Rho-kinase inhibition reduces DNA fragmentation in human sperm cryopreservation

Abstract

Human sperm cryopreservation is essential for assisted reproductive technology; however, the freeze–thaw process reduces motility and increases DNA fragmentation. Rho-associated coiled-coil kinase (ROCK) inhibitors improve post-thaw survival in several cell types, yet their application to human sperm remains underexplored. We evaluated whether ROCK inhibition reduces DNA fragmentation during cryopreservation by liquid nitrogen vapor slow freezing or vitrification. Each semen sample was divided into four aliquots: 2 were cryopreserved via liquid nitrogen vapor slow freezing with or without ROCK inhibitor and 2 underwent vitrification with or without ROCK inhibitor. After 1 week of cryostorage, samples were thawed at the room temperature, and DNA fragmentation was quantified using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. DNA fragmentation decreased with ROCK inhibitor versus control after liquid nitrogen vapor slow freezing (mean ± standard deviation [s.d.]: 10.47% ± 6.77% vs 13.54% ± 7.0%; P = 0.002) and after vitrification (mean ± s.d.: 12.51% ± 5.84% vs 16.5% ± 10.77%; P = 0.006). ROCK inhibition thus reduces post-thaw sperm DNA fragmentation with both cryopreservation methods, particularly with liquid nitrogen vapor slow freezing, supporting its potential to improve sperm storage for assisted reproductive technology.

INTRODUCTION

Human sperm cryopreservation is a well-established approach for gamete banking within assisted reproductive technology programs.1 Its principal advantage is preserving male fertility in men with testicular cancer before chemotherapy or radiotherapy, which can cause testicular failure or ejaculatory dysfunction.2,3 Cryopreservation is also widely used for azoospermic patients undergoing surgical sperm retrieval, thereby avoiding repeated biopsies or aspirations.4,5 Donor sperm is employed to manage severe male-factor infertility (e.g., azoospermia) and to facilitate assisted reproduction for female–female same-sex couples. In donor programs, frozen semen is utilized to provide sufficient time to screen for sexually transmitted infections, such as human immunodeficiency virus (HIV) and hepatitis B virus, before clinical use.2

Cryopreservation methods include computerized slow freezing, liquid nitrogen vapor slow freezing, and vitrification.6 In computerized slow freezing, addition and removal of cryoprotectants across freezing–thawing steps expose spermatozoa to chemical and physical stressors including intracellular ice crystal formation cellular dehydration, osmotic shock, and altered membrane permeability.7,8 Vitrification induces a glass-like state without intracellular ice formation; however, it requires exposure to high cryoprotectant concentrations that can damage spermatozoa.9 Additional mechanisms implicated in cryo-induced DNA damage include oxidative stress, release of apoptotic enzymes, and endonuclease activation.10,11

The effects of freezing and thawing on sperm quality have been extensively characterized.12,13 Multiple studies report a marked decrease in sperm motility after cryopreservation8,13,14 along with elevated sperm DNA fragmentation.8,12 A recent study demonstrated that the freeze–thaw process also markedly alters chromatin architecture, disrupting nuclear organization and reducing chromatin compaction, especially with slow freeze–thaw protocols.15 Elevated sperm DNA fragmentation correlates with impaired fertilization, higher miscarriage rates, and lower pregnancy rates after in vitro fertilization or intracytoplasmic sperm injection.16,17,18

Approaches to minimize apoptosis during cryopreservation include antioxidant supplementation with vitamin C and vitamin E, Tes and Tris (TEST) yolk buffer (TYB) with glycerol,9 zinc supplementation,19 and sperm preparation before storage.20 However, no method completely prevents cryoinjury or constitutes a standardized, optimal protocol to date.

