Ovarian stimulation

Controlled ovarian stimulation plays an important role in improving outcomes from IVF. However, exogenous gonadotrophins may impair oocyte and embryo development as well as endometrial receptivity, increase chromosomal aneuploidy, and alter epigenetic modifications, leading to detrimental effects on perinatal outcomes and long-term health . In addition, the primary imprinting process occurs at a relatively late stage in oogenesis; gonadotropic hormones used during superovulation to mature many oocytes simultaneously may harm imprint acquisition in oocytes. Alternatively, oocytes that have not completed the imprinting process might be prematurely released, or oocytes of poor quality that would not have ovulated without treatment might mature .

Evidence shows that in Finland, singleton girls undergoing ART treatment had more major heart anomalies due to ovulation induction than the controls who conceived naturally . Similarly, the risks of birth defects were higher in ovulation induction than natural conception . Systolic blood pressure and subscapular skinfold thickness were elevated in IVF children when compared with children conceived by natural cycle IVF without ovarian stimulation and subfertile couples who conceived naturally . Notably, the levels of T4 and FT4 in IVF children positively correlated with maternal serum levels of estradiol during the first trimester of pregnancy . These show that the process of ovarian induction may contribute to these risks.

IVF and embryo culture

An optimal embryo culture medium is important for embryonic development and subsequently, the success of IVF treatment. It is controversial as to which embryo culture medium is most appropriate . The use of different culture media in clinical practice and prolonged embryo culture to blastocyst stage of human embryos might affect the imprinting process during the preimplantation period . Several studies using mouse models have demonstrated that genomic imprinting in preimplantation embryos can be disturbed by specific culture conditions . Increasing evidence suggests that the type of culture medium may impact birth weight in IVF children . Schieve et al. found that singletons conceived by ART were at an increased risk of low birth weight, overall adjusted risk ratio being 1.8. Additionally, Shi and Haaf found increased rates of abnormal methylation patterns in mouse embryos after superovulation and after the use of certain embryo culture media.

In vitro maturation

In vitro maturation (IVM) refers to maturation in culture of immature oocytes that may or may not have been exposed to short courses of gonadotropins . Although positive results have been reported by some clinics, in vitro maturation has not yet become a mainstream fertility treatment and still considered experimental. The most important reason is the lower chance of a live birth per treatment with IVM when compared with conventional in vitro fertilization . At the same time, the genetic risks of IVM are not very clear. Although so far, no elevated risks for fetal malformations have been reported, no doubts of an increased incidence of congenital abnormalities have been raised and no alterations were found in the metabolism profile of adult mouse offspring born from in vitro matured oocytes compared with that from in vivo matured oocytes ; however, more epidemiological investigations and animal experiments are necessary.

Epigenetic risks of IVF

Epigenetics is defined as heritable changes in gene expression without alterations in DNA sequence . Epigenetic modifications, including DNA methylation, histone modifications, micro-RNAs, and higher order packaging of DNA around nucleosomes may affect the temporal and spatial gene expression patterns essential in embryonic, fetal, and postnatal development . DNA methylation is the most widely studied epigenetic mechanism. It can inhibit gene expression, while DNA demethylation can activate the expression. There are two waves of DNA methylation and demethylation during gametogenesis and early preimplantation development . Thus, ovarian stimulation, assisted fertilization, and other in vitro manipulations involved in IVF may impair the establishment of the DNA methylation in gametes with the maintenance of DNA methylation within preimplantation embryos .

It is well known that disruption of genomic imprinting causes several human genetic syndromes, such as Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome, and Angelman syndrome (AS) . The incidence of imprinting disorders in the general population is very low (1 in 15,000 newborns), less than 0.01%. So the imprinting disorder in offspring conceived by ART is not as severe as supposed . In our previous studies, we found long-lasting changes in Igf2/H19 in both liver and skeletal muscle of mice conceived by IVF, which may be associated with epigenetic changes in DNA methylation and expression of miR-483 .

