Methods for determining human sperm quality
The present invention relates generally to the fields of reproductive medicine. More specifically, the present invention relates to methods and kits for determining the human sperm quality.
1. A method for assessing sperm fertility rate in an infertile or hypofertile subject or a subject with difficulty to conceive for more than one year, comprising the steps of:
i) liquefying a semen sample obtained from said subject at 37° C. for 30 minutes,
ii) isolating spermatozoa from said semen sample through a single-step density gradient centrifugation,
iii) washing said isolated spermatozoa with tris-buffered saline,
iv) homogenizing said isolated spermatozoa with a lysis buffer to obtain a spermatozoa protein lysate,
v) measuring the expression level of one or both of a kinase anchoring protein 4 (AKAP4) and Hexokinase 1 in said protein lysate, and
vi) comparing said measured expression level with a reference value, wherein detecting a differential in the expression of the one or both of AKAP4 and Hexokinase 1 between the sample and the reference value is indicative of sperm fertility rate.
2. The method according to claim 1 wherein said subject is classified as a normozoospermic subject.
3. The method according to claim 1 , wherein said tris-buffered saline comprises 0.1 mM of HCl, 100 mM of NaCl, and has a pH equal to 7.6.
4. The method according to claim 1 , wherein step v) is performed by
analyzing said spermatozoa protein lysate obtained in step iv) in a 1D SDS-PAGE with blue staining,
providing a numerical analysis of said blue stained gel,
selecting protein bands between Low molecular weights 15 to 30 kDA and protein bands between High molecular weights 80 and 110 kDa,
representing each band by a densitometric curve and calculating the area under the densitometric curve, and
calculating a ratio between the area under the densitometric curve from Low molecular weight and High molecular weight band intensities.
5. The method according to claim 4 wherein a ratio between 1 to 2 is indicative of good sperm quality, and a ratio above 2 is indicative of bad sperm quality.
6. The method according to claim 3 , wherein step v) is performed by
analyzing said spermatozoa protein lysate obtained in step iv) in a 1D SDS-PAGE with blue staining,
providing a numerical analysis of said blue stained gel,
selecting protein bands between Low molecular weights 15 to 30 kDA and protein bands between High molecular weights 80 and 110 kDa,
representing each band by a densitometric curve and calculating the area under the densitometric curve, and
calculating a ratio between the area under the densitometric curve from Low molecular weight and High molecular weight band intensities.
7. A method enabling an infertile or hypofertile subject or a subject with difficulty to conceive for more than one year to determine the most successful assisted-reproductive technology comprising the steps of:
i) liquefying a semen sample obtained from said subject at 37° C. for 30 minutes,
ii) isolating spermatozoa from said semen sample through a single-step density gradient centrifugation,
iii) washing said isolated spermatozoa with tris-buffered saline,
iv) homogenizing said isolated spermatozoa with a lysis buffer to obtain a spermatozoa protein lysate,
v) measuring the expression level of one or both of AKAP4 and Hexokinase 1 in said protein lysate, and
vi) comparing said measured expression level with a reference value, wherein detecting a differential in the expression of the one or both of AKAP4 and Hexokinase 1 between the sample and the reference value is indicative of a given assisted reproductive technology.
8. The method according to claim 7 , wherein step v) is performed by
analyzing said spermatozoa protein lysate obtained in step iv) in a 1D SDS-PAGE with blue staining,
providing a numerical analysis of said blue stained gel,
selecting protein bands between Low molecular weights 15 to 30 kDA and protein bands between High molecular weights 80 and 110 kDa,
representing each band by a densitometric curve and calculating the area under the densitometric curve, and
calculating a ratio between the area under the densitometric curve from Low molecular weight and High molecular weight band intensities,
wherein when said ratio is above 2 choosing intrauterine insemination (IUI) as an assisted-reproductive technology.
9. The method according to claim 7 , wherein said tris-buffered saline comprises 0.1 mM of HCl, 100 mM of NaCl and has a pH equal to 7.6.
10. The method according to claim 7 , wherein said subject is classified as a normozoospermic subject.