DNA-fragmented sperm ability to bind to the zona pellucida

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Abstract

BACKGROUND: A high percentage of the infertile couples are classified as having unexplained infertility. At the same time, male partners may have both normozoospermia and high level of sperm DNA fragmentation. The investigation of the selectivity of the oocyte zona pellucida for spermatozoa with fragmented DNA is an extremely promising direction. The novel data could improve sperm selection methods, which in turn will increase the efficiency of IVF cycles.

AIM: The aim of this study was to assess the ability of spermatozoa with fragmented DNA to bind to the zona pellucida.

MATERIALS AND METHODS: The material for the study was ejaculate samples from patients with normozoospermia (n = 5) and sperm donors (n = 4), as well as the zona pellucida of immature oocytes (n = 25) from nine patients. Sperm preparation was carried out by centrifugation in a density gradient of silicone particles and the swim-up method. Sperm DNA fragmentation was assessed using the method of fluorescent labeling of single- and double-stranded DNA breaks (TUNEL assay). Manipulations with gametes and the zona pellucida were performed using micromanipulation equipment and a femtosecond laser.

RESULTS: Spermatozoa with fragmented DNA can effectively bind to the oocyte zona pellucida. However, among the zona pellucida-bound spermatozoa, the proportion of cells with fragmented DNA was significantly lower than among the unbound ones. The data obtained suggest the key role of the oocyte zona pellucida in the selection of spermatozoa with intact DNA.

CONCLUSIONS: Spermatozoa with fragmented DNA retain the ability to bind to the oocyte zona pellucida. The use of the selective binding of spermatozoa with intact DNA to the zona pellucida can serve as a method for selecting gametes for intracytoplasmic injection of spermatozoa into the oocyte. Data on the selective properties of the oocyte zona pellucida can serve to select the optimal strategy in cases of “hidden” male factor, in which men with normozoospermia experience impaired fertility, in particular, due to sperm DNA fragmentation.

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About the authors

Mariia A. Ishchuk

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

Email: mashamazilina@gmail.com
ORCID iD: 0000-0002-4443-4287
SPIN-code: 1237-6373
Russian Federation, Saint Petersburg

Evgeniia M. Komarova

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

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

Cand. Sci. (Biol.)

Russian Federation, Saint Petersburg

Elena A. Lesik

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

Email: lesike@yandex.ru
ORCID iD: 0000-0003-1611-6318
SPIN-code: 6102-4690

Cand. Sci. (Biol.)

Russian Federation, Saint Petersburg

Irina D. Mekina

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

Author for correspondence.
Email: irendf@mail.ru
ORCID iD: 0000-0002-0813-5845
SPIN-code: 4682-8590

Cand. Sci. (Biol.)

Russian Federation, Saint Petersburg

Anna A. Pendina

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

Email: pendina@mail.ru
ORCID iD: 0000-0001-9182-9188
SPIN-code: 3123-2133

Cand. Sci. (Biol.)

Russian Federation, Saint Petersburg

Alexander M. Gzgzyan

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

Email: agzgzyan@gmail.com
ORCID iD: 0000-0003-3917-9493
SPIN-code: 6412-4801

MD, Dr. Sci. (Med), Professor

Russian Federation, Saint Petersburg

Natalia I. Tapilskaya

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

Email: tapnatalia@mail.ru
ORCID iD: 0000-0001-5309-0087
SPIN-code: 3605-0473

MD, Dr. Sci. (Med.), Professor

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

MD, Dr. Sci. (Med.)

Russian Federation, Saint Petersburg

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Supplementary files

Supplementary Files
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1. JATS XML
2. Fig. 1. The flowchart of the study. GV, germinal vesicle stage; MI, metaphase I of meiosis; MII, metaphase II of meiosis

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3. Fig. 2. The number of zona pellucida-bound spermatozoa in patients 1–9

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4. Fig. 3. Halves of the oocyte zona pellucida after washing from unbound spermatozoa: a, following the addition of the donor’s ejaculate; b, following the addition of the patient’s ejaculate. Phase contrast, zoom ×400

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5. Fig. 4. Sperm DNA fragmentation level among the zona pellucida-bound and unbound spermatozoa. ZP — zona pellucida (Me, Q1–Q3; a box and whisker plot; Mann–Whitney U test, p = 0.049)

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СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
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