The ejaculates of healthy fertile men (n=25) aged from 22-38 years old (y.o.) [(30.6±1.1 y.o. (mean ± SEM)], and of subfertile men (n=78) aged 25-48 y.o. (34.1±0.8 y.o.) were studied. The identification of normal and subfertile samples was based on the spermogram parameters described below. As the investigation was performed with participation of the Reproduction Center, the preliminary basis for the identification of samples as subfertile was also the inability for the donors to have children. The durations of ‘barren marriages’ ranged from 6 months to 15 years. No reproductive organ diseases were diagnosed in donors with abnormal spermograms, the donors did not abuse alcohol and were not smokers. The fertile samples were taken from the donors who had children in the previous 3 years.

First, we studied the influence of MW irradiation on spermatozoa from healthy donors. The whole ejaculate after complete liquefaction were examined for the standard spermogram parameters. Each sample was divided into three parts: Control _F, Exp1 _F and Exp2 _F. The Control _F was not exposed to the MW irradiation, Exp1 _F was exposed to the MW irradiation as a whole ejaculate, and Exp2 _F was centrifugated at 12,580 rcf for 15 min at room temperature, and divided into seminal plasma and sperm cells, after which the seminal plasma and spermatozoa were exposed to MW irradiation separately from each other. The Control _F and Exp1 _F group, after MW exposure, were then also centrifuged as above, and divided into seminal plasma and spermatozoa. The seminal plasma and spermatozoa from all three experiments were used for the specific assays. The spermogram parameters were also evaluated in the Exp1 _F and Exp2 _F groups after MW exposure. Next, the effect of MW treatment on spermatozoa with regard to metabolic processes was assessed. The ejaculates from donors with pathospermia (Control _S, Exp1 _S) were exposed to the MW treatment and processed similarly to those of healthy donors. Figs. 2 and S1 outlines the study design briefly.

The present study was performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) (18) and was approved by the Local Ethics Committee of the Astrakhan State Medical University of the Health Ministry of the Russian Federation (protocol no. 2; 21st May 2021). Informed consent for participation in the study and use of their samples was obtained from all participants.

The electromagnetic field was produced by a generator of monochromatic electromagnetic waves ‘Yav-1-7.1’ (Scientific Production Association, Istok). The MW parameters (λ=7.1 mm, υ=42.194 GHz) as well as the duration of the experiment have previously been determined to induce the intended bioeffects without a heating effect, and are recommended for clinical and experimental use (4,5,19). The absence of a thermal effect ensured the specificity of biological action (5,19,20).

The assessment of the primary standard spermogram parameters was performed according to the generally accepted methods recommended by WHO and leading experts (21,22). Microscopic studies of sperm were performed on a transmission electron microscope; morphology and motility characteristics were explored (21,22). The motile sperm cells were counted using a Goryaev chamber. All these parameters were determinants for the verifying the samples as normal or subfertile ones at the zero point of the study. The biochemical investigations included membrane resistance tests, acrosin activity assay and apoptosis investigation by evaluation of PS externalization by AnnexinV-propidium iodide (PI) staining.

The sperm membrane resistance to the 1% sodium chloride solution (Milovanov test) was explored (23). Briefly, the ejaculate was diluted gradually with 1% sodium chloride solution and incubated for 3 min at 37˚C with counting of motile sperm cells. The first dilution (1:500) was performed by adding 10 ml 1% sodium chloride solution to 0.02 ml ejaculate, next dilutions (1:1,000, 1:2,000, 1:4,000, 1:6,000, etc.) were obtained by adding 0.5 ml volumes of 1% NaCl solution to the corresponding volumes of the first diluted (1:500) solution. The number of motile sperms were counted in ejaculate samples before and 3 min after each dilution under a light microscope (magnification, x100), using a Goryaev chamber, and accordance with methods recommended by WHO and leading experts (21,22). The diluting was performed gradually starting from 1:500 up to the complete cessation of spermatozoa motility (23). The higher the dilution at which the motility remained, the greater the membrane resistance was considered to be. The membrane resistance was evaluated in equivalent units (EU) corresponding to the degree of dilution; for example, if the highest dilution maintaining sperm motility was 1:4,000, then the resistance was considered 4,000 EU. The mean ± SEM were then calculated.

The membrane resistance to acetic acid was evaluated in the Joel test (23). The ejaculate was diluted with 0.5% acetic acid solution (1:1) and incubated at 37˚C. At 0 h (before the dilution) and every 10 min after dilution, the number of motile spermatozoa was counted in the ejaculate samples, up to the complete cessation of motility. The duration of motility in these conditions was a marker of membrane resistance.

