Comparative Study of Semen Cryopreservation Techniques and Their Impact on Post-Thaw Sperm Quality

Abstract

Semen cryopreservation is critical for assisted reproductive technologies (ART) and fertility preservation, yet cryoinjury during freezing-thawing remains a significant challenge. This study conducted a comparative analysis of slow freezing and vitrification techniques to evaluate their impact on post-thaw sperm quality parameters. Thirty normozoospermia samples from healthy donors were processed using slow freezing (10% glycerol, controlled-rate cooling) and vitrification (15% glycerol + 0.5M sucrose, ultra-rapid cooling). Post-thaw assessments included motility (computer-assisted sperm analysis), viability (eosin-nigrosine staining), morphology (Papanicolaou staining), and DNA integrity (sperm chromatin structure assay). Vitrification significantly outperformed slow freezing across all metrics: total motility (55.1% vs 36.5%, p<0.001), viability (70.9% vs 48.3%, p<0.001), normal morphology (7.0% vs 4.5%, p<0.01), and DNA fragmentation index (18.5% vs 34.6%, p<0.001). The absence of ice crystal formation in vitrification likely accounts for reduced cryoinjury, preserving membrane integrity and DNA stability. These findings advocate for vitrification as the preferred method in clinical settings, offering enhanced preservation of sperm functionality critical for ART success. Despite limitations in sample size and demographic diversity, this study provides robust evidence supporting protocol standardization and further research into long-term outcomes of vitrified sperm in reproductive applications.

Introduction

Semen cryopreservation, a cornerstone of assisted reproductive technologies (ART) and fertility preservation, enables long-term storage of viable spermatozoa at cryogenic temperatures (−196°C−196°C) but faces a critical barrier: cryoinjury, where ice crystallization, osmotic shock, and dehydration during freeze-thaw cycles impair sperm motility, membrane integrity, and DNA stability.

Traditional slow freezing—the clinical gold standard—relies on controlled cooling (0.5°C/min0.5°C/min) and cryoprotectants like glycerol to mitigate intracellular ice formation, yet extracellular ice crystals persist, inflicting mechanical damage that reduces post-thaw viability by 30–50%. In contrast, vitrification, an innovative ultra-rapid cooling method (>10,000°C/min>10,000°C/min), bypasses ice formation entirely by solidifying water into a glass-like state, theoretically minimizing cryoinjury; however, its reliance on high cryoprotectant concentrations (e.g., 15% glycerol, 0.5M sucrose) introduces toxicity risks, and inconsistent protocol standardization has hindered widespread clinical adoption.

This study directly compares these techniques, analysing post-thaw outcomes in motility (CASA), viability (eosin-nigrosine), morphology (Papanicolaou), and DNA integrity (SCSA) across 30 normozoospermia samples, with preliminary data revealing vitrification’s superiority: 55.1% vs. 36.5% total motility (p<0.001p<0.001), 70.9% vs. 48.3% viability (p<0.001p<0.001), and 18.5% vs. 34.6% DNA fragmentation (p<0.001p<0.001), attributable to its ice-free process. By addressing the paucity of robust comparative data, this work challenges the entrenched slow freezing paradigm, advocating for vitrification’s integration into ART protocols despite its technical complexities. The findings hold transformative potential for fertility preservation patients—particularly those facing gonadotoxic therapies—by optimizing post-thaw sperm quality, thereby enhancing IVF/ICSI success rates. Furthermore, this research underscores the urgency of refining vitrification protocols to balance cryoprotectant efficacy with toxicity, paving the way for standardized, globally accessible cryopreservation strategies that prioritize both biological integrity and reproductive equity.

I. LITERATURE REVIEW

1. Historical Context And Clinical Adoption

The advent of sperm cryopreservation marked a paradigm shift in reproductive medicine, with its origins tracing back to Polge et al.'s groundbreaking 1949 discovery of glycerol’s cryoprotective properties. This breakthrough enabled the long-term preservation of living cells at ultra-low temperatures, effectively addressing the challenge of ice crystal formation. By the mid-1950s, clinicians began experimenting with frozen human semen for artificial insemination, and by 1973, Sherman documented the first successful clinical application of cryopreserved sperm. Initially, the primary beneficiaries were men undergoing radiotherapy or chemotherapy, as Sanger et al. (1992) emphasized the necessity of cryobanking for cancer patients, workers exposed to occupational hazards, and individuals electively delaying parenthood.The World Health Organization’s 1999 manual standardized semen analysis protocols, establishing key post-thaw quality benchmarks, including motility (>40%), morphology (>4% normal forms), and viability (>58%) (WHO, 1999). Despite these advancements, Royere et al. (1996) highlighted persistent variability in post-thaw recovery rates, attributing inconsistencies to differences in cryoprotectant formulations, cooling rate fluctuations, and individual sperm resilience.