Ovarian stimulation

Controlled ovarian stimulation plays an important role in improving outcomes from IVF. However, exogenous gonadotrophins may impair oocyte and embryo development as well as endometrial receptivity, increase chromosomal aneuploidy, and alter epigenetic modifications, leading to detrimental effects on perinatal outcomes and long-term health . In addition, the primary imprinting process occurs at a relatively late stage in oogenesis; gonadotropic hormones used during superovulation to mature many oocytes simultaneously may harm imprint acquisition in oocytes. Alternatively, oocytes that have not completed the imprinting process might be prematurely released, or oocytes of poor quality that would not have ovulated without treatment might mature .

Evidence shows that in Finland, singleton girls undergoing ART treatment had more major heart anomalies due to ovulation induction than the controls who conceived naturally . Similarly, the risks of birth defects were higher in ovulation induction than natural conception . Systolic blood pressure and subscapular skinfold thickness were elevated in IVF children when compared with children conceived by natural cycle IVF without ovarian stimulation and subfertile couples who conceived naturally . Notably, the levels of T4 and FT4 in IVF children positively correlated with maternal serum levels of estradiol during the first trimester of pregnancy . These show that the process of ovarian induction may contribute to these risks.

IVF and embryo culture

An optimal embryo culture medium is important for embryonic development and subsequently, the success of IVF treatment. It is controversial as to which embryo culture medium is most appropriate . The use of different culture media in clinical practice and prolonged embryo culture to blastocyst stage of human embryos might affect the imprinting process during the preimplantation period . Several studies using mouse models have demonstrated that genomic imprinting in preimplantation embryos can be disturbed by specific culture conditions . Increasing evidence suggests that the type of culture medium may impact birth weight in IVF children . Schieve et al. found that singletons conceived by ART were at an increased risk of low birth weight, overall adjusted risk ratio being 1.8. Additionally, Shi and Haaf found increased rates of abnormal methylation patterns in mouse embryos after superovulation and after the use of certain embryo culture media.

In vitro maturation

In vitro maturation (IVM) refers to maturation in culture of immature oocytes that may or may not have been exposed to short courses of gonadotropins . Although positive results have been reported by some clinics, in vitro maturation has not yet become a mainstream fertility treatment and still considered experimental. The most important reason is the lower chance of a live birth per treatment with IVM when compared with conventional in vitro fertilization . At the same time, the genetic risks of IVM are not very clear. Although so far, no elevated risks for fetal malformations have been reported, no doubts of an increased incidence of congenital abnormalities have been raised and no alterations were found in the metabolism profile of adult mouse offspring born from in vitro matured oocytes compared with that from in vivo matured oocytes ; however, more epidemiological investigations and animal experiments are necessary.

Epigenetic risks of IVF

Epigenetics is defined as heritable changes in gene expression without alterations in DNA sequence . Epigenetic modifications, including DNA methylation, histone modifications, micro-RNAs, and higher order packaging of DNA around nucleosomes may affect the temporal and spatial gene expression patterns essential in embryonic, fetal, and postnatal development . DNA methylation is the most widely studied epigenetic mechanism. It can inhibit gene expression, while DNA demethylation can activate the expression. There are two waves of DNA methylation and demethylation during gametogenesis and early preimplantation development . Thus, ovarian stimulation, assisted fertilization, and other in vitro manipulations involved in IVF may impair the establishment of the DNA methylation in gametes with the maintenance of DNA methylation within preimplantation embryos .

It is well known that disruption of genomic imprinting causes several human genetic syndromes, such as Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome, and Angelman syndrome (AS) . The incidence of imprinting disorders in the general population is very low (1 in 15,000 newborns), less than 0.01%. So the imprinting disorder in offspring conceived by ART is not as severe as supposed . In our previous studies, we found long-lasting changes in Igf2/H19 in both liver and skeletal muscle of mice conceived by IVF, which may be associated with epigenetic changes in DNA methylation and expression of miR-483 .

