Introduction

Artificial oocyte activation (AOA) supports fertilization by temporarily elevating calcium (Ca2+) concentrations in oocytes and is an effective treatment for oocyte activation deficiency (OAD). The three AOA methods include chemical, electrical, and mechanical stimulations. We previously demonstrated that the live birth rate was significantly higher for couples with fertilization rates < 50% through ICSI-alone in the intracytoplasmic sperm injection (ICSI)-AOA (ICSI-AOA) group (18.0% [57/316]) than in the ICSI-alone group (4.7% [4/85]) across 4,893 oocytes, 198 couples, and 649 oocyte retrieval cycles at 18 Japanese facilities1. Recent reports indicate that clinical pregnancy rates were higher for infertile couples with fertilization failure history using ICSI-AOA (41.6%, n = 35/84) than using ICSI-alone (8.3%, n = 5/60)2. Several studies have reported that ICSI-AOA using a Ca2+ ionophore (A23187 or ionomycin), classified as chemical stimulation, is effective for addressing male factor fertilization failure3,4.

AOA cannot replicate the normal patterns of calcium oscillation5raising concerns about longitudinal neurodevelopment in children6. Although a meta-analysis on AOA showed that the risk of adverse obstetric, neonatal, or congenital outcomes in children born via ICSI-AOA is comparable to those born via ICSI-alone (relative risk = 1.27, 95% confidence interval [CI] = 0.70–2.28)7aberrant DNA methylation and gene expression changes have been reported in rodent offspring treated with ionomycin8,9 and in human cleavage-stage embryos exposed to A23187 10, suggesting that ICSI-AOA poses a potential risk of neurodevelopmental and psychiatric disorders5,10.

Recent studies have reported aberrant DNA methylation in imprinted genes (e.g., H19, SNRPN)—associated with Beckwith-Wiedemann, Angelman, and Prader-Willi syndromes—in human embryos following AOA11. While these epigenetic changes appear to resolve during later developmental stages, the study included only seven live births after ICSI-AOA, leaving long-term implications uncertain. To date, no phenotypic abnormalities have been documented in children born following AOA; however, existing evidence remains limited by small sample sizes and short follow-up periods. Given the expanding clinical application of AOA, rigorous long-term safety assessments are essential. These should include molecular investigations of neurodevelopment and imprint stability, complemented by extended longitudinal follow-up of children.

A 2020 survey by the Japanese Ministry of Health, Labour, and Welfare reported that 67.3% of assisted reproduction facilities had adopted AOA12and it has been covered by insurance since 2022. In the United Kingdom, the Human Fertilization and Embryology Authority (HFEA) removed AOA from its list of treatment add-on interventions lacking sufficient evidence of effectiveness in 2023 due to growing evidence supporting its efficacy and classification as a clinical-grade procedure13. The HFEA specified that AOA use should be cautiously limited to cases with specific clinical indications, including OAD, fertilization rates < 30%, or cephalic round globozoospermia14. Additionally, the European Society of Human Reproduction and Embryology Special Interest Group has advised caution regarding ICSI-AOA, avoiding the use of the term “recommendation”14 due to insufficient evidence regarding its safety.

Therefore, we aimed to investigate the impact of ICSI-AOA on paediatric neurodevelopment. A cross-sectional design was employed, involving Keio University and eight affiliated institutions under the Japanese Institute for Standardization of Assisted Reproductive Technology (JISART), supported by Tokyo Medical University and the University of Tokyo. Neurodevelopmental assessment was conducted using the Japanese version of the Ages and Stages Questionnaires, 3rd Edition (J-ASQ-3), which evaluates communication, gross motor, fine motor, problem-solving, and personal-social skills.

