Hippo signaling in the ovary and polycystic ovarian syndrome.

PURPOSE: To provide a commentary on our understanding of the role that the Hippo signaling pathway may play in patients with polycystic ovarian syndrome (PCOS) and how this understanding may impact the diagnosis of PCOS. METHODS: We assessed publications discussing the role of the Hippo signaling pathway in the ovary. In particular, we discuss how Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, has been used in treatment of patients with primary ovarian insufficiency. Furthermore, we discuss our own data on variations in Hippo signaling pathway gene expression in cumulus cells isolated from women undergoing IVF with a previous diagnosis of PCOS. RESULTS AND CONCLUSIONS: Aberrant Hippo signaling in PCOS patients is likely a contributing mechanism to the multifactorial etiology of the disease. Given the challenge of discerning the underlying etiology of oligo-ovulation in some patients, especially those with normal body mass indices, and the need for customized stimulation protocols for PCOS patients who have an increased risk of over-response and higher percentage of immature oocyte yield, it is important to identify these patients prior to treatment. Hippo gene expression fingerprints could potentially be used to more accurately define patients with PCOS. Additionally, targeting this pathway with pharmacologic agents could lead to non-surgical therapeutic options for PCOS.

No change in live birthweight of IVF singleton deliveries over an 18-year period despite significant clinical and laboratory changes.

STUDY QUESTION: Has live birthweight changed over 18 years of autologous fresh and frozen IVF? SUMMARY ANSWER: Regardless of changes in clinical care and laboratory practice over 18 years, birthweight has remained stable. WHAT IS KNOWN ALREADY: Birthweight has historically been used as a marker of neonatal health. Frozen embryo transfers lead to heavier live birthweights compared with fresh embryo transfers. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 7295 singletons from autologous fresh (n = 6265) and frozen (n = 1030) IVF cycles from 1996 to 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients undergoing autologous IVF cycles between 1996 and 2013 resulting in a singleton live born with a birthweight recorded were included. One-way ANOVA and t-tests compared mean live birthweight in fresh and frozen cycles in 6-month increments over 18 years. Linear regression analysis was performed to investigate predictors of birthweight. MAIN RESULTS AND THE ROLE OF CHANCE: Mean birthweight after fresh (3283 ± 601 g) and frozen (3462 ± 621 g) cycles were significantly different (P < 0.001). ANOVA demonstrated no significant difference in mean weight from fresh or frozen cycles over 6-month intervals. No difference in weight was noted between Days 3 and 5 transfers or between ICSI and standard IVF. No difference was found across known changes when comparing media, laboratory location, cryopreservation method or gonadotrophins. LIMITATIONS, REASONS FOR CAUTION: Limitations include the small number of frozen low birthweight neonates. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that changes in IVF practice, with the exception of fresh or frozen embryo transfer, have little impact on mean live birthweight. STUDY FUNDING/COMPETING INTERESTS: No funding was received for this study. The authors have no conflicting interests. TRIAL REGISTRATION NUMBER: Not applicable.

Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.