Evaluation of Epigenetic Factors in Surrogacy: A Mini-Review

10.30699/jogcr.


Introduction
Surrogacy is one of the Assisted Reproductive Technologies (ARTs) in which the intended parents, who are unable to conceive in usual ways, allocate the gestation and birth to another woman known as the surrogate mother.The surrogate mother carries the fetus during the pregnancy, gives birth to the child, and then delivers the baby to the intended parents.There is no genetic link between the surrogate mother and the child.The embryo entering the surrogate mother's uterus is results from the sperm and oocytes of the original parents or donors using the In Vitro Fertilization (IVF) (1-4).Infertility, medical conditions, sexual identity disorders, similar problems, and social issues are important factors in selecting this method.Women with severe Mullerian duct anomalies or congenital uterine or vaginal anomalies are the main candidates for this method.Other potential candidates include individuals with Mayer-Rokitansky-Küster-Hauser syndrome, who have a female genotype but phenotypically, have congenital uterine or vaginal ageneses or anomalies, and patients with androgen insensitivity syndrome.Also, women who have had a previous hysterectomy, women with Turner syndrome, women with a history of multiple miscarriages, and women who have not benefited from long-term infertility treatments.A history of severe cardiovascular disease or medical conditions that expose the fetus to high teratogen levels are other reasons for selecting this method (5, 6).
The surrogacy process includes ovarian hyperstimulation, ovulation induction, oocyte retrieval, IVF, embryo (blastocyst) culture, embryo selection, and embryo transfer into the uterus of the surrogate mother.This process is closely related to the IVF technique.Epigenetic means beyond genetic that involves multiple processes of hereditary alterations in gene expression without DNA sequence changes.DNA methylation, histone modification, and changes in non-coding RNAs (ncRNAs) are the central epigenetic mechanisms (7).Epigenetic alterations by internal and external factors can also be inherited by future generations.In mammals, embryogenesis cannot be adequately done without epigenetic mechanisms.Embryonic cells are prone to epigenetic alterations because gametes' epigenetic programming occurs during early differentiation in gametogenesis and before implantation (8).In the present mini-review, we intended to briefly mention the various studies and effective aspects of epigenetic alterations in ARTs, especially surrogacy, as well as reviewing the available studies in this field.

Methods
In this mini-review, all the data were compiled from electronic databases including Google Scholar, PubMed, Scopus, and Web of Science (ISI).The search was conducted without a time restriction, up to February 2021 using the following keywords: "Reproductive Technologies", "Surrogacy", "Epigenetic", "DNA methylation" and "embryo".

Epigenetic Alterations in Prenatal Life
Epigenetics plays a critical role in ovulation, spermatogenesis, and embryonic growth, development, and health.Epigenetic dysregulation can result in silencing or improper expression of certain genes, thereby leading to disturbances.Human studies have shown that the biology of the surrogate mother can reprogram the embryo's epigenome and that any disturbance in the early stages of life, especially the critical period of prenatal life, will have programmed effects on lifelong health (9).This fact proves the biological link between the surrogate mother and the child in addition to the proven emotional relationship, and the surrogate mother can alter the child at the epigenetic level.Epigenetic alterations in ARTs may lead to clinical problems, including failure in embryo implantation, miscarriage, intrauterine growth retardation with placental dysfunction, phenotypical changes, and even genomic imprinting syndromes (2, 10).

