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RESEARCH QUESTION: Is artificial oocyte activation (AOA) effective for patients with unexplained low or no fertilization following IVF/intracytoplasmic sperm injection (ICSI)? DESIGN: All IVF/ICSI cases resulting in total fertilization failure or fertilization rate ≤25% at Ninewells Assisted Conception Unit, Dundee between January 2014 and December 2021 (n = 231) were reviewed contemporaneously. After exclusion of obvious stimulation, egg, sperm and/or assisted reproductive technology laboratory factors, patients with at least one cycle of IVF/ICSI resulting in apparently unexplained fertilization abnormalities were offered research investigations, including sperm immunocytochemistry for phospholipase C zeta (PLCζ) protein expression. This retrospective case-control cohort study evaluated laboratory and clinical outcomes for 39 couples (15 attended for sperm studies research) that subsequently undertook ICSI-AOA with Ca2+ ionophore. RESULTS: Comparing preceding IVF/ICSI and subsequent ICSI-AOA for each patient, the number of eggs collected was similar; however, ICSI-AOA resulted in a significantly improved fertilization rate (57.2% versus 7.1%; P < 0.0001). The uplift for a subset of 10 patients identified with PLCζ deficiency was 66.3% versus 4.6% (P < 0.0001). Overall, ICSI-AOA resulted in a higher number of fresh embryo transfers (94.6% versus 33.3%; P < 0.0001), a significantly higher clinical pregnancy rate (CPR) and live birth rate (LBR; 18.9% versus 2.6%; P = 0.02), a significant increase in cycles with surplus embryos suitable for cryostorage (43.6% versus 0%; P < 0.0001), and increased cumulative CPR (41.0% versus 2.6%; P < 0.0001) and LBR (38.5% versus 2.6%; P < 0.0001). CONCLUSION: AOA is a powerful tool that can transform clinical outcomes for couples experiencing apparently unexplained fertilization abnormalities. PLCζ assays have the potential to be valuable diagnostic tools to determine patient selection for ICSI-AOA, and research efforts should continue to focus on their development.

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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.

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Objective To investigate the clinical phenotype,sperm characteristics,genetic etiology and the outcomes of assisted repro-ductive technology of 2 minority patients(P1 Hui and P2 Uyghur)with globozoospermia.Methods The clinical data and various pa-rameters of semen examination in the 2 minority patients with globozoospermia were analyzed.The ultrastructure of their sperms was ob-served,and the genetic etiology of the patients was analyzed by whole exome sequencing(WES)and qPCR.Intracytoplasmic sperm in-jection(ICSI)combined with artificial oocyte activation(AOA)were applied for assisted reproduction thyrapy.The outcomes of assis-ted reproduction in the two couples were observed.Results Homozygous 109681 bp deletion in DPY19L2 gene was found in both the patients.The homozygous deletion of DPY19L2 gene in P2 patient came from his inbred parents.Low sperm motility and high sperm DNA fragmentation rate were observed in the P1 patient,the and the rate of round-headed sperm was 100％in morphology.The ab-sence of sperm acrosome and ultrastructural defects of plasma membrane,mitochondria and microtubules were found under the electron microscope.In the P2 patient,sperm motility and sperm DNA fragmentation rate patient were normal,and the sperm morphology was 100％round-headed sperm.Under the electron microscope,the main defects of sperm were small and round head with acrosomal ab-sence,structural damage,but the ultrastructural defects of organelles were rare,such as plasma membrane,mitochondria and microtu-bules.Both couples of the two patients received ICSI+AOA for assisted pregnancy,and the ICSI fertilization rate was 62.5％for P1 pa-tient and 75％for P2 patient.Both the patients successfully obtained clinical pregnancy.Conclusion The abnormal DPY19L2 gene is the main genetic cause in the globozoospermia patients with different ethnic backgrounds.Both structural damage and ultrastructural de-fects of organelles such as plasma membrane,mitochondrial and microtubule damage could be found in globozoospermia.ICSI combined with AOA should be an effective assisted reproductive thyrapy strategy for the patients with globozoospermia.

