Fertilization failure assay identifies stage-specific arrests in couples with total fertilization failure.

Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. NCT03354013.

Study question What role do genetic factors play in infertile men acting fertilization and cleavage failures? Summary answer We identified novel variants in DNAH10, DNAH12, CFAP47, and PLCZ1 associated with sperm structural defects, contributing to male infertility through fertilization and cleavage failures. What is known already Male infertility is a significant global issue affecting a substantial number of individuals. Although numerous pathogenic candidate genes have been identified, the genetic causes underlying fertilization and cleavage failures in infertile men remain largely unexplored. Study design, size, duration This study involved whole-exome sequencing of 20 unrelated patients experiencing multiple IVF failures due to fertilization and cleavage issues at our IVF center. Sperm analyses were conducted using transmission electron microscopy and light microscopy. Abnormal embryos were assessed through morphological evaluation, whole-genome amplification, and next-generation sequencing. Candidate gene variants were validated via Sanger sequencing, followed by bioinformatics analysis, protein structural modeling, and functional assays. Participants/materials, setting, methods Twenty unrelated male patients with a history of recurrent IVF failures attributed to fertilization and cleavage defects. Main results and the role of chance We identified novel compound heterozygous variants in DNAH10 (c.2569C>T: p.Arg857*; c.4102A>G: p.Met1368Val) and DNAH12 (c.2439delG: p.Thr814Leufs*8; c.10254G>T: p.Trp3418Cys) in patients with fertilization failures during conventional IVF. Additionally, a hemizygous missense variant in CFAP47 (c.8006C>T: p.Thr2669Met) was linked to cleavage failures, and a homozygous missense variant in PLCZ1 (c.589C>T: p.Arg197Cys) was associated with polyspermy and fertilization failures. A quantitative parental contamination test using next-generation sequencing confirmed the involvement of PLCZ1 in polyspermy-related fertilization failures. Intracytoplasmic sperm injection (ICSI) emerged as a potential effective treatment for patients with DNAH10 and DNAH12 variants, while combining ICSI with assisted oocyte activation (AOA) may improve reproductive outcomes for those with CFAP47 and PLCZ1 deficiencies. Limitations, reasons for caution In this study, we uncovered the relationship between the genotype and phenotype of novel causative variants associated with sperm head deformities in humans. However, the precise molecular mechanisms by which these genes contribute to sperm head development remain unclear and warrant further investigation in future research. Wider implications of the findings This genetic evidence enhances our understanding of the mechanisms behind fertilization and cleavage failures in male infertility. It provides valuable insights that can inform personalized treatment strategies and improve reproductive outcomes for affected individuals. Trial registration number No