Apoptotic markers used to assess human spermatozoa focus on DNA integrity and chromatin structure. The common techniques include the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay, single-cell gel electrophoresis, sperm chromatin structure assay, and sperm chromatin dispersion test.21,22,23,24 Among these, the TUNEL assay is widely regarded as a precise method for directly measuring DNA damage. The assay labels the 3’-OH termini of fragmented DNA with dUTP via the terminal deoxynucleotidyl transferase enzyme, enabling simultaneous detection of single- and double-strand breaks by flow cytometry or fluorescence microscopy.22,25 Cryopreservation-induced sperm DNA damage has been demonstrated using the TUNEL assay.22,23,24

Mitochondria are central to sperm function. In human sperm cryopreservation studies, reduced mitochondrial membrane potential (MMP) correlates with decreased motility, underscoring mitochondria’s role in regulating sperm function.26 Conversely, increased MMP is associated with improved motility parameters and enhanced fertility potential.27 Several attempts to improve post-thaw outcomes have employed mitochondria-targeted antioxidants.28

Rho-associated coiled-coil kinase (ROCK), a member of the serine–threonine kinase family, was identified as a downstream target of the small guanosine triphosphase (GTP)-binding protein Rho.29 ROCK has been detected in sperm from multiple mammalian species, including boar, rat, bull, sheep, elephant, and human.30,31,32,33,34 ROCK regulates cellular growth, adhesion, migration, metabolism, and apoptosis by controlling actin-cytoskeletal assembly and cell contraction.35,36 ROCK inhibitors have improved cryosurvival of human neural stem cells37 and human embryonic stem cells.38,39 In in-vitro-produced bovine blastocysts, post-warming oocyte treatment with a ROCK inhibitor increased oocyte survival. In vitrified–warmed bovine oocytes, ROCK inhibition also enhanced developmental competence to the blastocyst stage, with reduced apoptosis and normalization of microtubule-organizing center function.40

Few studies have examined the effects of ROCK inhibitors on post-thaw human sperm. We therefore evaluated whether supplementing 2 semen cryopreservation techniques with a ROCK inhibitor preserves sperm DNA integrity. The primary outcome was DNA fragmentation as assessed by TUNEL, a marker of late apoptosis. The secondary outcomes were sperm motility and MMP, with the latter assessed by 5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, after cryopreservation with the ROCK inhibitor. These findings will clarify whether ROCK inhibition protects sperm from cryoinjury.

MATERIALS AND METHODS

Study design and ethics

This experimental study was conducted at Reproductive and Biology Unit, Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University (Bangkok, Thailand) in accordance with the Declaration of Helsinki. The Siriraj Institutional Review Board approved the protocol (Approval No. SIRB 089/2557) prior to enrollment. All methods adhered to relevant guidelines and regulations.

Participants and eligibility

A total of 29 men from infertile couples at the Reproductive and Biology Unit, Faculty of Medicine Siriraj Hospital undergoing semen analysis between December 2013 and March 2014 were enrolled after providing informed consent. Inclusion criteria were healthy men aged ≥18 years with normal semen analysis results. Semen was collected via masturbation after 2–7 days of abstinence. Eligible samples had sperm concentration >15 × 10⁶ sperm per ml, ≥32% progressive motility, >4% normal morphology as per Kruger’s strict criteria,41 and leukocytes <1 × 10⁶ ml⁻¹. Exclusion criteria were semen volume <2 ml, active sexually transmitted infections (e.g., HIV, syphilis, or hepatitis B), genital tract infection (e.g., active prostatitis or urethritis), or surgically retrieved semen.

Semen analysis and sample preparation

Samples liquefied at room temperature and were analyzed within 30 min. Semen volume, sperm concentration, sperm motility, and sperm viability were assessed following the 5^th edition of World Health Organization guidelines.42 Morphology was evaluated using Kruger’s strict criteria.41 Fresh samples were also assessed for DNA fragmentation via TUNEL and for MMP via JC-1 staining.

Experimental allocation and cryopreservation protocols

Each sample was divided into four aliquots (Figure 1). Two aliquots underwent liquid nitrogen vapor slow freezing (with or without ROCK inhibitor) and two underwent vitrification (with or without ROCK inhibitor).

Figure 1.

Study schema for sample allocation. Each semen sample was allocated to four groups: liquid nitrogen vapor slow freezing with or without ROCK inhibitor and vitrification with or without ROCK inhibitor. The ROCK inhibitor was Y-27632 at 10 µmol l⁻¹. ROCK: Rho-associated coiled-coil kinase; PR: progressive; WBC: white blood cell; MMP: mitochondrial membrane potential; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; HIV: human immunodeficiency virus; JC-1: 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide; STD: sexually transmitted disease.

