Introduction

The use of assisted reproductive technology (ART), including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), has increased by over 20% over the past 10 years [1]. Although the vast majority of offspring conceived through IVF or ICSI are in good health, prior studies have indicated that ART is associated with an increased risk of birth defects [2,3,4], such as dysfunction of the cardiovascular, metabolic, and neurological systems [5], pediatric neoplasms (e.g., leukemia and Hodgkin's lymphoma) [6], and epigenetic diseases [7]. Additionally, the use of ICSI further increases this risk [3]. Moreover, numerous research findings suggest that frozen embryo transfer cycles may increase the risk of having a large-for-gestational-age baby and the risk of higher birth weight than the fresh IVF/ICSI strategy [8,9,10,11,12,13]. According to the "early origins hypothesis," popularized by Barker [14], the environment experienced during fetal development plays a crucial role in influencing the long-term health outcomes of an individual.

The term "epigenetics" is used to describe the information and mechanisms that exist beyond the DNA sequence itself. Epigenetic regulation plays a pivotal role in genome function. Epigenetic marks encompass a range of mechanisms, including modifications to the chromatin structure (such as acetylation, methylation, and ubiquitination of histone proteins), the presence of non-coding RNA molecules (including long non-coding RNAs, microRNAs, and PIWI-interacting RNAs), and direct modifications to the DNA itself (such as DNA methylation and hydroxymethylation). The field of epigenetics has emerged as a pivotal area of investigation in the context of chronic diseases including cancer, obesity, diabetes, and neurodegenerative disorders [15]. Genomic imprinting is an epigenetic process in which one allele of a gene is selectively silenced in accordance with the parent of origin. The genes that undergo this process are designated as imprinted genes. Most imprinted genes are located in clusters within the genome and are regulated by epigenetic modifications in their imprinted control regions. These epigenetic markers serve to determine which allele is expressed. Disruptions in the imprinting of these genes have been linked to the development of genetic disorders and developmental abnormalities [16]. In addition, epigenetic modifications are sensitive to environmental factors and extensive epigenetic reprogramming events occur during gametogenesis and early embryonic development [17].

One viewpoint is that laboratory procedures can influence the epigenome of gametes and embryos. This suspicion is correlated with the observation that ART procedures occur precisely during the period characterized by significant changes in the organization of the epigenome [18]. Moreover, previous studies on animal models suggested an increased incidence of imprinting errors in offspring conceived through ART compared to natural conception, due to in vitro culture conditions, gamete or embryo manipulation, ovulation induction, or hormonal stimulation [19, 20]. However, reports on the effects of IVF on imprinted genes have been heterogeneous [21], and the question of whether this increased risk is associated with specific procedures utilized in ART or whether it is related to the inherent biological factors associated with infertility remains unresolved.

To gain a more nuanced understanding of the influence of ART on the epigenetic landscape of human development, we conducted a study involving fetal tissue obtained following multifetal pregnancy reduction (MFPR) conceived using different modes of ART. The primary objective was to examine alterations in DNA methylation patterns occurring within the promoter region and the imprinting control element (ICE) of the H19 gene, given its involvement in fetal growth and development.

Material and methods

Study samples

Written informed consent was obtained from all participating pregnant women following multifetal pregnancy reduction conceived by ART. The study was reviewed and approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University for 30 participants (Ethical number: ZDSFY-2017-YLS-70) and was conducted in accordance with the guidelines of the Committee. This study included 29 samples from 29 women with multifetal pregnancies who underwent MFPR between January 2018 and October 2019 at the Reproductive Center of the Third Affiliated Hospital of Zhengzhou University. One sample was excluded from the study because it was not an IVF cycle. These samples were divided into four groups according to ART type (IVF or ICSI) and embryo status (fresh or thawed). Specifically, 9 samples were obtained by conventional IVF from fresh cycles (c-IVF group), 4 by ICSI from fresh cycles (ICSI group), 10 by conventional IVF from thawed cycles (cryo-IVF), and 6 by ICSI from thawed cycles (cryo-ICSI). All pregnancies were reduced to singleton or twin pregnancies at approximately seven weeks of gestation by ultrasound-guided transvaginal mechanical rupture or injection of KCl solution. After multifetal pregnancy reduction, fetal tissues were stored at -196 °C in liquid nitrogen until further analysis. Although it is feasible to procure two reduction fetal tissues from a pregnant woman, the reduction fetal tissue is subjected to testing as a single sample. This approach guarantees that the final analysis comprises one sample per pregnant woman.

