[Isolation and identification of spermatids in semen of male infertile patients].
OBJECTIVE
To develop a method by which large and purified populations of spermatids can be isolated in semen of male infertile patients.


METHODS
A total of fifteen ejaculates containing cellular elements from infertile patients with various andrological pathologies were obtained after a 24-hour abstinence. A modified discontinuous Percoll gradient (15%, 22%, 30%, 40%, 50%, 60%) centrifugation method was used to isolate the spermatids. After centrifugation at 2,000 r/min for 30 min at 18 degrees C, the single Percoll fractions were separated and analyzed in order to select the one with the greatest purity of spermatid. The germinal cells in each isolated fraction were counted using a Macro sperm counting chamber, then the contents of spermatids were determined by morphology (Wright-Giemsa staining method) and flow cytometry (FCM) analysis, while the contaminated leukocytes were assessed by anti-CD45 immunocytochemistry.


RESULTS
After Percoll centrifuged, six single fractions were obtained. Morphology and FCM analysis showed that the 22% fraction contained mostly spermatids [(91.85 +/- 5.18)%, P < 0.005] and the mean density in this fraction was (1.010 +/- 0.786) x 10(5)/ml. While in the 30% fraction, various immature spermatogenic cells including spermatids were present and leukocytes mostly presented in the 60% fraction.


CONCLUSIONS
A large population of relatively purified spermatids can be isolated from the ejaculates of infertile patients by using this modified discontinuous Percoll gradient centrifugation method.
Zhu
Pei-Yuan
PY
Laboratory of Reproduction & Genetics, Nanjing General Hospital of Nanjing Command, Nanjing, Jiangsu 210002, China.
Huang
Yu-Feng
YF
Cui
Ying-Xia
YX
Hu
Yu-An
YA
Xu
Jian-Ping
JP
D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D007248:Infertility, Male / Q000473:pathology*; D017493:Leukocyte Common Antigens / Q000032:analysis; D007962:Leukocytes / Q000737:chemistry / Q000166:cytology; D008297:Male; D012661:Semen / Q000166:cytology*; D013087:Spermatids / Q000737:chemistry / Q000166:cytology*
D017493:Leukocyte Common Antigens