Sperm DNA fragmentation and hydroxymethylation in normozoospermic and pathospermic patients



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Abstract

Background. The search for new criteria allowing to assess the ejaculate quality based on the structural and functional characteristics of the sperm genome is of high importance for reproductive science.

Aim – analysis of DNA integrity and 5-hydroxymethylcytosine (5hmC) content in the same ejaculated spermatozoa obtained from normozoospermic and pathospermic patients.

Materials and methods. The study included ejaculate samples from 63 patients with normozoospermia (n=33) and pathospermia (n=30). Microscope slide preparations were made to detect DNA fragmentation by TUNEL, record digital images, detect 5hmC by indirect immunofluorescence, and record digital images of the same fields of view again. A total of 126,000 spermatozoa were analyzed - 2,000 in each sample.

Results. A significant proportion of spermatozoa (72.8–94.2%) in all samples were characterized by the absence of DNA fragmentation and very low 5hmC levels. The frequency of spermatozoa with DNA fragmentation, increased hydroxymethylation level, or both characteristics varied between samples and equaled 0.05–13.8%, 0.15–11.5%, and 0.99–13.38%, respectively. The frequency of spermatozoa with DNA fragmentation and/or hyperhydroxymethylation did not differ between ejaculates of normozoospermic and pathospermic patients. A relationship between DNA fragmentation and hyperhydroxymethylation and their nonrandom combination in ejaculated spermatozoa was confirmed.

Conclusions. The non-random combination of DNA fragmentation and hyperhydroxymethylation in spermatozoa and the relationship between these characteristics suggest their joint trigger, which is most likely oxidative stress. The presence of other factors leading to DNA damage and increase of 5hmC level explains the non-100% coincidence of these characteristics in spermatozoa. Evaluation of DNA fragmentation and the level of its hydroxymethylation in spermatozoa seems promising for ejaculate quality assessment and identifying potential causes of idiopathic infertility.

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About the authors

Yanina M. Sagurova

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: yanina.sagurova96@mail.ru
ORCID iD: 0000-0003-4947-8171
SPIN-code: 8908-7033

мл. научн. сотр.

Russian Federation, Saint Petersburg

Olga A. Efimova

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: efimova_o82@mail.ru
ORCID iD: 0000-0003-4495-0983
SPIN-code: 6959-5014
Scopus Author ID: 14013324600
ResearcherId: F-5764-2014

Cand. Sci. (Biol.)

Russian Federation, Saint Petersburg

Mikhail I. Krapivin

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: krapivin-mihail@mail.ru
ORCID iD: 0000-0002-1693-5973
SPIN-code: 4989-1932
Scopus Author ID: 56507166200

Junior Researcher

Russian Federation, Saint Petersburg

Mariia A. Ishchuk

Научно-исследовательский институт акушерства, гинекологии и репродуктологии им. Д.О. Отта

Email: mashamazilina@gmail.com
ORCID iD: 0000-0002-4443-4287
SPIN-code: 1237-6373

мл. научн. сотр.

Russian Federation, Санкт-Петербург

Dmitry A. Staroverov

Научно-исследовательский институт акушерства, гинекологии и репродуктологии им. Д.О. Отта

Email: st110982@student.spbu.ru
ORCID iD: 0009-0004-9716-4964
SPIN-code: 3438-7974

Лаборант-исследователь

Russian Federation, Санкт-Петербург

Ekaterina D. Trusova

Научно-исследовательский институт акушерства, гинекологии и репродуктологии им. Д.О. Отта

Email: trusova.ek@mail.ru
ORCID iD: 0009-0005-6529-5799

Лаборант-исследователь

Russian Federation, Санкт-Петербург

Evgeniia M. Komarova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: evgmkomarova@gmail.com
ORCID iD: 0000-0002-9988-9879
SPIN-code: 1056-7821
Scopus Author ID: 57191625749

Cand. Sci. (Biol.), head of Laboratory of preimplantation development

Russian Federation, Saint Petersburg

Olesya N. Bespalova

The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O. Ott

Email: shiggerra@mail.ru
ORCID iD: 0000-0002-6542-5953
SPIN-code: 4732-8089
Scopus Author ID: D-3880-2018

MD, Dr. Sci. (Med.)

Russian Federation, 3 Mendeleevskaya Line, Saint Petersburg, 199034

Anna A. Pendina

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology; Saint Petersburg State University

Author for correspondence.
Email: pendina@mail.ru
ORCID iD: 0000-0001-9182-9188
SPIN-code: 3123-2133
Scopus Author ID: 6506976983

Cand. Sci. (Biol.), senior research associate

Russian Federation, Saint Petersburg; Saint Petersburg

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