Results

Cohort characteristics

Overall, 158 cases were analysed, among which 81 and 77 were in the ICSI-alone and ICSI-AOA groups, respectively, with one twin pregnancy in the ICSI-AOA group. The median maternal age at delivery (ICSI, 35.0 [interquartile range = 32.0–38.0] vs. ICSI-AOA, 36.0 [33.0–39.0] years) and paternal age (ICSI, 38.0 [34.5–41.0] vs. ICSI-AOA, 37.0 [33.0–40.0] years), the proportion of mothers employed as office workers (ICSI, 17.3% vs. ICSI-AOA, 28.9%), and maternal educational background were comparable (Table 1). Household income, which was categorized into five ranges from < 3 to > 10 million yen, did not differ significantly between the groups. Fathers were significantly more likely to be employed as teachers in the ICSI-AOA group (9.2%) than in the ICSI-alone group (1.2%).

Table 1 Parental and children Characteristics.
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We examined the influence of sibling presence and social engagement on neurodevelopment by analysing singleton birth rates, sibling composition, and levels of social engagement between the groups. The singleton birth rates were 98.7% and 100% in the ICSI-AOA and ICSI-alone groups, respectively, showing comparable rates. No significant differences in sibling composition were observed between the groups. The proportion of children participating in social activities was similar between the ICSI-AOA (77.9%) and ICSI-alone (85.2%) groups (Table 1).

J-ASQ-3 evaluation between ages 12 and 60 months

All 81 and 77 children in the ICSI-alone and ICSI-AOA groups, respectively, completed the J-ASQ-3 questionnaire without any missing responses. The participants were categorized into nine groups based on age, each completing the corresponding age-appropriate J-ASQ-3 questionnaire. Specifically, the ICSI-alone and ICSI-AOA groups showed no significant differences in any of the five J-ASQ-3 subdomain scores—communication, gross motor, fine motor, problem-solving, or personal-social skills—across all age groups (Fig. 1). Among the 45 fully adjusted mean differences evaluated, a single P-value (the personal-social skills score at 48 months) reached the threshold for statistical significance between the ICSI-AOA and ICSI-alone groups (adjusted mean difference = 7.76; 95% CI = 0.47–15.05; P = 0.038) (Table 2). However, Q-Q plot analysis demonstrated that this value was well within the expected null distribution, indicating no evidence of systematic deviation or a true effect (Supplementary Fig. S1). This result underscores the observed data consistency with the expected random variation, reinforcing the findings’ overall reliability. Subsequently, the number of cases below the cutoff and within the monitoring zone for the five J-ASQ-3 subdomain scores were compared, revealing no significant developmental differences in the five subdomains between the ICSI-alone and ICSI-AOA groups (Table 3).

Fig. 1
figure 1

J-ASQ-3 cores at age 12–60 months | Total raw scores on each J-ASQ-3 domain (communication, gross motor, fine motor, problem-solving, and personal-social) for children born via ICSI alone and ICSI-AOA at 12, 18, 24, 30, 36, 42, 48, 54, and 60 months of age. No significant group differences are found in any of the J-ASQ-3 domains (A–E). Error bars represent 95% confidence intervals, indicating variation within the sample. J-ASQ-3 Japanese version of the Ages and Stages Questionnaire 3rd Edition, ICSI intracytoplasmic sperm injection, AOA artificial oocyte activation.

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Table 2 Comparison of the J-ASQ-3 scores between the ICSI-alone and ICSI-AOA Groups.
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Table 3 Comparison of the numbers below cutoff and in monitoring zones based on the J-ASQ-3 scores between the ICSI-alone and ICSI-AOA Groups.
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J-ASQ-3 evaluation between A23187 and ionophore

Each AOA method poses distinct neurodevelopmental risks. Therefore, we evaluate the J-ASQ-3 scores associated with each reagent used for AOA. The AOA protocol comprised 59, 15, and 3 cases treated with A23187, ionomycin, and electrical stimulation, respectively. No mechanical stimulation, SrCl₂, or phospholipase C zeta (PLCζ) cases were reported (Supplementary Table S1). The analysis showed no significant differences in any of the five J-ASQ-3 subdomain scores across all age categories between the ionomycin and A23187 groups (Fig. 2). Similarly, the number of cases below the cutoff and within the monitoring zone for the five J-ASQ-3 subdomain scores was similar between the A23187 and ionomycin groups.

