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Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown-Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%–4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%–6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strain require further investigation.

Abstract Background Actin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. In this study, we aim to explore the additional functions of ACTL7A beyond the process of acrosome biogenesis and investigate the possible underlying mechanisms. Methods Nuclear morphology analysis was used to observe the sperm head shape of ACTL7A-mutated patients. Actl7a knock-out (KO) mouse model was generated. Immunofluorescence and transmission electron microscopy (TEM) were performed to analyze the structure of spermatids during spermiogenesis. Tandem mass tags labeling quantitative proteomics strategy was employed to explore the underlying molecular mechanisms. The expression levels of key proteins in the pathway were analyzed by western blotting. Intracytoplasmic sperm injection (ICSI)-artificial oocyte activation (AOA) technology was utilized to overcome fertilization failure in male mice with a complete knockout of Actl7a. Results The new phenotype of small head sperm associated with loss of ACTL7A in patients was discovered, and further confirmed in Actl7a-KO mice. Immunofluorescence and TEM analyses revealed that the deletion of ACTL7A damaged the formation of acrosome-acroplaxome-manchette complex, leading to abnormalities in the shaping of sperm heads. Moreover, a proteomic analysis of testes from WT and Actl7a-KO mice revealed that differentially expressed genes were notably enriched in PI3K/AKT/mTOR signaling pathway which is strongly associated with autophagy. Inhibition of autophagy via PI3K/AKT/mTOR signaling pathway activation leading to PDLIM1 accumulation might elucidate the hindered development of manchette in Actl7a-KO mice. Remarkably, AOA successfully overcame fertilization failure and allowed for the successful production of healthy offspring from the Actl7a complete knockout male mice. Conclusions Loss of ACTL7A causes small head sperm as a result of defective acrosome-acroplaxome-manchette complex via autophagy inhibition. ICSI-AOA is an effective technique to rescue male infertility resulting from ACTL7A deletion. These findings provide essential evidence for the diagnosis and treatment of patients suffering from infertility.

IntroductionAttempts to artificially activate unfertilized oocytes at 24 h post intracytoplasmic sperm injection (ICSI) have generally resulted in poor outcomes. This study aims to explore a new strategy for early judgement and rescue activation of unfertilized oocytes at 5 h post ICSI to avoid unexpected fertilization failure (UFF) or unexpected low fertilization (ULF) in ICSI cycles.MethodsFirstly, time-lapse data from 278 ICSI cycles were retrospectively analyzed to establish an indicator for fertilization failure prediction. Secondly, 14 UFF and 20 ULF cycles were enrolled for an observational study, early rescue oocyte activation (EROA) was performed on oocytes without post-ICSI Pb2 extrusion to investigate fertilization efficiency, embryo development and clinical outcomes.ResultsThe average time to Pb2 extrusion post-ICSI was 3.03±1.21 h, 95.54% of oocytes had extruded Pb2 before 5 h, and the sensitivity and specificity for monitoring Pb2 extrusion at 5 h by time-lapse imaging to predict fertilization were 99.59% and 99.78%, respectively. Early rescue activation of oocytes with no Pb2 extrusion resulted in acceptable fertilization and embryo developmental outcomes, in terms of the fertilization rate (75.00, 72.99%), 2PN fertilization rate (61.36, 56.93%), good-quality embryo rate (42.59, 50.00%), blastocyst formation rate (48.28, 46.03%), good-quality blastocyst rate (34.48, 33.33%), and oocyte utilization rate (36.36, 27.74%), for both UFF and ULF cycles. The clinical pregnancy, embryo implantation, and early miscarriage rates in the rescue oocyte activation group did not significantly differ from those in the Pb2 extrusion group. Fourteen unexpected fertilization failures and 20 low fertilization ICSI cycles were rescued and resulted in clinical pregnancy rates of 40.00% (4/10) and 57.14% (8/14), respectively. ConclusionsThis study demonstrates that monitoring Pb2 extrusion by time-lapse imaging can accurately predict fertilization outcomes, suggesting that early rescue oocyte activation at 5 h post ICSI is an effective strategy for avoiding unexpected fertilization failure and low fertilization in ICSI cycles.