Study question Does the use of calcium ionophore improve fertilisation, blastulation, and clinical pregnancy rates in patients with prior failed or poor blastulation in a cycle-to-cycle comparison? Summary answer Calcium ionophore improved fertilisation and blastulation rates in failed fertilisation cases, showing potential clinical pregnancy benefits; however, no significant was observed on live birth rates. What is known already Fertilisation failure after ICSI, despite normal semen parameters, is commonly caused by oocyte activation deficiency (OAD), impairing calcium oscillations necessary for embryonic development. Calcium ionophores have been proposed to overcome this deficiency by stimulating oocyte activation, potentially improving fertilisation outcomes. While previous studies have investigated the use of calcium ionophores to address poor fertilisation rates, their impact on blastocyst formation rates remains unclear. With limited Randomised control trails to support its use in poor and sub-optimal outcomes following ARTs. This study aims to explore the effects of calcium ionophore on improving fertilisation, blastocyst formation, and ART outcomes. Study design, size, duration This retrospective study was conducted at a single centre over ten years (2013–2023) to assess the use of calcium ionophore. A total of 649 cycles were analysed, of which 36 cycles had failed fertilization and 613 cycles with low blastulation rates in the first cycle where calcium ionophore was not used. These were compared to subsequent cycles where calcium ionophore was used, analysing outcomes of fertilisation, blastulation, pregnancy, and clinical pregnancy, and live birth. Participants/materials, setting, methods A total of 649 cycles were divided into two main categories. First, no fertilization, second, no calcium ionophore used in the first cycle, and calcium ionophore used in subsequent cycles. Data were collected from 2013 to 2023, with less than six months between cycles. Surgically retrieved cycles were excluded. Statistical analysis was performed using the Chi-Square test in SPSS to determine the p-values. Main results and the role of chance In the failed fertilisation category, the subsequent cycle where calcium ionophore was used, involving 146 mature oocytes. The fertilisation rate significantly improved to 54.8% (80/146, p = <0.05), and the blastulation rate increased to 38.8% (31/80, p = <0.05). These results indicate that calcium ionophore positively impacted fertilisation and early embryo development in patients with a prior failed fertilisation cycle. In the low blastulation category, 613 cycles were analysed, comparing 250 cycles without calcium ionophore to 363 cycles where it was used. The fertilisation rates were similar (65.5% vs. 64.9%, p = <0.05), but the blastulation rate was statically significant with calcium ionophore (27.6% vs. 38.9%, p < 0.05). Interestingly, there was a trend in lower pregnancy rate in the calcium ionophore group (39.1% vs. 26.2%, p < 0.05), however, the clinical pregnancy rate was higher (35.8% vs. 50.3%, p = 0.1), suggesting that calcium ionophore may enhance embryo quality, leading to improved implantation potential. The live birth rate remained statistically unchanged (77.6% vs. 80.8%, p = 0.876), indicating that while calcium ionophore improved early-stage embryo development, its effect on final pregnancy outcomes was not significant. These findings suggest that calcium ionophore is beneficial in improving fertilisation and blastulation rates, in cases of failed fertilisation, with a potential impact on clinical pregnancy outcomes Limitations, reasons for caution The retrospective nature, potential selection bias, and limited data on long-term neonatal outcomes, requiring further randomised studies. Wider implications of the findings These results suggest that calcium ionophore improves fertilisation and blastulation rates, especially in patients with prior failed fertilisation. Despite a decrease in embryo transfer rates, clinical pregnancy rates was maintained. The live birth rate remained stable, indicating that calcium ionophore enhances early embryonic development without affecting final pregnancy outcomes. Trial registration number No

From October 1992 to December 1994, 2732 cycles of treatment by intracytoplasmic sperm injection (ICSI) were carried out in couples mainly with severe male-factor infertility. The overall fertilization rate in these 2732 cycles was 71% of intact oocytes. However, in 76 (72 couples) of these cycles, none of the injected oocytes became fertilized, so the total fertilization failure rate was 3% (76/2732 cycles). Details of these 76 cycles were analysed. The results show that total fertilization failure after ICSI may be explained by different factors related to (i) semen characteristics (only immotile or round-headed spermatozoa for ICSI) or (ii) the oocytes (number, abnormal morphology, damage after ICSI). Of 26 couples, 22 achieved fertilization in their subsequent ICSI cycles. In conclusion, total fertilization failure after ICSI for the treatment of severe male-factor infertility was mainly caused by the poor viability of the spermatozoa used for injection; it was also associated with a low number and poor quality of oocytes. Repeated ICSI treatment may be useful or necessary in couples with total fertilization failure.

