Heterozygous ZNHIT3 variants within the 17q12 recurrent deletion region are associated with Mayer-Rokitansky-Kuster Hauser (MRKH) syndrome.

The molecular basis of mullerian aplasia, also known as Mayer-Rokitansky-Kuster Hauser (MRKH) or congenital absence of the uterus and vagina, is largely unknown. We applied a multifaceted genetic approach to studying the pathogenesis of MRKH including exome sequencing of trios and duos, genome sequencing of families, qPCR, RT-PCR, and Sanger sequencing to detect intragenic deletions, insertions, splice variants, single nucleotide variants, and rearrangements in 132 persons with MRKH. We identified two heterozygous variants in ZNHIT3 localized to a commonly involved CNV region at chromosome 17q12 in two different families with MRKH. One is a frameshift, truncating variant that is predicted to interfere with steroid hormone binding of the LxxLL sequence of the C-terminal region. The second variant is a double missense/stopgain variant. Both variants impair protein expression in vitro. In addition, four more probands with MRKH harbored the stopgain variant without the nearby missense variant. In total, 6/132 (4.5%) of patients studied, including five with associated anomalies (type 2 MRKH), had ZNHIT3 variants that impair function in vitro. Our findings implicate ZNHIT3 as an important gene associated with MRKH within the 17q12 CNV region.

Rare structural variants, aneuploidies, and mosaicism in individuals with Mullerian aplasia detected by optical genome mapping.

The molecular basis of Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome remains largely unknown. Pathogenic variants in WNT4 and HNF1B have been confirmed in a small percent of individuals. A variety of copy number variants have been reported, but causal gene(s) remain to be identified. We hypothesized that rare structural variants (SVs) would be present in some individuals with MRKH, which could explain the genetic basis of the syndrome. Large molecular weight DNA was extracted from lymphoblastoid cells from 87 individuals with MRKH and available parents. Optical genome mapping (OGM) was performed to identify SVs, which were confirmed by another method (quantitative PCR, chromosomal microarray, karyotype, or fluorescent in situ hybridization) when possible. Thirty-four SVs that overlapped coding regions of genes with potential involvement in MRKH were identified, 14 of which were confirmed by a second method. These 14 SVs were present in 17/87 (19.5%) of probands with MRKH and included seven deletions, three duplications, one new translocation in 5/50 cells-t(7;14)(q32;q32), confirmation of a previously identified translocation-t(3;16)(p22.3;p13.3), and two aneuploidies. Of interest, three cases of mosaicism (3.4% of probands) were identified-25% mosaicism for trisomy 12, 45,X(75%)/46,XX (25%), and 10% mosaicism for a 7;14 translocation. Our study constitutes the first systematic investigation of SVs by OGM in individuals with MRKH. We propose that OGM is a promising method that enables a comprehensive investigation of a variety of SVs in a single assay including cryptic translocations and mosaic aneuploidies. These observations suggest that mosaicism could play a role in the genesis of MRKH.

Long-Term Follow-Up and Treatment of a Female With Complete Estrogen Insensitivity.

CONTEXT: We previously reported the first female with a causative ESR1 gene variant, who exhibited absent puberty and high estrogens. At age 15 years, she presented with lower abdominal pain, absent breast development, primary amenorrhea, and multicystic ovaries. The natural history of complete estrogen insensitivity (CEI) in women is unknown. OBJECTIVE: The purpose of this report is to present the neuroendocrine phenotype of CEI, identify potential ligands, and determine the effect of targeted treatment. DESIGN: We have characterized gonadotropin pulsatility and followed this patient's endocrine profile and bone density over 8 years. Seventy-five different compounds were tested for transactivation of the variant receptor. A personalized medicine approach was tailored to our patient. SETTING: Academic medical center. PATIENT OR OTHER PARTICIPANTS: A 24-year-old adopted white female with CEI. INTERVENTION(S): The patient was treated with diethylstilbestrol (DES) for approximately 2.5 years. MAIN OUTCOME MEASURE(S): Induction of secondary sexual characteristics. RESULTS: Luteinizing hormone (LH) pulse studies demonstrated normal pulsatile LH secretion, elevated mean LH, and mildly elevated mean follicle-stimulating hormone (FSH) in the presence of markedly increased estrogens. DES transactivated the variant ESR1 in vitro. However, DES treatment did not induce secondary sexual characteristics in our patient. CONCLUSIONS: Treatment with DES was not successful in our patient. She remains hypoestrogenic despite the presence of ovarian cysts with a hypoestrogenic vaginal smear, absent breast development, and low bone mineral mass. Findings suggest additional receptor mechanistic actions are required to elicit clinical hormone responses.