Abstract Background Artificial oocyte activation (AOA) is used to improve fertilization rate following fertilization failure after intracytoplasmic sperm injection (ICSI). Several studies have also shown that AOA may be involved in embryo development. Women with poor ovarian response are more likely to encounter in vitro fertilization (IVF) failure due to poor embryo quality. The aim of this study was to investigate whether AOA could improve embryo quality in older patients with diminished ovarian reserve undergoing IVF-ICSI cycles. Methods The retrospective cohort study consisted of 308 patients who fulfilled the POSEIDON Group 4 criteria and received IVF-ICSI cycles. The study group included 91 patients receiving AOA with calcium ionophores following ICSI. A total of 168 patients in the control group underwent ICSI without AOA. The baseline and cycle characteristics and embryo quality were compared between the two groups. Results At baseline, there were more IVF attempts, greater primary infertility, higher basal FSH levels and lower anti-Müllerian hormone (AMH) levels in the AOA group than in the non-AOA group. In terms of embryo quality, there were higher cleavage rates and top-quality Day 3 embryo (TQE) rates, as well as higher percentages of more than 1 TQE and TQE rates ≥50 in the AOA group than in the non-AOA group. The multivariate analysis revealed that AOA was positively associated with more than 1 TQE (adjusted OR 3.24, 95% CI 1.63–6.45, P = 0.001) and a TQE rate ≥ 50 (adjusted OR 2.14, 95% CI 1.20–3.80, P = 0.010). When the study population was divided into 2 subgroups based on the age of 40 years old, the beneficial effects of AOA on embryo quality were only observed in the subgroup of age ≥ 40 years old. Conclusions Our data suggest that AOA with calcium ionophores may improve embryo quality in older patients with diminished ovarian reserve undergoing IVF-ICSI cycles, especially in women aged ≥40 years.

Research questionDoes artificial oocyte activation (AOA) by a calcium ionophore (ionomycin) improve the previous fertilization failure or poor embryo development of intracytoplasmic sperm injection (ICSI) account for male factor infertility or other infertility causes?DesignThis retrospective study involved 114 patients receiving ICSI-AOA in Shanghai First Maternity and Infant Hospital with previous ICSI fertilization failure or poor embryo development. The previous ICSI cycles of the same patients without AOA served as the control group. The fertilization rates, cleavage rates, transferable embryo rates and blastocyst formation rates of the two groups were compared. Additionally, the clinical pregnancy, implantation rate and live birth rates were also compared to assess the efficiency and safety of AOA. Furthermore, two subgroup analyses were performed in this study based on the cause of infertility and the reason for AOA. The fertilization rate, embryonic development potential and clinical outcome were compared among groups.ResultsAmong 114 ICSI-AOA cycles, the fertilization rate, top-quality embryo rate, implantation rate, clinical pregnancy per patient and live birth rate per patient were improved significantly compared with previous ICSI cycles (p

