Shining a light on male infertility: the power of near-infrared photobiomodulation therapy for human asthenozoospermia

doi:10.21203/rs.3.rs-4767335/v1

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Shining a light on male infertility: the power of near-infrared photobiomodulation therapy for human asthenozoospermia

https://doi.org/10.21203/rs.3.rs-4767335/v1

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Sperm motility is a crucial factor in male fertility. Photobiomodulation (PBM) has been reported to increase sperm motility but lacks a standardized protocol. We collected asthenozoospermic (n=70) and normozoospermic (n=20) semen. The asthenozoospermic samples were irradiated with an 810 nm diode laser at 0.25W, 0.5W, 1W, and 2W. Sperm motility was assessed immediately, after 30 and 60 minutes. A sample size calculator, unpaired t-test and one-way ANOVA with post-hoc Tukey HSD tests were used for statistics. One and 2 W were the most effective outputs in increasing progressive motility compared to control (p<0.001). The maximum effect was immediately after 1 W-PBM (p<0.001), and decreased after 60 minutes (p<0.001). Time physiologically decreased vitality (p<0.001), but less in the 1W-PBM samples (p<0.05). 1W-PBM did not affect chromatin condensation. Asthenozoospermic samples displayed an impairment of 80% in oxygen consumption and ATP production and a slight inefficiency of oxidative phosphorylation compared to normozoospermic samples (p<0.001). 1W-PBM partially restored the functionality of aerobic metabolism (p<0.001) by recovery of oxidative phosphorylation efficiency. PBM did not affect lactate dehydrogenase (glycolysis pathway). No irradiated samples increased accumulated malondialdehyde, a marker of lipidic peroxidation. In conclusion, PBM improves progressive motility in asthenozoospermia through increased mitochondrial energetic metabolism without harmful oxidative stress.

Biological sciences/Cell biology

Physical sciences/Optics and photonics

Health sciences/Diseases/Reproductive disorders

low level laser therapy

asthenozoospermia

sperm motility

mitochondrial metabolism

DNA fragmentation

sperm vitality

male fertility

Infertility is a global public health problem that affects about 10% of couples. Approximately 50% of cases involve a male factor alone (30%) or combined with a female factor (20%) ¹. The prevalence of male infertility is steadily increasing worldwide as semen quality and other markers of male reproductive health have declined considerably over the past decade ². Sperm motility is a crucial parameter in male fertility, and it is affected by energy consumption ³.

Mature spermatozoa consist of a head with a nucleus, a neck, and a flagellum that allows motility. It comprises three parts; mitochondria are present only in the midpiece ⁴. They are tightly interconnected and helically wound around the axoneme, forming the mitochondrial sheath. In humans, 10–12 mitochondrial cores are formed by 72–80 oblong mitochondria ⁵. The primary function of mitochondria is to produce energy. Mammalian sperm uses ATP for motility, capacitation, hyperactivation, acrosomal reaction, and oocyte penetration. However, there is still debate about the metabolic pathways responsible for energy production in spermatozoa ^4,6. Indeed, oxidative phosphorylation (OxPhos) and anaerobic glycolysis are not mutually exclusive, and sperms can use both pathways. Makai and Travis ⁷ have suggested that ATP production in the mitochondrial membrane may not be sufficient for diffusion throughout the flagellum. It has been postulated that mitochondria produce ATP through OxPhos in the midpiece and that anaerobic glycolysis provides ATP in the midpiece. Piomboni et al. ⁸ reviewed the literature on mitochondrial energy production and its role in human sperm motility and found a link between structural abnormalities and impaired sperm mitochondrial function in asthenozoospermia.

The possibility of energizing mitochondria through the irradiation of visible or near-infrared light has attracted increasing interest over the last 40 years, starting with the first evidence suggested by Karu et al. ^9,10 and later by Passarella et al. ¹¹ with a wavelength of about 630 nm. Recently, the possibility of stimulating photoacceptors in mitochondrial complexes with different wavelengths in the near-infrared has been characterized. All the research showed increased ATP production through the photo-energization of cytochromes. In particular, a diode laser light of 810 nm or 980 nm mainly modulated complex IV and, to a lesser extent, III, while 1064 nm stimulated complexes I, III, and IV ^12–16. Medical and veterinary use of this phototherapy is known as photobiomodulation (PBM), formerly referred to as low-level laser therapy. It is used to support recovery from various diseases often associated with mitochondrial dysfunction ^17–21.

