Assisted reproductive technologies do not alter mutation frequency or spectrum

Abstract

Assisted reproductive technologies (ARTs) have now contributed to the birth of >3 million babies worldwide, but concerns remain regarding the safety of these methods. We have used a transgenic mouse model to examine the effects of ARTs on the frequency and spectrum of point mutations in midgestation mouse fetuses produced by either natural reproduction or various methods of ART, including preimplantation culture, embryo transfer, in vitro fertilization, intracytoplasmic sperm injection, and round spermatid injection. Our results show that there is no significant difference in the frequency or spectrum of de novo point mutations found in naturally conceived fetuses and fetuses produced by in vitro fertilization, intracytoplasmic sperm injection, or round spermatid injection. These results, based on analyses of a transgenic mouse system, indicate that with respect to maintenance of genetic integrity, ARTs appear to be safe.

Fig. 1.

Developmental scheme for each type of embryo/fetus. A timeline of development to the midgestation stage (10.5 dpc) is shown along with a schematic representation of relative portions of development in vivo (white bar) and in vitro (black bar) for each type of reproduction examined. Control fetuses were produced by natural mating plus natural endogenous gestation (Natural). Test fetuses were produced by natural mating followed by recovery and in vitro culture in CZB to the two-cell stage (Natural/CZB 2-Cell) or the morula/blastocyst stage in either CZB media (Natural/CZB Mor/Blast) or Whitten's media (Natural/WM Mor/Blast), or fetuses were produced by IVF, ICSI, or ROSI fertilization, followed in each case by culture to the morulat/blastocyst stage, embryo transfer, and subsequent development in vivo to midgestation (10.5 dpc). Because implantation of mouse embryos occurs at 4.5 dpc regardless of the stage of embryos transferred, a curved line is used in this figure to show the overlap period between portions of development in vitro and in vivo.

Fig. 2.

DNA methylation in the lacI transgene. Bisulfite genomic sequencing was used to assess the extent of DNA methylation in the lacI transgene in 10.5 dpc fetuses generated by natural mating + natural development (natural), IVF, ICSI, and ROSI. In each case, 7–10 different PCR amplicons were examined. The map at the top of the figure shows the location of 42 CpG sites within the region encoding the DNA-binding domain relative to the transcriptional start site (bent arrow) in the lacI transgene. Filled circles indicate methylated cytosines, and open circles indicate unmethylated cytosines. In all cases, the transgene was heavily methylated (95–97%).