IntroductionThe application of microdissection testicular sperm extraction (micro-TESE) to retrieve the sperm of patients with non-obstructive azoospermia (NOA) has greatly increased. Patients with NOA often have poor quality sperm. Unfortunately, there are few studies on artificial oocyte activation (AOA) performed on patients who successfully retrieved motile and immotile sperm by micro-TESE after intracytoplasmic sperm injection (ICSI). Therefore, this study sought to obtain more comprehensive evidence-based data and embryo development outcomes to aid consultation of patients with NOA who opted to receive assisted reproductive techniques and to determine whether AOA needs to be performed in different motile sperm after ICSI.MethodsThis retrospective study involved 235 patients with NOA who underwent micro-TESE to retrieve adequate sperm for ICSI between January 2018 and December 2020. A total of 331 ICSI cycles were performed in the 235 couples. Embryological, clinical, and neonatal outcomes were demonstrated comprehensively between motile sperm and immotile sperm using AOA and non-AOA treatment.ResultsMotile sperm injection with AOA (group 1) showed significantly higher fertility rate (72.77% vs. 67.59%, p=0.005), 2 pronucleus (2PN) fertility rate (64.33% vs. 60.22%, p=0.036), and miscarriage rate (17.65% vs. 2.44%, p=0.018) compared with motile sperm injection with non-AOA (group 2). Group 1 had comparable available embryo rate (41.29% vs. 40.74%, p=0.817), good embryo rate (13.44% vs. 15.44%, p=0.265), and without an embryo for transfer rate (10.85% vs. 9.90%, p=0.815) compared with group 2. Immotile sperm injection with AOA (group 3) displayed significantly higher fertility rate (78.56% vs. 67.59%, p=0.000), 2PN fertility rate (67.36% vs. 60.22%, p=0.001), without an embryo for transfer rate (23.76% vs. 9.90%, p=0.008), and miscarriage rate (20.00% vs. 2.44%, p=0.014), but significantly lower available embryo rate (26.63% vs.40.74%, p=0.000) and good embryo rate (15.44% vs. 6.99%, p=0.000) compared with group 2. In groups 1, 2, and 3, the rates of implantation (34.87%, 31.85% and 28.00%, respectively; p=0.408), clinical pregnancy (43.87%, 41.00%, and 34.48%, respectively; p=0.360) and live birth (36.13%, 40.00%, and 27.59%, respectively; p=0.194) were similar.DiscussionFor those patients with NOA from whom adequate sperm were retrieved for ICSI, AOA could improve fertilization rate, but not embryo quality and live birth outcomes. For patients with NOA and only immotile sperm, AOA can help achieve acceptable fertilization rate and live birth outcomes. AOA is recommended for patients with NOA only when immotile sperm are injected.

Abstract Background The application of artificial oocyte activation (AOA) after intracytoplasmic sperm injection (ICSI) is successful in mitigating fertilization failure problems in assisted reproductive technology (ART). Nevertheless, there is no relevant study to investigate whether AOA procedures increase developmental risk by disturbing subsequent gene expression at different embryonic development stages. Methods We used a mouse model to explore the influence of AOA treatment on pre- and post-implantation events. Firstly, the developmental potential of embryos with or without AOA treatment were assessed by the rates of fertilization and blastocyst formation. Secondly, transcriptome high-throughput sequencing was performed among the three groups (ICSI, ICSI-AOA and dICSI-AOA groups). The hierarchical clustering and Principal Component Analysis (PCA) analysis were used. Subsequently, Igf2r/Airn methylation analysis were detected using methylation-specific PCR sequencing following bisulfite treatment. Finally, birth rate and birth weight were examined following mouse embryo transfer. Results The rates of fertilization and blastocyst formation were significantly lower in oocyte activation-deficient sperm injection group (dICSI group) when compared with the ICSI group (30.8 % vs. 84.4 %, 10.0 % vs. 41.5 %). There were 133 differentially expressed genes (DEGs) between the ICSI-AOA group and ICSI group, and 266 DEGs between the dICSI-AOA group and ICSI group. In addition, the imprinted gene, Igf2r is up regulated in AOA treatment group compared to control group. The Igf2r/Airn imprinted expression model demonstrates that AOA treatment stimulates maternal allele-specific mehtylation spreads at differentially methylated region 2, followed by the initiation of paternal imprinted Airn long non-coding (lnc) RNA, resulting in the up regulated expression of Igf2r. Furthermore, the birth weight of newborn mice originating from AOA group was significantly lower compared to that of ICSI group. The pups born following AOA treatment did not show any other abnormalities during early development. All offspring mated successfully with fertile controls. Conclusions AOA treatment affects imprinted gene Igf2r expression and mehtylation states in mouse pre- and post-implantation embryo, which is regulated by the imprinted Airn. Nevertheless, no significant differences were found in post-natal growth of the pups in the present study. It is hoped that this study could provide valuable insights of AOA technology in assisted reproduction biology.