The 1980s and 1990s witnessed an exponential rise in cryobanking demand, largely driven by advances in assisted reproductive technologies (ART) and increasing awareness of iatrogenic infertility risks. The first live birth using frozen sperm was reported by Bunge and Sherman (1953), followed by Critser et al. (1987), who demonstrated that cryopreserved sperm retained fertilization potential in zona-free hamster ova penetration assays. However, clinical outcomes remained suboptimal compared to fresh semen. Richter et al. (1984) noted pregnancy rates of 12% per cycle with cryopreserved donor sperm compared to 18% with fresh samples, a disparity linked to cryoinjury mechanisms such as oxidative stress and DNA fragmentation (Said et al., 2010). These findings underscored the need for optimized cryopreservation protocols to mitigate cellular damage during freezing and thawing.

1. Cryopreservation Techniques: Slow Freezing And Vitrification

1. Slow Freezing

Slow freezing, the conventional method since the 1950s, involves a gradual cooling process at a rate of 1–10°C/min to −80°C, followed by immersion in liquid nitrogen at −196°C. This method facilitates extracellular ice formation while minimizing intracellular crystallization via osmotic dehydration (Mazur, 2004). Taylor et al. (1982) reported that slow freezing preserves 30–50% of baseline motility, though DNA fragmentation increases by 15–20% compared to fresh samples, as quantified through TUNEL assays (Hammadeh et al., 2001). While clinically reliable, slow freezing has drawbacks. Ragni et al. (1990) observed comparable pregnancy rates between slow-frozen and fresh sperm in ovarian stimulation cycles (23% vs. 27%). However, reliance on extracellular cryoprotectants such as glycerol introduces osmotic stress, which disrupts sperm membrane integrity. Check et al. (1991) found that membrane lipid peroxidation increased by 35% post-thaw, impairing sperm-oocyte fusion. Cross and Hanks (1991) also observed a 40% reduction in fertilization capacity due to acrosomal enzyme leakage during thawing.

1. Vitrification

Vitrification, an ultra-rapid cooling technique developed in the 2000s, involves solidifying sperm into a glass-like state without ice formation. O’Neill et al. (2019) demonstrated that vitrification preserves 58% motility and 72% viability in normozoospermic samples, outperforming slow freezing (42% and 55%, respectively). The elimination of ice crystal formation mitigates mechanical damage to organelles and DNA. Tvrdá et al. (2021) reported 12% DNA fragmentation in vitrified sperm, compared to 25% in slow-frozen counterparts. Despite its advantages, vitrification faces clinical adoption challenges due to technical complexity. It necessitates high cryoprotectant concentrations (≥6M glycerol), which induce oxidative stress and mitochondrial dysfunction. Critser et al. (1987) observed a 40% decline in mitochondrial membrane potential at these concentrations, correlating with reduced ATP production. Spis et al. (2019) achieved live births using vitrified testicular sperm in azoospermic men but emphasized the necessity of standardized protocols to ensure reproducibility.

1. Cryoprotectants: Balancing Protection And Toxicity

1. Permeating Agents

Glycerol has remained the cornerstone of sperm cryopreservation since the 1950s, functioning by reducing intracellular water content via osmosis, thereby lowering the freezing point and minimizing ice nucleation. Centola et al. (1992) determined that a 7% glycerol concentration optimized post-thaw motility at 45%, while higher concentrations compromised mitochondrial membrane potential. O’Connell et al. (2002) found that a 10% glycerol concentration reduced ATP synthesis by 60%, illustrating the delicate balance between cryoprotection and cytotoxicity.

Dimethyl sulfoxide (DMSO), another permeating agent, exhibits comparable efficacy but induces greater DNA oxidation at concentrations exceeding 5% (Lucena & Obando, 1986). Emerging alternatives include ethylene glycol, which stabilizes membranes at lower concentrations (3–4%), although clinical data remain limited (Hammerstedt & Graham, 1992).

1. Non-Permeating Agents

Non-permeating agents such as sucrose and trehalose act as extracellular stabilizers, mitigating osmotic shock during freezing. TEST-yolk buffer (TYB), incorporating egg yolk phospholipids, improves motility retention by 15–20% compared to glycerol alone (Hallak et al., 2000). However, concerns about microbial contamination from animal-derived components have prompted interest in synthetic alternatives. Jeyendran et al. (1995) proposed hydroxyethyl starch (HES) as a safer option, demonstrating a 90% reduction in bacterial contamination in a study involving 200 samples.

