X‐chromosome inactivation and telomere size in newborns resulting from intracytoplasmic sperm injection

In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are generally considered tobe safe technologies for treatment of infertility. However, there is evidence for: (i) a small increase in chromosome abnormalities, especially involving the sex chromosomes, after ICSI [Bonduelle et al., 1998] (ii) an increase in pre-maturity and decrease in mean birthweight in singleton babies born after IVF or ICSI [Hansen et al., 2002;Winston andHardy, 2002; Powell, 2003], and (iii) a slight increase in birth defects after both IVF and ICSI [Hansen et al., 2002; Winston and Hardy, 2002; Powell, 2003]. Recent reports of an increase in imprinting errors (loss of normal parent-of-origin specific gene expression) after IVF/ ICSI have heightened concern for the safety of assisted reproduction procedures [Gosden et al., 2003; Niemitz and Feinberg, 2004]. All these findings may be a consequence of the selected population utilizing assisted reproductive technologies, if the underlying cause of sub-fertility is somehow related to increased abnormalities in eggs or sperm.However, one cannot exclude that the technology itself affects risk of birth defects by altering the normal environment during germ cell maturation or early embryo development. If thatwere the case, then subtle perturbations in development may be relatively common but only rarely yield a detectable phenotype such as intrauterine growth restriction or an imprinting disorder. We propose that assessment of developmentally regulated features of chromosomes, such as X-chromosome inactivation (XCI) and telomere length, can be used as additional measures for the assessment of early embryo development in the context of assisted reproduction. Both these changes are completed by the late blastocyst stage and thus may be sensitive to perturbations in epigenetic programming due to embryo culture. To assess this, we evaluated XCI skewing ratios and/or telomere length in DNA extracted from cord blood from a series of 48 female and 19 male newborns (including three sets of nonidentical twins) conceived after ICSI. University of British Columbia Ethics Committee approval was obtained before initiating the study. Akaryotype fromumbilical cord bloodwas available for all studied cases. One of these infants was a reciprocal translocation carrier [46,X,t(X;20)] [Ma et al., 2003]. The androgen receptor (AR) PCR assay of XCI was used as described previously [Beever et al., 2003]. Informative results were obtained for the AR assay of XCI in 44 of the 48 cord blood samples. The remaining four samples were either homozygous or had alleles too close to accurately evaluate. Control values for XCI skewing in 74 blood samples from 0–19 year olds were taken from a previous study in our laboratory [Hatakeyama et al., 2004]. The observed distribution of skewing is presented in Figure 1. There was no significant difference between ICSI samples and controls inmean level of skewing (65.1 vs. 69.8), or in the frequency of skewing 75% (18%vs. 35%) or 90% (4.6% vs. 10.8%). If the X;20 translocation case is removed from the analysis (since skewed XCI is expected in such translocations independent of ICSI) then the frequency of extreme skewing in the ICSI group would be only 2.3% (n.s.) and the mean level of skewing of 64.3 is significantly less than observed in controls (P1⁄4 0.025; t-test). Our control sample consisted of cases aged 0–19, and while there is no significant change in skewing between ages 0–19 [Hatakeyama et al., 2004], a slight effect of age in this range cannot be excluded. The distribution of XCI skewing in the general population is thought to be largely due to chance deviations from 50:50 as a consequence of the limited number of embryonic precursor cells present (4–20) at the time anX-chromosome is committed to inactivation within each cell of the developing blastocyst [Puck et al., 1992; Monteiro et al., 1998]. For example, extremely skewed XCI defined as >90% inactivation of one X, is present in only about 7% of young females [Beever et al., 2003] but is present in over 50% of chromosomally normal fetuses or newborns associated with high levels of placental trisomy [Penaherrera et al., 2000]. As the distribution of XCI skewing in the ICSI population was similar or less than that in the control population, we can assume that there is no major reduction in size of the embryonic precursor pool in the ICSI conceived blastocyst as compared to normally conceived blastocysts. Thus, we also infer that there are not likely to be increased levels of chromosome mosaicism in viable ICSI blastocysts as compared to viable normally-conceived blastocysts.Reduced skewing in the ICSI group could be explained by selection against slower growing embryos during the ICSI procedure (e.g., by selecting only the best growing embryos for transfer back to the mother’s uterus). Clearly, the present sample size is too small to exclude that abnormal events may occur ina small number of cases, orhaveaneffect that ismostly limited to non-viable embryos. Telomere regeneration is another programmed developmental change occurring primarily at the early embryo to blastocyst stage of development. Studies of bovine embryos have shown that telomerase activity ismaintained throughout Grant sponsor: The Hospital for Sick Children Foundation (grant to SM); Grant number: XG 02-086; Grant sponsor: Canadian Institutes of Health Research Grant (to WPR); Grant number: MOP-15667.