Apoptosis in germ cell in vitro in middle-aged and older men with chronic prostatitis under exposure to reprotoxicants (by the example of benzene)

Introduction. The issues of studying the causes and development mechanism of chronic prostatitis as the most important factor in the formation of male infertility are extremely relevant nowadays. Confirmed reprotoxicants including aromatic hydrocarbons inhibit spermatogenesis and lead to androgen deficiency.

Purpose: to identify the features of spermatozoa apoptosis in vitro in middle-aged and older men with chronic prostatitis under exposure to reprotoxicants (using benzene as an example).

Material and methods. A study of the ejaculate in thirty men with chronic prostatitis was conducted. 19 men were under 36 years old, 11 men were over 44 years old. Intracellular and membrane cell death markers (AnnexinV-FITC⁺PI^–, AnnexinV-FITC⁺PI⁺, Bax, caspase-3) in semen samples were identified by flow cytometry. Seminal fluid samples without benzene were control; samples with the addition of 0.001 µ/ml of benzene were experimental. Spontaneous and benzene-induced samples were incubated for 72 hours at 37 °C.

Results. The in vitro system revealed that the addition of 0.001 µg/ml benzene to the ejaculate of men under 36 years of age reduces the content of AnnexinV-FITC⁺PI^–-spermatozoa (p < 0.05). The content of CD95⁺- and AnnexinV-FITC⁺PI^–-spermatozoa in the sperm of men over 44 years of age was significantly reduced against the background of an increase in Bax compared with the results of patients under 36 years of age (p < 0.05).

Limitations. The study limitations are the use of specific cellular material — gametes, as well as indicators of cell differentiation clusters reflecting the cell death.

Conclusion. In vitro experiment verification features of sperm lethal program in men with chronic prostatitis revealed the signs of apoptosis inhibition associated with age and a chemical modifier — benzene. We revealed the signs of the violations in mitochondrial regulation and phosphatidylserine realization of programmed germ cell death in older men relative to young men were revealed. It is recommended to use the content of Bax, CD95⁺- and AnnexinV-FITC⁺PI^–-spermatozoa in an ejaculate as the diagnostic predictors of sperm fertility disorders in men = with chronic prostatitis under benzene exposure.

Compliance with ethical standards. The study was carried out in accordance with the norms of the Helsinki Declaration of the WMA “Ethical principles of medical research with human participation as a subject” (1964, 2013). The study was approved by the LEC of the Federal Scientific Center for Medical and Preventive Health Risk Management Technologies (protocol No. 1 of 22.01.2020). All participants signed a voluntary consent to participate in the study.

Contribution of the authors:
Dolgikh O.V. — concept and design of the study, editing;
Alekseev V.B. — concept and design of the study, editing;
Dianova D.G. — concept and design of the study, writing the text;
Alikina I.N. — collection and processing of material;
Nikonoshina N.A. — writing the text.
All authors are responsible for the integrity of all parts of the manuscript and approval of the manuscript final version.

Acknowledgment. The study had no sponsorship.

Conflict of interest. The authors declare no conflict of interest.

Received: July 28, 2023
Accepted: October 11, 2023
Published: December 23, 2023