CpG islands prediction and next-generation sequencing-based bisulfite sequencing PCR (BSP)

Five CpG islands were identified in the promoter region of the H19 gene using a web-based application called the CpG Island Predictor Analysis Platform (CpGPAP). Among these CpG islands, three (H19-1, H19-2, and H19-3) within the promoter region were selected for more detailed quantitative analysis of methylation-related changes in conjunction with the ICE of the H19 gene. The promoter region's H19-1 was chosen as the primary outcome of our study, while the H19-2 and H19-3 regions of the promoter region, as well as the ICE of the H19 gene, were selected as secondary outcomes for investigation.

Gene-specific DNA methylation was analyzed using the next-generation BSP method following a previously published protocol [22,23,24,25]. Briefly, BSP primers were designed using online MethPrimer software and are listed in Table 1. One microgram of genomic DNA from fetal tissue was converted using the ZYMO EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA), and one-twentieth of the eluted products were used as templates for PCR amplification with 35 cycles using the KAPA 2G Robust HotStart PCR Kit (Kapa Biosystems, Wilmington, MA, USA). For each fetal tissue sample, BSP products from multiple genes were pooled equally, 5'-phosphorylated, 3'-dA-tailed, and ligated to the barcoded adapter using T4 DNA ligase (NEB). Barcoded libraries from all fetal tissue samples were sequenced on an Illumina platform.

Table 1 Primer design for bisulfite sequencing PCR
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For each fetal tissue sample, the bisulfite sequencing reads underwent initial processing to remove adapters and low-quality reads using Trimmomatic-0.36 software. After filtering out low-quality reads and removing adapter sequences, the clean sequencing reads were aligned to the target sequences using the Bsmap software (v2.73) with default parameters. Bsmap uses genome hashing and bitwise masking techniques to achieve accurate and efficient bisulfite mapping. Methylation levels were determined as the fraction of reading counts of 'C' in the total read counts of both 'C' and 'T' for each covered C site. Methylated cytosine was identified using a binomial distribution, following the method described by Lister et al. [26]. This involved calculating the probability mass function for each methylation context (CpG, CHH, and CHG). Only CG sites covered by at least 200 reads in at least one sample were included in further analysis.

Statistical analyses

Initially, continuous variables, including maternal age and gestational age, as well as the distribution of methylation at four objective CpG islands, were analyzed using either one-way analysis of variance (for variables with a normal distribution) or the Kruskal–Wallis rank-sum test (for variables with a non-normal distribution). Categorical variables, such as the time of embryo transfer, were analyzed using the chi-squared test. Furthermore, the cases were stratified by each covariate (Fig. 2) to gain insight into the potential threshold effects and curvilinear relationships. To assess the relationship between gene-specific DNA methylation and ART, while controlling for potential confounding variables, we used linear regression models to derive adjusted beta (β) and 95% confidence intervals (CI). This approach was designed to exclude the potential effects of covariation. All statistical analyses were conducted using the R software (http://www.R-project.org) and the Empower Stats software (www.empowerstats.com, X&Y Solutions, Inc., Boston, MA). All tests were two-sided, and a p-value of less than 0.05 was considered statistically significant. Potential confounding variables were selected based on the established knowledge and factors that could potentially influence DNA methylation.

Results

The methylation levels of 29 fetal tissue samples after MFPR from various modes of ART conception were measured using next-generation sequencing-based BSP. Subsequently, we compared the DNA methylation levels of three CpG islands in the H19 promoter and ICE of imprinted genes in differentially methylated regions (DMRs) associated with the H19 gene. The primer design for the BSP is described in Table 1. The fundamental attributes of the covariates are presented in Table 2. The methylation levels of the three CpG islands in the H19 promoter and ICE in the DMRs of H19 are shown in Table 3. The outcome measures demonstrated notable disparities between the four groups for the primary objective H19-1 and secondary objective H19-2 CpG islands, with statistical significance indicated by p-values of 0.037 for both.