Study question Are there differences in fertilization and pregnancy rates post-TESE-ICSI between motile and immotile sperm, and do these vary in treatments with and without calcium ionophore? Summary answer Post-TESE-ICSI data reveals differences in fertilization and pregnancy rates when comparing motile vs. immotile sperm and treatments with and without calcium ionophore after TESE-ICSI. What is known already Testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) poses a challenge for every IVF laboratory. This is particularly true in patients exhibiting azoospermia, where only a few and mostly immotile sperm are present in testicular tissue. In these cases, a calcium ionophore treatment for activation of the oocyte may help to overcome poor prognosis fertilization outcome, and thereby enhancing the likelihood to achieve a transfer leading to a successful pregnancy. Although both techniques, TESE-ICSI and activating substances are commonly applied, detailed analyses for individual counselling of couples are missing. Study design, size, duration This study analyzed 787 TESE-ICSI cycles (2015-2023) regarding patient age, number of attempts, oocyte maturity, fertilization and pregnancy rate after fresh embryo transfer. Cycles were split into four groups: (A) at least one motile sperm was injected (n = 493); (B) only immotile sperm were used (n = 294); (C) calcium ionophore (n = 474); (D) no calcium ionophore (n = 313). 17 cycles were excluded from the study because no sperm were found on the day of TESE-ICSI and oocytes were vitrified. Participants/materials, setting, methods In our IVF clinic, calcium ionophore treatments are only offered if a pre-screening of TESE samples reveals the absence of motile spermatozoa or if a previously conducted TESE-ICSI attempt resulted in a low fertilization rate (<30%) or a total fertilization failure. Statistical analyses was performed to compare the motile and immotile sperm group and the groups with or without calcium ionophore treatment. Main results and the role of chance No differences were found in all groups (A-D) regarding age of female or male patient and number of collected oocytes per group. However, our results demonstrate a difference in the mean number of ICSI attempts when comparing motile sperm and immotile sperm (2.0±1.3 vs. 2.3±1.6, p < 0.05), and when comparing attempts with calcium ionophore treatment and those without (2.4±1.6 vs. 1.8±1.8, p < 0.0005). The mean fertilization rate (2PN stage) was higher in the motile sperm group (A) compared to the immotile group (B) (57.8% vs. 28.4%, p < 0.0005) and also higher in attempts of group (D) in comparison to attempts of group C (56.8% vs. 40.2%, p < 0.0005). While fertilization failure occurred less often in group (A) (3.2%) it occurred to a higher rate in group (B) (19.4%), followed by group (C) (12.0%). Biochemical (BPR) and clinical pregnancy rates (CPR) showed a significant difference between the motile and immotile sperm group (BCR: 33.2% vs. 12.3%, p < 0.0005; CPR: 25.6% vs. 9.3%, p < 0.0005), but no differences between the two calcium ionophore groups. Limitations, reasons for caution This analysis was done retrospectively. A potential subgroup analysis of groups (A) and (B) into calcium ionophore use and of groups (C) and (D) into motile or immotile sperm will reveal the effect of calcium ionophore on success rates regardless of sperm motility. Wider implications of the findings Our data provides insight into success rates of TESE-ICSI cycles, especially if only immotile sperm were present. This will serve as a helpful guide for physicians when counseling patients about their success rates and to optimize the outcome in cases with poor prognosis by using a calcium ionophore treatment. Trial registration number not applicable

In this study we investigated whether morphology and chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We examined unfertilized oocytes, using the fluorochrome Hoechst 33342, to determine whether a relationship exists between failure of fertilization and sperm chromatin quality. Sperm chromatin packaging quality was assessed using the chromomycin A3 (CMA3) fluorochrome, and the presence of DNA damage in spermatozoa, using in-situ nick translation, Normal males present sperm parameters with a normal morphology of > 20%, CMA3 fluorescence of < 30% and exhibit endogenous nicks in < 10% of their spermatozoa. When patients were separated according to these values no difference was observed in their fertilization rates after ICSI. When the unfertilized ICSI oocytes were examined, we found that patients with CMA3 fluorescence of <30% and nicks in < 10% of their spermatozoa had only 17.5 and 21.6% respectively of their unfertilized oocytes containing spermatozoa that remained condensed. In contrast, patients with higher CMA3 and nick values had a significantly higher number, 41.2 and 48.9%, of their unfertilized oocytes containing condensed spermatozoa. sperm morphology did not show any such pattern. The percentage of spermatozoa which had initiated decondensation in unfertilized oocytes was not influenced by morphology, CMA3 fluorescence or nicks. In light of these results we postulate that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation after ICSI and result in failure of fertilization.