Prenatal DNA Methylation
Conserved non-exon elements rich in CpG dinucleotides in the human genome are the main targets for epigenetic alterations.DNA methylation is the most common epigenetic alteration, in which a methyl group is added to the C5 position of the nucleotide cytosine (11).This alteration does not change the DNA sequence.If this methylated region is close to the gene, it can lead to underexpression or even silencing (12, 13).In the active mitotic cells, DNA methyltransferases (DNMTs) perform the methylation using donor groups, including methionine, S-adenosyl methionine, choline, vitamin B6, vitamin B12, zinc, betaine, folic acid, etc. (14).Methylated CpG dinucleotides condensate and inactivate the chromatin and subsequently suppress the transcription.These effects are directly exerted by interfering with the activities of transcription factors and indirectly exerted by using the Methyl-CpG-Binding Domains (MBDs), which adsorbs the Histone Deacetylases (HDACs) and Histone Methyltransferases (HMTs) (15).In early embryogenesis, epigenetic silencing of genes from paternal or maternal origins is mediated by the maintenance methylation activity of DNMT1, while the tissue-specific gene expression and postnatal DNA methylation require spontaneous methylation activity of DNMT3a and DNMT3b (16, 17).
Genetic imprinting is an epigenetic alteration that causes the preferential rather than definitive expression of the parental allele.During the critical period of genomic imprinting, which is from conception to 2 yr old age and is known as the 1000 days, epigenetic alterations can play a critical role in development, thereby influencing the risk of future development of chronic diseases such as cardiovascular diseases, diabetes, obesity, etc. (18).The imprinted genes are often tissue-specific and are abundantly found in regions close to CpG islands or CG-rich sequences.Genomic imprinting is removed in primordial gametes during gametogenesis and is then re-established gender-specifically in the next stages.This reprogramming is essential for maintaining the inheritance pattern in imprinted loci (19).
DNA of the spermatozoa is methylated differentially at several maternal and paternally imprinted sites, and also has a unique pattern of general methylation.ARTs such as Intra Cytoplasmic Sperm Injection (ICSI) and IVF may increase the risk of epigenetic abnormalities and impact embryonic growth and development by using immature spermatozoa, which may not be appropriately imprinted or may not have a maintained methylation pattern (Figure 1) (20).
Delayed oocyte growth and maturation, which prevents imprinting maintenance at the right time, ovulation induction, or use of older oocytes, which are abundantly acetylated during mitosis, can result in epigenetic impairments (21).Human and murine studies have suggested that gonadotropins used in ART can release metaphase II oocytes with defective or unstable imprinting patterns and induce molecular changes in oocytes with adverse effects on genomic imprinting maintenance in embryogenesis (22, 23).Oocytes from ovarian hyperstimulation are immature and require in vitro culture to achieve maturity.Genomic imprinting analyses in oocytes from ovulation induction showed abnormalities in 4 imprinted murine genes of PEG1 (paternally expressed genes 1), KCNQ1OT1 (KCNQ1 opposite strand/antisense transcript 1), Zac (Zinc-activated ion channel), and H19 mice in comparison with the oocytes from natural ovulation (24).In addition, human studies have reported that imprinted genes, including SNRPN (small nuclear ribonucleoprotein polypeptide N), H19, PEG1/MEST (mesoderm-specific transcript), KCNQ1OT1, and their regulatory regions are prone to abnormal changes in methylation or gene expression pattern in some pre-implanted embryos (25, 26).A previous study showed hypomethylation in the group using ARTs compared to the group with natural conception by investigating several genomic arrays (27).Also, while investigating ovulation induction in human models, another study reported that oocytes from ovulation induction in ARTs showed hypomethylation of PEG1 and H19, which was compatible with murine models (28).Also, there are other similar findings including the abnormal methylation of the differentially methylated regions (DMRs) in gene H19, which are normally methylated in the maternal allele, the demethylation of DMRs in the maternal LITI locus, which is normally methylated, in Beckwith-Wiedemann syndrome, and the decreased level of HDAC and incomplete acetylation of H3K9 in in-vitro-cultured oocytes (29, 30).
There is defective SNRPN imprinting in the Prader-Willi and Angelman syndromes.The SNRPN methylation level is higher in children conceived with ISCI than IVF and normal conception.However, the difference in the methylation level is slight in children born with IVF compared to spontaneous pregnancies (31).The glucose tolerance gene expression level is different in children born with IVF and normal pregnancy, and children born with IVF have different lipid profiles, fasting blood sugar, body fat distribution, and cardiovascular performance.These findings confirm the relationship between epigenetic alterations and increased risk of cardiovascular diseases (32, 33).