The spermatozoa were washed using PBS (pH 6.5) to remove the seminal plasma and extracted with 0.2 M acetate buffer (pH 2.4). The activity of free acrosine (EC 3.4.21.10) was determined spectrophotometrically as described by Schill (24). Briefly, benzoylarginine ethyl ester was hydrolyzed by acrosine producing ethanol, the latter was then assayed in an alcohol dehydrogenase reaction with formation of NADH(H⁺) detected at 366 nm (24) on the Novaspec III spectrophotometer (Amersham Biosciences). The total activity of acrosin was determined following rapid thawing (at 23˚C for 30 min) of the pre-frozen (at -196˚C liquid nitrogen) ejaculate. The pro-enzyme activity was calculated by subtracting the activity of free acrosine from the total acrosine activity. Acrosin activity was expressed in international units per 1 million spermatozoa (µU/10⁶ cells) (24,25).

Sperm cells were washed to remove the seminal plasma and incubated in Menezo B-2 medium (BioMerieux) in a humidified incubator with 5% CO₂ at 37˚C. The apoptotic process of gametes was assessed using fluorescein-labeled Annexin V (AnV), which binds to PS residues on the membrane surface of apoptotic cells, and PI, a fluorescent DNA dye that allows differentiation of cells with damaged membranes from those with intact membranes. The FITC Annexin V Apoptosis Detection Kit was purchased from BD Biosciences, Inc, and used according to the manufacturer's protocol. During the early stage of apoptosis, cells bind AnV, but like living cells, are impermeable to PI, therefore, they are AnV+/PI-. The percentage of such cells among the total number of cells was calculated (10,11). The microscopic studies were performed on a fluorescent microscope at a magnification of x100. The AnV+/PI-(green fluorescence without red fluorescence) cells were counted using a Goryaev chamber.

PA was extracted from seminal plasma using n-butanol (1:1; pH=10) for 1 h at room temperature, and after evaporation of the butanol phase, the remaining solution was electrophoretically separated in 1.5% agar gel with 0.1 M citric acid buffer (pH=3.4-3.6) for 1 h at room temperature, with a voltage of 200 V and an amperage of 40 mA (Patent for invention RUS no. 2225981 dated 28.02.2002). Spm and Spd were visualized as pinkish-violet spots after staining with ninhydrin and identified using standard solutions (Fluka). The amount of PA in the sperm plasma of each man was determined using scanning electrophoregrams, transferring the image into a digital format on a PC using a specific computer program ‘PN 5108’ (certificate of registration of a computer program: RUS 2003612170 dated 21.07.2003). The concentrations of Spm and Spd were calculated using a classical calibration curve (plots reflecting the concentration-dependent peak areas for the standard PA solutions). The method was tested with natural semen samples (n=10) and showed the linearity within the working range of 0.05-40 µmol/ml, the detection limit for seminal plasma PA was 0.064 µmol/ml, variability interval 0.4 µmol/ml, maximum relative error ≤15%.

Statistical evaluation was performed after checking the distribution for normality (Shapiro-Wilk test) in Statistica version 7.0 (StatSoft Inc.). Comparisons between the studied values were assessed using an unpaired Student's t-test (P<0.05) or a one-way ANOVA followed by a post-hoc Tukey's test for multiple comparisons in Microsoft Excel 2016 (Microsoft Corporation) or SPSS version 24 (IBM Corp.), respectively. Data are presented as the mean ± SEM. Assessment of the correlation between parameters was performed using a Pearson correlation coefficient analysis (r). P<0.05 was considered to indicate a statistically significant difference.

The standard spermogram characteristics were obtained for the sperm cells from Exp1-2 _F and Control _F samples (Fig. S2A). An extended cytological study of the spermatozoa of fertile men did not reveal significant changes in the number of the head (amorphous, small, tapered, round, double-headed forms, etc.), neck (‘bent’ neck, thin midpiece, etc.) or tail defects (two-tailed forms, a short tail, etc.) in the experimental samples after exposure to low-intensity MW irradiation compared to the Control Samples (P>0.05, Table SI). Also, no alterations in the motility of the spermatozoa were observed after MW treatment of fertile men spermatozoa both in Exp1 _F and Exp2 _F (P>0.05, Table SII).

The evaluation of the functional state of sperm membranes by their resistance to 1% sodium chloride solution revealed that a short-term exposure to MW radiation resulted in a 26% increase in membrane resistance when applied to the whole ejaculate (Exp1 _F), and in a 17% increase when the cells were separated from the plasma (Exp2 _F). These findings are summarized in Table I.

The Joel test showed a significant increase in spermatozoa resistance to acetic acid after the MW treatment. The differences in both the Control _F and the Exp _F were insignificant when incubated for <10 min, but after 20-30 min of incubation, the differences became statistically significant (P<0.05). Generally, the motility of sperm in samples after MW exposure was maintained for ~10 min longer than in Control _F samples (Table SIII).

The next step of the present study was the determination of apoptotic markers in spermatozoa. The results are presented in Table I; Fig. S3A and B, E and F. The content of АnV+/PI-sperms in the Control _F group was 10.47±0.50%. The percentage of these early apoptotic gametes was reliably reduced by almost 20% (P<0.01) in Exp1 _F after MW exposure. The changes in the content of АnV+/PI-spermatozoa in the Exp2 _F did not significantly differ rom the Control _F (P>0.05). To make the results of the short-term MW impact assessment more demonstrative, the apoptosis index (AI) was calculated as follows: AI=Apoptotic percentage the in experimental group/apoptotic percentage in the Control _F group.