Fig. 2
figure 2

J-ASQ-3 scores for comparing neurodevelopment in children conceived with ICSI-AOA using A23817 or ionophores | Total raw scores on each J-ASQ-3 domain (communication, gross motor, fine motor, problem-solving, and personal-social) for children born with ICSI-AOA using A23817 or ionophores at 12, 18, 24, 30, 36, 42, 48, 54, and 60 months of age. Both groups have no significant differences at all age points and in all domains (A–E). Data points represent mean scores; the bars represent 95% confidence intervals and indicate within-sample variation. J-ASQ-3 Japanese version of the Ages and Stages Questionnaire 3rd Edition; ICSI intracytoplasmic sperm injection, AOA artificial oocyte activation.

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Validation of risk factors in children scoring below the monitoring zone on the J-ASQ-3

Identifying factors associated with neurodevelopmental delays is essential for recognizing at-risk children and enabling timely interventions. We identified populations at high risk of developmental delay requiring evaluation or follow-up within each subdomain. Children scoring below the monitoring zone on the J-ASQ-3 (Supplementary Fig. S2) were identified, and their backgrounds, including maternal and paternal age, child’s sex, presence of siblings, group living status, gestational age at delivery, birth weight, and delivery mode, were compared with those of children scoring within the normal range. Among these factors, maternal age, sibling composition, and child’s sex were significantly associated with scores below the monitoring zone: higher maternal age (35.0 [32.0–38.0] vs. 37.0 [33.0–39.8] years; OR = 1.10 [1.01–1.20], P = 0.037), absence of siblings (OR = 4.00 [0.20–0.78], P = 0.007), and male sex (OR = 2.57 [1.31–5.05], P = 0.006) (Supplementary Table S2).

Discussion

This is the first multicentre cross-sectional study to evaluate the long-term safety of ICSI, followed by AOA on neurodevelopment in a large cohort of children. Our findings suggest that the neurodevelopmental safety of ICSI-AOA is comparable to that of ICSI-alone in terms of social and motor development in 12–60 months. Analysis of scores below the cutoff and within the monitoring zone revealed no significant differences across all subdomains. No significant differences in the J-ASQ-3 scores were observed between the AOA methods using A23187 and ionomycin, suggesting that variations in the AOA techniques do not adversely affect neurodevelopmental outcomes.

Previous studies evaluating the language abilities of children aged 3–10 years conceived via ICSI-AOA, using the Reynell Developmental Language Scales, found that 4 of the 20 children scored below the 10th percentile in specific subdomains15. The analysis of children scoring below the cutoff or requiring monitoring revealed no significant differences, suggesting that AOA does not negatively impact communication. Key methodological differences likely account for the discrepancies in these findings. A previous study assessed a population with a high proportion of twins, conducted evaluations over an extended single-day period, and tested children in an unfamiliar environment with unfamiliar individuals present15. However, our study used parent-reported assessments conducted in approximately 15 min within the comfort of their homes, likely providing a more accurate reflection of the children’s true abilities. Given experimental evidence linking altered calcium signalling during fertilization to changes in gene expression and early neurodevelopment, larger cohort studies with long-term follow-up are needed to clarify the potential implications of AOA.

This study did not include children born after AOA with SrCl2 or PLCζ. Precision medicine approaches have been explored to detect OAD in advance, targeting sperm-related (including PLCζ and PLCζ I mutations) and oocyte-related (including wee1-like protein kinase 2, PAT1 homolog 2, tubulin beta-8 chain, and transducin-like enhancer protein 6 mutations) factors16. However, an effective method for selecting the appropriate AOA protocol is yet to be established. SrCl2 and PLCζ are considered potent agents for oocyte activation; however, their clinical application remains limited. The activation mechanism of SrCl2 is well characterized in murine oocytes but remains poorly understood in human oocytes17. Similarly, PLCζ shows significant variability in expression levels and functional capacity among individuals, complicating the development of standardized therapeutic dosing regimens18. These limitations contribute to insufficient data supporting the efficacy and safety of SrCl2 and PLCζ in human applications, hindering their broader clinical adoption. Ca2+ ionophores have well-defined activation mechanisms, and substantial clinical evidence supports their efficacy and early postnatal safety. They have become the preferred agents for AOA in routine clinical practice12 leading to a larger proportion of infants being born following their use.

Recent literature highlights concerns about neurodevelopmental delays in children born during the coronavirus disease 2019 (COVID-19) pandemic, particularly regarding language and communication skills13,19,20,21. These delays are attributed to multifactorial influences, including social restrictions (e.g., lockdowns), economic instability, and increased parental stress. Maternal COVID-19 is associated with cytokine storms, which can impair foetal development and induce DNA damage21. Since the pandemic was declared in March 2020, assuming that all children included in this study were exposed to its social consequences is reasonable. Many participants experienced significant early developmental stages during the pandemic. Reduced social interactions, limited access to developmental resources, and heightened family stress may have exerted similar developmental effects on both groups. This shared context may explain the lack of significant differences in communication scores between the ICSI-AOA and ICSI-alone groups in our study and could influence outcomes in future studies using the ASQ-3.

Early intervention is a well-established method of improving developmental outcomes in children with developmental delays. A study on the Early Start Denver Model, conducted over 2 years involving children diagnosed with autism spectrum disorder (ASD) at 18–30 months, showed improvements in Intelligence Quotient, adaptive behaviour, and electroencephalograph patterns that were similar to those of typically developing children22,23. The American Academy of Pediatrics recommends combining standardized screening tests for ASD in children aged 9–30 months to support early identification of developmental disorders24,25,26. Although the J-ASQ-3 used in this study is a screening tool rather than a diagnostic instrument, reevaluating children below the monitoring zone and initiating early intervention may promote developmental improvements. We compared confounding variables associated with the J-ASQ-3 scores in children who scored below the cutoff, those in the monitoring zone, and typically developing children to explore the factors contributing to developmental risk beyond AOA. While no significant differences were found in the below-cutoff group, an analysis of children in the monitoring zone revealed that having siblings was associated with a lower adjusted OR for being in the monitoring zone (adjusted OR = 0.40, 95% CI = 0.20–0.78). Male children had a higher adjusted OR than female children (adjusted OR = 2.57, 95% CI = 1.31–5.05). These results support the known higher risk of developmental disorders in males27 and suggest that having siblings promotes developmental progress28. Overall, this analysis indicates that AOA is possibly a minimal risk factor for developmental disorders in this cohort.

In our cohort, paternal age did not differ significantly between the conventional ICSI and ICSI-AOA groups (median 37.0 vs. 38.5 years; P = 0.91). Moreover, paternal age was not predictive of scoring below the ASQ-3 monitoring threshold. Although recent evidence suggests that advanced paternal age may subtly influence offspring neurodevelopment29these data indicate that it is unlikely to have confounded the association between AOA and neurodevelopmental outcomes.

Epigenetic mechanisms govern gene expression, cellular function, and developmental processes, and DNA methylation plays a central role in normal embryonic development. Aberrant DNA methylation patterns in infants have been linked to an increased risk of ASD9. A comparative analysis of 962 assisted reproduction technology (ART)-conceived and 983 naturally conceived singleton newborns revealed a global trend of hypomethylation in ART-conceived individuals, with 607 differentially methylated CpG sites identified, affecting genes associated with growth and neurodevelopment30. Although these findings highlight the potential epigenetic risks associated with ART, whether specific ART procedures contribute to these alterations remains unclear. Our analysis showed that children conceived with AOA exhibited no neurodevelopmental differences compared to those conceived without AOA. These differences suggest that AOA, despite its mechanistic role in overcoming incomplete oocyte activation, a process known to disrupt epigenetic reprogramming during zygotic genome activation31does not introduce additional neurodevelopmental risks.

To further explore the observed trend, it is noteworthy that in our cohort, the ICSI-AOA group showed numerically low J-ASQ-3 communication scores at 12, 18, and 24 months of age. However, this difference was no longer apparent by 30 months, suggesting any early developmental lag may be transient. This pattern, combined with the absence of significant between-group differences in the proportion of children falling below the threshold or requiring monitoring, suggests that AOA is unlikely to have lasting adverse effects on communication development. Nevertheless, these findings warrant cautious interpretation, and ongoing developmental surveillance remains important.

This study demonstrated AOA’s safety for neurodevelopment in children up to 60 months, reinforcing the recent decision by the HFEA13 and the fact-finding survey’s results in Japan, which recommended upgrading AOA to clinical grade based on accumulating evidence32. As evidence of AOA’s efficacy and safety continues to increase, its evaluation in clinical practice is expected to improve, potentially expanding its indications. However, adhering to the current indications for AOA use is crucial, particularly in clinical settings, given the limited understanding of its long-term effects.

Limitations

This study employed a cross-sectional design, assessing children aged 12–60 months using the J-ASQ-3, with no longitudinal follow-up. While heterogeneity and potential biases from environmental and genetic factors could not be eliminated, minimal bias was suggested, as no significant differences in social or environmental backgrounds were observed, except for a higher number of fathers with teaching professions in the ICSI-AOA group than in the ICSI-alone group. The COVID-19 pandemic may have affected developmental outcomes, given its impact on early childhood environments. Finally, this study lacked adequate data to compare various AOA methods, underscoring the necessity of longitudinal multicentre studies to assess the long-term safety and effectiveness of alternative techniques29.

Conclusions

This is the first study to demonstrate the long-term safety of ICSI, followed by AOA on neurodevelopment in a large cohort of children. Based on the findings, no significant concerns regarding any adverse effects of AOA on childhood neurodevelopment have been identified. These data add to the body of evidence available to physicians when considering approaches for couples experiencing fertilization failure. However, more detailed studies on the long-term childhood development born after all AOA types are needed before any definitive conclusions can be drawn.

Participants and methods

Study design and participants

We collected data on mothers who gave birth via ICSI-alone or ICSI-AOA at Keio University Hospital and eight JISART facilities between August 2018 and June 2023. Based on responses from the eight participating facilities, A23187-mediated AOA was performed using either a HEPES-buffered calcium ionophore (CIM-10, Kitazato Corporation, Japan) or A23187 obtained from SIGMA (C7522, USA), Gynemed (GM508 CultActive, Germany), or FujiFilm (019-20111, Japan). GM508 CultActive was supplied as a ready-to-use A23187 ionophore solution, with its concentration undisclosed. When preparing other A23187 solutions, stock concentrations were diluted in HEPES-buffered medium with DMSO to achieve final working concentrations of either 5 µM or 10 µM. In all cases, AOA was initiated within 30 min after ICSI and consisted of a single exposure at 37 °C for 5–15 min. Ionomycin (I-0634, Sigma-Aldrich, Belgium) was also used as a calcium ionophore. In this protocol, oocytes were exposed to a 5 µM ionomycin solution for 10 min, starting 20 min after ICSI.

The neurodevelopmental survey focused on children aged 12–60 months who were conceived via ICSI-alone or ICSI-AOA between August 2018 and January 2023. The exclusion criteria included cases involving testicular sperm extraction, mothers aged ≥ 43 years at the time of embryo transfer, and double embryo transfers using ICSI-alone and ICSI-AOA. No age restrictions were applied for fathers. Prior to completing the J-ASQ-3 and an additional questionnaire, written informed consent was obtained from all participants. To specifically assess the impact of ICSI-AOA on neurodevelopmental outcomes, we established a control group matched by offspring age. For each child born following ICSI-AOA, one age-matched control child was selected from the IVF database of the corresponding facility. These children were conceived and delivered during the same time period to ensure comparable durations of postnatal follow-up. All procedures were conducted in accordance with the relevant guidelines and regulations.

On December 8, 2023, all facilities were instructed to collect relevant cases and complete the case report forms. Among the 375 identified women, 326 children aged 12–60 months were selected. Questionnaires were distributed equally to both groups (163 ICSI-AOA and 163 ICSI) totalling 326 children on January 5, 2024, with February 18, 2024, as the submission deadline. However, participants were not informed about the survey in advance; therefore, a 1,000-yen gift card was included with the questionnaire.

Valid responses were obtained from 158 children (158/266, 59.4%), comprising 77 of 128 (60.2%) in the ICSI-AOA group and 81 of 138 (58.7%) in the ICSI group. Among the remaining cases: 60 questionnaires were undelivered, 104 were not returned, 3 were declined, and 1 was excluded for not meeting inclusion criteria. Parents were also asked to complete a paper-based questionnaire on factors influencing child development, including sibling composition and social activities (e.g., daycare or kindergarten), parental educational background, occupation, and household income.

Assessment of neurodevelopment

The J-ASQ-3 is a level 1 screening tool that evaluates five developmental domains. This questionnaire, completed by parents or caregivers, is known for its high reliability and validity. It has been translated into several languages and is widely used in clinical and research settings internationally33,34,35,36 .The J-ASQ-3 is a Japanese translation of the ASQ-3, with items adapted to Japanese culture and developmental standards for children in Japan37. The cutoff is defined as scores ≤-2.0 standard deviations (SD) below the age-specific mean, indicating a strong suspicion of developmental delay, whereas the monitoring zone (-1.0 to -2.0 SD) suggests the need for evaluation or follow-up. In this study, we defined scores ≤ − 1.0 SD as “below monitoring” to include both the monitoring zone and below-cutoff categories, due to the limited number of children scoring below the cutoff. A binomial logistic regression analysis was performed using AOA presence, child’s sex, gestational age, and J-ASQ-3 age as independent variables, with children below the cutoff or in the monitoring zone as the dependent variable.

Statistical analysis

All statistical analyses were conducted using IBM SPSS Statistics for Windows, version 28 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 9 (GraphPad Software, Inc., La Jolla, CA, USA), with statistical significance considered at P < 0.05. Power analyses, based on the primary outcome, were used to investigate the mean differences in the J-ASQ-3 subdomain scores between children conceived with and without AOA. Covariance analysis was used to determine the mean differences in the J-ASQ-3 subdomains between the ICSI-alone and ICSI-AOA groups. We used quantile-quantile plots (Q-Q plots) analysis to assess the overall distribution of P-values derived from the analysis of 45 adjusted mean differences in J-ASQ-3 scores between ICSI-alone and ICSI-AOA. This method was selected to rigorously evaluate whether the observed P-value distribution aligns with the expected null distribution, enabling the identification of potential deviations due to systematic biases or random variation. The Q-Q plot analysis strengthens our multiple comparison framework robustness by providing a visual and statistical assessment of conformity.

The percentages of children identified as being below the monitoring threshold and cutoff levels indicative of delays, as defined by the ASQ-3 developer, were computed for each subdomain and the overall J-ASQ-3 outcome. Additionally, the odds ratios (ORs) were calculated using a binary logistic regression model. The independent variables included AOA presence, sex, gestational age at birth, and age in months during the J-ASQ-3 assessment. All results were reported with 95% confidence intervals (CIs).