The sperm chromatin structure assay (SCSA) was used to measure over 500 human semen samples from two independent studies: Study I, 402 samples from 165 presumably fertile couples wishing to achieve pregnancy over 12 menstrual cycles; Study II, samples from 115 patients seeking fertility counselling. The SCSA measures susceptibility to DNA denaturation in situ in spermatozoa exposed to acid for 30 s, followed by acridine orange staining. SCSA data from the male partners of 73 couples (group 1) achieving pregnancy during months 1‐3 of Study I were used as the standard of ‘sperm chromatin compatible with high fertility’ and were significantly different from those of 40 couples (group 3) achieving pregnancy in months 4‐12 (P < 0.01) and those of male partners of 31 couples (group 4) not achieving pregnancy (P < 0.001). Group 2 contained couples who had a miscarriage. SCSA values for Study II were almost twice that of the Study I fertility standards. Within-couple repeatability tended to be less for group 3 than for groups 1, 2 or 4. Based on logistic regression, spermatozoa with denatured DNA (cells outside the main population, COMPa t) were the best predictor for whether a couple would not achieve pregnancy. Some 84% of males in group 1 had COMPat <15%, while no couples achieved pregnancy in group 1 with ‡30% COMPat, a threshold level considered not compatible with good fertility. Using selected cut-off values for chromatin integrity, the SCSA data predicted seven of 18 miscarriages (39%).

Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic

Intact, viable X and Y chromosome-bearing sperm populations of the rabbit were separated according to DNA content with a flow cytometer/cell sorter. Reanalysis for DNA of an aliquot from each sorted population showed purities of 86% for X-bearing sperm and 81% for Y-bearing sperm populations. Sorted sperm were surgically inseminated into the uterus of rabbits. From does inseminated with sorted X-bearing sperm, 94% of the offspring born were females. From does inseminated with sorted Y-bearing sperm from the same ejaculates, 81% of the offspring were males. The probability of the phenotypic sex ratios differing from 50:50 were p less than 0.0003 for X-sorted sperm and p less than 0.004 for Y-sorted sperm. Thus, the phenotypic sex ratio at birth was accurately predicted from the flow-cytometrically measured proportion of X- and Y-bearing sperm used for insemination.

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Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting.

Sex preselection: High-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Thousands of offspring have now been produced via artificial insemination with spermatozoa sexed by flow cytometry and cell sorting. We are unaware of any other practical approach to sexing spermatozoa that maintains fertility. Accuracy of sexing usually is 85-95% in most species, but somewhat lower with human spermatozoa. Spermatozoa are sexed in series, one at a time, at routine rates of about 3000 live spermatozoa of each sex per second for most species, and nearly twice that rate under optimal conditions for some species. Owing to various constraints and statistical considerations, there appears to be an upper theoretical limit to sexing spermatozoa of about 10,000 live spermatozoa of each sex per second with current methodology. About a quarter of the spermatozoa processed are sexed; the rest are discarded in the process or lost due to logistical constraints. Spermatozoa undergo some damage during sorting, although much less in terms of viability than with routine cryopreservation; fertility is lower with sexed than control spermatozoa. Offspring from sexed spermatozoa appear to have no more abnormalities than do controls, and both groups grow and thrive similarly. Despite high costs and complex procedures, sexing spermatozoa, usually followed by cryopreservation, is being used commercially for cattle and horse production in several countries, and is used to produce girls to avoid X-chromosome-linked genetic diseases.

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Current status of sexing mammalian spermatozoa

Sexing mammalian sperm for production of offspring: the state-of-the-art.

A new and improved method of preparing mammalian spermatozoa for high resolution flow cytometric DNA analysis and flow sorting is described. Ejaculated or cryopreserved sperm were briefly sonicated to remove tails and then stained with Hoechst 33342. This simple procedure was found superior to more severe treatments of dimethylsulfoxide washes, fixation in 80% ethanol, and protease digestion of the sperm membranes and tails by papain. Flow cytometric DNA analyses of sperm samples subjected to varying sonication times indicated that X and Y chromosome-bearing sperm populations could be well resolved with as little as 15-sec sonication. In addition, a comparison of sonicated samples stained with four concentrations of bisbenzimide (Hoechst 33342) or 4′,6-diamidino-2-phenylindole (DAPI) indicated that 2.5 or 5.0 μg/ml of Hoechst was sufficient to resolve the X and Y sperm populations. In order to quantitatively describe the flow cytometric data, several indices (sample quality, orientation and splitting) were developed.

Flow cytometry of X and Y chromosome-bearing sperm for DNA using an improved preparation method and staining with Hoechst 33342.

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation < 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.

J. P. Flook

Papers

Flow cytometry of X and Y chromosome-bearing sperm for DNA using an improved preparation method and staining with Hoechst 33342.

Flow sorting of X and Y chromosome-bearing spermatozoa into two populations.