Comparative Genomic Mapping Implicates LRRK2 for Intellectual Disability and Autism at 12q12, and HDHD1, as Well as PNPLA4, for X-Linked Intellectual Disability at Xp22.31.

We report a genomic and phenotypic delineation for two chromosome regions with candidate genes for syndromic intellectual disability at 12q12 and Xp22.31, segregating independently in one family with four affected members. Fine mapping of three affected members, along with six unreported small informative CNVs, narrowed down the candidate chromosomal interval to one gene LRRK2 at 12q12. Expression studies revealed high levels of LRRK2 transcripts in the whole human brain, cerebral cortex and hippocampus. RT-qPCR assays revealed that LRRK2 transcripts were dramatically reduced in our microdeletion patient DGDP289A compared to his healthy grandfather with no deletion. The decreased expression of LRRK2 may affect protein-protein interactions between LRRK2 and its binding partners, of which eight have previously been linked to intellectual disability. These findings corroborate with a role for LRRK2 in cognitive development, and, thus, we propose that intellectual disability and autism, displayed in the 12q12 microdeletions, are likely caused by LRRK2. Using another affected member, DGDP289B, with a microdeletion at Xp22.31, in this family, we performed the genomic and clinical delineation with six published and nine unreported cases. We propose HDHD1 and PNPLA4 for X-linked intellectual disability in this region, since their high transcript levels in the human brain substantiate their role in intellectual functioning.

A presentation of ovarian fibrothecoma in a middle-aged female with recurrent massive ascites and postmenopausal bleeding: A case report.

Fibrothecomas are mostly benign ovarian tumors. We describe a rare case of fibrothecoma in a 59-year-old woman with a history of recurrent massive ascites and postmenopausal bleeding. Over 2 years she has undergone more than 50 paracenteses with 8-11 L withdrawn each time. She presented with a newly diagnosed pelvic mass and bilateral hydrosalpinges demonstrated on ultrasound and were confirmed with computed tomography. The pelvic magnetic resonance imaging showed a large complex cystic mass arising from the left ovary measuring 16.6 × 12.1 × 8.6 cm3 with an area of irregular mural enhancement. The patient underwent left salpingo-oophorectomy with benign frozen section results. The final pathology was consistent with cystic degeneration of fibrothecoma.

Umbilical Cord Blood Mesenchymal Stem Cells as an Infertility Treatment for Chemotherapy Induced Premature Ovarian Insufficiency.

BACKGROUND: Premature ovarian insufficiency (POI) is a challenging disease, with limited treatment options at the moment. Umbilical cord blood mesenchymal stem cells (UCMSCs) have demonstrated promising regenerative abilities in several diseases including POI. MATERIALS AND METHOD: A pre-clinical murine case versus vehicle control randomized study. Two experiments ran in parallel in each of the three groups. The first was to prove the ability of UCMSCs in restoring ovarian functions. The second was to prove improved fertility. A total of 36 mice were randomly assigned; 6 mice into each of 3 groups for two experiments. Group 1 (control), group 2 (sham chemotherapy), group 3 (stem cells). RESULTS: In the first experiment, post-UCMSCs treatment (group 3) showed signs of restored ovarian function in the form of increased ovarian weight and estrogen-dependent organs (liver, uterus), increased follicular number, and a significant decrease in FSH serum levels (p < 0.05) compared to group 2, and anti-Mullerian hormone (AMH) serum levels increased (p < 0.05) in group 3 versus group 2. Immuno-histochemistry analysis demonstrated a higher expression of AMH, follicle stimulating hormone receptor (FSHR) and Inhibin A in the growing follicles of group 3 versus group 2. In the second experiment, post-UCMSCs treatment (group 3) pregnancy rates were higher than group 2, however, they were still lower than group 1. CONCLUSION: We demonstrated the ability of UCMSCs to restore fertility in female cancer survivors with POI and as another source of stem cells with therapeutic potentials.

Minimal difference in phenotype between adolescents and young adults with polycystic ovary syndrome.

