and endocrine features were compared across groups using t-tests and mixed models.
Women with obesity exhibited fewer recruitment events versus non-obese women (1±0.6 vs. 2±0.9; p=0.010). They also had fewer selectable follicles (4±3 vs. 8±6 follicles/participant, p=0.022) which grew faster but remained static longer and regressed slower (all growth rates, p<0.05) compared to their non-obese counterparts. Anti-Mullerian hormone levels were lower in the obese versus non-obese group (4.40±3.01 vs. 5.94±2.49ng/mL, p=0.023). Of the selectable follicles, fewer progressed to ≥10mm in the obese versus non-obese group (1±0.7 vs. 2±0.8 follicles, p=0.030) with ultimately fewer anovulatory follicles achieving dominance (0±0.7 vs. 1±0.9 follicle, p=0.029). Ovulatory follicles in the obese group were selected at smaller diameters (7.5±1.6 vs. 9.5±1.9mm, p=0.001) versus their non-obese counterparts. However, the kinetics of dominant follicle growth were similar between groups. Post-ovulation, progesterone concentrations across the luteal phase were lower in women with obesity (4.95±3.34 vs. 7.85±5.74ng/mL, p=0.001) and the prevalence of luteal phase defects (LPDs) higher versus non-obese women, defined by either integrated (76 vs. 29%, p=0.002) or maximum (71 vs. 24%, p=0.002) progesterone levels.
Eumenorrheic women with obesity display evidence of suppressed follicle dynamics as evidenced by fewer recruitment events, selectable follicles, and anovulatory dominant follicles.  LPDs are more prevalent in women with obesity during natural ovulatory cycles which may underlie subfertility in this population. A smaller size at selection suggests premature responsiveness to luteinizing hormone which may result in suboptimal luteinization. Further studies are needed to elucidate the degree to which these defects impact fertility and fecundity in our increasingly obese demographic.

Women with obesity display defects in follicular recruitment, selection, and dominance alongside luteal phase dysfunction during natural ovulatory cycles

Background: SKYLIGHT 2 (NCT04003142) investigated safety and efficacy of fezolinetant (a neurokinin-3 receptor antagonist) on frequency and severity of moderate-to-severe vasomotor symptoms (VMS) and sleep disturbance. In this analysis, persistence of fezolinetant efficacy was investigated over 52 weeks.
Methods: This double-blind Phase 3 study randomized women aged ≥40–65 years with moderate-to-severe VMS associated with menopause (average of ≥7 hot flashes/day) to once-daily placebo or fezolinetant 30mg or 45mg for 12 weeks. Women completing 12 weeks entered an extension period, with those on placebo re-randomized to fezolinetant 30mg or 45mg (placebo/fezolinetant), and those originally on fezolinetant remaining on their dose for an additional 40-weeks. Fezolinetant efficacy was evaluated vs placebo for 12-weeks through change in VMS frequency, VMS severity, and Patient-reported Outcomes Measurement Information System Sleep Disturbance–Short Form 8b (PROMIS) Total Score. Persistence of efficacy for fezolinetant was evaluated descriptively (without statistical comparisons) over the extension period. Results: The analysis comprised 484 women (fezolinetant 30mg n=166, fezolinetant 45mg n=167, placebo/fezolinetant 30mg n=76, placebo/fezolinetant 45mg n=75). Improvement in VMS frequency and severity observed through week 12 (statistically significant differences vs placebo) was maintained throughout the 52-week total study period for those receiving fezolinetant. Fezolinetant demonstrated further reductions in VMS frequency and severity from baseline to beyond week 12. For VMS frequency, there was a least squares (LS) mean (SE) baseline-to-week 12 reduction of –6.83 (0.39) VMS/day for fezolinetant 30mg and –7.50 (0.39) for 45mg, and a mean (SD) baseline-to-week 52 reduction of –8.03 (4.53) for fezolinetant 30mg and –8.48 (3.98) for 45mg. For VMS severity, LS mean (SE) baseline-to-week 12 reduction was –0.64 (0.06) for 30mg and –0.77 (0.06) for 45mg, and mean (SD) baseline-to-week 52 reduction was –0.83 (0.82) for fezolinetant 30mg, and –0.95 (0.78) for 45mg. Women re-randomized from placebo to fezolinetant experienced a reduction in frequency and severity of VMS consistent with that in women receiving fezolinetant throughout the study. Fezolinetant also reduced PROMIS-assessed sleep disturbance, with a LS mean (SE) baseline-to-week 12 reduction of –4.1 (0.5) for fezolinetant 30mg and –5.5 (0.5) for 45mg (statistically significant difference vs placebo for fezolinetant 45mg) and a mean (SD) baseline-to-week 52 reduction of – 6.3 (7.3) for fezolinetant 30mg and –5.7 (7.9) for 45mg. The safety profile observed for the 40-week extension period was consistent with that of the 12-week placebo-controlled period. Conclusion: Fezolinetant 30mg and 45mg once daily were efficacious for treatment of moderate-to-severe VMS associated with menopause. Efficacy was persistent and reductions in VMS frequency were maintained during the extension period, at levels consistent with weeks 1 through 12. Fezolinetant 45mg improved sleep at week 12 and improvement was maintained through the active treatment extension period. No safety signals of concern were apparent for either fezolinetant dose.

Fezolinetant for Treatment of Moderate-to-severe Vasomotor Symptoms Associated with Menopause: Results from a 52-week Study (Skylight 2)

Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor.  Upon binding to endogenous and exogenous ligands, ERα dimerizes and binds to regulatory regions across the genome known as enhancers.  Enhancers act as nucleation sites for the recruitment of coregulatory proteins and ultimately regulate target gene transcription.  We have previously used global run-on sequencing (GRO-seq) to detect transcription at active enhancers that generates enhancer RNAs (eRNAs).  eRNAs are poorly characterized noncoding RNAs and are thought to regulate enhancer function and gene expression.  To better understand the functions of these eRNAs, we annotated the estrogen-regulated eRNA transcriptome in ERα-positive breast cancer cells using precision nuclear run-on of capped RNA (PRO-cap) and polyA-depleted RNA-seq.  We then cloned selected eRNAs, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9.  eRNAs promoted expression of cognate, but not heterologous, target genes by increasing ERα recruitment and simulating p300-catalyzed H3K27 acetylation at the enhancer.  We identified a ~40 nucleotide functional eRNA regulatory motif (FERM) present in many estrogen-regulated eRNAs that is necessary and sufficient to promote gene expression by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation.  The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers, to exert its effect on enhancer assembly and dynamics.  Overexpression of a targeted eRNA controlling expression of an oncogene increased cell proliferation, implicating eRNAs in regulating breast cancer cell growth.  Together, we provide a genome-wide annotation of estrogen-regulated eRNAs, demonstrate the mechanisms by which targeted eRNAs promote target gene expression in vivo, identify the FERM element within eRNAs that is critical for regulating target gene expression, and show the biological implications of oncogenic eRNAs in breast cancer growth.  Collectively, our results demonstrate the biological and mechanistic roles of eRNAs in breast cancer.
  This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K.

Development and Validation of a Short Version of the Quality of Life DSD Questionnaire (QoL-DSD) for Parents of Young Children with Disorders/Differences of Sex Development

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