Table 2 Baseline characteristics of fetal tissue obtained from c-IVF/ICSI/cryo-IVF/cryo-ICSI conceived pregnancies
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Table 3 Methylation levels of the three CpG islands in the H19 promoter and ICE within the DMRs of H19
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Univariate regression analysis revealed that, in contrast to the c-IVF group, fetal tissues obtained through ICSI therapy (β = -2.634, 95% CI = -4.605 to -0.664, p = 0.0147) or cryo-ICSI (β = -2.176, 95% CI = -3.904 to -0.448, p = 0.0208) exhibited hypomethylation at the primary outcome H19-1 CpG sites. Furthermore, significantly lower methylation levels were observed at H19-2 CpG sites in the cryo-IVF group (β = -2.040, 95% CI = -3.741 to -0.340, p = 0.0269) and cryo-ICSI groups (β = -2.583, 95% CI = -4.534 to -0.633, p = 0.0156) than in the c-IVF group (Fig. 1a). Additionally, the methylation values of H19-2 CpG sites significantly increased in the middle (2.106%) and high (1.935%) maternal age trisections compared to those in the low trisection (95% CI = 0.315–3.897, p = 0.0294; 95% CI = 0.106–3.763, p = 0.0481, respectively) (Fig. 1b). The methylation of the primary outcome H19-1 CpG sites in the high tertile gestational age group also improved by 2.579% compared to that in the low tertile group (95% CI = 0.341 to 4.816, p = 0.0325) (Fig. 1c).

Fig. 1
figure 1

Crude association of methylation levels within the analyzed regions with basal characteristics. a Comparative methylation of ICSI, cryo-IVF, and cryo-ICSI compared to the c-IVF group. The horizontal dashed line depicts methylation levels of the corresponding regions in the c-IVF group. The midpoints of the three vertically colored line segments signify alterations in the average methylation level of the respective region in the group compared to the c-IVF group, that is, the linear regression coefficient β value. b Impact of maternal age on methylation patterns in specific regions. The horizontal dotted line represents the average methylation level in the low-tertile maternal age group. The midpoints of the two vertically colored line segments represent alterations in the average methylation levels observed in the middle tertile and high tertile groups relative to the low tertile group. c Impact of gestational age on methylation patterns in specific regions. The horizontal dotted line represents the average methylation level in the low tertile gestational age group. The midpoints of the two vertically colored line segments indicate the changes in average methylation levels observed in the middle tertile and high tertile gestational age groups compared with those in the low tertile group. d Impact of different transfer days on methylation patterns in specific regions of the samples. The horizontal dashed line represents the average methylation level in the embryos transferred on D3. The midpoint of the vertical red line segment represents the change in the average methylation level observed in the group of embryos transferred on D5 compared to that in the D3 group. The midpoints of these vertically colored line segments represent the linear regression coefficient β value, which quantifies the magnitude and direction of the methylation changes. The upward position of the midpoint relative to the dashed line signifies an increase in the methylation level in that group, indicating a positive value of β. Conversely, a negative value of β indicates a decrease. The length of each line segment represents the 95% confidence interval of the corresponding β values. D3, day 3; D5, day 5

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As depicted in Fig. 2, upon stratification by maternal age, it was evident that the methylation levels in the H19-2 CpG sites of thawed embryos (cryo-IVF and cryo-ICSI) decreased by 3.627% and 3.557%, respectively, in the middle tertile of maternal age (95% CI = -5.734 to -1.521, p = 0.0118; 95% CI = -5.663 to -1.450, p = 0.0130, respectively) (Fig. 2a). When stratified by gestational age, significantly decreased methylation values of the primary outcome H19-1 CpG sites were observed in the ICSI, cryo-IVF, and cryo-ICSI groups in the middle tertile (β = -4.874, 95% CI = -7.545 to -2.203, p = 0.0050; β = -2.229, 95% CI = -3.832 to -0.627, p = 0.0213; β = -2.948, 95% CI = -4.638 to -1.259, p = 0.0065, respectively) (Fig. 2b). In the high tertile of gestational age, both cryo-IVF and cryo-ICSI groups showed significant reductions in methylation levels at the primary outcome H19-1 specific CpG sites of the H19 promoter (β = -2.830, 95% CI = -4.947 to -0.713, p = 0.0306 and β = -3.275, 95% CI = -5.920 to -0.629, p = 0.0415, respectively). The embryo cryopreservation group (including cryo-IVF and cryo-ICSI) also exhibited decreased methylation levels at two other second-outcome CpG sites (H19-2 and H19-3) in the H19 promoter compared to the c-IVF group (p < 0.05 for each CpG site), and the ICSI group exhibited decreased methylation of H19-3 CpG sites (β = -3.381, 95% CI = -5.804 to -0.957, p = 0.0257). Moreover, the cryo-IVF group demonstrated a decreased methylation level at the ICE CpG sites compared to the c-IVF group (p = 0.0075) in the high tertile gestational age group (Fig. 2c). Additionally, our findings indicated that when stratified by the timing of embryo transfer, the H19-1 CpG sites of the ICSI groups (ICSI and cryo-ICSI groups) were hypomethylated on day 3 (β = -2.819, 95% CI = -5.234 to -0.404, p = 0.0361; β = -2.461, 95% CI = -4.495 to -0.428, p = 0.0305, respectively), and the methylation level at the CpG sites of ICE was lower in the cryo-IVF group than in the c-IVF group (β = -3.793, 95% CI = -6.258 to -1.329, p = 0.0082) (Fig. 2d). (Some trisections of covariates did not display due to small samples of no more than 10 cases.)