This index shows the ratio of the AnV+/PI- gametes after exposure to the MW radiation (Exp1 _F) to the number of such cells in the Control _F group. AI values <1 (as in the Exp1 _F group) were indicative of inhibition of apoptosis in gametes. The sodium chloride resistance of sperm membranes after MW exposure (Exp1 _F) correlated inversely with the content of AnV+/PI- spermatozoa in ejaculates (r=-0.5; P<0.01) and with AI (r=-0.4; P<0.05).

The determination of the enzymatic activity of acrosine is used as a diagnostic test for evaluating spermatozoa fertilization in vitro (24,25). Serine proteinase EC 3.4.21.10 acrosine is one of the key acrosomal enzymes and plays an important role in the process of oocyte fertilization (25). However, there is no data on the functioning of the acrosine system under the influence of MW-irradiation in the available literature. The activity of free acrosine was significantly lower in the experimental samples, particularly in Exp1 _F, and was 87% of that observed in the Control _F group (Table II). The total activity of acrosin did not change compared with the Control _F group. The pro-enzyme activity of acrosin in ejaculate samples after MW-exposure compared to the control did not increase significantly, increasing by ~4% (Table II). The ratio of pro-acrosin/free acrosin was calculated, and its value increased on average by almost 20% in Exp1 _F compared with the Control _F (Table II).

The next step of the study was to reveal the role of the seminal plasma PA on the effect of MW on spermatozoa. The assessment of the PA content was performed on Control _F, Exp1 _F and Exp2 _F groups. The Control Samples showed that the concentration of Spm was 1.197±0.111 µmol/ml, and Spd was 1.265±0.150 µg/ml (Table III, Fig. 3). The MW treatment caused a significant decrease in the concentrations of both PAs in the Exp1 _F group (P<0.05). The Spd concentration changed to a greater degree, so these alterations were not just quantitative, but also involved the profile of PA content. The change in the spectrum of PA levels in the seminal plasma after MW exposure is illustrated by the shift in the Spm/Spd ratio (0.95→1.22). The PA concentrations after MW treatment were not altered in the absence of spermatozoa (Exp2 _F, P>0.05, Table III). However strong correlations (r=0.9, P<0.05) between the number of apoptotic cells and PA levels were observed for the Control _F and Exp1 _F samples.

The PA electrophoregram and densitogram of seminal plasma of a fertile man before and after the exposure of the ejaculate to low-intensity MW are shown in Fig. 3. Two separate spots can be seen on the platelets corresponding to Spd and Spm visualized in the ninhydrin reaction. An additional spot of unknown substance appears in the electrophoregram after MW exposure. This substance is present not far from the PA, therefore it may be considered as a substance similar to PAs. At the same time its velocity of migration is lower than that of PA, suggesting it has a different molecular weight/charge ratio. Presumably this spot might belong to an acetylated Spd or Spm. Unfortunately, it was not possible to determine the identity of the molecule accounting for this spot due to a lack of standard components, and will thus form the focus of future investigations.

The results gained for the fertile samples provide evidence of the positive action of MW treatment, such as the increase in membrane stability and improved functionality of spermatozoa (lowered acrosin activity, inhibition of apoptosis). This effect was observed mainly for the complete ejaculates against the background of seminal plasma PA levels lowering, and was absent or less pronounced in the sperm cells exposed to MW in the absence of seminal plasma. The PAs did not ‘disappear’ from the spermatozoa-free plasma (Exp2 _F) after MW treatment. Taken together these results reveal the involvement of the PA system on the effect of MW upon sperm cells.

The investigation of the influence of MW irradiation on spermatozoa of subfertile samples was performed in order to reveal any potential therapeutic effects (Fig. S2B; Fig. S3C and D). The complete ejaculates were tested only (Control _S and Exp1 _S).

Due to the large variability in the parameters of spermograms of the patients in the group of pathospermia, the changes in the morphology of the gametes, in the functional state of their membranes and in PS externalization as a marker of sperm apoptosis, were not statistically significant (all P>0.05). The reaction of subfertile spermatozoa to MW differed from that of the normozoospermia samples. In particular, the number of progressively motile gametes increased to 24% (Exp1 _S) from 16% (Control _S), while the number of non-progressively motile spermatozoa decreased from 20% in Control _S to 12% in Exp1 _S (P<0.05, Table IV). The quantity of immotile spermatozoa did not change significantly (P>0.05, Table IV).

First the levels of PA in the spermoplasm of subfertile men revealed that their levels drastically differed from those of fertile men, in particular the residual concentration of Spm and Spd was 58 and 15% from that of healthy donors, respectively. The study of the dynamics of the PA in the subfertile spermoplasm after MW treatment revealed two types of responses. In the first type, there was no significant change in the levels of PA, there was only a slight tendency to an increase in the levels of Spd, although this increase was not significant. In the second type of response, both Spm and Spd levels were significantly decreased, which resembled the response in normozoospermia and led to an increase in the Spm/Spd ratio.