Reports on animals suggest that PBM may improve spermatogenesis ^22–24 or increase sperm motility ^25–31 in fish, mice, dogs, boar, ram, and cattle models.

Several studies conducted in vitro on human spermatozoa highlighted the potential benefits of PBM on sperm function ^20,32–35. Almost all studies have examined the positive impact of PBM therapy on the increase in the motility of human sperm from healthy subjects ^36–42. Some studies included sperm from oligozoospermic and asthenozoospermic patients and showed that irradiation significantly increased sperm motility also in abnormal sperm ^{37,39,42–44}. In two studies, PBM therapy before cryopreservation, even in normozoospermic semen samples, played a protective role against the detrimental effects of cryopreservation by preserving the functional parameters of spermatozoa ^45,46.

Overall, as often happens in the field of PBM therapy, standardization of application protocols remains a challenge due to the heterogeneity of population characteristics and sample size, the type of intervention, and the different LED/laser light parameters (in terms of wavelength, output power, distance, energy density, and other characteristics of the radiation source), duration of exposure and manifestation of effects. In addition, the biological mechanisms of PBM on sperm are not fully known, and limited safety assessments are available sometimes with conflicting data. For instance, Lenzi et al. ³⁶ have shown that an increase in progressive sperm motility after laser irradiation (647 nm) was associated with a faster rate of sperm ATP consumption, suggesting that laser irradiation may have an “energy modulation effect” on normal sperm. However, later, it was observed that near-infrared radiation resulted in a loss of cell viability, increased lipid peroxidation leading to diminished membrane function, and higher susceptibility of sperm DNA to fragmentation, resulting in induction of apoptosis ⁴⁷.

Our work investigated how PBM with 810 nm wavelength impacts the sperm of men with asthenozoospermia, even considering the possible harmful side effects. To validate an optimized protocol, we first identified the laser power that maximized sperm parameters. Then, we analyzed the impact of PBM on sperm motility and its duration after irradiation. The effectiveness of the procedure was tested by monitoring the OxPhos and the lactate dehydrogenase (LDH) involved in the glycolytic energy pathway. Safety was assessed through analysis of sperm vitality, DNA fragmentation, and lipidic peroxidation.

2.1 Technical characteristics of the equipment used and the irradiation setup

The irradiations were done with the ENEA GaAl-As diode laser model (Garda Laser S.A.S., Verona, Italy). The device allowed irradiating at a wavelength of 810 nm ± 2 for 60 seconds in continuous wave mode. The irradiation power was set at 0.25, 0.5, 1.0, or 2.0 W to generate an energy of 15.0, 30.0, 60.0, or 120.0 J (power density of 0.25, 0.5, 1.0, or 2.0 W/cm²; fluence of 15.0, 30.0, 60.0 or 120.0 J/cm²). The control samples' power was set to 0.0 W. Irradiations were delivered using a flat profiled handpiece (FT-HP). Our previous FT-HP characterization showed that it can provide a uniform and consistent energy distribution over a spot area of 1 cm², independent of distance ^48,49. A 635 nm red light pointer (negligible power, <0.5 mW) was used in both treatments to visualize the exposed area and maintain experimental blinding. A Pronto-250 power meter (Gentec Electro-Optics, Inc. G2E Quebec City, Canada) was used to ensure the accuracy of the irradiated laser parameters. The irradiations were carried out with the handpiece fixed to a stand and in contact mode with the surface of the multiwells plate (diameter of a single well approximately 1.1 cm). All irradiations were performed with the multiwells plate placed on a Metal Velvet light-absorbing plate (Acktar Ltd., 8643 Kiryat-Gat, Israel). The extremely low reflectance of the plates, which is guaranteed to be equal to or less than one percent in the near-infrared band, significantly reduces reflections from the top of the bench. Any undesirable thermal effects were avoided by monitoring the exposure with a FLIR ONE Pro-iOS thermal camera (FLIR Systems, Inc., Portland, OR, USA) (dynamic range: -20°C/+400°C; resolution 0.1°C).

