Since the birth of the first in vitro fertilization (IVF)-conceived child in 1978 (1), assisted reproductive technology (ART) has enabled the conception of over 10 million children worldwide (2). However, substantial research has raised concerns about the health outcomes of ART-conceived offspring, highlighting risks such as cardiovascular, musculoskeletal, chromosomal defects, urogenital diseases, and cancers (3–5).

More recent studies have suggested potential links between specific ART procedures, like intracytoplasmic sperm injection (ICSI), and neurodevelopmental disorders, bringing into question the safety of these techniques (6). Globally, the number of children born following ART with frozen- thawed embryo transfer (FET) has surpassed those born through fresh embryo transfer in many regions (7, 8). This trend is further driven by the increasing adoption of extended embryo culture, particularly under single embryo transfer policies (9). However, both FET and extended embryo culture may induce epigenetic changes, influenced by variables such as temperature, gas concentration, and pH fluctuations during the procedures. These changes could have significant implications for child health, particularly concerning increased birth weights and a heightened risk of large-for-gestational-age (LGA) outcomes after frozen embryo transfers (10–14). Consequently, it is essential to verify the long-term health outcomes for children conceived through FET or extended embryo culture.

In response to these concerns, this study seeks to investigate the impact of different embryo transfer strategies on the health outcomes of children conceived through IVF. Utilizing a comprehensive dataset that includes various patient demographics and medical variables, the study aims to clarify potential associations between embryo transfer techniques (including FET and extended embryo culture) and singleton health outcomes up to early childhood (2–5 years of age) in Taiwan. The findings will provide essential insights into the implications of ART procedures on child health, informing both clinical practice and policy decisions surrounding IVF and ART.

This retrospective cohort study was conducted in Taiwan and approved by the institutional review board of Kaohsiung Medical University Chung-Ho Memorial Hospital, IRB-No. KMUHIRB-E(I)-20210222, which waived the requirement for informed consent because the data were encrypted and deidentified.

More than 99% of the citizens of Taiwan have participated in the National Health Insurance program since 1995, and the national population registry data set is linked to the national ART and birth certification data set. Couples who entered the IVF treatment in Taiwan have been completely recorded in the Taiwan national ART database. The ART database in Taiwan was established in the year 1998. It collects case data of individuals who undergo assisted reproduction procedures at the respective reproductive institutions, excluding assisted insemination between spouses. Medical information in the national population registry data set is recorded at the time of visit to outpatient public or private clinics. The databases have undergone de-identification processes, including the removal of directly identifiable fields such as names and addresses. Sensitive fields such as identification numbers, institution codes, insurance policy unit codes, tax identification numbers, dates of birth, medical dates, and admission dates have been masked to comply with the strong data protection standards of FIPS 140–2 Level 3 international security standards. The related data can only be used within the independent operating area set up by the authority, and any disclosed statistical results are carefully reviewed to ensure that there is no possibility of identifying specific individuals through the data application or disclosure methods.

Table 1 presents the Characteristics of the Study Population. The final cohort study included 10,803 eligible singletons born through ART embryo transfer and 894,615 through natural conception.

Before applying Inverse Probability of Treatment Weighting (IPTW), significant differences were observed between the groups. In both fresh and frozen embryo transfer scenarios, the blastocyst groups had a higher proportion of younger mothers and fathers, while the cleavage stage groups had a higher percentage of older individuals. Among women under 35 years old, natural conception had the highest proportion, followed by the blastocyst stage, and then the cleavage stage (50.15% in fresh blastocyst vs. 35.9% in fresh cleavage stage; 45.82% in frozen blastocyst vs. 35.91% in frozen cleavage stage; 71.42% in natural conception). Conversely, for women aged 40 and above, the cleavage stage had the highest proportion, followed by the blastocyst stage, and then natural conception (13.7% in fresh cleavage stage vs. 6.49% in fresh blastocyst stage; 14.69% in frozen cleavage stage vs. 9.48% in frozen blastocyst stage, 3.66% in natural conception). The same pattern was observed for paternal age: fathers under 40 years old were most prevalent in natural conception, followed by the blastocyst stage, and then the cleavage stage (76.94% in fresh blastocyst vs. 72.23% in fresh cleavage stage; 77.49% in frozen blastocyst vs. 71.63% in frozen cleavage stage, 87.79% in natural conception).

In terms of pregnancy risk factors, pregnancy-induced hypertension was more common in the frozen groups (3.99% in frozen blastocyst, 4.87% in frozen cleavage stage) compared to the fresh groups (2.69% in fresh blastocyst, 3.9% in fresh cleavage stage) and natural conception (1.30%). Gestational diabetes mellitus was more prevalent in the cleavage stage groups (6.08% in fresh cleavage vs. 4.78% in fresh blastocyst; 6.01% in frozen cleavage vs. 4.66% in frozen blastocyst, 2.00% in natural conception). Additionally, gestational complications, such as placenta previa, placenta abruption, and preterm premature rupture of membranes (PPROM), were more frequent in ART groups compared to natural conception.