Prenatal Histone Modifications
Post-translational histone modifications are other epigenetic mechanisms that have been widely studied.
The variability of alterations and amino acid sequences of histones, including lysine and arginine methylation and acetylation, serine and threonine phosphorylation, and ubiquitination, can be considered as a type of coding (34).The enzyme HDAC1, which plays a critical role in the chromatin restructuring, is significantly decreased in oocytes, the first stage of cleavage, and two-cell embryos that have undergone in vitro maturation.This suggests that in vitro maturation results in decreasing transcription in genes such as HDAC1 (35).A previous study reported a defective chromatin restructuring due to impaired histone acetylation in in-vitro-cultured oocytes (36).Another study showed that in vitro culture caused alteration at the histone levels and in the expression of the genes Igf2 (insulin-like growth factor 2) and Oct4 (octamerbinding transcription factor 4) in mice, confirming the impact of culture media (37).The same group reported in another study that expression of human imprinted genes H19 and 3MEST and histone H3 lysine 9 (H3K9) acetylation in in-vitro-cultured blastocysts was significantly increased compared to in-vivo-cultured blastocysts.However, the histone H3 lysine 9 (H3K9) methylation was significantly decreased (38).

Prenatal Functions of ncRNAs
NcRNAs control many aspects of the activities of gene regulatory networks, including transcription and post-translational regulations.MicroRNAs (miRNAs) are a major group of these RNAs.They are small, noncoding endogenous molecules that are approximately 21 to 23 nucleotides in length and suppress gene expression by mRNA translation inhibition or mRNA degradation (39, 40).The surrogate mother is related to the embryo through the transplacental transfer of microchimeric molecules (small chemical molecules), nutrients, and antibodies (41, 42).During embryonic development, there are several miRNAs in the endometrial fluid of women carrying an embryo from a donated oocyte.These molecules encode information on the expression regulation of embryonic and, eventually, fetal genes.This indicates the impact of the surrogate mother's genome on the embryo's genetic development.This effect determines which of the child's genes will be expressed (43).
There are several reports of the remarkable effect of maternal lifestyle, including smoking, nicotine or caffeine addiction, alcohol consumption, psychological stress, etc., on the epigenetic alterations in neurological disorders (44-46).A previous study compared the miRNA expression levels in 25 placentas obtained at birth from women with a history of tobacco use during pregnancy and reported a significant decrease in miR-16, miR-21, and miR-146a levels due to maternal tobacco use (47).Their result suggested that the lower expression of miR-16 and miR-21 in the placenta does associate with reduced birth weight.In addition, miR-146a was significantly reduced following nicotine use in the immortal human trophoblastic cell lines (a cell line derived from villi cells in the third-trimester), while levels of other miRNAs were not changed.Down regulation of these miRNA upregulates the targets of these miRNA and may have further effects downstream for both placenta and fetus.Another study compared levels of miR-233 and miR-155 in the placentas and umbilical cords of a group of mothers with nicotine exposure and found increased levels of miR-233 (48).Ovarian hyperstimulation in mice was associated with decreased expression of miRNAs, including miR-122, miR-144, and miR-211, which are involved in neuronal migration and differentiation.However, further studies are needed to illustrate the relationship between epigenetic alterations and the risk of neurological disorder development in ARTs.