Sperm cryopreservation methods: liquid nitrogen vapor slow freezing

Each semen sample was divided into two equal aliquots. The first aliquot was mixed 1:1 with cryoprotective medium (Quinn’s Advantage Sperm Freezing Medium; SAGE In Vitro Fertilization Inc., Trumbull, CT, USA). The second aliquot was mixed 1:1 with the same medium supplemented with 10 µmol l⁻¹ ROCK inhibitor Y-27632 (Sigma–Aldrich, Poole, UK). Mixtures equilibrated at room temperature for 10 min and were loaded into cryovials (NUNC Cryotube 1.0 ml; Thermo Fisher Scientific Inc., Waltham, MA, USA). Cryovials were suspended horizontally 15 cm above liquid nitrogen for 30 min to cool samples to −80°C and they were then plunged into liquid nitrogen (−196 °C) for storage.

After 1 week, cryovials were thawed at room temperature for 10 min. Post-thaw sperm were layered onto a discontinuous Percoll gradient consisting of 1 ml of 40% Percoll overlaying 1 ml of 80% Percoll in a 15 ml conical tube (BD Falcon; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Samples were centrifuged (Beckman Coulter, Brea, CA, USA) at 300g for 20 min. The supernatant was discarded and the pellet was resuspended in 2 ml sperm washing medium (Quinn’s Sperm Washing Medium; SAGE In Vitro Fertilization Inc.). After centrifugation (Beckman Coulter) at 200g for 10 min, the pellet was washed again using the same protocol. Post-thaw motility, DNA fragmentation via TUNEL, and MMP via JC-1 were assessed using the same protocols as for fresh semen.

Sperm cryopreservation methods: vitrification

Each ejaculate sample (500 µl) was prepared by density gradient centrifugation (Beckman Coulter) and mixed 1:1 with 1% human serum albumin (HSA) in human tubal fluid (HTF). The mixture was divided equally, centrifuged at 380g for 5 min, and the supernatant was discarded. One pellet was resuspended in 150 µl 0.5 mol l⁻¹ sucrose in 1% HSA. The other was resuspended in 150 µl 0.5 mol l⁻¹ sucrose in 1% HSA containing 10 µmol l⁻¹ ROCK inhibitor. Suspensions were incubated at 37°C in 5% CO₂ for 5 min.

Vitrification followed the method from Isachenko et al.43 A metal strainer was immersed in liquid nitrogen with a cryovial positioned at the bottom (NUNC Cryotube 1.0 ml; Thermo Fisher Scientific Inc.). Drops of sperm suspension (30 µl each) were dispensed directly into liquid nitrogen using a micropipette held at a 45° angle and 10 cm above the liquid nitrogen surface. Each cryovial containing solid spheres was packaged and stored in liquid nitrogen.

After 1 week, five spheres were thawed by sequential submersion into 5 ml of HTF with 1% HSA prewarmed to 37°C. Gentle vortexing was applied for 5–10 s. The post-thaw suspension was maintained at 37°C in 5% CO₂ for 10 min before being centrifuged (Beckman Coulter) at 380g for 10 min. The final pellet was resuspended in 50 µl HTF-HSA.

Determination of sperm motility

Sperm motility was assessed using a computer-assisted semen analyzer (HTM-IVOS; Hamilton Thorne Biosciences, Beverly, MA, USA) equipped with the clinical human motility program, version 12. A disposable counting chamber (20-µm depth; Leja, Nieuw-Vennep, the Netherlands) was used. Motility was reported as the percentage of progressively motile sperm.

Determination of sperm DNA fragmentation by TUNEL

Semen smears were air-dried at the room temperature and then fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at 15°C–25°C. Slides were rinsed with PBS and permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice (2°C–8°C). After washing twice with PBS, slides were incubated in a humidified chamber at 37°C for 60 min with 50 µl of terminal deoxynucleotidyl transferase enzyme and fluorescein-labeled nucleotides (In Situ Cell Death Detection Kit, Fluorescein; Roche, Mannheim, Germany). Slides were washed three times with PBS and air-dried. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) II solution (Abbott Laboratories, Abbott Park, IL, USA). A total of 200 spermatozoa per sample were evaluated using a fluorescence microscope (Imager-Z1; Zeiss, Göttingen, Germany) with dual band-pass filters at 1000× magnification. All sperm nuclei appeared blue under the DAPI filter. TUNEL-positive spermatozoa (those with fragmented DNA) emitted bright green fluorescence when excited at 490 nm (Figure 2).