2.2 Study population and study design. We collected semen samples from donors undergoing semen analysis for fertility diagnosis at the SS Physiopathology of Human Reproduction, IRCCS Ospedale Policlinico San Martino, Genova, Italy, from December 2023 to May 2024. The study complied with the Declaration of Helsinki ⁵⁰ and was approved by the Ethics Committee of Regione Liguria (ID 13852). Written informed consent was obtained from all donors, and participation was voluntary. The following parameters were checked based on WHO criteria ⁵¹: semen volume, sperm count per milliliter, sperm motility, and percentage of sperm with normal morphology. Inclusion criteria: asthenozoospermia and normozoospermia. Exclusion criteria: semen with sperm agglutinations, round cells, bacterial contamination, severe oligospermia, genetic syndrome.

PBM therapy for sperm was carried out in native ejaculates. Each asthenozoospermic sample was divided into 5 equal aliquots, four of which were irradiated at different powers (0.25 W, 0.5 W, 1 W, 2 W), respectively. One aliquot served as a control and received laser treatment with 0.0 W. A second operator trained in laser therapy then treated the samples. Sperm motility was assessed in a blinded manner immediately after irradiation (T0), after 30 (T30) and 60 minutes (T60), by other experienced operators. Sperm chromatin dispersion and membrane integrity were assessed blindly in untreated and 1 W irradiated asthenozoospermic samples after 60 minutes. Energetic metabolism, oxidative stress, and oxidative damage were analyzed in normozoospermic samples as a reference for analyzing metabolism and control and 1 W irradiated asthenozoospermic samples. Figure 1 summarizes the experimental setup.

2.3 Semen collection.

All individuals collected semen by masturbation into a sterile container after 2 to 5 days of abstinence. Sperm samples were liquefied at room temperature for 30–60 minutes.

2.4 Sperm motility.

Sperm motility was assessed using a Makler chamber. The sperm score for motility evaluation was expressed as follows: a = rapid progressive motility, b = slow progressive motility, c = in situ motility, and d = immobility. The progressive motility rate was calculated as the percentage of a + b.

2.5 Sperm vitality. To check for the safety of the sperm-PBM treatment, sperm vitality was estimated 60 minutes after irradiation by assessing the membrane integrity of the cells by dye exclusion (dead cells have damaged plasma membranes that allow entry of membrane-impermeant stains). Briefly, a 50-μl aliquot of semen was mixed with an equal volume of eosin (Life test, AB Analitica, Italy); after 30 seconds, it was smeared on a glass slide and immediately examined under an Eclipse Si microscope (Nikon Europe B.V., Amstelveen, The Netherlands) at 20X magnification. Spermatozoa with red or dark pink heads were considered dead, whereas spermatozoa with white heads were considered alive. A minimum of 200 sperms per sample was counted, and the percentage of live sperms was calculated.

2.5 Sperm DNA fragmentation. For further confirmation of the safety of PBM treatment, measurement of sperm DNA fragmentation was performed 60 minutes after irradiation. We used Halosperm^®G2 kit (Halotech DNA SL, Madrid, Spain) based on the Sperm Chromatin Dispersion (SCD) technique. Fresh sperms are immersed in an inert agarose microgel on a pretreated slide. An initial acid treatment denatures DNA in those sperms with fragmented DNA. Following this, a controlled DNA denaturation process facilitates the subsequent removal of most of the nuclear proteins in each spermatozoon. In this way, normal spermatozoa create halos formed by loops of DNA at the head of the sperm, which are not present in those with damaged DNA. When no massive DNA breakage is present, nucleoids from sperm with fragmented DNA either, do not show a dispersion halo or the halo is minimal. Spermatozoa without DNA fragmentation shows a dispersion halo, and spermatozoa with fragmented DNA do not show a dispersion halo, or the halo is minimal. A minimum of 300 sperms per sample were counted using conventional microscopy (Eclipse Si, Nikon) at 20X magnification, and the percentage of sperms with fragmented DNA was calculated. Values of DNA fragmentation above 30% were considered pathological.

