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Updated: Sep 27, 2026
The Examination of Peroxidase-Positive Leukocytes in Semen
Published on: January 19, 2024
[Isolation and identification of spermatids in semen of male infertile patients]
Pei-Yuan Zhu1, Yu-Feng Huang, Ying-Xia Cui
1Laboratory of Reproduction & Genetics, Nanjing General Hospital of Nanjing Command, Nanjing, Jiangsu 210002, China.
Objectives:
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.
Insights
This study presents a new method to isolate purified spermatids from infertile men. The modified Percoll gradient centrifugation effectively yields large populations of spermatids for potential use in fertility treatments.
Area of Science:
- Reproductive Biology
- Andrology
- Cell Biology
Background:
- Male infertility affects a significant portion of the population.
- Isolation of pure spermatids is crucial for advanced reproductive technologies.
- Current methods for spermatid isolation face challenges in purity and yield.
Purpose of the Study:
- To develop and validate a method for isolating large, purified populations of spermatids.
- To improve the efficiency of spermatid isolation from semen of infertile patients.
Main Methods:
- Utilized a modified discontinuous Percoll gradient centrifugation technique.
- Analyzed ejaculates from infertile patients with various andrological pathologies.
- Employed morphology, flow cytometry (FCM), and immunocytochemistry for cell analysis.
Main Results:
- The 22% Percoll fraction yielded the highest purity of spermatids (91.85 +/- 5.18%).
- Flow cytometry and morphology confirmed spermatid enrichment in the 22% fraction.
- Leukocytes were primarily found in the 60% fraction, indicating successful separation.
Conclusions:
- The modified discontinuous Percoll gradient centrifugation is effective for isolating purified spermatids.
- This method offers a viable approach for obtaining sufficient spermatid populations from infertile men.
- The technique holds potential for improving assisted reproductive technologies.
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