Figure 2.

TUNEL staining of human spermatozoa. TUNEL-positive nuclei (DNA fragmentation) show bright green fluorescence (arrow), whereas TUNEL-negative nuclei (intact DNA) appear blue. Scale bar = 20 µm. TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

For positive controls, spermatozoa were treated with 1 U ml⁻¹ recombinant DNase I in buffer containing 10 mmol l⁻¹ Tris-HCl (pH 7.4), 10 mmol l⁻¹ NaCl, 5 mmol l⁻¹ MnCl₂, 0.1 mmol l⁻¹ CaCl₂, and 25 mmol l⁻¹ KCl. Samples were incubated at 37°C for 30 min. For negative controls, the terminal deoxynucleotidyl transferase enzyme was omitted from the labeling solution. Both controls were included in each experimental batch. DNA fragmentation was reported as the percentage of TUNEL-positive spermatozoa.

Determination of MMP

MMP was measured using the lipophilic cationic dye JC-1 (MitoProbe JC-1 Assay Kit; Molecular Probes, Eugene, OR, USA), following the manufacturer’s instructions. Sperm suspension (1 ml) was incubated with 2 µmol l⁻¹ JC-1 at 37°C in a 5% CO₂ incubator for 30 min. Stained spermatozoa were examined by epifluorescence microscopy (Axiolab DRB [KT 450905]; Zeiss) at 200× magnification. JC-1 monomers, indicating low MMP, emitted green fluorescence at 527 nm. JC-1 aggregates formed within mitochondria with high MMP emitted red fluorescence at 590 nm. In sperm with intact MMP, JC-1 accumulated in mitochondria, producing red fluorescence. In sperm with depolarized or damaged mitochondria, JC-1 remained monomeric, producing diffuse green fluorescence. A total of 200 spermatozoa were evaluated per slide (Figure 3).

Figure 3.

Mitochondrial membrane potential evaluated by JC-1 with (a) low MMP (JC-1 monomers, green) and (b) intact MMP (JC-1 aggregates, yellow midpiece from merged green and red). Scale bars = 20 µm. JC-1: 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide; MMP: mitochondrial membrane potential.

Statistical analyses

Statistical analyses were performed using PASW Statistics, version 18 (SPSS Inc, Chicago, IL, USA). Data were expressed as mean (± standard deviation [s.d.]). Normality of each dataset was assessed using the Kolmogorov–Smirnov test. Paired t-tests were used to compare the outcomes between control (without ROCK inhibitor) and treatment (with ROCK inhibitor) groups within each cryopreservation method. For multiple comparisons, repeated-measures analysis of variance (ANOVA) with Bonferroni post hoc testing was applied. P < 0.05 was defined as statistical significance.

Sample size calculation

The sample size was calculated based on a pilot study in which mean (±s.d.) post-thaw DNA fragmentation after liquid nitrogen vapor slow freezing was 12.0% (±5.7%) in controls and 8.4% (±4.2%) with ROCK inhibitor. Assuming a two-sided alpha of 0.05 and 90% power, 29 participants were required to detect this difference.

RESULTS

Participant characteristics

The study included 29 infertile men (age [mean ± s.d.]: 38.7 ± 5.4 years; range: 30–52 years). The abstinence period (mean ± s.d.) was 4.1 ± 1.2 days. Baseline semen parameters are summarized in Table 1; Sperm concentration (mean ± s.d.) was 67.3 × 10⁶ ml⁻¹ ± 31.1 × 10⁶ ml⁻¹, and progressive motility (mean ± s.d.) was 50.9% ± 7.1%. In fresh sperm, TUNEL-quantified DNA fragmentation index (mean ± s.d.) was 3.9% ± 3.0%, and MMP (mean ± s.d.) measured by JC-1 was 85.7% ± 16.1%.

Table 1.