2.6 OxPhos function evaluation

The OxPhos function, in terms of ATP synthesis and oxygen consumption rate (OCR), was assessed in 50 mg of total protein of normozoospermic samples, asthenozoospermic samples, PBM-treated asthenozoospermic samples immediately or after 30 minutes from the laser administration permeabilized with 0.01% digitonin and resuspended in phosphate buffered saline (PBS).

ATP synthesis was measured by a GloMax^® 20/20 Luminometer (Promega, Milan, Italy) at 30-second intervals over 2 minutes using the luciferin/luciferase methods. OCR was measured using an amperometric electrode (Unisense Microrespiration, Unisense A/S, Aarhus, Denmark) in a sealed chamber. For both assays, 10 mM pyruvate and 5 mM malate were used as respiring substrates and 0.1 mM ADP was added just before the start of the evaluations ⁵².

To assess the OxPhos efficiency the P/O value was calculated as the ratio between the produced ATP and the consumed oxygen. Values around 2.5 indicates a whole coupling between energy synthesis and respiration; lower values indicate an uncoupling status ^53,54.

2.7 Energy status evaluation

The energy status has been evaluated as the ratio between intracellular concentrations of ATP and AMP. ATP and AMP were quantified using the enzyme coupling method, employing 50 μg of total protein for each assay. ATP measurement involved monitoring NADP reduction at 340 nm and AMP level was evaluated following the NADH oxidation at 340 nm, as previously described ⁵⁵.

2.8 Lactate dehydrogenase activity assay

Lactate dehydrogenase (LDH; EC 1.1.1.27) activity was assessed using 20 μg of total protein by tracking NADH oxidation at 340 nm with a double-beam spectrophotometer (UNICAM UV2, Analytical S.n.c, Langhirano, Italy). The reaction mixture consisted of 100 mM Tris-HCl pH 7.4, 0.2 mM NADH, and 5 mM pyruvate ⁵⁶.

2.9 Malondialdehyde intracellular concentration assay

Malondialdehyde (MDA) concentration was determined using the thiobarbituric acid reactive substances (TBARS) assay, as a marker of lipid peroxidation to evaluate oxidative damage. The reaction was monitored spectrophotometrically at 532 nm, as previously described ⁵⁶.

2.10 Statistics. According to Rosner ⁵⁷, the sample size for the PBM therapy screening was calculated using the sample size calculator available at ClinCalc.com. Based on the preliminary data and considering a beta of 0.2, an alpha significance level of 0.05, and a power of 0.8 for the initial screening trials with the 0.25, 0.5, 1, and 2 W powers, a minimum of 18 patients was considered necessary to detect a clinically significant difference. One-way analysis of variance (ANOVA) was used to analyze the data. Tukey's multiple comparison test was then performed. The software used was Prism 8 (GraphPad software, Boston (MA), USA). The comparison between the means of two independent samples was performed using an unpaired t-test. The data are presented as the mean ± standard deviation (SD). The statistical significance was determined at a probability level of p<0.05.

3.1 Patient and Baseline Characteristics

Progressive motility screening (0.25W-2W) was performed in 20 asthenozoospermic patients according to the results of the sample size calculation. Afterward, the study was performed on a total of 70 asthenozoospermic patients using 810 nm PBM therapy at 1W power. In the energy metabolism and oxidative stress studies, semen from 20 normozoospermic patients was used as a baseline for comparison. Overall, the cohort consisted of 100% Caucasian males, aged 34.1 ± 8.8 years for the asthenozoospermic patients and 35.1 ± 9.4 years for the normozoospermic patients, with no significant difference between groups. In May 2024, patient recruitment and sample analysis were completed.

3.2 Effect of different powers of the laser on sperm motility. All treated samples exhibited impaired sperm motility, with less than 30% initial progressive motility according to WHO criteria ⁵¹. Impaired non-irradiated samples were used for comparison.