Fig. 2
figure 2

Association between the methylation levels within the analyzed regions and modes of ART conception stratified by each covariate. a Comparative methylation compared to c-IVF in the middle tertile maternal age subgroup (n = 11). b Comparative methylation compared to c-IVF in the middle tertile gestational age subgroup (n = 14). c Comparative methylation compared to c-IVF in the high tertile gestational age subgroup (n = 12). d Comparative methylation compared to c-IVF from the Day 3 embryo subgroup (n = 20). The horizontal dashed lines represent average methylation levels in the corresponding regions in the c-IVF group. The purple segments represent the 95% confidence intervals of the linear regression coefficient β values for the ICSI group. The green segments represent 95% confidence intervals of the linear regression coefficient β values for the cryo-IVF group. The yellow segments represent 95% confidence intervals of the linear regression coefficient β values for the cryo-ICSI group. The midpoints of these three line segments represent the changes in the average methylation levels relative to those in the c-IVF group, which are represented by the linear regression coefficient β values. Significant differences between groups are indicated by p-values in the corresponding regions of the segments. Some subgroups of covariates are not displayed because of the small sample size of no more than 10 cases

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Prior to adjustment for maternal age, gestational age, and time of embryo transfer, the primary outcome methylation level of H19-1 CpG sites was found to be decreased by 2.634% and 2.176% in the ICSI (95% CI = -4.605 to -0.664, p = 0.0147) and cryo-ICSI (95% CI = -3.904 to -0.448, p = 0.0208) groups, respectively, in comparison to the c-IVF group. The second outcome, methylation of H19-2 CpG, decreased by 2.040% and 2.583% in the cryo-IVF (95% CI = -3.741 to -0.340, p = 0.0269) and cryo-ICSI (95% CI = -4.534 to -0.633, p = 0.0156) groups, respectively. Following adjustment, the DNA methylation level of H19-1 CpG sites declined in the ICSI group (β = -3.189, 95% CI = -5.034 to -1.345, p = 0.0026). The results also indicated that the cryo-IVF and cryo-ICSI groups exhibited significantly lower DNA methylation levels than the c-IVF group (β = -2.150, 95% CI = -3.706 to -0.593, p = 0.0129; β = -2.238, 95% CI = -3.817 to -0.659, p = 0.0110, respectively). Furthermore, hypomethylation of H19-2 CpG sites was observed in the cryo-IVF (β = -2.132, 95% CI = -4.071 to -0.192, p = 0.0425) and cryo-ICSI groups (β = -2.598, 95% CI = -4.566 to -0.630, p = 0.0168). Although no significant differences were observed in the methylation levels of the H19-3 CpG sites among the four groups, a reduction in methylation at the ICE CpG sites was observed in the cryo-IVF group (β = -2.697, 95% CI = -5.231 to -0.163, p = 0.0488) compared to that in the c-IVF group (Fig. 3).