Baseline semen parameters (n=29)

Parameters	Value (mean±s.d.)	Range
Semen volume (ml)	3.0±0.9	2–4
Semen pH	7.7±0.2	7.5–8.0
Sperm concentration (106 ml−1)	67.3±31.1	18–180
Total sperm count (106)	197±112	72–560
Progressive motility (%)	50.9±7.1	40–65
Vitality (%)	76.0 ±7.0	58–88
Normal morphology rate (%)	12.9±6.0	4–28
WBC count (106 ml−1)	0.3±0.2	0.1–0.8
DNA fragmentation index (%)	3.9±3.0	1–14
MMP (%)	85.7±16.1	21.5–98.5

DNA: deoxyribonucleic acid; MMP: mitochondrial membrane potential; s.d.: standard deviation; WBC: white blood cell

Effect of ROCK inhibitor: liquid nitrogen vapor slow freezing

After liquid nitrogen vapor slow freezing, motility was significantly higher with ROCK inhibitor than without (mean ± s.d.: 82.8% ± 5.7% vs 77.5% ± 8.1%; P < 0.001; Figure 4a). DNA fragmentation was significantly lower with ROCK inhibitor (mean ± s.d.: 10.5% ± 6.8% vs 13.5% ± 7.0%; P = 0.002; Figure 4b). MMP did not differ significantly (mean ± s.d.: 84.4% ± 21.3% vs 81.2% ± 24.4%; P = 0.497; Figure 4c).

Figure 4.

Post-thaw outcomes of (a) progressive motility, (b) DNA fragmentation index, and (c) MMP with and without ROCK inhibitor. DNA: deoxyribonucleic acid; JC-1: 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide; MMP: mitochondrial membrane potential; ROCK: Rho-associated coiled-coil kinase; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Effect of ROCK inhibitor: vitrification

After vitrification, motility did not differ significantly with ROCK inhibitor (mean ± s.d.: 50.0% ± 23.7% vs 45.1% ± 21.5%; P = 0.163; Figure 4a). DNA fragmentation was significantly lower with ROCK inhibitor (mean ± s.d.: 12.5% ± 5.8% vs 16.5% ± 10.8%; P = 0.006; Figure 4b). MMP did not differ significantly (mean ± s.d.: 50.1% ± 19.5% vs 53.2% ± 20.3%; P = 0.394; Figure 4c).

Effect of cryopreservation on DNA fragmentation

Relative to fresh sperm, cryopreservation significantly increased DNA fragmentation with both liquid nitrogen vapor slow freezing and vitrification (P < 0.001 for each; Figure 5a). There was no significant difference between the two freezing methods (P = 0.566; Figure 5a). With ROCK inhibitor, DNA fragmentation remained significantly higher than that in fresh sperm for both cryopreserved groups (P < 0.001 for each; Figure 5b), with no significant difference between freezing methods (P = 0.48; Figure 5b).

Figure 5.

DNA fragmentation by TUNEL across freezing conditions. Fresh, liquid nitrogen vapor slow freezing and vitrification (a) without ROCK inhibitor, and (b) with ROCK inhibitor. ^***P < 0.001. ROCK: Rho-associated coiled-coil kinase; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

DISCUSSION

Sperm cryopreservation is a well-established technique for infertility treatment and gamete storage. Its goal is to preserve sperm while maintaining structure and DNA integrity, such that post-thaw cells are equivalent to pre-freeze cells. ROCK inhibitors have been incorporated into cryopreservation protocols for several cell types to enhance post-thaw survival.32,39 The present study evaluated whether a ROCK inhibitor protects human spermatozoa during cryopreservation. We assessed sperm motility, DNA fragmentation, and MMP using two freezing methods: liquid nitrogen vapor slow freezing and vitrification.

Post-thaw progressive motility was significantly higher with ROCK inhibitor than control in the liquid nitrogen vapor slow freezing group, and DNA fragmentation was significantly lower. These findings suggest a cryoprotective effect of ROCK inhibitor that preserves motility and maintains DNA integrity. Cryopreservation adversely affects sperm quality through chemical and physical stressors, including intracellular ice formation, excessive dehydration, osmotic shock, mitochondrial dysfunction, and DNA damage.8,12,13,20 ROCK-1, a member of the Rho-associated coiled-coil-containing kinase family, localizes predominantly to the sperm head and less to the tail in several mammalian species, and it regulates acrosomal exocytosis.30 Therefore, ROCK inhibition during freezing may stabilize the plasma membrane and lessen cryoinjury. Our results align with reports of similar protection in feline and bovine sperm.32,33

MMP did not differ significantly between groups, possibly because midpiece mitochondria are tightly ensheathed by plasma membrane with minimal cytoplasm. This configuration may reduce susceptibility to cryoinjury, thereby obscuring any protective effects of the ROCK inhibitor.