The effect of 810 nm laser on sperm progressive motility immediately after irradiation at different powers (0.25-2 W) is reported in Figure 2. The results show that only 1 W and 2 W significantly increased progressive motility relative to the control, inducing an increment of 50%.

3.3. PBM effects on sperm motility. Based on the above screening protocol results, 1W and 2W have been identified as the laser power capable of maximizing sperm motility parameters. Further experiments were performed at 1 W, which we previously demonstrated to be effective and safe in an urchin sea in vitro model ⁵⁸. PBM improved progressive motility according to the post-exposure time: the maximum effect with respect to control was obtained immediately after irradiation (T0) (29% versus 36%, p<0.001) and kept high within 30 minutes (T30) (29% versus 35%, p<0.01) with a decrease after 60 minutes (T60) (27% versus 31%, p> 0.05) (Figure 3A).

Compared to controls, the immotile fraction of sperms significantly reduced with an average percentage decrease of -11% at T0 and T30 (p< 0.05) and -9% at T60 (p> 0.05) (Figure 3B).

3.4 PBM does not affect sperm membrane integrity. Figure 4A shows the presence of viable and nonviable sperm after eosin staining. Figure 4B shows the viability test results on the samples at the baseline and controls and the irradiated samples at 60 minutes after irradiation. Time physiologically decreased vitality with respect to the baseline (control T0) (p<0.001). However, the process is less evident in the PBM samples (laser T60) than in the control T60 (p<0.05).

3.5 PBM does not affect sperm DNA integrity. Our analysis showed that chromatin dispersion remained stable after 60 minutes from PBM (p>0.05) (Figure 5). We did not detect significant differences in the DNA fragmentation level after PBM compared to untreated sperm. Interestingly, even 5 samples with a positive SCD test (>30%) at the baseline (41.2% ± 5.0%) did not display an increased value of chromatin dispersion after irradiation (41.1% ± 5.1%).

3.6 PBM increases OxPhos activity in asthenozoospermic samples

To verify whether the increased motility observed in asthenozoospermic samples after PBM could be due to an enhancement in cellular energy metabolism, the OxPhos function was assessed by comparing the results obtained with normozoospermic samples. The data reported in Figure 6 show that, under basal conditions, asthenozoospermic samples produce less ATP due to reduced oxygen consumption than normozoospermic samples, suggesting a deficit in OxPhos. This decrease only regards energy yield, as mitochondrial efficiency, indicated by the P/O ratio, appears similar to that observed in normozoospermic samples. The reduction in aerobic metabolism in asthenozoospermic samples is partially reversed after treatment with PBM, as both ATP synthesis and OCR increase by approximately 50% compared to the untreated sample immediately after irradiation and in the following 30 minutes. This increase does not alter the coupling between chemical energy synthesis and cellular respiration, as P/O values remain consistently around 2.5.

3.7 The PBM-induced OxPhos increment in asthenozoospermic samples leads to an enhanced cellular energy state.

Following the increase in OxPhos, it was examined whether the cellular energy state also increased by evaluating the ATP and AMP intracellular concentrations and their ratio (Figure 7). The data show that asthenozoospermic samples are characterized by low ATP levels and high AMP concentration compared to normozoospermic samples, resulting in a low cellular energy state that could explain the reduced motility. The increase in OxPhos in asthenozoospermic samples induced by PBM causes a 1.5-fold increase in intracellular ATP concentration and a reduction of about 50% in AMP levels, leading to a 3.5-fold increase in the ATP/AMP ratio. This effect is observed both immediately after irradiation and 30 minutes post-treatment. However, it is a reversible effect, as 60 minutes after laser administration, ATP, AMP levels, and the ATP/AMP ratio return to values similar to those of untreated asthenozoospermic samples. This finding is not surprising, as it has been demonstrated that the effect of PBM on isolated mitochondria is reversible ⁵⁹ However, the increase in the energy state of asthenozoospermic samples could ensure improved motility for a sufficient time to allow fertilization.

3.8 The altered OxPhos activity in asthenozoospermic samples is not compensated by an enhancement in anaerobic glycolytic metabolism.