OBJECTIVE: To test the hypothesis that the polycystic ovary syndrome (PCOS) phenotype, or its component features, is less severe in adolescents than in young adult patients, in a referred (clinical) population. DESIGN: Cross-sectional study. SETTING: Tertiary-care academic medical center. PATIENT(S): Two hundred seventy-four adolescents and young adults aged 13.0-24.9 years with PCOS according to the National Institute of Health 1990 criteria. Patients were categorized as adolescents (AD: 13.0-18.9 years; n = 91) and young adults (YA: 19.0-24.9 years; n = 183). Adolescents were further categorized as early adolescents (Early-AD: 13.0-15.9 years; n = 31) and late adolescents (Late-AD: 16.0-18.9 years; n = 60). INTERVENTION(S): History, physical examination, hormonal assays with the use of standardized protocols. MAIN OUTCOME MEASURE(S): Unadjusted and adjusted odds ratios (ORs; adjusted for body mass index [BMI] when applicable) were calculated for biochemical hyperandrogenism (HA), hirsutism (HIR), acne, and degree of oligo/amenorrhea (OA). PCOS phenotypes were classified as HIR+HA+OA, HA+OA, and HIR+OA. RESULT(S): Our analysis demonstrated minimal significant difference in the prevalence of the three PCOS phenotypes, or component features, between AD and YA patients. The risks for obesity were higher for YA versus AD, and the risk of acne was lower for YA versus AD. There was no significant difference between Early-AD and Late-AD. BMI-adjusted models did not significantly modify the main findings. CONCLUSION(S): The present study suggests that the PCOS phenotype is established in early adolescence, remains constant into adulthood, and is not related to BMI.

The ontogeny of myometrial stem cells in OCT4-GFP transgenic mouse model.

BACKGROUND: Myometrium, the muscular wall of the uterus, is an active organ markedly remodeled during a woman's reproductive life, especially during pregnancy. Different studies using the 5-bromo-2'-deoxyuridine and side population methods in murine and human myometrium have suggested the presence of somatic stem cells in this tissue because of its remarkable regenerative capacity. Recently, our group has developed a surface-marker (Stro1/CD44)-specific approach to isolate and characterize myometrial somatic stem cells (SSCs) from humans and rats. OBJECTIVE: In this study, we aimed to identify and localize the putative myometrial stem cell population in the murine uterus by using the specific surface markers, Nanog/CD44. METHODS: Uteri from OCT4-GFP transgenic mice at different early-life time points were analyzed via single and double immunohistochemistry to co-localize myometrial stem cell marker CD44 with other general stemmness markers, e.g., Nanog and Oct-4. Finally, we correlated the frequency of myometrial stem cells in vivo with the expression of sex steroid hormone receptors, estrogen receptor α (ERα), and progesterone receptors A and B (PR A&B). RESULTS: Nanog+/CD44+ stem cells were present in murine myometrium. Both stem cell markers were shown to co-localize with Oct-4 expression. Time-course experiments demonstrated that their percentages were significantly lower at the pre-sexual age of 1 week than at the sexually mature ages of 3 to 24 weeks. Importantly, both ERα and PR A&B were abundantly expressed in the myometrium at ages 1, 3 and 4 weeks. CONCLUSIONS: We demonstrated that murine CD44+ myometrial cells have features of somatic stem cells with the expression of typical undifferentiated markers. Furthermore, our results suggest that myometrial stem cells are sex steroid hormone dependent, likely via paracrine pathway, and increase in numbers with reproductive maturity and rise in serum estrogen and progesterone levels around 3 weeks of age in mice. The abundance and early onset expression of ER/PR emphasize the vulnerability of neonatal myometrium to environmental endocrine disruptors which can potentially lead to permanent reprograming and adult onset of myometrial disorders such as uterine fibroids.

Human Mesenchymal Stem Cells Partially Reverse Infertility in Chemotherapy-Induced Ovarian Failure.

INTRODUCTION: Chemotherapy is the most commonly used modality to treat human cancers; however, in many cases it causes irreversible ovarian failure. In this work, we plan to evaluate the restorative function of human bone marrow mesenchymal stem cells (BMSCs) in a chemotherapy-induced ovarian failure mouse model. METHODS: Acclimatized 4 to 6 week-old female mice (C57BL/6) were assigned randomly to a vehicle-treated control group (group 1), chemotherapy-treated group followed by vehicle alone (group 2), or chemotherapy-treated group followed by stem cell intraovarian injection (group 3). Outcomes were evaluated using immunohistochemistry (IHC), serum hormonal assays, and estrous cycle monitoring and breeding potential. RESULTS: Post BMSCs administration, group 3 promptly showed detectable vaginal smears with estrogenic changes. Increase in total body weight, ovarian weight, and weight of estrogen-responsive organs (uterus and liver) was observed at 2 weeks and continued to end of the experiment. Hematoxylin and Eosin histological evaluation of the ovaries demonstrated a higher mean follicle count in group 3 than in group 2. Group 3 had lower follicle-stimulating hormone (FSH) levels ( P = .03) and higher anti-Müllerian hormone serum (AMH) levels ( P = .0005) than group 2. The IHC analysis demonstrated higher expression of AMH, FSH receptor, inhibin A, and inhibin B in growing follicles of group 3 versus group 2. Tracking studies demonstrated that human BMSCs evenly repopulated the growing follicles in treated ovaries. Importantly, breeding data showed significant increases in the pregnancies numbers, 2 pregnancies in group 1 and 12 in group 3 ( P = .02). CONCLUSIONS: Intraovarian administered BMSCs are able to restore ovarian hormone production and reactivate folliculogenesis in chemotherapy-induced ovarian failure mouse model.

