The possibility that ex vivo
manipulations associated with ART may induce or predispose epimutations during early development is a persistent concern in the field (5
). Given the intrinsic heritability of epigenetic modifications, any epimutation generated during embryogenesis has the potential to be propagated to a large proportion of somatic cells throughout the lifetime of the organism and thus could cause or predispose developmental defects or disease phenotypes. However, ICSI and related ART procedures involve a wide array of manipulations including gonadotropin stimulation to induce superovulation, injection of a sperm into an oocyte, in vitro
culture of the resulting preimplantation embryo and transfer of the preimplantation embryo into a recipient uterus. The different techniques used in ICSI coincide chronologically with different stages of the epigenetic reprogramming process that occurs during gametogenesis and preimplantation development. Therefore, deciphering which aspect(s) of the ICSI procedure is involved in the genesis of epimutations may provide useful mechanistic insights into the normal epigenetic reprogramming process, which could, in turn, facilitate improvements in ART procedures to minimize the occurrence of epimutations in offspring generated by these fertility treatments.
As expected, all of the naturally conceived juvenile control mice displayed normal allele-specific DNA methylation and expression profiles, as did all of the adult mice produced by natural reproduction as reported in our earlier study (18
). However, three of the six ICSI-derived juvenile mice showed epimutations or epimutant phenotypes at different imprinted loci in different somatic tissues. Thus, the frequency of epimutations and/or epimutant phenotypes observed in ICSI-derived juvenile mice (50%) was identical to that we previously observed in ICSI-derived adult mice (18
) (Table ). This suggests that ICSI-derived mice accumulate epimutations during early development and that these defects can be maintained indefinitely in somatic tissues.
In at least half of the instances, we observed both epimutations and epimutant phenotypes associated with the same allele in the same tissue of the same mouse; however, in other cases, we observed either an epimutation with no associated epimutant phenotype or vice versa (Table ). Importantly, our analysis of allele-specific DNA methylation was limited to a portion of the DMR associated with each imprinted gene, such that abnormal hypomethylation or persistent hypermethylation in other parts of the DMR or in other associated regulatory sequences of the gene may have been responsible for cases in which we observed apparent discordance between allele-specific methylation and expression patterns. Alternatively, other types of epimutations (e.g. abnormal histone modifications) could have been responsible for epimutant phenotypes in the absence of epimutations detected at the level of DNA methylation.
Our detection of epimutations in ICSI- and SCNT-derived mice, but not in any of the naturally conceived control mice, suggests that one or more aspects associated with the ICSI and/or SCNT procedures either directly causes or indirectly predisposes the occurrence of epimutations. Extensive genome-wide epigenetic reprogramming normally occurs immediately after fertilization in naturally conceived embryos, so we wondered if the ICSI or SCNT procedures might disrupt this process. The ICSI and SCNT procedures differ in this regard, because embryos produced by ICSI are derived from gametes bearing genomes that have undergone normal germline-specific epigenetic programming, whereas embryos produced by SCNT utilize genetic information from a donor somatic cell that has not undergone this programming prior to transfer into the enucleated oocyte. The fact that we observed a slightly lower incidence of epimutations in mice produced by SCNT compared with those produced by ICSI suggests that the disruption of genome-wide epigenetic reprogramming in the zygote was not a primary cause of the epimutations we observed in the ICSI mice.
Embryo culture and embryo transfer were two manipulations common to the procedures we used to generate SCNT and ICSI mice, and both have previously been shown to be a source of abnormal epigenetic effects in mice (3
). A key difference between the ICSI and SCNT procedures is that during SCNT, the oocyte genome that was previously exposed to gonadotropin stimulation to induce superovulation was removed and replaced with a somatic cell nucleus that had not been exposed to gonadotropins, whereas during the ICSI process, the oocyte genome that was exposed to gonadotropin stimulation was retained and propagated to all cells in the ensuing embryo. Thus, although gonadotropin stimulation was used to induce the superovulation of oocytes that were subsequently used to produce both the ICSI- and the SCNT-derived offspring, any direct effects of this treatment on epigenetic programming were likely to be retained and potentially more impactful in the ICSI mice than in the SCNT mice.
The small sample size in each category of mice we investigated precluded us from determining if the slightly greater incidence of epimutations observed in ICSI mice compared with that in SCNT mice was statistically significant. Therefore, to further assess the potential contribution of gonadotropin stimulation to the occurrence of epimutations, we subjected female mice to ovarian stimulation followed by natural mating and analyzed epigenetic profiles in the ensuing juvenile offspring. We found that the use of gonadotropin stimulation did indeed lead to the occurrence of epimutations in somatic tissues in six of the eight superovulation-derived mice. These results support the suggestion that gonadotropin stimulation without subsequent sperm injection can perturb allele-specific methylation and/or expression profiles at multiple imprinted loci in a manner similar to that seen in mice produced by ICSI.
The mechanisms by which epimutations are induced in ART-derived offspring remain largely unknown. Hormonal stimulation may disrupt imprint acquisition during oogenesis by forcing oocytes to develop more rapidly than normal or by ‘rescuing’ lower quality oocytes that might otherwise have never matured (33
). Alternatively, exposure to exogenous gonadotropins can promote molecular changes in the oocyte that alter the maintenance of genomic imprints during subsequent embryogenesis (13
). Our results support the latter hypothesis, because the epimutations we observed were consistently manifest as a loss of methylation and/or a gain of expression of the normally hypermethylated, repressed allele, regardless of whether that was the paternal or maternal allele of the specific imprinted gene investigated. This concept is consistent with a recent report, demonstrating that individual oocytes exposed to gonadotropin stimulation initially exhibited normal methylation profiles at imprinted loci, but nevertheless gave rise to embryos in which epimutations were detected (14
Our studies provide the first evidence of an additional novel effect of exposure to gonadotropin—delayed reprogramming of the maternal allele of a paternally imprinted gene (H19
) during the development of the male germ line. Thus, reprogramming of the maternal DMR of the H19
gene in spermatogonia was delayed in 100% (seven of seven) of the juvenile male mice we investigated that were derived from oocytes exposed to exogenous gonadotropins, indicating that this is a consistent effect of this exposure. In the case of the H19
gene, this represented delayed reprogramming of the maternal DMR while reprogramming of the paternal DMR appeared to proceed normally. Thus, this appears to represent a delay in the acquisition of normal imprinting that is manifest uniquely upon the maternal allele—the same allele that was directly exposed to gonadotropin stimulation during the superovulation process. Importantly, as demonstrated in our previous study (18
), complete reprogramming of both alleles was, nevertheless, achieved by the adult stages in spermatogenic cells of ICSI-derived mice such that epimutations induced by the ICSI procedure were not transmitted to the subsequent generation.
Many developed countries now offer a wide range of ART methodologies for the treatment of various types of subfertility or infertility, and the stimulation of folliculogenesis by exogenous gonadotropins is an integral component of essentially every one of these procedures. Collectively, the data presented here suggest that gonadotropin stimulation used to promote folliculogenesis and oocyte maturation can impact normal epigenetic programming by inducing the formation of epimutations and/or epimutant phenotypes in offspring produced from stimulated oocytes. Thus, it appears that multiple aspects of the ART process, including the gonadotropin stimulation of folliculogenesis, embryo culture and/or embryo transfer, have the potential to induce epimutations in offspring produced by these methods (19
). Taken together, the results presented in this study and several other studies (3
) compel further investigation into the occurrence, effects and causative mechanisms of epimutations in offspring produced with the assistance of gonadotropin stimulation of folliculogenesis and other aspects of the ART process.