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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.

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Research question: Does 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? Design: This 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. Results: Among 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<0.05 to P< 0.001), and the miscarriage rate in the AOA group was significantly lower than that of the control group (p<0.001). In the AOA subgroups based on the cause of infertility, the fertilization rates of each subgroup were significantly improved compared with previous control cycles except for the mixed factor infertility subgroup (p<0.05 to p<0.001). In the AOA subgroups based on the reason for AOA, the fertilization rates of each subgroup were significantly increased compared with those in their previous ICSI cycle without AOA (p<0.001); however, there was no significant difference in the top-quality embryo rate. No significant improvement was found in the implantation rates and the clinical pregnancy rate in each subgroup except for the poor embryo development subgroup. In the 114 AOA cycles, 35 healthy infants (21 singletons and 7 twins) were delivered without major congenital birth defects or malformations. Conclusion: This study showed that AOA with the calcium ionophore ionomycin can improve the reproductive outcomes of patients with previous fertilization failure and poor embryo development after ICSI.

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Fertilization failure refers to the failure in the pronucleus formation, evaluating 16-18 h post in vitro fertilization or intracytoplasmic sperm injection. It can be caused by sperm, oocytes, and sperm-oocyte interaction and lead to great financial and physical stress to the patients. Recent advancements in genetics, molecular biology, and clinical-assisted reproductive technology have greatly enhanced research into the causes and treatment of fertilization failure. Here, we review the causes that have been reported to lead to fertilization failure in fertilization processes, including the sperm acrosome reaction, penetration of the cumulus and zona pellucida, recognition and fusion of the sperm and oocyte membranes, oocyte activation, and pronucleus formation. Additionally, we summarize the progress of corresponding treatment methods of fertilization failure. This review will provide the latest research advances in the genetic aspects of fertilization failure and will benefit both researchers and clinical practitioners in reproduction and genetics.

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This large multi-center retrospective study examined whether artificial oocyte activation (AOA) using Ca2+ ionophore following ICSI improves the live birth rate for couples with previous ICSI cycles of unexplained low fertilization rate. In this large-scale multi-center retrospective study conducted in Japan, data were collected from Keio University and 17 collaborating institutions of the Japanese Institution for Standardizing Assisted Reproductive Technology. Between January 2015 and December 2019, 198 couples were included in this study. Oocytes for both the intervention and control groups were procured from the same pool of couples. Oocytes obtained from ICSI cycles with no or low fertilization rate (<50%) with unknown causes were included in the control (conventional ICSI) group while oocytes procured from ICSI cycles followed by performing AOA were assigned to the intervention (ICSI-AOA) group. Those fertilized with surgically retrieved sperm were excluded. ICSI-AOA efficacy and safety were evaluated by comparing these two groups. Live birth rate was the primary outcome. The ICSI-AOA group (2,920 oocytes) showed a significantly higher live birth per embryo transfer rate (18.0% [57/316]) compared to that of the conventional ICSI group with no or low fertilization rate (1,973 oocytes; 4.7% [4/85]) (odds ratio 4.5, 95% confidence interval 1.6-12.6; P<0.05). A higher live birth rate was observed in younger patients without a history of oocyte retrieval. Miscarriage, preterm delivery, and fetal congenital malformation rates were similar between the two groups. ICSI-AOA may reduce fertilization failure without increasing risks during the perinatal period. AOA may be offered to couples with an ICSI fertilization rate < 50%.

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Introduction: The 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. Methods: This 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. Results: Motile 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. Discussion: For 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.