Perspectives on Polycystic Ovary Syndrome: Is Polycystic Ovary Syndrome Research Underfunded?

Context: Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic abnormality with a worldwide prevalence of 4% to 21%, depending on diagnostic criteria. The National Institutes of Health (NIH) is the largest single funding agency in the world; it invests nearly $30.0 billion annually in biomedical research. Evidence Acquisition: Using the NIH Research Portfolio Online Reporting tool, we searched for all grants awarded by the NIH for PCOS and three other disorders with similar degrees of morbidity and similar or lower mortality and prevalence [rheumatoid arthritis (RA), tuberculosis (TB), and systemic lupus erythematosus (SLE)]. Evidence Synthesis: We compared funding by the NIH for PCOS, RA, TB, and SLE research for the years 2006 to 2015, inclusive. Conclusion: PCOS, compared with RA, TB, and SLE, was relatively less funded (total mean 10-year funding was $215.12 million vs $454.39 million, $773.77 million, and $609.52 million, respectively). Funding for PCOS was largely provided by one NIH Institute/Center (ICs) vs at least two ICs for SLE and RA; more individual Research Project Grants were awarded for RA, SLE, and TB than for PCOS, whereas PCOS funding was more likely to be through General Clinical Research Centers Program or Specialized Centers Program awards. Our data suggest that PCOS research may be underfunded considering its prevalence, economic burden, metabolic morbidity, and negative impact on quality of life. Greater education of NIH leaders, including those at the National Heart, Lung, and Blood Institute and National Institutes of Diabetes and Digestive and Kidney Diseases; other federal and state agency leads; elected leaders; and the general public by professional societies, the scientific community, and patient advocates regarding this disorder is needed.

Criteria, prevalence, and phenotypes of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a highly prevalent disorder effecting reproductive-aged women worldwide. This article addresses the evolution of the criteria used to diagnosis PCOS; reviews recent advances in the phenotypic approach, specifically in the context of the extended Rotterdam criteria; discusses limitations of the current criteria used to diagnosis, particularly when studying adolescents and women in the peri- and postmenopause; and describes significant strides made in understanding the epidemiology of PCOS. This review recognizes that although there is a high prevalence of PCOS, there is increased variability when using Rotterdam 2003 criteria, owing to limitations in population sampling and approaches used to define PCOS phenotypes. Last, we discuss the distribution of PCOS phenotypes, their morbidity, and the role that referral bias plays in the epidemiology of this syndrome.

Role of vitamin D in uterine fibroid biology.

OBJECTIVE: To provide a detailed summary of current scientific knowledge on uterine fibroids (leiomyomas) in vitro and in in vivo animal models, as well as to postulate the potential role of vitamin D3 as an effective, inexpensive, safe, long-term treatment option for uterine fibroids. DESIGN: PubMed search articles were used to identify the most relevant studies on uterine fibroids, as well as effects of vitamin D3 on uterine fibroid cells and fibroid tumor growth in in vivo animal models. SETTING: University research laboratory. PATIENT(S): Not applicable. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Not applicable. RESULT(S): Despite numerous publications available on uterine fibroids, information about the role that vitamin D3 plays in the regulation of uterine fibroids is limited. Most of the recent vitamin D3-related studies on uterine fibroids were published from our group. Recent studies have demonstrated that vitamin D deficiency plays a significant role in the development of uterine fibroids. Our recent studies have demonstrated that vitamin D3 reduces leiomyoma cell proliferation in vitro and leiomyoma tumor growth in in vivo animal models. These results postulate the potential role of vitamin D3 for an effective, safe, nonsurgical medical treatment option for uterine fibroids. CONCLUSION(S): This article reviews human and animal studies and uncovers new possibilities for understanding the vitamin D-based therapeutic option for an effective, safe, long-term treatment of uterine fibroids. On the basis of these results, a clinical trial with vitamin D3 or a hypocalcemic analog, paricalcitol, may be warranted for nonsurgical medical treatment of uterine fibroids.