Abstract Background Fertilization failure after intracytoplasmic sperm injection continues to affect couples and the etiology is not well-understood. Case presentation We characterized a couple with 2-year history of primary unexplained infertility. Three different assisted reproduction attempts (IVF + rescue ICSI, ICSI and ICSI-AOA) showed repeated fertilization failure for MII oocyte retrieval after controlled ovarian hyperstimulation. After whole-exome sequencing and sanger sequencing of the couple and their family members, variant pathogenicity was assessed using SIFT, PolyPhen2, Mutation Taster, and Human Splicing Finder software. We identified novel compound heterozygous mutations, c.1535 + 3A > G and c.946C > T (p. Leu316Phe), in WEE2 in the female proband. Trios analysis of the variations revealed an autosomal recessive pattern. c.1535 + 3A > G in WEE2 was predicted to break the wild-type donor site and affect splicing, and the missense mutation c.946C > T (p. Leu316Phe) of WEE2 was predicted to be pathogenic. Conclusion A novel compound heterozygous mutation in WEE2 was identified in an infertile female who experienced repeated fertilization failure even after ICSI-AOA. These novel mutations in WEE2 provided genetic evidence for fertilization failure.

Background: Ca2+ signaling pathway is suggested to play an essential role in mediating oocyte maturation. Aims: The aim of this study was to evaluate intracellular Ca2+ of resistant immature oocytes that failed to resume meiosis following subsequent in vitro culture reach metaphase II after calcium ionophore A23187 activation. Settings and Design: This in vitro analytical experimental study was conducted at Animal Science Laboratory of Indonesian Medical Education and Research Institute (IMERI), Human Reproductive Infertility and Family Planning of IMERI, and Electrophysiology Imaging of Terpadu Laboratory, Faculty of Medicine, University of Indonesia. Methods: A total of 308 oocytes classed as resistant immature following in vitro culture were randomly allocated to control (n = 113) and treatment groups (n = 195). The oocyte activation group was exposed to A23187 solution for 15 min and then washed extensively. Maturation was evaluated by observing the first polar body extrusion 20‒24 h after A23187 exposure. Ca2+ imaging was conducted using a confocal laser scanning microscope to identify the dynamic of Ca2+ response. Statistical Analysis: SPSS 20, Chi-square, and Mann–Whitney U-test were used in this study. Results: Activation of resistant immature oocytes with A23187 significantly increased the number of oocyte maturation compared with the control group (P

Introduction: Several factors are involved in failed fertilization following intracytoplasmic sperm injection (ICSI), which could be related to sperm, oocyte factors and/or both. Failure in oocyte activation is considered as the most important factor in failed fertilization after ICSI. To overcome this shortcoming, artificial oocyte activation (AOA) after ICSI has been suggested. Commonly, ionomycin and strontium chloride are used as the most efficient agents for oocyte activation in the clinic. In this study, for the first time, the clinical results of ionomycin and strontium chloride were compared on sister oocytes of couples who previously have had fertilization failure after ICSI. Methods: In this intervention study, 14 couples with male factor infertility and previous failed fertilization after ICSI were included in this study. Oocytes of their wives were divided into two groups. Half of the oocytes after ICSI were treated with ionomicin and other half were treated with strontium chloride. Then, fertilization rate, embryo quality and pregnancy status were compared through SPSS 18 software, Paired sample t-test and chi-square test. Results: Percentage of fertilization rate was significantly higher in oocytes that were activated using strontium chloride compared to those that were activated with ionomycin (p

Fertilization failure is common in patients with round-headed sperm, a form of globozoospermia. Artificial oocyte activation is able to assist oocyte fertilization after sperm injection in these patients. Comparisons between oocyte fertilization with or without calcium ionophore have been reported in patients with round-headed sperm. However, no comparison has been reported between round-headed sperm injection followed by calcium ionophone activation and normal sperm injection. In this case report, half of oocytes from a patient were injected with her partner’s round-headed sperm followed by calcium ionophore activation, and the other half of oocytes were injected with a donor sperm without calcium ionophore activation. The injected oocytes were cultured to examine fertilization, embryo development, and embryonic aneuploidies in the resulting blastocysts. The fertilization rate was lower in round-headed sperm injected oocytes (3/6) than that in donor sperm injected oocytes (5/6), but rates of blastocyst and aneuploidies were similar in the resulting embryos between the two groups. A euploid blastocyst resulted from round-headed sperm injection was transferred, and a healthy baby was delivered. These results indicate that calcium ionophore treatment can assist oocyte activation in patients with round-headed sperm, but its efficiency to activate oocytes is lower than that induced by a normal sperm injection. However, embryo development and chromosome integrity may not be affected by calcium ionophore treatment.