Usually, if OxPhos is defective, cells attempt to compensate for the lack of ATP synthesis and the conversion of NADH to NAD⁺ by increasing anaerobic glycolysis. However, asthenozoospermic samples do not seem to employ this strategy, as the levels of lactate dehydrogenase (LDH), the enzyme that catalyzes the conversion of pyruvate to lactate, are not higher than those in normozoospermic samples, nor does PBM treatment alter its activity (Figure 8).

3.9 PBM does not induce an accumulation of oxidative damage in asthenozoospermic samples. Considering that aerobic metabolism is always associated with the production of oxidative stress, even under conditions of complete coupling between ATP synthesis and respiration, the concentration of malondialdehyde (MDA), a marker of lipid peroxidation, was assessed. The data shown in Figure 9 indicate that asthenozoospermic samples under basal conditions display a higher concentration of MDA compared to normozoospermic samples. However, the MDA level does not increase following PBM treatment, suggesting that the increase in asthenozoospermic samples OxPhos does not induce further oxidative damage.

The positive contribution of PBM on sperm parameters has been reported in several studies ^20,32–34. However, while PBM offers a translational potential to treat spermatozoa with poor motility, there is no common standard, making it difficult to directly compare the various data and draw a firm conclusion about the safety of PBM on semen samples in clinical practice. Our results showed that laser irradiation of human asthenozoospermic samples significantly improves progressive motility through increased mitochondrial energetic metabolism without harmful oxidative stress nor negative impact on sperm membrane integrity and chromatin condensation. We used a diode laser light of 810 nm that we previously demonstrated to induce PBM on Paramecium primaurelia and mammalian mitochondrial activity without generating thermal damage ^13,60,61. It also did not negatively affect fertilization and early embryo development after irradiation of sea urchin eggs and spermatozoa ⁵⁸. Moreover, other Authors reported promising results at 810 nm in human normozoospermic semen samples ⁴¹. After identifying 1 W and 2 W as the most influential powers in increasing progressive motility and decreasing the percentage of immotile sperm, we chose 1 W for the following experiments. Indeed, the therapy showed preliminary aspects of efficacy and safety based on our evidence in vitro on protozoa and in vivo on sea urchins and humans. Indeed, 810 nm 1 W for 60 s over an area of 1 cm²increased the ATP production and the movement of the cilia in Paramecium primaurelia ^62,63; the mechanisms underlying the movement of the cilia in protozoa have homologies with the flagellar movement of spermatozoa ^64,65. It also improved fertilization rates in healthy sea urchin spermatozoa ⁵⁸, and allowed complete recovery in patients with Bell's palsy who were unresponsive to drugs ⁶⁶. No adverse effects were reported after follow-up.

We focused exclusively on a population of asthenozoospermic patients to avoid possible confounding factors. Specifically, we considered that normozoospermic samples could not benefit from laser therapy or do so in a reduced way. On the other hand, the low sperm number in oligozoospermic samples may make it challenging to count enough sperm to appreciate the laser effect on motility. To the best of our knowledge, this is the first study that included only fresh abnormal sperm and enrolled the largest cohort of semen samples from asthenozoospermic patients.

Semen parameters of asthenozoospermic patients were shifted above a threshold of 30% progressive motility, representing the standard criteria for normozoospermic, while in parallel, the fraction of immotile sperm decreased. Our results are in accordance with previous publications that reported enhanced sperm motility in semen samples treated with an infrared range of PBM ^36,39,41.

The underlying pathways of PBM therapy are not well established and may vary among different sperm states (fresh versus frozen, normal versus abnormal). At present, at least four mechanisms are believed to be related to the response of sperm to PBM: i) changes in mitochondrial function, ii) generation and/or release of reactive oxygen species (ROS) from sperm cells; iii) formation of nitric oxide, iv) activation of opsins coupled to G-proteins ³⁴. In this study, we investigated the involvement of mitochondria since they serve as the primary energy source for sperm movement, and in asthenozoospermia, sperm motility is impaired due to mitochondrial malfunction or structural defects of the tail ⁸. In addition, the release of nitric oxide and the effects on the opsins are mainly the result of an interaction in the visible range of 450 to 650 nm ^19,67. Our PBM protocol increased mitochondrial energy metabolism, providing biochemical evidence that mitochondrial activation is implicated in the increased sperm motility observed.