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Artificial oocyte activation (AOA) is considered an effective method to improve clinical outcomes in patients with some forms of male factor infertility and does not increase the risk of birth defects. However, the effects of AOA on patients with multiple morphological abnormalities of the sperm flagella (MMAF) caused by a DNAH1 mutation are still unknown. To explore the effects, our study analyzed a case with MMAF due to DNAH1 homozygous mutation that underwent testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI). The case had 28 MII oocytes. The 28 oocytes were divided randomly and equally into AOA and non-AOA groups. Ionomycin was used for AOA. We compared the clinical outcomes of two groups and selected three blastulation failure embryos from each group for transcriptome analysis (Data can be accessed through GSE216618). Differentially expressed genes (DEGs) were determined with an adjusted p-value <0.05 and a |log2-fold change| ≥1. The comparison of clinical outcomes showed that the two pronuclei (2PN) rate and grade 1-2 embryo rate at day 3 were not significantly different between the two groups. Transcriptome analyses of blastulation failed embryos showed that the use of AOA had potential risks of chromosome structure defects, transcriptional regulation defects, and epigenetic defects. In conclusion, when the case with MMAF due to DNAH1 mutation underwent TESE-ICSI, ionomycin-induced oocyte activation could not improve the clinical outcomes and introduced the risks of chromosome structure defect, transcriptional regulation defect, and epigenetic defect.

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RESEARCH QUESTION: Do fertilization rates differ between intracytoplasmic sperm injection (ICSI) cycles treated with artificial oocyte activation (AOA) using 10 µmol/l ionomycin or commercial A23187 in women at risk of failed or impaired fertilization? DESIGN: This single-centre, 7-year retrospective cohort study included 157 couples with a history of total fertilization failure (TFF, 0%) or low fertilization (<30%) after ICSI, or with severe oligo-astheno-teratozoospermia (OAT) in the male partner. Couples and underwent 171 ICSI-AOA cycles using either 10 µmol/l ionomycin or commercial A23187. The embryological and clinical outcomes were compared. RESULTS: Fertilization rates in the ionomycin group were significantly higher than those in the A23187 group for all three subgroups (TFF, 46.9% versus 28.4%, P = 0.002; low fertilization, 67.7% versus 49.2%, P < 0.001; severe OAT, 66.4% versus 31.6%, P < 0.001). AOA with ionomycin significantly increased the day 3 cleavage rate (P = 0.009) when compared with A23187 in the low fertilization group, but not in the TFF or severe OAT group (both P > 0.05). The rates of day 3 good-quality embryos, clinical pregnancy, implantation and live birth, and the cumulative live birth, did not differ between the two groups (all P > 0.05). A total of 64 live births resulted in 72 healthy babies born. CONCLUSIONS: AOA with 10 µmol/l ionomycin may be more effective than commercial A23187 in improving oocyte activation in patients at risk of failed or impaired fertilization, especially in cases of sperm-related defects.

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Introduction: Attempts 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. Methods: Firstly, 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. Results: The 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. Conclusions: This 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.

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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.

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Parthenogenesis is a common reproductive strategy among lower animals that involves the development of an embryo from an oocyte, without any contribution from spermatozoon. This phenomenon does not occur naturally in placental mammals. However, the mammalian oocytes can be artificially activated in vitro using mechanical, electrical, and chemical stimuli which can develop up to the blastocyst stage. In this chapter, we describe the protocol for generating haploid and diploid parthenotes from mouse oocytes using strontium as the activating agent under in vitro conditions.

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RESEARCH QUESTION: Does a double ionophore application improve the outcome of cycles in which single ionophore application was unsuccessful? DESIGN: This retrospective intervention study (duration 4.5 years) included 79 patients with suspected chronic failed oocyte activation (<30% fertilizations) and/or poor embryo development (developmental arrest, 24 h developmental delay, blastulation rate <15%) in both preceding cycles, the first without ionophore and the second with single ionophore treatment. Within the study period, all patients with failed ionophore treatments (single applications of ready-to-use calcimycin for 15 min) were offered an adapted protocol in the subsequent cycle (study cycle) in which the same ionophore was applied twice (separated by 30 min). Tests for paired data (control and study cycle) were used to reduce the effect of confounders. RESULTS: The overall fertilization rate did not differ between the study and control cycles. Cleavage (P = 0.020) and blastocyst formation (P = 0.018) rates improved significantly in the study cycles. Implantation (P = 0.001), biochemical (P < 0.001) and clinical pregnancy (P < 0.001) rates were also significantly higher in the study cycles. The study cycles resulted in 29 live births and all 32 babies born were healthy. CONCLUSIONS: This study suggests that double ionophore application may improve blastocyst formation and clinical pregnancy rates in cases of failed single ionophore treatment, irrespective of whether the ionophore was used to overcome fertilization failure or poor embryo development. Fertilization rate was only increased in cases with a history of fertilization failure. Because single ionophore treatment was used in only one previous cycle it cannot be ruled out that some improvement in clinical outcomes would also have been achieved by using single instead of double ionophore treatment again in the subsequent attempt.