ICSI procedure

The ICSI procedure involves fertilization by injection of a single sperm directly into an oocyte, often with spermatozoa with impaired mobility and abnormal morphology. There are, however, several concerns about the safety of ICSI as well as the potential risks for the offspring. First, the selection of morphologically normal sperm under the microscope during ICSI might not be as rigorous in vivo . Use of sperm with impaired mobility and morphology may lead to an increased incidence of abnormalities in offspring. Second, infertile men with oligozoospermia or azoospermia have an increased incidence of chromosomal anomalies, including Y-chromosome deletions and abnormal karyotypes that may be transmitted to their children . ICSI also evades natural selection at the oocyte membrane, which occurs both during natural conception and in conventional IVF, and allows genetically and structurally abnormal sperm to fertilize eggs and pass abnormal genetic materials to the children . Moreover, ICSI involves physical disruption of the cell membrane of the oocyte and introduction of extraneous material into the oocyte, together with the sperm, which is more invasive than conventional IVF . Finally, point mutations could occur as a result of chemical and environmental exposures in vitro . Polyvinylpyrrolidone (PVP) has been used successfully in ICSI to increase the viscosity of sperm solution, thus facilitating the handling and immobilization of sperm. However, the adverse effects of PVP have been reported that it could induce significant damage to sperm membranes. Moreover, PVP solution can be detected in injected embryos and exert a profound negative influence, which may be associated with chromosomal abnormalities in pregnancy derived from ICSI embryos .

Genetic effect of male infertility

Nearly half of all cases of male infertility are thought to be associated with genetic defects . It is clear that cytogenetic abnormalities (both somatic and meiotic) are a major cause of male infertility. Somatic chromosomal abnormalities are relatively common in humans. These include numerical chromosomal abnormalities, such as an extra chromosome, or structural abnormalities, such as a translocation .

Bonduelle et al. found a significantly higher rate of de novo chromosomal anomalies in ICSI offspring, relating mainly to a higher number of sex chromosomal anomalies and partly to a higher number of autosomal structural anomalies. Kong et al. conducted a study of genome-wide mutation rate and found that the diversity in mutation rate of single nucleotide polymorphism (SNP) is dominated by the age of the father at conception of the child. These observations shed light on the importance of the father’s age on the risk of diseases such as schizophrenia and autism. In humans, although many chromosomal defects and mutations are eliminated at preimplantation stages or as spontaneous miscarriages, ART, especially ICSI may facilitate transmission of these anomalies by enhancing fertility . In mouse, Ramos-Ibeas et al. had shown that ICSI with DNA-fragmented sperm can reduce the fertility of male offspring and lead to epigenetic perturbations.

One major indication for ICSI is congenital bilateral absence of vas deferens (CBAVD). Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (OMIM 602421 ) are believed to be responsible for these diseases . Two dysfunctional alleles are required. CFTR mutations are most commonly associated with CBAVD include ΔF508 and R117H. Other CFTR alleles are rarer. Among infertile patients with CBAVD, the incidence of CFTR mutation carriers is estimated to be 20-fold greater than that in the general population . ICSI in the group of CBAVD male patients has the risk of producing affected offspring when the female partner is a carrier. If the male requiring ICSI is homozygous for two dysfunctional CFTR alleles and the partner is heterozygous, the likelihood of any given offspring having two mutant alleles is 50%. If both are mutant, the usual genotype is allele compound heterozygosity. The phenotype of the offspring is likely to be less severe than in ΔF508 homozygosity, reflecting the genetic principle that phenotype reflects the less severe of two recessive alleles .

