Limitations of gene editing assessments in human preimplantation embryos.

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.

Correction of a pathogenic gene mutation in human embryos.

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

Functional Human Oocytes Generated by Transfer of Polar Body Genomes.

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

Age does not influence the effect of embryo fragmentation on successful blastocyst development.

We evaluated the rate of blastocyst development in day 3 embryos with appropriate cellular division and investigated whether maternal age modified the effect of embryo fragmentation on blastulation. Our data showed a significant negative correlation between the degree of embryo fragmentation and rate of blastocyst development, but age did not exert an effect on the degree of fragmentation in embryos with appropriate cleaving status, nor did it modify the significant effect embryo fragmentation had on blastocyst formation.

Postmenopausal hyperandrogenism caused by a benign cystic teratoma: a case report.

BACKGROUND: Mature, benign cystic teratomas of the ovary are common in reproductive-age women, but they are very rarely associated with androgen production and subsequent development of hirsutism or virilization. We describe a case of postmenopausal hirsutism and hyperandrogenism caused by a mature cystic teratoma as well as the 7 previously reported cases. CASE: A 55-year-old, postmenopausal woman presented with hirsutism and unilateral lower extremity edema. Pelvic ultrasound showed a complex cystic mass in the left ovary measuring 6.0 x 7.0 x 10 cm, and laboratory evaluations revealed progressively increasing testosterone levels. The patient underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy, with resection of a large, complex mass originating in the left ovary. The pathology department found 2 left, mature ovarian cystic teratomas containing a layer of Leydig cells. Postoperatively the patient experienced rapid normalization of the elevated testosterone level. CONCLUSION: Although rare, ovarian production of androgens resulting in hirsutism or virilization can occur with a hormonally active mature cystic teratoma.