Sperm Gender Check

Methods To Separate Sex-Based Sperm Populations.

Sub Link: Shape and Size
Sub Link: Sperm Motility
Sub Link: Sperm Density
Sub Link: Electrophobicity
Sub Link: Surface Properties (HY Antigen)
Sub Link: Difference in Chromosomal Content of X and Y Spermatozoa
Sub Link: Take Home Message
Sub Link: References

The primary reason for separating gender-specific sperm is to prevent couples from passing on sex-linked genetic diseases. A less compelling, but often desirable, application is sex selection for the purpose of family gender balancing. Several researchers have used different methods to separate X and Y sperm in relation to the other, however, the efficacy and practical validity of these methods is debatable, and even disproven in some cases.

In the 1960’s, a small, round head was thought to indicate a Y sperm while a larger, elongated head was considered an X sperm under microscopic evaluation (1,2). However, using a more sophisticated microscope later demonstrated that no such real differences existed (3).

Sperm Motility

The smaller and faster Y sperm have been thought to reach the bottom of an albumin gradient (4). However, the quinacrine fluorescent staining method used to differentiate between the two sperm populations was later proven to be nonspecific (5,6).

It is unclear whether Y spermatozoa move faster than X spermatozoa. However, it is now thought to be unlikely, given that if Y spermatozoa do in fact move faster than X spermatozoa, there would be a far greater difference between the male and female populations worldwide, with an almost zero chance of a couple having a daughter.

Sperm Density

The heavier X sperm are thought to sediment to the bottom of a multilayer gradient under mild centrifugation over time (7). 


X and Y sperm have been thought to exhibit differences in their electrophobicity (8,9); however, the method used to differentiate the two sperm populations (quinacrine fluorescent staining) has been proven to be nonspecific (7,8).

Surface Properties (HY Antigen)

HY antigen is a male tissue-specific antigen and is a fundamental part of the membrane of most male cells (10,11). However, it has been reported that the anti-HY antibody also binds to X spermatozoa and thus cannot be used to differentiate between X and Y spermatozoa.

Difference in Chromosomal Content of X and Y Spermatozoa

X-chromosome-bearing sperm have approximately 2.8% more total DNA than Y-chromosome-bearing sperm in humans (12,13). The intensity of the fluorescence signal emitted by DNA-specific fluorochrome-stained sperm allows the differentiation of X- from Y-bearing sperm. This difference in DNA content allows flow cytometric sorting to be done to produce enriched populations of X- or Y-bearing sperm, using the MicroSort® sorting technique.

Take Home Message

The only consistent de novo difference identified between X and Y spermatozoa to date is in their DNA content, which might be responsible for the differential expression of some genes and proteins and the occurrence of certain diseases in a sex-specific manner. However, it is unclear whether this difference in the DNA content results in other physical, chemical, and functional differences between X and Y spermatozoa. Moreover, the ambiguity in the existing findings might be due to the use of non-specific or less-specific methods for distinguishing between X and Y spermatozoa. 


  1. Shettles, L. B. (1960). Nuclear morphology of human spermatozoa. Nature 186, 648–649. doi: 10.1038/186648a0 
  2. Shettles, L. B. (1961). Human spermatozoa shape in relation to sex ratios. Fertil. Steril. 12, 502–508. doi: 10.1016/s0015-0282(16)34321-7
  3. Carvalho, J. O., Silva, L. P., Sartori, R., and Dode, M. A. (2013). Nanoscale differences in the shape and size of X and Y chromosome-bearing bovine sperm heads assessed by atomic force microscopy. PLoS One 8:e59387. doi: 10.1371/journal.pone.0059387
  4. Ericsson, R. J., Langevin, C. N., and Nishino, M. (1973). Isolation of fractions rich in human Y sperm. Nature 246, 421–424. doi: 10.1038/246421a0
  5. Flaherty, S. P., and Matthews, C. D. (1996). Application of modern molecular techniques to evaluate sperm sex selection methods. Mol. Hum. Reprod. 2, 937–942. doi: 10.1093/molehr/2.12.937
  6. Cui, K. H. (1997). Size differences between human X and Y spermatozoa and pre-fertilization diagnosis. Mol. Hum. Reprod. 3, 61–67. doi: 10.1093/molehr/ 3.1.61
  7. Iizuka, R., Kaneko, S., Aoki, R., and Kobayashi, T. (1987). Sexing of human sperm by discontinuous Percoll density gradient and its clinical application. Hum. Reprod. 2, 573–575. doi: 10.1093/oxfordjournals.humrep.a136591 
  8. Kaneko, S., Oshio, S., Kobayashi, T., Iizuka, R., and Mohri, H. (1984). Human Xand Y-bearing sperm differ in cell surface sialic acid content. Biochem. Biophys. Res. Commun. 124, 950–955. doi: 10.1016/0006-291x(84)91050-7Hum. Reprod. 2, 573–575. doi: 10.1093/oxfordjournals.humrep.a136591
  9. Kaneko, S., Iizuka, R., Oshio, S., Nakajima, H., Oshio, S., and Mohri, H. (1993). Separation of human X- and Y-bearing sperm using free-flow electrophoresis. Proc. Jpn. Acad. Ser. B 59, 276–279. doi: 10.2183/pjab.59.276 
  10. Ohno, S., and Wachtel, S. S. (1978). On the selective elimination of Y-bearing sperm. Immunogenetics 7, 13–16. doi: 10.1007/BF01843982
  11. Sills, E. S., Kirman, I., Colombero, L. T., Hariprashad, J., Rosenwaks, Z., and Palermo, G. D. (1998). H-Y antigen expression patterns in human X- and Y-chromosome-bearing spermatozoa. Am. J. Reprod. Immunol. 40, 43–47. doi: 10.1111/j.1600-0897.1998.tb00387.x
  12. Johnson, L. A., Welch, G. R., Keyvanfar, K., Dorfmann, A., Fugger, E. F., and Schulman, J. D. (1993). Gender preselection in humans? Flow cytometric separation of X and Y spermatozoa for the prevention of X-linked diseases. Hum. Reprod. 8, 1733–1739. doi: 10.1093/oxfordjournals.humrep.a137925
  13. Sumner AT, Robinson JA. A difference in dry mass between the heads of X- and Y-bearing human spermatozoa. J ReprodFertil. 1976;48:9–15. doi: 10.1530/jrf.0.0480009.