Dependable methods were developed for preimplantation sexing of human IVF embryos, for use in clinical settings where prospective parents are at high risk for transmission of X-linked diseases. Using single cultured cells and blastomeres from human embryos as model systems, a multiplex protocol was developed for rapid analysis via nested polymerase chain reaction (PCR). Reliability was enhanced by co-amplification of conserved amelogenin gene segments from both X and Y chromosomes, as well as Y-linked DYZ1 repetitive elements. Each cell was manually isolated and individually washed to avoid potential contaminants. Multiplex amplification allowed recognition of spurious amplification failures specific to particular amelogenin single-copy targets. The X-linked internal control and multiple Y-linked markers allowed recognition and exclusion of most aberrant samples, thus averting potential misdiagnosis. The optimized single-cell protocol reduced experimental sexing errors to < 2% (1/60), but also revealed potential pitfalls of single-cell analysis. With human triploid embryos, separate sampling of individual blastomeres provided concordant female or male signals. Slight modification adapted the procedure for diagnosis of biopsy material from blastocyst stage embryos, allowing separate analysis of multiple tubes containing multiple cells for improved reliability.
We report the world's first clinical pregnancy resulting from DNA-based enrichment for X-bearing human spermatozoa, for prevention of X-linked hydrocephalus. Sperm separation was followed by embryo biopsy and nested multiplex polymerase chain reaction (PCR) for gender determination. Enriched populations of X-bearing spermatozoa ranging from 80 to 89% pure as determined by fluorescence in-situ hybridization (FISH) resulted in in-vitro fertilization (IVF) rates indistinguishable from normal IVF procedures (65%). In two separate biopsy procedures, 7/9 and 15/16 of the resulting embryos were determined to be female by multiplex PCR. Embryo transfer resulted in a karyotypically normal female fetus. This technique should be widely applicable to gender selection for the prevention of genetic disorders.
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