Sub Link: Quinacrine method
Sub Link: Fluorescence In Situ Hybridization (FISH) method
Sub Link: Flow Cytometry “Microsort”
Sub Link: References

SpermGenderCheck method: Real-Time PCR method (qPCR) 

• SpermGender offers an accurate, quantitative determination of the number of X-bearing and Y-bearing spermatozoa in a semen sample, thus allowing the ratio of X:Y sperm to be determined.

• The testing method uses highly specific oligonucleotide primers and probes that detect the X chromosome or the Y chromosome separately or together simultaneously.

• The dead sperm cells used for the qPCR test are not used later in fertilizing an oocyte, or for intrauterine insemination (IUI) or in vitro fertilization (IVF) procedures. They are used only to reveal the normal ratio of X-bearing to Y-bearing sperm in a semen sample, and therefore to help determine the likelihood of that individual producing a male child or a female child.

Quinacrine method

• This is a simple method, but it requires a fluorescence microscope

• The method is nonspecific, and therefore does not produce a reliable or accurate result. (Flaherty and Matthews, 1996; Cui, 1997). 

• It can produce false positives and negatives in interphase cells (Thomsen and Niebuhr, 1986).

• It has been known to produce inaccurate results in human spermatozoa. (Wyrobek et al, 1984)

Fluorescence In Situ Hybridization (FISH) method

• Although it is performed routinely in many laboratories, it can have false positives or negatives; not all cells necessarily hybridize to the FISH probes, thus inaccuracies can result.

• It is also an expensive and time-consuming process that requires a fluorescence microscope.

Flow Cytometry “Microsort”

• This method is particularly expensive and time-consuming.

• It works by separating X- from Y-bearing sperm using flow cytometry, after the sperm cells are stained with a special fluorescent dye (Hoechst 33342), and based on the concept that X-sperm contain 2.8% more DNA than Y-sperm. This allows them to be separated from each other in a flow cytometer (Caroppo E. 2013; Said et al., 2011).
  • Example: The MicroSort procedure 
    • This procedure is only offered by select laboratories outside the United States.
    • It requires a prolonged period of time to accomplish sorting.
    • Hoechst 33342 fluorescent dye may be harmful – the exposure required (about 6 or 7 hours) for sorting with fluorescence may result in genetic mutations to the sperm.

Sorted samples are tested for gender specificity using fluorescence in situ hybridization as mentioned above.

References:

• Beckett TA, Martin RH and Hoar DI. (1989) Assessment of the Sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and Y-bodies. Fertil. Steril., 52, 829-835.

• Thomsen JL and Niebuhr E. (1986) The frequency of false-positive and false-negative results in the detection of Y-chromosomes in interphase nuclei. Hum. Genet., 73, 27-30.

• van Kooij, RJ. and van Oost A. (1992) Determination of sex ratio of spermatozoa with a deoxynbonucleic acid-probe and quinacrine staining: a comparison. Fertil. Steril, 58, 384-386.

• Wyrobek A, Watchmaker, G, Gordon L., Carrano AV, Ashworth L, and Brandriff B. (1984) Aneuploidy and quinacrine-positive spots in human sperm. [Abstr.] Am. J. Hum. Genet., 36(4), Supplement 118s.

• Cui, K. H. (1997). Size differences between human X and Y spermatozoa and prefertilization diagnosis. Mol. Hum. Reprod. 3, 61–67. doi: 10.1093/molehr/ 3.1.61

• 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

• Caroppo E. (2013) Sperm sorting for selection of healthy sperm: is it safe and useful?. Fertil Steril 100; 695-6

• Said T and Land J (2011) Effects of advanced selection methods on sperm quality and ART outcome: A systematic review. Hum Reprod Update 17; 719-33