After implementing IPTW, significant disparities in maternal age and pregnancy risk factors were no longer observed. However, paternal age remained younger in the natural conception group. The distribution of individuals across different groups after IPTW adjustment was as follows: 3,125 for fresh cleavage stage, 1,332 for fresh blastocyst, 1,465 for frozen cleavage stage, 4,708 for frozen blastocyst, and 878,643 for natural conception. The Standardized Mean Differences (SMD) for these variables indicated that balance in maternal age and pregnancy risk factor was achieved after IPTW adjustment.

Table 2 presents the perinatal outcomes and incidence rates of various major and minor diseases in children conceived via different ART methods and natural conception. The table compares fresh cleavage stage transfer (Day 2-3), frozen cleavage stage transfer (Day 5-6), fresh blastocyst transfer (Day 5-6), frozen blastocyst transfer (Day 5-6), and natural conception. Each disease is listed with its corresponding ICD-10 and ICD-9 codes, the number of cases (N), and the percentage (%).

Table 3 compare four different embryo transfer methods and stages (fresh blastocyst, frozen blastocyst, frozen cleavage, and fresh cleavage) to natural conception, while Table 4 compare these four methods against each other. Odds ratios (ORs) are used to assess perinatal outcomes, while hazard ratios (HRs) indicate differences in disease incidence rates. A p-value of < 0.005 is considered statistically significant. When compared to natural conception ( Table 3 ), the use of ART is associated with an increased risk for preterm labor, low birth weight, and several major and minor diseases. However, no significant differences were observed for orofacial cleft, croup, and jaundice. Notably, the HR for attention-deficit/hyperactivity disorder (ADHD) is markedly elevated in ART-conceived children, particularly in the fresh blastocyst group, which demonstrated the highest HR (HR 19.49, 95% CI 6.71-56.61, p<0.0001) relative to natural conception. Conversely, the incidence of genitourinary conditions, such as urinary tract infections and pyelonephritis, is lower in the ART groups compared to those conceived naturally. In this study, melanoma, malignant neoplasms of the skin, and leukemia were rare outcomes of interest. Across all groups, the number of cases for these conditions was zero, except for a single leukemia case observed in the frozen blastocyst group. Due to the absence of events in most groups, it was not possible to calculate hazard ratios (HRs) using Cox proportional hazards regression.

Comparing the four different embryo transfer methods and stages ( Table 4 ), large for gestational age (LGA) outcomes were more likely in frozen embryo transfers compared to fresh transfers, with ORs of 1.669 for frozen cleavage stage versus fresh cleavage stage and 1.678 for frozen blastocyst versus fresh blastocyst transfers. Frozen embryo transfers generally exhibit safety profiles similar to those of fresh embryo transfers, with blastocyst transfers showing comparable outcomes to cleavage stage transfers for major diseases. The only significant finding was an elevated HR for developmental delay (HR 2.212, 95% CI 1.29-3.8, p-value 0.0040) in children conceived via fresh blastocyst transfers compared to fresh cleavage stage transfers. In terms of minor diseases, frozen blastocyst transfers were associated with a higher incidence of atopic dermatitis (HR 1.57, 95% CI 1.20-2.07, p-value 0.0011) and croup (HR 1.92, 95% CI 1.32- 2.79, p-value 0.0007) compared to frozen cleavage stage transfers. Furthermore, when comparing frozen and fresh embryo transfers, frozen transfers were linked to a reduced rate of respiratory diseases (OR 0.84, 95% CI 0.75-0.95, p-value 0.0011). Overall, no significant differences were observed in the incidence of major or minor diseases across the different embryo transfer methods and stages.

This study contributes to the growing body of literature (16–21) and recent systematic reviews (22, 23) on health outcomes in children conceived through assisted reproductive technologies, highlighting both the benefits and potential risks associated with various embryo transfer methods. The “freeze-all” strategy has been linked to reduced odds of ovarian hyperstimulation syndrome, with pregnancy and neonatal outcomes comparable to conventional methods (14, 20, 24–26). Furthermore, extending in-vitro culture to the blastocyst stage has been found to increase live birth rates, especially in patients with a favorable prognosis (27–29). While these approaches have demonstrated some advantages, their benefit has recently been called into question. A major limitation in previous studies has been the inability to simultaneously assess both the developmental stage at embryo transfer and the effects of cryopreservation. Our aim is to explore whether embryo handling procedures, including cryopreservation and stage of development, impact children’s health outcomes.