Klinefelter syndrome (KS) is the most frequent abnormality of sex chromosomes with an estimated prevalence ranging from 1:500 to 1:700 in new born males . Non-mosaic Klinefelter (47,XXY) and mosaic KS (46,XY/47,XXY) are the most common chromosomal abnormalities observed in azoospermic males. About 90% of the cases are due to the 47,XXY karyotype, while the remaining 10% show a 46,XY/47,XXY mosaicism or higher grade X aneuploidies . Some of their gametes could be 24,XX or 24,XY due to secondary non-disjunction. Given half the products of meiosis I should be disomic, XXY men are theoretically at increased risk of sex chromosomal abnormalities. However, in XXY, 3.4% of gametes are 24,XY, rather than the theoretical 50%. Although less than expected, this number is still higher than the 1% disomic (XY) sperm observed in XY infertile males or the 0.2% in fertile XY males . Although most patients with KS are infertile, there are a few case reports of pregnancy without the use of ART, typically in mosaic cases. Sperm recovered from testicular biopsies of men with non-mosaic KS have been used to fertilize oocytes by ICSI techniques. With the introduction of ICSI, men with XXY karyotype have an increased chance of fathering their children . A number of human offspring have been fathered by men with non-mosaic KS . Although all these offspring are normal (46,XY or 46,XX), preimplantation genetic diagnosis (PGD) or prenatal diagnosis (PND) is strongly recommended .

The Y chromosome is attractive in the study of male-factor infertility, in that it contains many genes involved in spermatogenesis and development of male gonads. Y chromosome microdeletion is a frequent cause of infertility in males. The prevalence of microdeletions in azoospermic men ranges from 10% to 15%. In oligozoospermic men, the prevalence of microdeletions is 5%–10% . Microdeletion occurs on the long arm of the Y chromosome, which contains the azoospermia factor region (AZF region) with three subregions: AZFa, AZFb, and AZFc. Deletions in these regions are specifically related to failure of spermatogenesis . USP9Y and DBY (also called DDX3Y) are the two genes located in the AZFa region. Removal of both the genes causes Sertoli cell-only syndrome, which is defined as a complete absence of germ cells . AZFb contains 32 genes and overlaps with AZFc. Therefore, deletion in this sub-region often removes certain genes from the AZFc region such as DAZ1 and DAZ2 and is responsible for meiotic arrest at the primary spermatocyte stage. Complete deletion of AZFc can occur in a preexisting partial deletion or a complete deletion of a preexisting normal gene . Complete AZFc microdeletion may result in Y chromosome loss and sex reversal, with the potential predisposition of the offspring to the 45,X or 45,X/46,XY karyotype. Partial AZFc deletions as a result of recombination between sub-amplicons such as gr/gr, b1/b3, and b2/b3 may be clinically relevant for male infertility by altering the normal spermatogenesis or reducing fertility in the offspring. All male offspring of a man with Y microdeletion carry the deletion. Indeed, deletions of the AZF region encoding the azoospermia gene, DAZ, have been shown to be transmitted to the male offspring. AZF deletions cannot be transmitted to daughters because they do not inherit the paternal Y chromosome . As ICSI bypasses the physiological mechanisms related to fertilization, the Y chromosome microdeletions are transmitted to the offspring. There has been a series of studies on the Y chromosome microdeletions that are transmitted vertically from father to son via ICSI . Our study also indicates that the incidence of Y chromosome microdeletions is increased in children conceived by IVF and ICSI, and that is higher in male offspring conceived by ICSI than by IVF . The incidence of microdeletion was higher in male offspring conceived through ICSI than in those conceived through IVF, suggesting that the process of microinjection may be involved in increased microdeletion, although no statistically significant difference was found between the two treatments. However, this remains controversial.

Epigenetic risks of ICSI

Great attention has been drawn to the epigenetic effect of ICSI in recent years. The potential epigenetic disruption in children born through ICSI including AS, BWS, Silver-Russell syndrome (SRS), and so on has been reported in a number of research studies.

