*3.2. Epigenetic Changes after ART Techniques Are Associated with Altered Gene Expression in the Placenta and Congenital Imprinting Disorders*

Epigenetic regulatory mechanisms can originate from several sources: direct DNA methylation, non-coding RNA, imprinting, and post-translational modification of histone proteins and chromatin remodeling [80]. According to some clinical studies, it was speculated that prolonged exposure to extracorporeal environment might predispose human embryos to disorders of genomic imprinting and to epigenetic modification [81]. Each stage of embryo development, from fertilization to blastocyst formation, also in humans, can undergo to epigenetic changes and consequent differential gene expression in the placenta [82,83]. In the pre-implantation period, the embryonic epigenome is entirely reprogrammed by specific methylations [84], leading to an increase in the risk of gene expression alterations under specific circumstances. For example, there is evidence that aberrant fetal DNA methylation might cause abnormal development of the placenta and recurrent spontaneous abortion [85].

We must consider that ART procedures occur simultaneously to this extensive epigenetic reprogramming and we cannot rule out that the stress involved in ART might affect the establishment and/or maintenance of genomic imprinting (Figure 2). Actually, more than 100 imprinted genes have been found to be strictly involved in the growth and development of embryos [15]. In placentas from IVF pregnancies, a different methylation pattern was observed in comparison to non-IVF gestations and environmental conditions were considered responsible for these findings (Figure 2) [82,83]. IVF disturbs the DNA

methylation of the imprinting control region of *H19/Igf2*, *Snrpn*, *Peg3*, and *Kcnq1ot1* genes, inducing morphological alterations in the placenta and an increased risk of adult metabolic syndrome [86]. Studies using animal models have demonstrated a dysregulation of several maternally and paternally expressed imprinted genes (e.g., *Kcnq1ot1* involved in placental growth, *Peg10* that is required for the differentiation of placental spongiotrophoblast and labyrinth, *Peg11/Rtl1* involved the development of placental labyrinth and nutrient passive transport, *Sfmbt2* playing a key role in the maintenance of trophoblast cell types) in the IVF group as compared to the control group [87–89]. Most of these genes play an important role in the proper placental morphology and function. Moreover, specific transcription factors, such as GATA binding protein 3 (GATA3), produced by trophectoderm at blastocyst stage, may be responsible for tissue or cell altered invasion and migration under the influence of the local environment [90]. methylation of the imprinting control region of *H19/Igf2, Snrpn, Peg3*, and *Kcnq1ot1* genes, inducing morphological alterations in the placenta and an increased risk of adult metabolic syndrome [86]. Studies using animal models have demonstrated a dysregulation of several maternally and paternally expressed imprinted genes (e.g., *Kcnq1ot1* involved in placental growth, *Peg10* that is required for the differentiation of placental spongiotrophoblast and labyrinth, *Peg11/Rtl1* involved the development of placental labyrinth and nutrient passive transport, *Sfmbt2* playing a key role in the maintenance of trophoblast cell types) in the IVF group as compared to the control group [87–89]. Most of these genes play an important role in the proper placental morphology and function. Moreover, specific transcription factors, such as GATA binding protein 3 (GATA3), produced by trophectoderm at blastocyst stage, may be responsible for tissue or cell altered invasion and migration under the influence of the local environment [90].

more than 100 imprinted genes have been found to be strictly involved in the growth and development of embryos [15]. In placentas from IVF pregnancies, a different methylation pattern was observed in comparison to non-IVF gestations and environmental conditions were considered responsible for these findings (Figure 2) [82,83]. IVF disturbs the DNA

*Int. J. Mol. Sci.* **2022**, *22*, x FOR PEER REVIEW 6 of 20

**Figure 2.** ART techniques affecting epigenetics in the pre-implantation embryo. Epigenetic dysregulation is involved in an abnormal placentation and genetic imprinting disorders. sFLT-1 denotes soluble fms-like tyrosine kinase 1, GATA3 GATA binding protein 3, BWS Beckwith–Wiedemann syndrome, AS Angelman syndrome, PWS Prader–Willi syndrome, SRS Silver–Russell syndrome. **Figure 2.** ART techniques affecting epigenetics in the pre-implantation embryo. Epigenetic dysregulation is involved in an abnormal placentation and genetic imprinting disorders. sFLT-1 denotes soluble fms-like tyrosine kinase 1, GATA3 GATA binding protein 3, BWS Beckwith–Wiedemann syndrome, AS Angelman syndrome, PWS Prader–Willi syndrome, SRS Silver–Russell syndrome.

