Novel WEE2 compound heterozygous mutations identified in patients with fertilization failure or poor fertilization.

PURPOSE: To study associations between novel WEE2 mutations and patients with fertilization failure or poor fertilization. METHODS: Thirty-one Chinese patients who underwent treatment with assisted reproductive technology and suffered from repeated (at least two times) total fertilization failure (TFF) or a low fertilization rate were enrolled. Genomic DNA was extracted from patients for whole-exome sequencing. Suspicious mutations were validated by Sanger sequencing. WEE2 protein levels in oocytes from affected patients were examined by immunofluorescence. Disruptive effects of mutations on WEE2 protein stability, subcellular localization, and kinase function were analyzed through western blotting, immunofluorescence, and flow cytometry in HeLa cells. RESULTS: Three of thirty-one (9.6%) enrolled patients had six compound heterozygous mutations of the WEE2 gene, and three of them were reported here for the first time (c.115_116insT, c.756_758delTGA, and c.C1459T). Oocytes from affected patients showed decreased WEE2 immunofluorescence signals. In vitro experiments showed that the mutant WEE2 gene caused reduced WEE2 protein levels or cellular compartment translocation in HeLa cells, leading to decreased levels of the phosphorylated Cdc2 protein. Compared with the wild-type WEE2 protein, the mutant WEE2 proteins were also found to have different effects on the cell cycle. CONCLUSION: Three novel compound heterozygous WEE2 variants were found in patients with pronucleus formation failure. This study provides new evidence that WEE2 mutations result in loss of function, which could result in fertilization failure.

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = -0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = -0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.

Kinetics of meiotic maturation in oocytes from unstimulated ovaries and duration of pronucleus presence and preimplantation development.

OBJECTIVE: To evaluate the meaning of meiotic maturation kinetics and duration of pronucleus presence (DPP) for parthenogenetic activation outcome. DESIGN: Retrospective study. SETTING: University hospital. PATIENT(S): Eight patients with endometrioid adenocarcinoma and 65 patients who underwent in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). INTERVENTION(S): After collection of oocytes from nonstimulated ovaries of patients with endometrioid adenocarcinoma, in vitro maturation (IVM) and parthenogenetic activation performed with time-lapse imaging; after ICSI, embryos similarly incubated with time-lapse imaging. MAIN OUTCOME MEASURE(S): Timing of the release of the first polar body (fPB), DPP, and developmental stage with IVM and parthenogenetic activation; after ICSI, assessment of DPP and preimplantation developmental stage. RESULT(S): With IVM, 55.2% of oocytes matured; 53.1% of fPBs were released within 24 hours, and 46.9% of fPBs were released after 24 hours. Regarding developmental stage, oocytes that released fPB later during IVM tended to develop more than oocytes that released the fPB within 24 hours. For embryos from parthenogenetic activation the DPP was statistically significantly shorter than the DPP of embryos from ICSI. With ICSI, the DPP was statistically significantly shorter in embryos that developed to ≥8 cells than embryos whose final development included ≤7 cells. The development rate in parthenogenetic activation was statistically significantly lower than that in ICSI. CONCLUSION(S): Embryo development is negatively affected by DPP that is too short or too long. When the DPP was short with parthenogenetic activation, embryo development did not proceed, indicating that DPP is an important determinant of parthenogenetic activation outcomes as with the timing of fPB release.

Novel WEE2 gene variants identified in patients with fertilization failure and female infertility.

OBJECTIVE: To determine whether variants in the WEE2 (WEE1 homolog 2, also known as WEE1B) gene, which has been known to function in the formation of pronuclei during fertilization, contribute to fertilization failure. DESIGN: Case-control genetic study. SETTING: University hospital. PATIENT(S): Ninety infertile women with repeated cycles of pronucleus formation failure undergoing in vitro fertilization and/or intracytoplasmic sperm injection treatment as well as 200 fertile control women. INTERVENTION(S): Genomic DNA was extracted from the peripheral blood. The whole exons of WEE2 were amplified by means of polymerase chain reaction and then Sanger sequencing was performed. MAIN OUTCOME MEASURE(S): Variants analysis of WEE2 gene. RESULT(S): We identified five subjects that were subjected to homozygous or compound-heterozygous variants of WEE2: case 1 (from a consanguineous family) with homozygous frameshift variant: c.293_294insCTGAGACACCAGCCCAACC (p.Pro98Pro fsX2); case 2 with homozygous missense variant: c.1576T>G (p.Tyr526Asp); and three cases with compound-heterozygous variants: case 3: c.991C>A (p.His331Asn) and c.1304_1307delCCAA (p.Thr435Met fsX31); case 4: c.341_342 del AA (p.Lys114Asn fsX20) and c.864G>C (p.Gln288His); and case 5: c.1A>G (p.0?) and c.1261G>A (p.Gly421Arg). Besides c.1576T>G (from case 2) and c.864G>C (from case 4), which have been previously reported as rare single nucleotide polymorphisms (SNPs), the other six variants were novel and predicted by software to be deleterious. The parental genotypes of case 1 and case 2 indicated that the detected homozygous variants were inherited in an autosomal recessive mode. All of the detected variants were absent from the control cohort. CONCLUSION(S): Novel variants found in WEE2, which is autosomal-recessive inherited, may be related to recurrent pronucleus formation failure and female infertility.

Evaluation of sperm subpopulation structure in relation to in vitro sperm-oocyte interaction of frozen-thawed semen from Holstein bulls.

The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.