To broaden the knowledge of PBM treatment of human sperm, it is essential to analyze side effects. To date, it is challenging to draw a firm conclusion about the safety of PBM on semen samples in male subjects because there are some significant differences in the data available in the literature in terms of population characteristics, sample size, and type of methods used to conduct safety tests. In general, most of the human sperm DNA damage during manipulation is oxidatively induced ⁶⁸ because spermatozoa are poor in enzymes that metabolize ROS, like catalase and glutathione peroxidase ^68,69 and have limited DNA repair capability ⁷⁰. This may be the reason why 810 nm PBM did not reduce oxidative stress damage in irradiated asthenozoospermic samples, as has been observed in other cell types ⁷¹.

Oxidative stress causes lipid peroxidation of the plasma membrane, which is rich in polyunsaturated fatty acids, leading to the breakdown of membrane‐dependent functions in these cells, including motility and sperm‐oocyte fusion ⁷⁰. Since we did not observe MDA accumulation, a marker of lipidic peroxidation, in irradiated asthenozoospermic samples, we argue that our PBM treatment does not increase oxidative stress in sperm. Noteworthy, to investigate this aspect without introducing potential biases into the system, we applied PBM only to native spermatozoa within the seminal plasma since manipulation (centrifugation, resuspension) of seminal fluids results in an artificially high level of ROS ^72,73. Moreover, seminal plasma is one of the most potent antioxidant fluids ⁷⁰.

Functionally, our laser therapy protocol did not adversely affect the integrity of the sperm membrane, preserving vitality and chromatin condensation, and did not introduce DNA fragmentation or worsen it when already present.

All these safety aspects are essential for applying PBM in clinical practice as an asthenozoospermia therapy. One of the putative applications would be a laser-induced improvement of sperm motility to increase pregnancy rates achieved by intrauterine insemination. The percentage of motile sperm that has the ability for forward progression is one of the most critical prognostic indicators of fertilization, which can increase the likelihood of pregnancy ⁷⁴. Bypassing time, sperm cells decrease their ability to move ⁷⁵. Boosting the availability of total motile sperm can improve the chance of pregnancy. This finding could be helpful in cases of moderate male infertility to encourage the use of intrauterine insemination as a cost-effective first-line treatment ⁷⁶. On the one hand, it is noteworthy that in our experiments, the effect of irradiation on sperm motility was kept high within 30 minutes after irradiation, unlike other studies where the effect decreased significantly after 10 minutes of phototherapy ⁷⁷. On the other hand, we have shown a significant increase in progressive sperm motility from 29% to 35%, but we cannot conclude that this increase predicts a better clinical outcome.

More benefits could be expected in selecting viable ejaculated or surgically retrieved testicular/epididymal spermatozoa in patients with immotile sperm before microinjection into the oocyte. Sperm motility is an essential selection criterion for embryologists during intracytoplasmic sperm injection (ICSI). A method of testing sperm viability is to induce sperm motility by increasing cyclic adenosine monophosphate (cAMP) levels by treating a semen sample with phosphodiesterase inhibitors, such as theophylline and pentoxifylline ⁷⁸. Although pentoxifylline is a popular in vitro sperm motility enhancer in many embryology laboratories, its application is not commonly approved in clinical practice because the consequences of pentoxifylline treatments in terms of potential damage to sperm DNA, oocytes, embryos, and newborns are contradictory ⁷⁹. In this context, stimulating sperm motility by PBM could advance sperm selection.

Another clinical application of PBM could be the treatment of frozen and thawed semen, especially in programs of fertility preservation of cancer patients before chemo- and radiotherapy. Despite its widespread use, the cryopreservation technique can cause damage to sperm through oxidative stress that induces lipid peroxidation, DNA fragmentation, and apoptosis ⁸⁰. In addition, sperm motility and fertilizing potential are reduced due to membrane, cytoskeletal, and acrosome damage ^81,82. The harmful side effects of the freezing and thawing process may be countered by PBM-preconditioning of fresh semen before the cryopreservation procedure, hopefully resulting in increased mitochondrial membrane potential and decreased levels of intracellular ROS and lipid peroxidation ^45,46.