Consequently, particular attention was paid to blastocyst stage transfer and its possible adverse effect on perinatal outcome. In this regard, there are data from animal studies demonstrating that in vitro fertilization and embryo culture can strongly impact the placental transcriptome [91,92]. In a multicenter double-blind randomized controlled trial, including 836 couples, a significative reduction of birthweight (158 g) and an increase of PTB (8,6% vs. 2,2%) was associated with one of the two culture media used for embryo culture [93]. Moreover, it has been demonstrated that in vitro culture conditions may obstacle mitochondrial maturation and oxidative phosphorylation, inducing cells to increase glycolysis with the aim of maintaining energetic homeostasis: this pathway is represented Consequently, particular attention was paid to blastocyst stage transfer and its possible adverse effect on perinatal outcome. In this regard, there are data from animal studies demonstrating that in vitro fertilization and embryo culture can strongly impact the placental transcriptome [91,92]. In a multicenter double-blind randomized controlled trial, including 836 couples, a significative reduction of birthweight (158 g) and an increase of PTB (8.6% vs. 2.2%) was associated with one of the two culture media used for embryo culture [93]. Moreover, it has been demonstrated that in vitro culture conditions may obstacle mitochondrial maturation and oxidative phosphorylation, inducing cells to increase glycolysis with the aim of maintaining energetic homeostasis: this pathway is represented by the so-called "Warburg effect" [94]. This adaptation pathway may induce epigenetic changes with possible adverse consequences on fetal growth (Figure 2) [94]. There is evidence that the longer the in vitro embryo culture lasts (i.e., blastocyst transfer in comparison to cleavage stage), the more epigenetic changes that can occur [81,94], especially in the developing placenta, although this is not surprising. In this respect, we should consider that

this organ develops from the outer trophoblastic layer and consequently is more sensible to epigenetic regulatory changes caused by environmental manipulation and influences during ART. Placental overgrowth was demonstrated by Vroman in the rat model, where embryo culture from 1-cell to blastocyst stage showed adverse outcomes. Reduced fetal weight and lower placental DNA methylation, together with the abovementioned increased levels of sFLT-1, were observed [37]. Additional evidence of aberrant DNA methylation following in vitro embryo culture has been obtained using the bovine model [95]. In this study, 2-, 8- and 16-cell stage embryos were flushed from the oviducts and allowed to further develop in vitro up to the blastocyst stage and their methylome investigated. It was clear that each step of the in vitro culture gave a contribution to epigenetic alterations and that the highest levels resulted around the time of early genomic activation of embryo. In this regard, a recent discovery demonstrated that human embryonic genomic activation initiates at one-cell stage [96].

However, it would be important to distinguish the potential contribution of epigenetic problems related to infertility from epigenetic alteration deriving from the culture conditions [97]. Besides the ART procedures themselves, differential methylation has also been identified in placentas of pregnancies conceived from couples with infertility utilizing different fertility treatments [82,98]. Differential DNA methylation in placentas from pregnancies conceived with different fertility treatments, even as early as the late first trimester, has been described [99].

Furthermore, ART can influence epigenetic regulation of placental formation and function by changing the embryonic environment, placental gene expression, and placental adaptive response to embryonic development [100]. In this respect, Zhang et al. [91] analyzed genes in placental samples obtained from the IVF and control groups, identifying seventeen upregulated and nine downregulated genes. Moreover, when compared to normal pregnancies, the transcriptome profile of first-trimester placental villi appeared significantly altered in IVF-ET pregnancies. A total of 3405 differentially regulated genes in the placenta following IVF (>2-fold change; *p* < 0.05) was identified by Zhao et al. [92], with 1910 upregulated and 1495 downregulated. The study revealed that these genes are involved in more than 50 biological processes and pathways: coagulation cascades, immune responses, transmembrane signaling, metabolism, cell cycle, stress control, invasion, and angiogenesis, all playing an important role in early pregnancy. From a clinical point of view, many studies have provided useful data on placental pathologies linked to the in vitro environment in which embryo develops. A Swedish population-based registry study from 2002 through 2013 showed an increased risk of placenta previa after blastocyst transfer compared to cleavage-stage transfer (OR 2.18; 95% CI 1.79–2.65) and spontaneous conception (OR 6.38; 95% CI 5.31–7.66) [101]. For FET, higher birthweight, and higher risk of LGA and VLGA were found in blastocyst vs. cleavage stage transfer [102–104]. Similar data were reported by Huang (2020), comparing results from day 3 vs. day 7 embryo in 4489 infertile women from 2006 to 2017 [105].

An example of the invasiveness of the in vitro reproductive machinery is the introduction in the clinical practice of the ICSI, which overcomes severe male infertility problems [106], injecting a single sperm into the cytoplasm of a mature oocyte. The mechanical introduction of one sperm escaping the natural control of the fertilization process by the oocyte has raised several criticisms. Reasonable concerns were expressed considering that animal studies on the safety of this practice were limited in comparison with those performed by Edwards before Louise Brown was born in 1978. The revision of some large experience and reviews did not report or demonstrated important consequences of this procedure on the newborn [107], and thus far, the cost–benefit ratio seems reasonable. However, abnormal spermatozoa prepared for ICSI showed improper methylation patterns at imprinting regions, leading to conceive embryos affected by genetic imprinting disorders. Not surprisingly, the alterations in the sperm might explain why ICSI is the fertilization procedure that induced the highest number of differentially expressed genes [108].

An increased reported incidence of autism spectrum disorders in babies born from ART might find some possible source in epigenetic alterations [82]. Numerous studies have reported an increased risk of congenital imprinting disorders in children conceived by ART (Figure 2) [109,110]. In 2019, a wide epidemiological study in Japan found 3.44-, 4.46-, and 8.91-fold increased frequencies of Prader–Willi syndrome (PWS), Beckwith–Wiedemann syndrome (BWS), and Silver–Russell syndrome (SRS) after ART, respectively [111]. In particular, BWS, which is a clinically and genetically heterogeneous overgrowth disease, is caused by epigenetic defects at the 11p15 chromosomal region and a four- to six-fold increased risk of its outcome in ART pregnancies has been reported [22].