With vitrification, post-thaw motility and MMP did not differ between ROCK inhibitor and control groups. However, DNA fragmentation was lower with ROCK inhibitor than control. Although vitrification was developed to minimize cryoinjury, it has not consistently improved motility or DNA fragmentation outcomes.12,43 These findings suggest that ROCK inhibition protects DNA integrity during vitrification despite no detectable effect on motility.

Both liquid nitrogen vapor slow freezing and vitrification significantly increased DNA fragmentation relative to fresh semen, consistent with prior reports.43 Adding ROCK inhibitor before freezing did not alter DNA fragmentation in fresh samples, indicating that the compound itself does not induce DNA damage. Although the ROCK inhibitor reduced DNA fragmentation in freeze–thawed samples, fragmentation remained significantly higher in both cryopreserved groups than that in fresh semen.

Several mechanisms may underlie the observed protection against DNA damage. ROCK-1 is predominantly localized in sperm heads, which contain compacted DNA, with lower expression in tails.30 ROCK activity increases when Rho binds to the Rho-binding domain, producing an activating conformational change. ROCK-1 can also be activated by caspase-3-mediated cleavage near the carboxyl terminus.44 A key downstream target is myosin light chain; ROCK phosphorylates the chain directly and promotes its phosphorylation indirectly by inhibiting myosin phosphatase. Myosin light chain activation drives cell contraction linked to apoptosis and membrane blebbing.37 Therefore, ROCK pathway inhibition may attenuate apoptotic signaling and limit DNA damage.

Multiple strategies have been explored to improve sperm cryopreservation, yet evidence remains limited and no approach has achieved broad clinical uptake. Media supplemented with various agents, including ROCK inhibitors, have improved post-thaw quality in prior reports. In this study, adding a ROCK inhibitor to cryoprotective media improved post-thaw integrity across physical and DNA integrity endpoints. Motility improved significantly with liquid nitrogen vapor slow freezing, and DNA preservation improved significantly with both liquid nitrogen vapor slow freezing and vitrification. These techniques remain widely used because they are simple, cost-effective, and operationally straightforward.

This study has several methodological limitations. We did not classify motility by velocity (e.g., rapid vs slow progressive motility). Thus, ROCK inhibition might have shifted sperm between velocity subpopulations, increasing rapidly progressive sperm while decreasing slow progressive sperm. In the liquid nitrogen vapor slow freezing group, post-thaw sperm underwent density gradient centrifugation before motility assessment to remove dead or amorphous cells that confound computer-assisted analysis. Consequently, the higher post-thaw motility compared to fresh samples may partly reflect selection of motile, morphologically normal sperm by this preparation step. The study was performed in 2013 under an approved protocol; semen analysis followed the 5^th edition of World Health Organization guidelines42 then in effect, which differ slightly from present reference ranges. Future studies should evaluate cryoprotective effects in abnormal semen profiles, including oligozoospermia, teratozoospermia, and asthenozoospermia.

To our knowledge, few human studies have examined ROCK inhibitors and sperm DNA fragmentation after cryopreservation; most data derive from animal models. We did not assess abnormal semen parameters such as oligozoospermia or elevated DNA fragmentation index, which represents a limitation. Future work should standardize procedural conditions and evaluate these phenotypes to provide more comprehensive insights. This study evaluates ROCK inhibition across distinct human sperm freezing techniques, an area that has been relatively under explored.

In conclusion, ROCK inhibition reduces DNA fragmentation during cryopreservation and improves motility with liquid nitrogen vapor slow freezing. These findings highlight its potential to enhance sperm storage for assisted reproductive technology.

AUTHOR CONTRIBUTIONS

PL and SP wrote the main manuscript and performed the research, including formal analysis and investigation. IT, JP, PL, and RC reviewed and edited the manuscript. SP provided conceptualization and supervision. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declared no competing interests.