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PURPOSE: To determine if 5mM calcium chloride dihydrate supplementation of the Polyvinylpyrrolidone (PVP) media at the time of ICSI (ICSI-Ca) improves fertilization, utilization, and clinical pregnancy rates compared to ICSI alone, particularly in patients with a history of low fertilization (< 50%). METHODS: Retrospective study between 2016 and 2021 at Monash IVF Victoria on a paired cohort of patients (n = 178 patients) where an ICSI cycle was analyzed coupled with the subsequent ICSI-Ca cycle. The paired cohort was further subdivided into a low-fertilization cohort (< 50% fertilization on previous cycles: n = 66 patients) compared to the remaining patients with fertilization ≥ 50% (n = 122). Exclusion criteria included donor cycles, PGT patients, surgical sperm retrieval, women ≥ 45 years old, patients with > 6 cycles, and patients with ≤ 5 inseminated oocytes. RESULTS: Calcium supplementation significantly increased both fertilization (28.8% ICSI vs 49.7% ICSI-Ca, P < 0.0001) and clinical pregnancy rate (4.9% ICSI vs 25.0% ICSI-Ca: P < 0.05) in the low-fertilization cohort but not in the normal-fertilization cohort. Interestingly, utilization rate significantly increased in the normal-fertilization cohort (32.6% ICSI vs ICSI-Ca: 44.9%, P < 0.01) but not in the low-fertilization cohort, although the number of embryos utilized per patient after ICSI-Ca increased in both groups. CONCLUSION: Calcium supplementation does not appear to be a detrimental addition to ICSI and may improve IVF outcomes, particularly for patients with a history of low fertilization. Further investigations including prospective case-matched studies or a RCT are required to confirm these findings.

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Intracytoplasmic sperm injection (ICSI) is an important treatment option for male infertility at pre-sent.However, a few patients still suffer from repeated ICSI fertilization failure because their sperm is unable to activate oocytes.Artificial oocyte activation (AOA) technology can improve the fertilization rate, pregnancy rate, live birth rate, etc., but it remains unknown whether AOA has short- and long-term effect on offspring.In this article, recent literature about the effect of AOA technology on perinatal outcomes, genetics, physical development and neurological development of offspring was summarized.This paper aims to provide reference for reproductive medicine workers and pediatricians in clinical practice.

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PURPOSE: To explore whether artificial oocyte activation (AOA) can improve embryo developmental potentiality and pregnancy outcomes for patients with a history of embryo developmental problem. METHODS: This was a retrospective study and candidate patients with embryo development problems were collected. A total of 1422 MII eggs from the enrolled 140 patients were randomized divided equally into 2 groups, half for the AOA group (AOA), and the rest of sibling mature eggs for the control group (non-AOA). The patients were further divided into two subgroups: (1) the rate of good-quality day 3 embryos was 0% (group 1, n = 66); (2) the rate of good-quality day 3 embryos ranged from 1 to 30% (group 2, n = 74). RESULTS: In the early embryonic growth, there were no significant differences in the outcomes of AOA and non-AOA groups in terms of normal fertilization rates, cleavage rates, day 3 good-quality embryo rates and available blastocyst rates (72.7% vs. 79.3%, 97.4% vs. 98.0%, 20.1% vs. 19.7%, 6.6% vs. 8.4% in group 1, respectively; 77.7% vs. 81.9%, 98.1% vs. 97.0%, 25.8% vs. 22.1%, 9.6% vs. 9.3% in group 2, respectively). In the late embryonic growth, no significant differences were found in biochemical and clinical pregnancy rates, implantation rates, miscarriage rates, and live-birth rates (50.0% vs. 45.2%, 45.2% vs. 40.5%, 37.3% vs. 31.3%, 10.5% vs. 11.8%, 40.5% vs. 35.7%, respectively) between two groups. In addition, neonatal outcomes were similar in both the groups as well. CONCLUSION: Our study demonstrated that the AOA using ionomycin 1 h after ICSI did not bring benefits to the early or late development of embryos derived from patients with a history of embryo developmental problems.