AS is a neurogenetic disorder that is characterized by severe mental retardation, impaired motor development, poor balance accompanied by jerky movements, absence of speech, and an unusually happy disposition. This disorder affects 1 in 20,000 children and is caused by genetic mutations that decrease the activity or expression of UBE3A gene on chromosome 15 . Case reports have suggested that infertile couples treated with ICSI have increased risk of imprinting defect causing AS . Ludwig et al. performed a cohort study and indicated that the risk of conceiving a child with an imprinting defect causing AS was significantly increased in couples who had undergone hormonal stimulation or ICSI. However, Manning et al. reported that a regular DNA-methylation pattern was found in the AS region among 92 children born through ICSI, and none of them had clinical symptoms of AS.

BWS is a rare imprinting disorder with an estimated incidence of 1 in 13,700 live births; it is a congenital overgrowth syndrome characterized by macroglossia, midline abdominal wall defects, macrosomia, neonatal hypoglycemia, and a number of other developmental anomalies. Moreover, children with BWS have an increased risk of developing embryonal tumors such as Wilm’s tumor, rhabdomyosarcoma, and hepatoblastoma . Patients with this disorder have abnormalities at chromosome 11p15. The imprinted genes implicated in the etiology of BWS have been mapped to the 11p15 imprinted region, including the paternally expressed genes IGF2 and the maternally expressed genes H19. Normally, the H19 and IGF2 genes are regulated by DMR1 in a coordinated manner . Gomes et al. reported a case with BWS conceived by ICSI and detected a different methylation pattern at the KvDMR in embryonic and extra-embryonic tissues. In this case, the ICSI procedure was performed in a couple without characteristics of BWS phenotype. Gomes and coworkers observed different maternal methylation patterns at KvDMR in DNA samples from different tissues (hypomethylation in amniotic fluid and umbilical cord blood and normal methylation in placenta). Lim et al. compared clinical phenotype and molecular features of 25 children with BWS who were born through ART (12 through IVF and 13 through ICSI) and that of 87 children with BWS who were born through spontaneous conception. The result of their study indicated that loss of maternal allele methylation at differentially methylated regions (DMRs) occurred in 37.5% of ART and 6.4% of non-ART BWS cases, which suggested that ART may be associated with disturbed normal genomic imprinting in the offspring. In a review by Vermeiden et al. , the result pointed out that there was a significant positive association between IVF/ICSI treatment and BWS, with an RR of 5.2 (95% CI 1.6–7.4). However, Bowdin et al. performed a cohort study among children born through ART with the purpose to detect cases with BWS and AS. They sent a questionnaire to 2492 families with IVF/ICSI children performed detailed clinical assessment of 47 children and found only 1 case of BWS and no cases of AS. Their findings suggest that the absolute risk of imprinting disorders in children conceived by ART is small (<1%).

Given that ICSI is invasive as it involves physical disruption of the cell membrane of the oocyte and bypasses some physiological steps of fertilization that occur in spontaneous conception, there are concerns on the safety of this procedure. Large longitudinal studies are needed to evaluate the long-term effects of ICSI to address whether ICSI may increase the risk of genetic diseases and imprinting disorders. Moreover, it remains to be determined whether the implicated association between ICSI and outcome of the offspring is related to the procedures or the genetics of the couples.

Embryo cryopreservation

The important causes of cell damage and cell death during cryopreservation are the formation of ice crystals and high concentration of salt in the remaining unfrozen fraction of the cells . So far, two main techniques, slow-freezing method and vitrification, are commonly used in ART. The differences between these two techniques are the concentration of cryoprotectants used and the rates in cooling and warming. Cryoprotectant could lower the freezing point of the cells. Slow-freezing method uses low concentration of cryoprotectants, low cooling rates, and fast warming rates, while vitrification uses high concentration of cryoprotectants with ultra-rapid cooling and warming rates, resulting in a glass-like (vitreous) state and thus, reducing chilling sensitivity and ice crystallization damage.