Effective Factors on Epigenetic Alteration in Surrogacy
For the first time, Barker suggested the fetal programming hypothesis.He reported that intrauterine events are much more important than postnatal events in terms of gene expression patterns and that chronic diseases, including cardiovascular diseases, obesity, diabetes, etc. are not often caused by only genetic or lifestyle factors.However, they are affected by intrauterine events during pregnancy (49).The fetus receives genetic material from the parents or gamete donors, but the surrogate mother can also affect the fetus.For example, she exerts these effects via diet, and physical activity is done as working or daily activities, the air she breathes, and surrounding sounds.Xenochemicals, including alcohol, tobacco smoke, biophenol, etc. are considered teratogens and can cross the placenta and cause fetal functional defects.Also, it has been reported that endogenous factors such as maternal stress in pregnancy and hypoxia have a significant effect on children's health during childhood and possibly adulthood (50, 51).All these factors should be considered when choosing the ideal surrogate mother.There are reports that mothers using donated oocytes have a significant effect on the fetus, and there are similarities between the child and other family members in families using donated oocytes.
Since there is a close relationship between monocarbon metabolism and diet, DNA methylation can be influenced by the maternal diet (52).It has been reported that children from mothers with a low-calorie diet during pregnancy have a higher risk of diabetes development later in life (44, 53).It has also been found that gestational diabetes alters the embryo/fetus's methylation pattern, placental tissue, and umbilical cord blood (54).Genes with altered methylation patterns due to maternal diabetes are involved in pathways of metabolic diseases, and it was suggested that maternal diabetes alters the child's metabolism epigenetically.It was reported that carbohydrate intake in early pregnancy and childhood obesity are associated with the DNA methylation level of the RXRA (retinoid X receptor alpha) gene promoter in the fetal umbilical cord (55).
Maternal pre-pregnancy obesity and gestational diabetes can significantly increase leptin gene methylation (56).A previous study showed that the normally methylated maternal gene MEST is significantly hypomethylated in fetuses from mothers with gestational diabetes (57).Pre-pregnancy folate intake was associated with several serum biomarker levels, including methionine, choline, and S-adenosyl methionine, and plays an essential role in lymphocyte DNA methylation (58, 59).Evaluation of the methylation levels of imprinted genes in umbilical cord blood from women taking folate supplementation after 12 weeks of gestation showed that the supplementation was associated with increased methylation of IGF2 and decreased methylation of PEG3 and LINE1 (60).
Various studies have discovered a second genome.This genome includes the human microbiome genomes actively interacting with sperm and oocyte genomes through metabolites and causes epigenetic alterations (61, 62).Microbial colonization, especially the microbial flora of the amniotic fluid, placenta, and uterus, can prenatally affect fecal microbial flora development (63).The postnatal impaired microbiome can be due to skin contact, route of delivery, diet, and antibiotic use.These effects play significant roles in the development of disease risk, including chronic cardiovascular diseases, diabetes, obesity, allergic diseases, asthma, and autoimmune diseases through epigenetic alterations (64, 65).Environmental factors, including chemical pollution, tobacco use, alcohol, radiation, temperature changes, and other external stresses can affect growth, metabolism, risk of disease development in future generations, and behavioral disorders such as schizophrenia through epigenetic programming (66, 67).These environmental factors can impair DNA methylation and DNA fragmentation, as in infertile men in whom DNA damage by oxidative stress leads to DNA methylation (68-70).
Antepartum exposure to high levels of stress, including environmental pollutions, physiological stress, or depression, can increase the risk of fetal somatic system growth retardation or adulthood disturbances.The effects of prenatal stress on fetal and childhood developments, including immune system performance, brain development, and behavior development, have been extensively investigated in animal studies.According to animal studies, maternal cortisol levels can adversely affect fetal hypothalamicadrenal axis development (71, 72).Stress leads to the maternal hypothalamic-adrenal axis activation and subsequent glucocorticoid secretion that can reach the fetus through placental transfer (73, 74).
Maternal cortisol levels increase during pregnancy; maternal stress also increases maternal cortisol and decreases the activity of placental enzymes that reduce glucocorticoid levels.These enzymes neutralize cortisol's harmful effects by converting it to an inactive form before reaching the fetus (75).
A previous study investigated the relationship between antepartum stress exposure of mother and methylation level in the promoter of gene NR3C1 (76).Increased depression in the third trimester was associated with increased methylation of binding sites NGFI-A in the neonatal gene NR3C1, which subsequently stimulates the hypothalamic-adrenal axis (77).Also, antepartum stress and anxiety were significantly associated with 1F promoter methylation in the CpG region of the NR3C1 gene (51).