Study question Does CultActive© improve reproductive outcomes of ICSI cycles in cases of low fertilization rate and normal fertilization rate? Summary answer The application of CultActive© after ICSI improves fertilization rates only in clinical indication of fertilization failure. What is known already Oocyte activation deficiency is attributed in the majority of cases to fertilization failure in ICSI cycles, and these can be corrected by increasing initial levels of calcium (Ca2+) using assisted oocyte activation techniques (AOA), such as the use of Ca2+ ionophores. These agents enhance intracellular calcium release and increase the membrane permeability facilitating the influx of extracellular Ca2+, which initiates the activation cascade. Previous results suggest that Ca2+ ionophore treatment can increase the live birth rate after failed ICSI cycles in couples with poor fertilization rates, embryo developmental arrest, and also increase the number of good quality embryos. Study design, size, duration This was a retrospective observational study. There were 30 patients included and the first group consisted of 30 ICSI cycles, 231 oocytes and 56 embryos (without AOA). The second group consisted of the same patients (n = 30) with 30 ICSI cycles using AOA, 219 oocytes and 47 embryos. Subgroups were defined according to clinical indications (normal fertilization rate group: ≥ 65 % and low fertilization rate group: &amp;lt;65% ). All data was collected from January-December 2022. Participants/materials, setting, methods 450 oocytes were assessed in the study. 231 oocytes belonged to the pre-AOA and 219 belonged to the AOA. The oocytes were exposed for 15 minutes after ICSI in a solution containing the Ca2+ ionophore A23187, CultActive© (Gynemed, Germany). Fertilization, good quality blastocyst formation and pregnancy rate were analyzed.Good quality blastocyst were defined as ≥ 3BB. Statistical significance was analyzed using the Paired Student T test (p &amp;lt; 0.05 was considered statistically significant). Main results and the role of chance The cohort of patients with normal fertilization rate (without clinical indications),was not significantly different. The AOA treatment gave a fertilization rate of 61% compared to 71% of the control cycles (p = 0.1398). There was no increase in the number of good quality blastocysts formation when the study group was compared to the control (p = 0.1660). In the cohort of patients with an indication of low fertilization, a significantly higher fertilization rate was recorded compared to the control (51% and 37%, respectively p = 0.0238). There were no significant difference in the quantity of good quality blastocysts compared with the control (p = 0.3328). Regardless of normal or low fertilization rates there was no significant difference in pregnancy rate when CultActive© was used in treatment (p &amp;gt; 0.05). Limitations, reasons for caution There are limitations in our study. First, the retrospective design and limited study population are major limitations. The data should be interpreted cautiously in the subgroup analyses due to potential bias, specifically the small population in the group without clinical indication. Wider implications of the findings AOA was only beneficial for couples with poor fertilization. Embryo quality and pregnancy rate did not improve with CultActiveive©. External variation was minimized by using the same patients in each cohort. This suggests AOA should not be used without clinical indication or for poor embryo quality/blastulation rate. Trial registration number not applicable

Live birth rates and miscarriage rates with the use of calcium ionophore in patients with prior failed or poor fertilization

PurposeTo study associations between novel WEE2 mutations and patients with fertilization failure or poor fertilization.MethodsThirty-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.ResultsThree 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.ConclusionThree 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.

Fertilization failure after IVF in 304 couples—A case-control study on predictors and long-term prognosis