Of course, some ethical concerns must be resolved before the use of PBM of sperm in assisted reproductive technologies (ART), whose ultimate goal is a healthy baby. Indeed, laser technology is now being applied in ART to reduce procedure times and increase the consistency of traditional techniques such as assisted hatching, embryo biopsy in the preimplantation genetic diagnosis program, and sperm immobilization ⁸³. It is also important to note that the energy used to enhance sperm motility in PBM is much lower than those used for, i.e., assisted hatching, since it is only used to stimulate the photosensitive enzyme cytochrome c oxidase and below the threshold for ionization. However, there is undoubtedly little published information about the potential adverse effects that laser application - even for PBM of sperm - could have on the developing human embryo, making further research necessary.

Although our study is a preliminary report, its strength is that our approach represents a first standardized protocol easily adaptable to the clinical environment. In fact, the short exposure time prevents samples from being exposed to sub-optimal conditions for too long, and the immediate effect, which lasts consistently up to 30 minutes (after 60 minutes, the effect returns to the level of untreated samples), ensures standardization of the procedure. We are aware that there are some limitations. First, we did not check the straightness of sperm movement and the flagellar waveform, which help evaluate sperm hyperactivation ⁸⁴. However, the complex parameters of the flagellar movement of hyperactivated spermatozoa are challenging to identify reliably and unambiguously through manual analysis. Motility stereotypes can be assessed by computer-aided sperm analysis (CASA). However, the comparability of measurements among different instruments is not yet known, and their biological value is not fully understood ⁵¹. A second limitation is that we did not evaluate the effectiveness of our PBM protocol in improving fertilization potential and the impact on embryo quality. This was due to the Italian law prohibiting the insemination of human oocytes outside the standard procedures recognized as non-experimental. However, in the sea urchin, a well-known model for studying the molecular mechanisms underlying fertilization and human embryonic development ⁸⁵, we have obtained an encouraging increase in sperm fertilization rates, without the occurrence of embryonic and larval anomalies ⁵⁸.

Laser irradiation at 810 nm of human sperm significantly improves progressive motility through increased mitochondrial energetic metabolism without harmful oxidative stress, nor negative impact on membrane integrity and chromatin condensation.

AUTHOR CONTRIBUTIONS: Conceptualization, Data curation, Formal analysis, S.S; S.R.; A.A.; P.S. Methodology, S.S; S.R.; E.M.; C.R.; A.A.; P.S. Funding acquisition, A.A.; P.A. Visualization, S.R.; M.B.; A.A.; P.S.; Writing-original draft, S.R.; A.A.; P.S; Writing-review and editing, S.S., A.A.; P.S.; Sofware and Resources, C.M.; M.B.

FUNDING: This work was partially supported by the Fondo per la Ricerca di Ateneo (FRA) University of Genova, code 100022-2023-AA-FRA_001 to A.A.

INSTITUTIONAL REVIEW BOARD STATEMENT: Ethics Committee of Regione Liguria (ID 13852).

INFORMED CONSENT STATEMENT: Written informed consent was obtained from all donors, and participation was voluntary.

DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available from the corresponding author upon reasonable request.

ACKNOWLEDGEMENTS: We thank Eng. Cucinotta of Garda Laser for his key role in providing the ENEA diode laser (GaAlAs) and for his commitment to the advancement of scientific research.

COMPETING INTERESTS STATEMENT: The authors declare no conflicts of interest. Garda Laser S.A.S. had no role in the study's design, data collection, analysis, interpretation, manuscript writing, or decision to publish the results.

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No competing interests reported.

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GRAPHICAL ABSTRACT In asthenozoospermic sperm of 70 patients, photobiomodulation at 1 W significantly improved progressive motility. Without increasing markers of oxidative stress such as malondialdehyde, it improves mitochondrial energy metabolism and partially restores oxidative phosphorylation efficiency. It does not affect the integrity of sperm DNA or sperm viability.

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Shining a light on male infertility: the power of near-infrared photobiomodulation therapy for human asthenozoospermia

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