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STUDY QUESTION: What are the genetic causes of total fertilization failure (TFF) in a proband suffering from male infertility? SUMMARY ANSWER: Novel compound heterozygous variants (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) in actin-like protein 7A (ACTL7A) were identified as a causative genetic factor for human TFF. WHAT IS KNOWN ALREADY: ACTL7A, an actin-related protein, is essential for spermatogenesis. ACTL7A variants have been reported to cause early embryonic arrest in humans but have not been studied in human TFF. STUDY DESIGN, SIZE, DURATION: We recruited a non-consanguineous family whose son was affected by infertility characterized by TFF after ICSI. Whole-exome sequencing was used to identify the potential pathogenic variants. Artificial oocyte activation (AOA) after ICSI was performed to overcome TFF and any resulting pregnancy was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sanger sequencing was performed to validate the variants. Pathogenicity of the identified variants was predicted by in silico tools. The ultrastructure of spermatozoa was studied by transmission electron microscopy (TEM). Immunofluorescence staining and western blotting were used to investigate the mechanism of the variants on the affected spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE: Novel compound heterozygous variants in ACTL7A (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) were identified in a family with TFF after ICSI. In silico analysis predicted that the variants lead to a disease-causing protein. TEM showed that the ACTL7A variants caused ultrastructural defects in the acrosome and perinuclear theca. Protein expression of ACTL7A and phospholipase C zeta, a key sperm-borne oocyte activation factor, was significantly reduced in the affected sperm compared to healthy controls, suggesting that the ACLT7A variants lead to an oocyte activation deficiency and TFF. AOA by calcium ionophore (A23187) after ICSI successfully rescued the TFF and achieved a live birth for the patient with ACTL7A variants. LIMITATIONS, REASONS FOR CAUTION: Given the rarity of sperm-associated TFF, only one family with an only child carrying the ACTL7A variants was found. In addition, the TFF phenotype was not assessed in two or more ICSI cycles, due to the intervention in ICSI with AOA after one failed ICSI cycle. Further studies should validate the ACTL7A variants and its effect on male infertility in larger independent cohorts. WIDER IMPLICATIONS OF THE FINDINGS: : Our findings revealed a critical role of ACTL7A in male fertility and identified bi-allelic variants in ACTL7A associated with human TFF, which expands the genetic spectrum of TFF and supports the genetic diagnosis of TFF patients. We also rescued TFF by AOA and obtained a healthy live birth, which provides a potentially effective intervention for patients with ACTL7A pathogenic variants. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81971374 and 81401267). No conflicts of interest were declared. TRIAL REGISTRATION NUMBER: N/A.

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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.

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PURPOSE: To evaluate whether ionophore application at the oocyte stage changes the morphokinetics of the associated embryos in cases of artificial oocyte activation. METHODS: In a prospective sibling oocyte approach, 78 ICSI patients with suspected fertilization problems had half of their MII-oocytes treated with a ready-to-use ionophore (calcimycin) immediately following ICSI (study group). Untreated ICSI eggs served as the control group. Primary analyses focused on morphokinetic behavior and the presence of irregular cleavages. The rates of fertilization, utilization, pregnancy, and live birth rate were also evaluated. RESULTS: Ionophore-treated oocytes showed a significantly earlier formation of pronuclei (t2PNa) and a better synchronized third cell cycle (s3) (P < .05). The rate of irregular cleavage was unaffected (P > .05). Ionophore treatment significantly improved the overall rates of fertilization (P < .01) and blastocyst utilization (P < .05). CONCLUSION: Ionophore application does not negatively affect cleavage timing nor is it associated with irregular cleavage.

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