BackgroundInfertility and pregnancy loss are major concerns in reproductive medicine, often linked to genetic factors affecting oocyte maturation. The WEE2 gene, which encodes an oocyte-specific kinase critical for meiosis, plays a vital role in fertilization. Variants in WEE2 have been implicated in oocyte maturation arrest and total fertilisation failure (TFF), particularly in women undergoing assisted reproductive technologies. This study aimed to investigate the association between WEE2 gene polymorphism and fertilization failure in women undergoing intracytoplasmic sperm injection (ICSI).Materials and MethodsThis prospective comparative study enrolled 137 infertile women undergoing ICSI procedures. Peripheral blood samples were collected from all participants for genetic analysis. Genotyping of the WEE2 gene variant rs1476640 was performed using a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay, which was specifically designed and optimised for this study.ResultsWEE2 gene polymorphism (rs1476640) showed a significant association with fertilization outcomes (p < 0.0001). The T allele was protective, while the C allele increased the risk of fertilization failure (OR = 9.06; 95% CI: 3.27–25.14). Significant differences were also observed in infertility duration, oocyte count, and endometrial thickness between pregnant and non-pregnant women (p < 0.05), but not in age or BMI.ConclusionThe WEE2 rs1476640 polymorphism is significantly associated with fertilization failure in ICSI-treated women. Screening for this variant may help identify patients at risk and support personalised infertility treatment strategies.

A Prospective Randomized Trial of ICSI Versus IVF for the Treatment of Unexplained Infertility

The zona pellucida (ZP) has multiple roles in reproductive processes, including oocyte maturation, fertilization and implantation. We used, for the first time, a genetic approach to study whether human ZP genes possess structural alterations in women with unsuccessful IVF trials. In theory, this may result in gradual reduction of sperm-zona interaction and eventually in total fertilization failure (TFF). Eighteen infertile women (TFFs) whose IVF did not result in any fertilized oocytes, whereas fertilization by ICSI was successful, were screened for mutations in ZP genes by means of conformation-sensitive gel electrophoresis. Twenty-three fertilizers in IVF (FIVFs) and 68 women with proven fertility (WPFs) constituted the two control groups. Altogether, 20 sequence variations were found in the ZP genes. Two variations in ZP3, one in the regulatory region (c. 1-87 T --> G) and one in exon 6 [c. 894 G --> A (p. K298)] existed more frequently in TFFs than in FIVF and WPF groups (P-values 0.027 and 0.008, respectively). Our study on ZP genes of infertile women revealed a high degree of sequence variations. This may reflect gradual reduction of fertility among TFFs, but the putative roles and influences of single variations can only be hypothesized.

ABSTRACTThe objective of this study was to explore the association of sperm mitochondrial ND2 (MT-ND2) gene variants with total fertilization failure (TFF). A retrospective comparative study of 246 cases of fresh in vitro fertilization (IVF) cycles or half-intracytoplasmic sperm injection cycles in the Han Chinese population was performed from July 2011 to May 2017. A total of 59 cases undergoing TFF, and 187 control cases with normal fertilization (fertilization rates >50%) were included. The sperm mitochondrial genovariation was determined using nested sequencing. A total of 32 homoplasmic variants and 47 heteroplasmic variants of MT-ND2 gene were observed in this study. There were no significant differences in the frequencies of the 32 homoplasmic variants of MT-ND2 gene between the TFF and control groups. A total of 53 pair-wise comparisons were performed, and the general characteristics of the IVF failure and control subjects were adjusted in logistic models. Data suggested that there were no significant differences in the frequencies of point 4914, 5320, and 5426 heteroplasmic variants of MT-ND2 gene between the TFF and control groups. In addition, no significant difference was observed in the frequency of mtDNA haplogroup D or haplogroup G between the IVF failure group and the normal fertilization group. This study suggests that the MT-ND2 gene variants might not be associated with TFF.Abbreviations: ATP: adenosine triphosphate; dNTP: deoxy-ribonucleoside triphosphate; FADH2: flavin adenine dinucleotide; FDR: false discovery rate; FSH: follicle-stimulating hormone; IVF: in vitro fertilization; LH: luteinizing hormone; MTATP6: mitochondrially encoded ATP synthase 6; MTCYB: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MT-ND2: mitochondrial ND2; NADH: nicotinamide adenine dinucleotide; ND2: NADH dehydrogenase subunit 2; OXPHOS: oxidative phosphorylation; PCR: single nucleotide polymorphisms; SNPs: single nucleotide polymorphisms; TFF: total fertilization failure

Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection