Impact of Laser-Assisted Hatching on Cognitive and Metabolic Development of Singletons Conceived Through Frozen-Thawed Embryo Transfer: A Matched Cohort Study.

Xin‐Yi Liu, Chun‐Xi Zhang, Yi‐Feng Liu and Dan Zhang designed the study and supervised the trial. Xin‐Yi Liu, Chun‐Xi Zhang and Yi‐Qing Wu obtained ethical approval. Xin‐Yi Liu, Chun‐Xi Zhang, Jian‐Peng Chen, Qi Liang, Jing‐Lei Xue, Ling‐Hui Li, Qi‐Qi Xu, Fang‐Hong Zhang, Ying‐Zhi Yang and Yue‐Mei Wang performed the study and conducted the follow‐up. Xin‐Yi Liu, Chun‐Xi Zhang and Yi‐Feng Liu conducted all analyses. Xin‐Yi Liu, Yi‐Feng Liu and Dan Zhang wrote the manuscript.

The present study was approved by the Institutional Ethical Review Board of Women's Hospital, School of Medicine, Zhejiang University (IRB‐20230071‐R). Written informed consents were obtained from the parents of the included children. The study was registered in the Chinese Clinical Trial Registry (ChiCTR2300071711).

This cohort study enrolled children conceived through FET at the reproductive centre of Women's Hospital, Zhejiang University School of Medicine between January 2016 and December 2019. Both IVF and ICSI cycles were included in this study. To avoid the effects of twins on the study results, only singleton live births were included. The exclusion criteria were as follows: (1) PGT; (2) chromosomal abnormalities of the mother or father; (3) sperm or egg donation; (4) death of offspring; and (5) participation in other clinical studies. In total, 114 children conceived through LAH were selected; of them, 19 were lost due to lack of contact and 12 refused participation. Ultimately, 83 parents consented to participating in the study. Two children could not provide the required cooperation to complete the intelligence test, and 41 did not attend the appointment at the end of the study because of work or children's illnesses. We created a 2:1 matched control cohort comprising women without a history of LAH use (non‐LAH) during FET and their children. The matching factors were age of children (within 1 year) and sex. To enhance statistical precision and control for possible confounding variables, children conceived without using LAH were matched in a 2:1 ratio based on their age and sex throughout the follow‐up period using frequency matching method. Participants in the LAH group were stratified by age and sex, resulting in distinct subgroups defined by these variables. An equivalent number of control participants from the eligible population were selected while maintaining the 2:1 ratio between the two groups. Eventually, a total of 40 women‐children in the LAH group and 80 women‐children in the non‐LAH group were enrolled in the study. The study was approved by the Institutional Ethical Review Board of Women's Hospital, Zhejiang University School of Medicine (IRB‐20230071‐R). Written informed consents were obtained from the parents of the children included in the study. In addition, the study was registered in the Chinese Clinical Trial Registry (ChiCTR2300071711). The LAH procedure involved placing thawed embryos on a heated stage and observing them under an inverted microscope. Precise laser targeting was employed to focus on the widest gap between the embryo and ZP, ensuring minimal disruption to blastomeres. Laser intensity was adjusted according to ZP's thickness, gradually thinning it and creating a zone spanning approximately one‐quarter of its length, with a depth equivalent to two‐thirds of its original thickness. Following LAH, the embryos were cultured in optimal conditions until transfer. Data on parental demographics and ART characteristics were collected from the medical records. The parental demographics included variables such as age, body mass index (BMI), educational level, occupation, smoking status and annual household income. Educational level was categorised into three groups—middle school or below, high school, and college or higher. Primary caregivers' occupations were classified into four groups based on the European Socioeconomic Classification (E‐SeC) [ 11 ]: E‐Sec 1–3 group (higher professionals and managers, higher clerical, services and sales workers), E‐Sec 4–6 group (small employers and self‐employed, farmers, lower supervisors and technicians), E‐Sec 7–9 group (lower clerical, services and sales workers, skilled workers, semi‐skilled and unskilled workers) and E‐Sec 10 group (unemployed). According to the 2023 per capita disposable income data released by the National Bureau of Statistics of China, the present study classified annual household incomes into four categories— 500000 CHY. Perinatal, delivery and children's lifestyle information, including pregnancy complications (anaemia, gestational diabetes and gestational hypertension), gestational age, birth weight, preterm birth (gestational age < 37 weeks), mode of delivery (caesarean section or vaginal delivery), neonatal intensive care unit (NICU) status, feeding patterns, duration of breastfeeding, daily sleep hours and daily exercise hours, was collected from standardised questionnaire interview. During the follow‐up, we collected routine anthropometric data, including height, weight, BMI and blood pressure. The Growth Standards for Children Under 7 Years of Age recommended by the Chinese National Health Commission in 2023 were utilised as the reference standard to mitigate the impact of sex and age variations on children's growth data. Standard deviation scores (SDS) for height, weight and BMI by age were recorded for each child, with specific thresholds used to represent overweight (+1 ≤ BMI_SDS < +2), obesity (BMI_SDS ≥ + 2) and retardation (height_SDS < ‐2). Hypertension was diagnosed when either systolic or diastolic blood pressure was above the P95 percentile for their age and sex [ 12 ]. Metabolic indicators encompassed fasting blood glucose, insulin levels, blood lipids (triglycerides, cholesterol, low‐density lipoprotein [LDL] and high‐density lipoprotein [HDL]), thyroid function (FT3, FT4, TSH) and vitamin D levels. The Chinese version of the Wechsler Preschool and Primary Scale of Intelligence, Fourth Edition (WPPSI‐IV) and the Adaptive Behavior Assessment System II (ABAS‐II) were selected for assessing cognitive and behavioural profiles. The WPPSI‐IV is a standardised measure of intelligence for children between 2 years and 6 months through to 7 years and 7 months of age [ 13 ]. The children are required to complete 13 subtests. The composite scores encompass verbal comprehension index (VCI), visual space index (VSI), fluid reasoning index (FRI), work memory index (WMI), processing speed index (PSI) and the final full‐scale intelligence quotient (FSIQ). The ABAS‐II is a questionnaire for assessing children's adaptive capacity. It assesses 10 skill areas and produces three key adaptation scores (conceptual, social and practical skills), as well as the final general adaptive composite score (GAI). SPSS statistics 27.0 (IBM) was used to analyse the data. All continuous data are presented as means ± standard deviation, and categorical data are presented as numbers (%). Independent samples t ‐tests were used to compare continuous variables between the groups, and the chi‐square or Fisher's exact test was used for comparing categorical variables. Multivariate linear regression model was used to evaluate the correlation between LAH use and the test scores of various dimensions of the WPPSI‐IV and ABAS‐II. The model was adjusted for parents' educational level, primary caregiver's occupation, annual household income level, maternal BMI, paternal smoking status, breastfeeding duration, daily exercise hours and sex of the child. All hypothesis tests were two‐sided, with values of p  < 0.05 considered statistically significant. The power calculation was conducted using PASS 15 software before the study according to a previous study [ 14 ]. To achieve a power of ≥ 0.80 (α = 0.05, ratio = 1:2), with a 6 points difference in FSIQ (standard deviation = 10) assumed between the two groups, 33 children who underwent LAH and 66 who did not undergo LAH were required for inclusion in this study.

The demographic and clinical characteristics of participants are presented in Table  1 . A significant difference in maternal age was observed between the LAH and non‐LAH groups, with women undergoing LAH more likely to be older (32.6 ± 4.8 years vs. 30.7 ± 3.4 years, p  = 0.026). The proportion of women with gestational diabetes mellitus was lower in the LAH group than in the non‐LAH group (10.0% vs. 22.5%, p  = 0.037). Paternal age, BMI, education level of parents, primary caretaker's occupation, paternal smoking status, level of annual household income, gestational week, birth weight, premature birth rate, caesarean section rate and NICU rate were similar between the two groups. The proportion of children in the LAH group with ≥ 1 h of daily exercise was lower than that of children in the non‐LAH group (55.3% vs. 77.8%, p  = 0.014). No significant differences in other lifestyle factors, such as feeding style, breastfeeding duration and daily sleep hours, were observed between the groups. Baseline characteristics of parents and offsprings. Abbreviations: BMI = body mass index, E‐SeC = European Socioeconomic Classification, GDM = gestational diabetes mellitus, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilisation, LAH = laser‐assisted hatching, NICU = neonatal intensive care unit.PCOS = polycystic ovarian syndrome. Daily sleep hours and daily exercise hours were missing for two LAH cases and eight non‐LAH cases. Congenital malformation was observed in two cases of atrial septal defect, once case of inverted nipple, and one case of concealed penis. History of serious disease was noted in five cases of severe pneumonia, three cases of Kawasaki disease, three cases of hernia, one case of pertussis, one case of adenoid hypertrophy, one case of haemangioma and one case of nasolacrimal duct cyst. The LAH offspring had similar height, weight, BMI, thyroid function, lipid profile, glucose metabolism index and vitamin D level as those of the non‐LAH offspring. The proportions of participants with retardation, overweight, obesity and hypertension were not significantly different between the two groups (Table  2 ). When stratified by sex, the female offspring conceived with LAH showed slightly elevated total cholesterol (TC) and LDL levels than those of female offspring conceived without LAH, whereas male offspring conceived with LAH exhibited a slight decrease in free triiodothyronine (FT3) levels compared to their non‐AH counterparts (Table  S1 ). Physical and metabolic characteristics of offspring. Abbreviations: BMI = body mass index, FBG = fasting blood glucose, FINS = fasting insulin, FT3 = free triiodothyronine, FT4 = free thyroxine, HDL = high‐density lipoprotein, LAH = laser‐assisted hatching, LDL = low‐density lipoprotein, TC = cholesterol, TG = triglyceride, TSH = thyroid stimulating hormone. A total of 38 of 40 children in the LAH group and 79 of 80 children in the non‐LAH group underwent fasting venous blood sample tests. In the WPPSI‐IV assessment, children in the LAH group scored 109.3 ± 12.55 on the FSIQ, whereas those in the non‐LAH group scored 108.1 ± 11.60, demonstrating no statistical difference between the two groups. The subscale scores (VCI, VSI, FRI, WMI and PSI) were similar in the groups. In the ABAS‐II assessment, no significant differences in the GAI, conceptual skills, social skills and practical skills scores were observed between the two groups. However, offspring conceived with LAH showed a higher VCI score compared to those conceived without LAH after adjusting for potential risk factors in the linear regression model [aβ = 6.09, 95% confidence interval = 1.44, 10.73], and no significant differences in the other subscales were observed (Table  3 ). Cognitive and behavioural profiles of offspring. Abbreviations: ABAS‐II = Adaptive Behaviour Assessment System II, CI = confidence interval, FRI = fluid reasoning index, FSIQ = full‐scale intelligence quotient, GAI = general adaptive composite score, LAH = laser‐assisted hatching, PSI = processing speed index, VCI = verbal comprehension index, VSI = visual space index, WMI = work memory index, WPPSI‐IV = Wechsler Preschool and Primary Scale of Intelligence, Fourth Edition. 1 Non‐LAH children did not complete all tests. Model adjusted for parents' educational level, primary caregiver's occupation, annual household income level, maternal BMI, paternal smoking status, breastfeeding duration, daily exercise hours and sex of the child.

In this cohort study of offspring conceived through ART followed up till preschool years, singletons born after LAH showed similar physical growth, metabolic indicators, cognitive function and adaptive behaviour to those born without LAH. Since the introduction of the concept of assisted hatching over 30 years ago [ 3 ], an increasing number of couples with infertility have successfully conceived and achieved live birth using assisted hatching techniques. The periconceptional period is the stage where key events of reproduction such as gametogenesis, fertilisation, resumption of mitotic cell cycles in the zygote and embryo implantation occur [ 15 ], marking the transition from the parental to the embryonic genome [ 16 ]. This period is a critical ‘window period’ for early life development. Embryos at this stage exhibit developmental plasticity; when exposed to adverse environmental conditions, they may undergo maladaptive changes that predispose them to diseases in the future [ 17 ]. The potential impact of high temperature exposure, reaching up to 130°C–160°C during LAH, on the development and long‐term health outcomes of offspring is of great concern for clinicians and parents. Previous studies have primarily focused on whether LAH affects neonatal outcomes, with most findings indicating no significant association between LAH and increased risk of preterm birth or low birth weight. However, the potential influence of this in vitro technique on the future development of offspring remains uncertain. Therefore, it is imperative to investigate whether LAH has any detrimental effects on the long‐term health of the offspring. According to the results of this study, no significant increase in the risk of retardation, obesity, hypertension or abnormal glucose and lipid metabolism was observed among offspring conceived through LAH. The higher maternal age observed in the LAH group compared to the non‐LAH group may attributed to the fact that LAH is more likely to be recommended for older women in clinical practice. Other baseline characteristics potentially associated with offspring physical development and metabolism, such as parental BMI [ 18 , 19 ], maternal history of PCOS [ 20 ], smoking status [ 21 ], feeding patterns [ 22 ] and socioeconomic factors were similar between the two groups, which further strengthens the reliability of our study findings. Despite the observed differences in TC and LDL levels among female offspring conceived through LAH and in FT3 levels among male offspring conceived through LAH, all measurements remained within normal ranges. Given the small sample sizes within each sex‐specific subgroup, caution is warranted in interpreting these findings because they may not demonstrate significant clinical relevance. The current data indicate that sex may modulate the effects on lipid metabolism, potentially underpinning an increased susceptibility to cardiovascular disease in adulthood [ 23 ]. This highlights the importance of rigorous monitoring of glucose and lipid profiles, particularly in female offspring. The intellectual development of children has emerged as a focus in the safety assessment of assisted reproduction techniques. In this study, the WPPSI‐IV and ABAS‐II were used to comprehensively evaluate children's intelligence development, in accordance with the definition of intellectual disability provided by the American Association on Intellectual and Developmental Disabilities, indicating significant limitations in both cognitive function and adaptive behaviour [ 24 ]. Previous studies have indicated that children's cognitive development can be affected by various factors, including genetic factors, regional economic development [ 25 ], parental education level [ 26 ], prenatal exposure to adverse conditions [ 18 , 27 ] and feeding pattern [ 26 , 28 ]. Moreover, our findings indicated that maternal prepregnancy BMI, parental educational level, occupation of the primary caregiver, annual household income, paternal smoking status, breastfeeding duration and the child's sex affect the WPPSI‐IV scores (Table  S2 ). After adjusting for these confounding factors in the model, the offspring in the LAH and non‐LAH groups showed comparable level of cognitive function and adaptive behaviour. Furthermore, we observed that offspring conceived through LAH demonstrated higher VCI scores, which is closely associated with cultural background, family environment and educational experiences of the participants. The trend may be attributed to the fact that parents in the LAH group typically underwent a more challenging process of ART and consequently prioritised their children's education. Therefore, our findings must be interpreted with caution. To explain this trend, further increases in sample size and language‐specific testing are necessary. Overall, this study comprehensively assessed the long‐term safety of offspring conceived through LAH in terms of physical development, metabolic function and neuro‐intelligence development. The findings of the study indicate that LAH did not confer an increased risk of developmental delay, metabolic abnormalities or intelligence disorders in the offspring. Consequently, it can be inferred that LAH is a relatively safe method of ART. These results assuage concerns regarding the well‐being of children born to parents with infertility who have undergone LAH and offer valuable insights for future clinical counselling and practice. To the best of our knowledge, this is the first follow‐up study to assess the health of offspring born after LAH until preschool years. Our cohort only included singletons to mitigate the potential impact of multiple births on offspring outcomes [ 29 ]. In addition, all LAH procedures were performed by highly trained embryologists at a single reproductive centre, thus minimising bias arising from variations in AH techniques and operator proficiency. Furthermore, trained researchers conducted follow‐up assessment at a single centre using specialised measurement methods and standardised test scales, eliminating bias resulting from differences in follow‐up protocols. Nevertheless, this study had some limitations that should be acknowledged. First, the major limitation of the present study is the inclusion of a relatively small sample size. Second, we only conducted a follow‐up on the health status of preschool children aged 4–6 years. However, the presence of some chronic diseases such as hypertension and abnormal glucose and lipid metabolism which tend to manifest in adulthood was not evaluated. Therefore, it is imperative to establish a comprehensive long‐term follow‐up system with multiple time points to provide robust evidence regarding the safety of LAH and other in vitro procedures in assisted reproduction.

The cognitive, metabolic and physical developmental outcomes of preschool children born through LAH were comparable with those of children not born through LAH, suggesting that LAH could be considered a safe ART method. However, further extended and regular follow‐up is necessary to validate these findings.

The World Health Organization estimates that approximately 17.5% of the adult population—roughly one in six individuals worldwide—are affected by infertility [ 1 ]. An increasing number of couples seek assisted reproductive technology (ART). ART has evolved rapidly over the past decades due to improved clinical decision‐making and innovations in ART‐related techniques, including intracytoplasmic sperm injection (ICSI), preimplantation genetic texting (PGT) and assisted hatching (AH). Despite the increasing success rates of ART, concerns remain about the impact of in vitro procedures, performed during critical period of gamete and early embryonic development, on the long‐term health of the offspring. Embryo implantation is a prerequisite for pregnancy [ 2 ], and for successful implantation, the embryo must escape from the zona pellucida (ZP) in a process called ‘hatching’. However, during in vitro fertilisation (IVF) and embryo transfer, controlled ovarian hyperstimulation, prolonged in vitro culture of embryos, embryo freezing and thawing may cause ZP abnormalities, such as thickening and hardening, resulting in hatching difficulties. AH helps embryos hatch by disrupting the ZP, with methods including mechanical hatching, chemical hatching and laser‐assisted hatching (LAH) [ 3 , 4 , 5 ]. Currently, LAH is most widely used owing to its short exposure time, precise localisation and high reproducibility. Nevertheless, ZP ablation temperature can reach 130°C–160°C [ 6 ], and the heat dissipated into the surrounding medium may adversely affect the quality and developmental potential of the embryo [ 6 ]. A previous study reported reduced blastocyst total cell counts in six‐to‐eight‐cell‐stage laser‐hatched embryos, potentially impacting the implantation and developmental potential of the embryos [ 7 ]. Although several studies have indicated the absence of significant deleterious effects of LAH on neonatal prognosis [ 8 , 9 ], concerns remain regarding the long‐term adverse health consequences for offspring conceived using this technique. A retrospective study using a phone questionnaire to assess child development in the first year of life suggested that LAH does not increase the risk of congenital malformations or developmental delay in singletons [ 10 ]. However, only limited indicators can be observed in infancy; therefore, it is necessary to follow‐up children conceived through LAH until they reach an age that allows a more definitive developmental assessment. The present study aimed to examine the impact of LAH on the physical, metabolic, cognitive and behavioural profiles of singletons conceived through frozen–thawed embryo transfer (FET) at the preschool age.

The authors declare no conflicts of interest.

Table S1. Table S2.