Empty follicle syndrome (EFS) is defined as the failure to aspirate oocytes from mature ovarian follicles during in vitro fertilization. Except for some cases caused by pharmacological or iatrogenic problems, the etiology of EFS remains enigmatic. In the present study, we describe a large family with a dominant inheritance pattern of female infertility characterized by recurrent EFS. Genome-wide linkage analyses and whole-exome sequencing revealed a paternally transmitted heterozygous missense mutation of c.400 G>A (p.Ala134Thr) in zona pellucida glycoprotein 3 (ZP3). The same mutation was identified in an unrelated EFS pedigree. Haplotype analysis revealed that the disease allele of these two families came from different origins. Furthermore, in a cohort of 21 cases of EFS, two were also found to have the ZP3 c.400 G>A mutation. Immunofluorescence and histological analysis indicated that the oocytes of the EFS female had degenerated and lacked the zona pellucida (ZP). ZP3 is a major component of the ZP filament. When mutant ZP3 was co-expressed with wild-type ZP3, the interaction between wild-type ZP3 and ZP2 was markedly decreased as a result of the binding of wild-type ZP3 and mutant ZP3, via dominant negative inhibition. As a result, the assembly of ZP was impeded and the communication between cumulus cells and the oocyte was prevented, resulting in oocyte degeneration. These results identified a genetic basis for EFS and oocyte degeneration and, moreover, might pave the way for genetic diagnosis of infertile females with this phenotype.During in vitro fertilization (IVF) treatment, oocyte retrieval is performed after ovarian stimulation via vaginal puncture. Cumulus-oocyte complexes (COCs), which consist of cumulus cells surrounding the centrally located oocyte, are isolated from the individual's follicular fluid. As was first described by Coulam et al. in 1986, empty follicle syndrome (EFS) is a condition in which the ovarian response to stimulation and follicular development seems normal but no oocytes are retrieved for fertilization.1 EFS can be classified as either false EFS (FEFS) or genuine EFS (GEFS). FEFS is mainly caused by pharmacological or iatrogenic problems; however, the etiology of GEFS, which is responsible for about 33% of EFS, still remains enigmatic. 2 It has been proposed that GEFS is caused by dysfunctional folliculogenesis, ovarian aging, or genetic factors including pericentric inversion of chromosome 2 and LHCGR (MIM: 152790) mutations. 3-7 A retrospective study of 12,359 individuals who underwent assisted reproductive technology (ART) revealed that the prevalence of GEFS was about 0.016%. 8 Without oocytes for fertilization, these individuals fail to achieve pregnancy after a demanding and expensive medical intervention, resulting in stress to both physicians and the individuals themselves.9
Applications of genome editing ultimately depend on DNA repair triggered by targeted doublestrand breaks (DSBs). However, repair mechanisms in human cells remain poorly understood and vary across different cell types. Here we report that DSBs selectively induced on a mutant allele in heterozygous human embryos are repaired by gene conversion using an intact wildtype homolog as a template in up to 40% of targeted embryos. We also show that targeting of homozygous loci facilitates an interplay of non-homologous end joining (NHEJ) and gene conversion and results in embryos which carry identical indel mutations on both loci. Additionally, conversion tracks may expand bidirectionally well beyond the target region leading to an extensive loss of heterozygosity (LOH). Our study demonstrates that gene conversion and NHEJ are two major DNA DSB repair mechanisms in preimplantation human embryos. While gene conversion could be applicable for gene correction, extensive LOH presents a serious safety concern.
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