Fig. 3
figure 3

Multivariate regression model for the association between methylation levels of the analyzed regions and ART conception mode. The horizontal dashed lines represent the average methylation levels in the corresponding regions in the c-IVF group. The midpoints of the colored line segments in the figure represent the β values of the corresponding analysis group. The distance of the midpoints from the dotted line reflects the change in the methylation level of the region compared to the c-IVF group. The corresponding line segment represents the 95% confidence interval of the β value. (a p = 0.0026, b p = 0.0129, c p = 0.0110, d p = 0.0147, e p = 0.0208, f p = 0.0425, g p = 0.0168, h p = 0.0269, i p = 0.0156, j p = 0.0488)

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Discussion

During the process of embryonic development, the phenomenon of imprinting is initially erased in primary germ cells (PGCs). It is subsequently re-established during gametogenesis and is maintained after fertilization and throughout the subsequent stages of development. During the preimplantation period, embryos undergo genome-wide demethylation and remethylation, whereas the differentially methylated regions (DMRs) of the imprinted genes remain intact. Some environmental conditions, including ovarian stimulation, IVF, embryo culture, embryo transfer, and embryo cryopreservation, have the potential to induce alterations in genomic imprinting regions, which can subsequently affect neonatal development during the period of methylation change [16]. The H19 gene is part of an imprinted cluster located downstream of IGF2 on the human chromosome 11p15. It encodes a 2.3 kb untranslated RNA that is highly expressed during embryogenesis [27]. Transcription of the maternal allele of the H19 gene is dependent on an ICE located two kilobases upstream of H19. This element undergoes methylation and is required for the silencing of H19 on the paternal chromosome, involving the transcriptional co-repressor CCCTC-binding factor (CTCF) [28]. The imprinted gene H19 is essential for the early stages of embryogenesis, implantation, growth and development, and gametogenesis. Following birth in mammals, H19 may play a pivotal regulatory role in many physiological and pathological processes, including hormone synthesis, reproductive system tumors, and skeletal muscle development [29]. Moreover, a study by Murphy SK indicates that the methylation patterns at the H19 DMRs remain relatively stable in different tissue types during early human development [30] This stability in methylation patterns suggests that H19 DMRs may serve as a target for detecting changes in methylation.

Previous research has frequently utilized preimplantation embryos, placental and cord blood samples, as well as blood cells and epithelial cells from neonates, to investigate the methylation status of specific genomic loci. However, the current study was uniquely designed to employ embryonic tissue from approximately 7-week-old embryos. This allowed for direct and objective observation of methylation changes at specific loci in embryos at around 7 weeks of gestational age, following embryo transfer. This study provides new insights into the dynamics of embryonic methylation patterns in vivo.

Table 3 shows the considerable differences in methylation levels at the H19-1 and H19-2 loci for the primary and secondary outcomes, respectively, among the four study groups. These differences were further supported by Fig. 1, which shows the correlation between methylation levels and basic traits. Furthermore, Fig. 3 presents a more robust analysis employing a multivariate regression model, thereby providing stronger evidence of observed differences. The study findings suggest that H19-1, the primary outcome, exhibits hypomethylation in fetal tissue derived from ICSI, regardless of whether the ICSI procedure was performed using fresh or freeze-thawed sperm, compared with the c-IVF (control in vitro fertilization) group. These findings suggest that while in vitro manipulation techniques, such as ICSI, have improved the success rates and outcomes of ART for couples facing fertility challenges, there may be potential adverse effects associated with these techniques on the epigenetic modifications of embryos.

Reduced H19 methylation is detrimental to embryonic development, as it can lead to the loss of imprinting, biallelic H19 expression, and disruption of normal embryonic growth. Hypomethylation is also associated with increased H19 expression, which negatively affects the expression of other important imprinted genes such as IGF2. Aberrant H19 methylation and expression may impair cellular differentiation during embryogenesis and indicate broader epigenetic instability, contributing to developmental abnormalities. Meta-analytic data showed lower H19 DMR methylation levels and a 9.91-fold higher risk of DMR aberration in infertile male patients than in controls, and lower H19 methylation was also associated with higher rates of recurrent miscarriage [31]. In addition, loss of H19 imprinting is associated with pathological pregnancies and complications such as spontaneous abortion, preeclampsia, eclampsia, and fetal growth restriction, likely due to abnormal trophoblast function. This loss of imprinting may be related to DNA methylation levels at the H19 promoter. Maintaining the correct H19 methylation pattern is critical for ensuring normal embryonic growth and differentiation.

