Fluorescence in situ hybridization studies on the sex chromosome constitution of human sperm

Abstract

More males are conceived than females and more males are born as a result of in vitro fertilization (IVF) and donor insemination procedures. All donor sperm samples are frozen for a minimum of 6 months before they are used. The ratio of more boys to girls has been consistently reported over the years. A similar finding has been noted in the Galway Fertility Unit at University College Hospital Galway, Ireland. Traditionally, it has been accepted that the reason for this excess is that a Y chromosome-bearing sperm swims faster than the X-bearing sperm because it contains less DNA and is therefore ''lighter.'' To test this hypothesis, semen samples were collected at the Galway Fertility Unit from men presenting for routine semen assessments. Each sample was divided into a number of aliquots. The first aliquot was assessed as the ''raw'' ejaculate to measure the initial ratio of X to Y. The second aliquot was prepared using Percoll density gradients, which allows for greater recovery of sperm with higher motility and improved sperm function. The final aliquot was frozen. The frozen sample was later thawed and prepared using Percoll. The prepared sperm were kept for 48 h and sampled at the time of preparation and at 24 h and 48 h to establish if there was any differential survival over time. The X:Y ratio was analyzed using the technique of fluorescence in situ hybridization (FISH). This allowed the sex chromosomes to be specifically stained and identified simultaneously. No difference was found in the X:Y ratio of the sperm. Therefore, any selection for the Y sperm must take place at some later stage.