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Purpose: To correct a potentially damaging mutation in haploid human embryonic stem cells., Methods: Exome sequencing was performed on DNA extracted from parthenogenetically derived embryonic stem cell line (pES12). An SLC10A2 gene mutation, which affects bile acid transport, was chosen as mutation of interest in this proof of concept study to attempt correction in human pluripotent haploid cells. Confirmation of the mutation was verified, and guide RNA and a correction template was designed in preparation of performing CRISPR. Haploid cells underwent serial fluorescence activated cell sorting (FACS) with Hoechst 33342 to create an increasingly haploid (1n) enriched culture. Nucleofection was performed on p. 37 and then cells were sorted for 1n DNA content with +GFP to identify the haploid cells that expressed Cas9 tagged with GFP., Results: 104,686 haploid GFP + cells were collected. Cells were cultured, individual colonies picked, and 48 clones were sent for Sanger sequencing. CRIPSR efficiency was 77.1%, with 7/48 (14.6%) clones resulting in a corrected SLC10A2 mutation. Confirmation of persistence of haploid cells was achieved with repeated FACS sorting and centromere quantification. Given the large number of passages and exposure to CRISPR, we also performed analysis of karyotypes and of off-target effects. Cells evaluated were karyotypically normal and there was no evident off target effects., Conclusions: CRISPR/Cas9 can be effectively utilized to edit mutations in haploid human embryonic stem cells. Establishment and maintenance of a haploid cell culture provides a novel way to utilize CRISPR/Cas9 in gene editing, particularly in the study of recessive alleles.

Background: Ovarian reserve (OR) testing with serum anti-Müllerian hormone (AMH) can provide information about a woman's fertility potential. The aim of this study was to assess interest and knowledge about OR testing and investigate the utility of measuring AMH in women of reproductive age deferring pregnancy., Methods: Women ages 27-37 years currently delaying childbearing were invited to take a survey regarding attitudes and knowledge about OR testing before and after an AMH measurement with explanation of their results., Results: Of 121 women who took the pre-test survey, 96% believed OR testing was beneficial. The median AMH of the 97 women who underwent testing was 3.3 ng/mL (IQR 1.9-5.4 ng/mL). Nineteen percent of women had AMH <10th percentile for age and 3% had an undetectable AMH. Although 83% of these women were using hormonal contraception, none had known risk factors for diminished ovarian reserve. Seventy-eight percent of women with low AMH levels for age planned to seek fertility preservation or pregnancy, while those with AMH levels within established age normograms were reassured. On the post-test survey, 100% reported benefit in knowing their AMH level. Follow-up testing, 6-8 months after the initial measurement, showed stable AMH levels for most participants., Conclusions: Women are interested in OR testing. Most women will be reassured by knowing their AMH level, whereas those with a lower AMH can be counseled on fertility preservation options or may attempt pregnancy earlier.

Objective: To assess the applicability of anti-Müllerian hormone (AMH) testing in the identification of women at risk for polycystic ovary syndrome (PCOS) when AMH is used in ovarian reserve screening in the general population., Methods: A secondary analysis was undertaken of a large cross-sectional study. Women aged 27-37years, presently delaying childbearing but interested in future fertility, completed an online questionnaire to assess knowledge and attitudes about ovarian reserve testing, and underwent serum AMH testing between October 2014 and April 2015 in New York, NY, USA. For the secondary analysis, women considered to have elevated AMH levels (≥4.7ng/mL) were invited for physical examination and transvaginal ultrasonography., Results: Among 97 women who underwent AMH testing, 32 (33.0%) had elevated AMH levels. Hyperandrogenism was reported by 8 (25.0%) women with elevated AMH and none with AMH concentrations lower than 4.7ng/mL (P<0.001). Irregular menstrual cycles before hormonal contraceptive use were reported by 16 (24.6%) of 65 women with AMH concentrations lower than 4.7ng/mL and 11 (34.4%) with elevated AMH (P=0.34). Of the 20 women with elevated AMH who returned for further evaluation, 16 (80.0%) had polycystic ovaries and 13 (65.0%) were diagnosed with PCOS (Rotterdam criteria)., Conclusion: When AMH levels are used as a screening test for fertility, elevated concentrations can identify women at risk for PCOS., (Copyright © 2016 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.)