This result is consistent with the findings reported by Loke et al., who indicated weak evidence of a relationship between ICSI and DNA methylation within the IGF2/H19 regulatory region. These findings suggest that processes associated with IVF/ICSI may contribute to the observed methylation differences [32]. In IVF, the sperm fuses with the oocyte, just as it does in vivo, bypassing certain steps that occur during ICSI. During ICSI, sperm chromatin enters the ooplasm along with the acrosome, cell membrane, and perinuclear material. These components do not enter the oocyte during normal fertilization and eventually disintegrate inside the oocyte. Anna Ajduk and Yasuhiro Yamauchi demonstrated that the dynamics of sperm chromatin remodeling differ between ICSI and IVF embryos. Chromatin remodeling in ICSI is more asynchronous compared than in IVF. Additionally, when sperm capacitation occurs before injection, remodeling asynchrony is enhanced, leading to delayed pronuclei formation and DNA synthesis. This interference with sperm chromatin remodeling may have consequences [33]. The asynchronization of chromatin remodeling in ICSI may potentially disrupt the normal epigenetic reprogramming that occurs during fertilization, resulting in altered DNA methylation patterns in subsequent embryos.

In 2009, Katari et al. investigated DNA methylation and gene expression in placental and cord blood samples from children conceived either through IVF or naturally. The researchers discovered that IVF conception was associated with lower methylation levels in the placenta and higher methylation levels in the cord blood at specific sites. These differences in methylation were also correlated with alterations in gene expression, affecting both imprinted and non-imprinted genes [34]. In another study conducted by Rancourt et al., researchers compared the methylation levels in the DMRs of imprinted genes. The results of this study indicated that there were no functionally relevant differences in methylation levels across five out of six imprinted DMRs in either the placenta or cord blood of infants conceived through ovulation induction or IVF when compared to infants conceived naturally. However, when examining specific DMRs, namely, KCNQ1, SNRPN, and H19, small but statistically significant differences in methylation levels were observed based on the method of conception. In cases of ovulation induction, the methylation level of H19 was 40.2% compared with 44.6% in spontaneous conceptions. Similarly, in IVF cases, the methylation level of H19 was 43.4% compared to 44.7% in spontaneous conceptions. Furthermore, the expression levels of genes associated with these control regions were correlated with the methylation levels of H19 [35]. These findings suggest that there may be epigenetic differences in the gametes or early embryos of couples undergoing infertility treatment or that assisted reproduction techniques themselves could impact DNA methylation and gene expression patterns. These differences or changes could potentially have long-term effects on gene expression [34]. However, these studies did not discuss the impact of IVF and ICSI on the methylation of H19 DMRs. In 2017, a study by Castillo-Fernandez et al. in Genome Medicine comparing naturally and IVF-conceived newborns showed notable changes in specific differentially methylated regions (DMRs) in the whole blood of IVF-conceived offspring. This study also confirmed previous findings of different methylation patterns in the H19/IGF2 region in cord blood mononuclear cells related to IVF. Researchers further suggest that factors such as ICSI or paternal infertility may contribute to these methylation changes. However, this study acknowledges its inability to definitively determine whether the observed methylation changes were primarily caused by IVF procedures, parental subfertility, or other factors [36].

It is worth noting that not all studies have found significant differences in DNA methylation patterns between ICSI and naturally fertilized embryos. For example, a study published by Zheng et al. in the journal Human Reproduction in 2013 investigated the methylation status of H19 and other imprinted genes in human spontaneous abortions (miscarriages) following ART and natural conception. The findings of this study revealed that the modes of conception (natural versus ART) and the specific fertilization methods (IVF and ICSI) used in ART do not have a substantial impact on the methylation patterns of imprinted genes. However, in this study, the researchers examined 62 chorionic villus samples and 13 muscle samples from cases of spontaneous abortion/stillbirth, as well as 73 chorionic villus samples from fetal reduction by transvaginal ultrasound after ART, to analyze the methylation status of 6 CpGs within the H19 gene [37]. A recent study published by Penova-Veselinovic et al. in Human Reproduction showed that in comparison to naturally conceived adolescents, there were no significant differences in the DNA methylation patterns in the whole blood of adolescents born through ART, such as IVF or ICSI. However, it is important to note that the study did not specifically investigate how ART procedures affect early embryo development or their potential impact on DNA methylation during this stage [38].

Based on our study, we inferred that artificial manipulation of the fertilization process during ICSI, as well as parental subfertility, could disrupt the normal epigenetic reprogramming that takes place during fertilization. This epigenetic instability can result in alterations in DNA methylation patterns that persist throughout embryonic development. If the aforementioned study fails to demonstrate the impact of conceptional treatment on H19 methylation, a subsequent study will unequivocally establish the influence of conceptional treatment on H19 methylation.