Evaluation of the Impact of in Vitro Culture Conditions on Embryo's Growth and Epigenome
There are extensively discussed risks and ambiguities regarding the effects of pre-implantation culture on embryonic physiology and epigenetics, despite the advantages of ARTs.Human studies have shown the association of ovarian hyperstimulation, high levels of gonadosteroids, blastocyst culture, culture media, and embryo cryopreservation in ARTs with intrauterine growth retardation and changes in birth weight pattern.This association is probably the result of epigenetic alterations (30, 78).In ARTs, exogenous gonadotropins are maintained at high levels during the critical period of implantation.There are several reports that exogenous gonadotropins may cause epigenetic alterations in four imprinted genes Peg1, Kcnq1ot1, Zac, and H19, and interfere with oocyte and embryonic development (1, 79).A previous study reported a relationship between imprinted genes and fetal growth retardation as well as placental disorders (20).
Since ARTs are simultaneous with the essential epigenetic reprogramming events, another important issue is the impact of culture media and culture conditions on an embryo's growth and epigenome.Human studies have reported that the culture media can cause several adverse effects, including defective implantation, low implantation rate, growth disorders, low quality of the embryo, decreased trophoblastic growth, and decreased a number of embryonic cells (2, 80, 81).It has been reported that the culture media causes a wide range of metabolic, developmental, and cellular changes in pre-implantation murine embryos that were effective on metabolic pathways (82).In the following, it was shown that the culture media could also affect human embryos in the pre-implantation stages (83).Studies found that the use of G5 and human tabul fluid culture media causes the different expression of the genes of several pathways in human embryos.Also, the addition of a growth factor to the culture media of growing embryos had unexpected effects on the gene expression pattern (81, 84).However, a murine study showed that the placenta and embryos' gross anatomy was not different in embryos cultured in different environments.However, no molecular studies were included in the last study (85).
The oxygen concentration in oocyte or embryonic cultures effectively affects metabolism, protein synthesis, and protein functions and plays a critical role in embryo implantation and survival (86).Oxygen levels in the fallopian tube, where natural fertilization occurs, have been reported to be 1%-9% in different mammalian species (87).Human embryos that are cultured in IVF at 20% oxygen concentration, which is similar to the atmosphere's oxygen concentration, can be very different from those in in vivo conditions (86).According to human studies, oxygen concentration levels similar to atmospheric oxygen concentration causes oxidative stress and oxygen-free radical activation (88, 89).Embryo culture under high O2 concentrations can hurt blastocyst growth, cell count, and embryonic metabolism in many species.Increased active oxygen levels can change protein levels and lipid functions and harm DNA and cell membrane integrity (Figure 1) (23).
The fetus receives genetic material from the parents or donors of sperm and oocyte, but factors including physiological stress, antepartum depression of the surrogate mother, food, daily and physical activities of the surrogate mother, and levels of environmental pollution exposure can increase the risk of fetal somatic system growth retardation or adulthood disturbances.Several human studies have shown that hormonal therapy can cause epigenetic reprogramming of gametes in the early stages of embryogenesis, and that epigenetic alterations in the resulting oocyte can be maintained during the embryonic period.These results suggest that the pre-implantation environment can affect neurological growth and development and lead to behavioral changes in adulthood.

Conclusion
This review emphasizes DNA methylation, histone modification, and changes in ncRNAs are the central epigenetic mechanisms that can be affected by in vitro embryo culture conditions and surrogate mother lifestyle.However, to make significant headway in understanding the impact of surrogacy on epigenetic modifications on the human conceptus, further detailed and long-term studies are required.Identification of epigenetic modifications on surrogacy may allow us to develop new interventions, preventive measures, and follow-up strategies.