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Controversy still surrounds the human serum albumin (HSA) method for separation of X- and Y-bearing human spermatozoa. There is doubt about whether the procedure does enrich sperm samples for the chosen sex chromosome. We have applied the HSA separation method in a clinic in Hong Kong, using the method as described by Ericsson et al. [Nature, 246, 421-424 (1973)] taking care to keep the sperm recovery to <5% of the initial number. Aliquots of separated spermatozoa were examined for X- and Y-bearing spermatozoa by fluorescent in-situ hybridization (FISH) using appropriate DNA probes. Of 18 couples wanting boys, 13 had single boys, one had twin boys, and one had twins comprising one boy and one girl. Only three single girls were born. This success rate of 83% is significantly different (P < 0.001) from the usual expected ratio. There were four miscarriages, one in the third and one in the fourth week of pregnancy. The times of the others are not definitely known, but are thought to have occurred early in pregnancy. We lack information on three couples. The FISH procedure showed no change in the normal and equal numbers of X- and Y-bearing spermatozoa after the HSA separation procedure. This study confirmed that the HSA sperm separation method can bias the number of babies in favour of males. However, the theory that it does so by enriching the sperm samples with Y-bearing spermatozoa appears to be incorrect and some other theory has to be postulated. It is tentatively proposed that passage through the HSA inactivates X-bearing spermatozoa more than Y-bearing spermatozoa, even though this is not apparent simply on inspection of sperm motility.
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