Characterization and distribution of HIV-infected cells in semen

Semen is a known vector for both human immunodeficiency virus (HIV) infection and transmission. However, the distribution and characteristics of HIV-infected cells in semen remain unclear. Investigating the possibility of transmission through the spermatozoon in semen is of great clinical significance to improve the strategies for exposure prevention and assisted reproduction for HIV-infected partners. Twenty-six HIV-infected patients, including twelve treatment-naïve (TN) patients and fourteen antiretroviral treated (ART) patients, were enrolled in this study. HIV p24 protein in spermatozoa was detected using imaging flow cytometry and immunohistochemistry, and HIV RNA was identified using next-generation RNAscope <i>in situ</i> hybridization. Additionally, we described the rates of HIV-positive spermatozoon and CD4⁺ T lymphocytes in semen, and found that p24⁺ spermatozoon were mainly CD4 negative regardless of whether the patients received ART. Of note, p24-positive cells in semen are predominantly spermatozoa, and we confirmed that motile spermatozoa carried HIV into peripheral blood mononuclear cells of healthy men <i>in vitro</i>. Our findings provide evidence regarding the risk of HIV-infected spermatozoa.