367 INCIDENCE OF CHROMOSOMAL ABNORMALITIES IN MALE AND FEMALE BOVINE EMBRYOS DERIVED FROM SEX-SORTED SPERM

Abstract

Previous studies from our laboratories have shown that developmental rates following IVF with frozen-thawed sex-sorted sperm are lower than with non-sorted sperm and that this may be related to a difference in the kinetics of development. In addition, it is known that the incidence of chromosomal abnormalities in in vitro produced bovine blastocysts is significantly greater than that observed in blastocysts derived in vivo (Viuff D et al. 1999 Biol. Reprod. 60 123-128). In this study, FISH with X- and Y-chromosome-specific probes was used to assess the extent of chromosome abnormalities in bovine blastocysts derived from IVF with X-sorted, Y-sorted, or unsorted semen from the same bull (n = 15 blastocysts per group). Individual blastocysts were transferred to a small drop of spreading solution (0.01 N HCl, 0.1% Tween 20) on a glass slide. Following digestion of the zona pellucida and cytoplasm, the isolated nuclei were fixed in 3:1 methanol:glacial acetic acid at 4°C for 24 h. Slides were then air dried, incubated at 60°C overnight and stored at -80°C until FISH analysis. The StarFISH X-Y FISH bovine sex test kit was used following minor modification and optimization of manufacturer protocols for use with blastocyst preparations. Slides were examined with standard fluorescence microscopy. Overall, 80% (36/45) of blastocysts were considered to be mixoploid i.e. were a mixture of normal diploid and aneuploid and/or non-diploid cells (10/15 for X-sorted, 14/15 for Y-sorted, and 12/15 for unsorted; P > 0.05). The severity of the chromosomal aberration was relatively low in all groups, however; in other words, only a small fraction of the total number of cells per embryo were chromosomally abnormal (1.64%, 5.62%, and 7.93% for X-sorted, Y-sorted, and unsorted, respectively). The most common chromosome abnormalities observed were diploid-triploid-tetraploid mixoploidies. Interestingly, 20% (3/15) of blastocysts derived from unsorted sperm were chimeric (XX/XY). If such chimeric embryos had been sexed by PCR they would have been ascribed as males. In addition, FISH with probes for non-sex chromosomes would not have revealed such chimerism. In conclusion, the incidence of chromosomal abnormalities was not different between blastocysts derived from sorted and unsorted sperm. The occurrence of mixed sex chimeras amongst blastocysts derived from unsorted sperm is interesting and requires further study. Whether it represents a previously undetected abnormality of IVF is as yet unknown.

MGHwas supported by a Posdoctoral Research Grant (2008-0198) from the Spanish Government.