Compared with the slow-freezing method, vitrification exposes the embryos to high concentrations of toxic cryoprotectants, although the post-warming survival rate and clinical pregnancy rate are also higher . Not many studies focused on the genetic and epigenetic risks of cryopreservation. Most of these studies are on animals, including zebrafish , mice , ovine , bovine , and rabbits . Owing to the ethical issues, similar studies are less on human embryos than those on human oocytes .

Genetic risks of embryo cryopreservation

Vitrification induces a transient increase in DNA breaks and a possible sporadic change in CpG methylation in mouse oocytes . DNA fragmentation is regarded as an indicator of DNA damage to assess the efficiency of cryopreservation . In recent years, several studies showed that the meiotic spindle is a sensitive target during cryopreservation. A paired randomized controlled trial indicated that there was no difference in the rate of embryonic aneuploidy between vitrified oocytes and control oocytes, as well as the ongoing pregnancy rate . However, larger incidence of abnormally shaped spindles was found in the vitrified blastocysts than in the fresh blastocysts .

Shaw et al. studied 19 genes known to be critical for early embryogenesis in frozen-thawed zygotes and fresh human embryos. The results showed that the expression levels of some genes were significantly changed after cryopreservation. These genes were associated with cell survival (BAX), stress response, trophectoderm formation, pluripotent stem cell maintenance (GAS5, SOX2, NANOG, and CDX2), genome activation, transcription, translation initiation, germ cell, and oocyte function (EIF1AX, NLRP5, ZAR1, ZSCAN1, and TSC2).

Epigenetic risks of embryo cryopreservation

DNA methylation plays a key role in transcriptional regulation of specific genes and elements, which is established through the activities of DNA methyltransferases (DNMTs) . A few studies investigated the effects of cryopreservation on DNA methylation status in human preimplantation embryos. Petrussa et al. compared embryos that had been cryopreserved on Day 3 using a slow-freezing DMSO protocol with control fresh embryos. The expression levels of de novo DNMT (DNMT3a and 3b) and the global DNA methylation levels were determined on Day 6. The results showed no difference between the two groups in terms of mean immunofluorescence intensity or protein expression. Interestingly, the switch toward nuclear DNMT3b expression was delayed from Day 5 to Day 6–7 in the cryopreserved embryos. There was no difference in global DNA methylation levels on Day 6, suggesting that the DNMT expression patterns could be disturbed after cryopreservation but reinstated in a timely manner . Another report studied two genes associated with BWS and found that vitrified GV oocytes acquire full DNA methylation in the maternal imprinted gene KCNQ1OT1 with the same efficiency as fresh oocytes and maintained the unmethylated state of paternal imprinted gene H19DMR (a paternal imprinting region) after IVM . There are several reports on the epigenetic status in other species. In rabbit blastocysts, vitrification did not alter the methylation levels of the OCT4 promoter . Oocyte cryopreservation did not affect the percentage of hypermethylated strands at DMRs of Snrpn, Igf2r (maternal imprinting region), and H19; Peg3 (paternal imprinting region) showed no difference in mouse blastocysts .

In addition to DNA methylation, histone modification is another crucial epigenetic mechanism. Tri-methylation of lysines 4 (K4) and 27 (K27) of histone H3 (H3K4me3 and H3K27me3) is associated with gene activation and repression, respectively. Maldonado et al. found that the levels observed for H3K4me3 were approximately 20% lower, while the levels for H3K27me3 were higher in the frozen-thawed bovine blastocysts. They hypothesized that the phenomenon was caused by cellular stress, especially oxidative stress. To test the hypothesis, they placed the blastocytes in normoxic (5%) or hyperoxic (20%) conditions and demonstrated that the levels of H3K4me2 and H3K9me2 were altered. Another study revealed that in vitro culture rather than prior vitrification and warming changed histone and chromatin status of mouse embryos . The present evidence demonstrated that cryopreservation does not impart great genetic and epigenetic risks. However, further studies are needed to confirm the effect of cryopreservation on genomic imprinting in human oocytes and embryos.