Development of a rapid, sensitive, and reproducible laboratory test kit for the assessment of plasma membrane integrity of human sperm.
OBJECTIVE
To develop a rapid, sensitive, and reproducible hypoosmotic swelling test kit for the assessment of plasma membrane integrity of human sperm in vitro.


METHODS
Prospective comparison of results with the World Health Organization (WHO) method, performed simultaneously.


METHODS
Infertility center in a major city in India.


METHODS
Couples who presented for infertility evaluation.


METHODS
The sperm tail-coiling pattern representing sperm plasma membrane integrity was analyzed by using different concentrations of NaCl and also with plain double distilled water (ddH(2)O). Hypoosmotic swelling solution with 2% NaCl in ddH(2)O equivalent to 68 Osm/L was selected for further analysis because it provided the highest and the qualitatively best type (g) of tail coiling among the various other options tried in comparison.


METHODS
A rapid laboratory test kit for the assessment of plasma membrane integrity of sperm was developed that is equally sensitive and reproducible as that described in the WHO protocol. The test was validated by using different normal and subnormal semen samples and in comparison with the standard WHO protocol.


RESULTS
After the screening with different hypoosmotic solutions by using semen samples from fertile volunteers, NaCl (2%) provided the highest and the best types of typical tail coiling, characteristic of sperm with good plasma membrane integrity. The study was then extended to 60 normozoospermic semen samples, which demonstrated a hypoosmotic swelling response of 69.5% +/- 5.23% and 71.5% +/- 4.89%, as per WHO or the present modified method, respectively (coefficient of correlation, r = 0.741). In 13 oligospermic and 18 teratozoospermic subjects, the hypoosmotic swelling response of sperm as per the new method were 36.4% +/- 5.75% and 34.2% +/- 7.78%, respectively, which were comparable to those obtained through the WHO method. There was a 15% drop in viability of sperm after the hypoosmotic challenge (5 min), irrespective of the hypoosmotic solution used. No other morphological alterations in sperm were observed after the hypoosmotic challenge.


CONCLUSIONS
The new test kit can be used routinely in laboratories for assessment of plasma membrane integrity of sperm in vitro.
Misro
Man Mohan
MM
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi, India. mm_misro@yahoo.com
Chaki
Sankar Prasad
SP
D002462:Cell Membrane / Q000473:pathology*; D048429:Cell Size; D002470:Cell Survival; D006801:Humans; D007038:Hypotonic Solutions / Q000737:chemistry; D007248:Infertility, Male / Q000175:diagnosis* / Q000473:pathology; D008297:Male; D009845:Oligospermia / Q000175:diagnosis / Q000473:pathology; D009997:Osmotic Pressure; D011446:Prospective Studies; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012965:Sodium Chloride / Q000737:chemistry; D013082:Sperm Tail / Q000473:pathology*; D014944:World Health Organization
D007038:Hypotonic Solutions; D011933:Reagent Kits, Diagnostic; D012965:Sodium Chloride