The study found that prior to accounting for factors such as maternal age, pregnancy age, and embryo transfer days, the methylation levels at the second outcome H19-2 site were lower in both the cryo-IVF and cryo-ICSI groups than in the c-IVF group. Further analysis showed consistent reductions in methylation levels at H19-2 CpG sites in thawed embryos from the cryo-IVF and cryo-ICSI groups within the middle tertile of the maternal age. The study also revealed lower methylation levels at three CpG sites (H19-1, H19-2, and H19-3) in the H19 gene of cryopreserved embryos in the high tertile of gestational age. The multivariate regression model provided stronger support for these observed differences after adjusting for various factors. This clearly indicated that the methylation levels of specific CpG sites in H19 were altered during the cryopreservation process. During cryopreservation, embryos are typically exposed to cryoprotectant solutions and rapidly cooled to prevent the formation of ice crystals. This process is performed to safeguard the delicate structures of the embryo; however, it can also lead to alterations in the methylation of the H19 gene. A study published by Cheng in the journal Fertility and Sterility in 2014 examined the potential impact of oocyte vitrification on DNA methylation in DMRs of three imprinted genes (H19, Peg3, and Snrpn) in mouse blastocysts generated through in vitro fertilization. Researchers have discovered that oocyte cryopreservation in mice is associated with a potential loss of DNA methylation of imprinted genes, including H19, Peg3, and Snrpn, in blastocysts [39].

In this study, it was established that the risks associated with ICSI and cryopreservation cycles surpass those associated with fresh IVF, yet the majority of these children generally exhibit standard physiological and developmental features. One conceivable explanation for the observed methylation differences in these children could be that embryonic and fetal development involves intricate, multifaceted processes that possess innate mechanisms for self-regulation and adaptation. Such mechanisms might enable the organism to actively rectify or compensate for specific epigenetic modifications such as methylation changes detected during prenatal development. Another potential cause is that it is crucial to consider the potential for epigenetic dysregulation to have latent or delayed consequences, which could lead to unrecognized long-term health issues in these children.

In our study of the association between maternal age and methylation at the CpG sites of H19-2, we discovered that the middle and high tertile groups had higher fetal methylation levels than the low tertile group. This suggests that other confounding factors such as blood glucose levels may contribute to increased methylation levels as maternal age increases. For instance, maternal glycemic status has been demonstrated to affect the methylation profile of the ADIPOQ gene in placental tissue [40], and maternal metabolic disorders such as gestational diabetes mellitus (GDM) have been associated with methylation levels at specific gene sites in the placenta [41]. Thus, it is conceivable that in the high tertile age group and middle tertile group, elevated blood glucose levels or other confounding factors interfered with the assessment of H19 gene methylation.

Despite the efforts made in this study, several limitations have been encountered. The small sample size of 29 cases resulted in the reduced statistical power of the test. Therefore, it is necessary to increase the sample size and conduct a more in-depth investigation for more comprehensive exploration of methylation alterations. Moreover, the absence of a naturally conceived control group, in which fertilization occurred naturally without superovulation, was another limitation. Stimulation can potentially affect embryonic development and the methylation of H19. This study did not include these investigations, making it an essential factor to consider when interpreting the effects of fertility treatments on epigenetic markers and embryonic development. Furthermore, the consequences of embryo cryopreservation and ICSI on H19 gene methylation in fetal tissue can vary depending on the method employed and the region analyzed. Lastly, the study only examined DNA methylation of specific CpG sites within the H19 gene and did not provide any information on the expression of the H19 gene nor did it analyze allele-specific gene expression. The underlying mechanisms connecting the observed methylation changes with the potential functional outcomes remain unknown. Future research should adopt a more comprehensive approach by integrating methylation analysis with complementary assessments of gene expression and other relevant molecular endpoints.

In conclusion, our findings provide further evidence of reduced methylation levels at CpG sites of the H19 gene in fetal tissues following MFPR achieved by ART, such as embryo cryopreservation and ICSI. This may be associated with an increased risk for birth defects. It is crucial to consider the potential benefits and risks of assisted reproductive technology (ART) in achieving pregnancy in couples, considering their individual circumstances. It is recommended that patients consult with healthcare professionals and genetic counselors for personalized guidance based on individual circumstances and medical history.