Our findings on neonatal outcomes align with prior research, indicating that transferring frozen-thawed embryos is associated with an increased risk of large-for-gestational-age (LGA) infants and cesarean sections, compared to fresh embryo transfers (14, 24, 30–32). All ART methods were associated with higher odds of preterm labor compared to natural conception. The increased preterm risk in ART cycles may be due to synchronization issues between the endometrium and embryos, as well as hormonal influences and inflammatory effects from continuous ovarian stimulation (33). This heightened risk of preterm birth and low birth weight may have implications for the long-term health of ART- conceived children.

The potential long-term consequences of high birth weight and LGA extend beyond infancy, potentially leading to obesity, diabetes, and cardiovascular disease later in life (34, 35). Recent studies from Finland have shown that boys born via frozen embryo transfer (FET) were heavier, with higher BMI and increased odds of being overweight compared to those born through fresh embryo transfer (ET) (36). Nevertheless, a 2023 systematic review from Italy found no significant differences between frozen and fresh transfers in terms of congenital malformations, neurodevelopmental disorders, growth, or chronic diseases (37). Similarly, another 2023 systematic review and network meta- analysis from Greece found no difference in the risk of congenital anomalies or adverse perinatal outcomes between blastocyst and cleavage stage transfers (22). Our study corroborates these findings, concluding that frozen and fresh embryo transfers, whether at the cleavage stage or blastocyst stage, result in comparable health outcomes in children.

Comparing natural conception, our investigation revealed that ART is associated with an increased risk of several conditions, most notably ADHD, particularly among children conceived through fresh blastocyst transfers. The neurodevelopmental risks of ICSI have been previously documented (4, 6), with potential mechanisms involving oxidative stress and DNA damage in selected sperm, or stress induced by procedural factors such as temperature, gas concentration, and pH value. The etiology of ADHD involves abnormalities in brain structure and function, as well as genetic influences, though the precise mechanisms remain unclear (38). Preterm birth has been established as a significant risk factor for ADHD (39, 40), and our study corroborates the association between ART procedures and increased rates of preterm birth and low birth weight. These findings highlight the importance of optimal perinatal care and early childhood interventions for children conceived through ART. Similarly, developmental delay was observed elevated in ART, which may be linked to multiple pregnancies and infertility-related factors, such as advanced parental age, regardless of ART use (41). The significant finding of increased developmental delay in children conceived via fresh blastocyst transfers compared to fresh cleavage-stage transfers may be partly attributed to the limited sample size. Notably, this elevated HR was not observed in frozen embryo transfers. The underlying mechanisms remain unclear, warranting further research to validate and clarify these findings.

Most large observational studies report a similar risk of cancer in children born after ART compared to the general population. However, a Danish study with a mean follow-up of 11.3 years found an elevated risk of childhood cancer, particularly leukemia, associated with the use of frozen embryo transfer (FET) (42). Similarly, a Nordic study identified an increased risk of epithelial tumors and melanoma following ART, as well as a higher risk of leukemia after FET (5). Freezing procedures may affect the embryonic cytoskeleton, DNA integrity, and the miRNA transcriptome (43–45). In the present study, the number of children aged 2–5 years with skin neoplasms, melanoma, and leukemia was limited, and no increased hazard ratio was observed. The conflicting results may partly be attributed to the low number of events in these disease and studies.

In our study, ART-conceived children exhibited higher rates of both major and minor diseases compared to those conceived naturally. However, specific ART techniques, including frozen embryo transfer and extended embryo culture, seem to have a limited impact on these outcomes. This may be explained by the underlying effect of parental infertility. A 2023 study from Australia supports this hypothesis, noting that the additional risk of congenital abnormalities was reduced and no longer statistically significant when comparing ART-conceived children to naturally conceived children born to parents with a history of infertility (46). This suggests that the increased risks observed in ART-conceived children may be partly explained by underlying parental infertility rather than ART procedures themselves.

In summary, while ART-conceived children face elevated risks for certain health conditions, particularly preterm birth and ADHD, the choice between frozen and fresh transfers or cleavage stage and blastocyst stage transfers does not appear to substantially influence these outcomes. These findings reinforce the importance of optimizing ART protocols and perinatal care, while also acknowledging the role of parental infertility in shaping the health of ART- conceived children.

This study underscores notable differences in health outcomes between fresh and frozen embryo transfers and between ART and natural conception. Compared to natural conception, ART is associated with higher risks, particularly for preterm birth, ADHD, and developmental delay. However, while ART-related risks may partly stem from underlying parental infertility, the overall effects of procedures such as extended culture or cryopreservation on neonatal outcomes appear minimal. Notably, the use of frozen embryos or extended culture does not seem to amplify these risks. These insights are valuable for guiding patient counseling for individuals considering ART.