1. Mechanisms Of Cryoinjury: Structural And Functional Impacts

Cryopreservation-induced damage primarily affects the sperm plasma membrane, acrosomal integrity, and DNA stability. Holt and North (1984) observed irreversible phase transitions in membrane lipid bilayers, leading to increased permeability to calcium and proteases. Cross and Hanks (1991) reported 40% acrosome loss in slow-frozen sperm, impairing oocyte penetration capacity. Furthermore, Sharma et al. (2021) found that slow freezing results in 25% DNA fragmentation compared to 12% in vitrified samples. Said et al. (2010) identified a 2.5-fold increase in caspase-3 expression post-thaw, suggesting apoptosis activation as a contributing factor.

1. Clinical Outcomes: Successes And Limitations

Fresh semen achieves higher per-cycle pregnancy rates (18% vs. 12%) in donor insemination, but cryopreservation offers the advantage of eliminating infectious risks (Wong et al., 1989). In IVF/ICSI, cryopreserved sperm achieve fertilization rates comparable to fresh samples (78% vs. 82%), as ICSI bypasses motility deficits (Yogev et al., 1999). Agrawal et al. (1995) reported a 65% pregnancy success rate in testicular cancer patients using cryopreserved sperm, despite an average post-thaw motility of 32%.

1. Unresolved Challenges And Future Directions

Concerns regarding epigenetic alterations and offspring health persist. Cryopreservation modifies sperm miRNA profiles, including downregulation of miR-34a, which regulates embryonic development (Shangguan et al., 2020). While long-term offspring health data are scarce, Spis et al. (2019) reported no congenital anomalies in 120 children born from vitrified sperm. Efforts to optimize vitrification protocols continue, with synthetic cryoprotectants such as carboxylated ε-poly-L-lysine showing promise (Zhu et al., 2021). Future research should focus on evaluating natural conception dynamics post-cryopreservation, as Lee et al. (2022) found a 50% reduction in cervical mucus migration capacity post-thaw.

II. METHODOLOGY

1. Study Design And Objectives

This study was designed to compare two semen cryopreservation techniques—slow freezing and vitrification—by evaluating key post-thaw sperm parameters, including motility, viability, morphology, and DNA integrity. The primary objective was to determine which method better preserves sperm quality, thereby contributing to advancements in assisted reproductive technologies (ART). A quantitative experimental approach was employed, wherein human semen samples underwent both cryopreservation techniques, and post-thaw sperm quality was assessed accordingly.

1. Ethical Considerations And Approval

Prior to the initiation of the study, ethical approval was obtained from the Rayat Bahra University, Punjab, and GFC Hospital, Kushpata, Ghatal, Paschim Medinipur, West Bengal. Participants provided written informed consent after being fully briefed on the study's purpose and the intended use of their sperm samples for research. The study adhered to the principles of the Declaration of Helsinki, ensuring the confidentiality and anonymity of all participants.

1. Sample Collection

Participants were selected based on specific inclusion and exclusion criteria to ensure a homogeneous study population. Eligible participants were healthy males aged 25 to 40 years with no history of infertility or significant medical conditions, and whose semen parameters met the World Health Organization (WHO, 2010) standards. Exclusion criteria included a history of chemotherapy or radiation therapy, infectious diseases such as HIV or Hepatitis B, and diagnosed conditions such as oligozoospermia, asthenozoospermia, or teratozoospermia.

To optimize semen quality, participants were instructed to abstain from ejaculation for 2 to 5 days before sample collection. Samples were collected via masturbation in a sterile container within a private facility and immediately processed in the laboratory. Each sample underwent a baseline semen analysis to confirm adherence to WHO standards, ensuring motility above 40%, concentration exceeding 15 million/ml, and normal morphology above 4%. The samples were then divided into two aliquots—one assigned to slow freezing and the other to vitrification.

1. Cryopreservation Procedures

1. Slow Freezing Method

Slow freezing, the conventional cryopreservation technique, was performed in a stepwise manner. Initially, the semen samples were diluted with a freezing medium containing 10% glycerol as a permeating cryoprotectant. This medium was added gradually in a 1:1 ratio at room temperature to prevent osmotic shock. The samples were then loaded into cryovials and cooled in a programmable freezer at a controlled rate of -1°C per minute until reaching -80°C. To facilitate ice crystal formation, manual seeding was performed by touching the cryovials with a cold rod at -6°C. Once the target temperature was achieved, the samples were transferred into liquid nitrogen at -196°C for long-term storage.

1. Vitrification Method

Vitrification involves ultra-rapid cooling to avoid ice crystal formation, thereby preserving sperm integrity. In this method, semen samples were mixed with a cryoprotectant containing 15% glycerol and 0.5 M sucrose in a stepwise manner, with a 5-minute equilibration period between additions to reduce toxicity. Following equilibration, the samples were placed in straws and directly plunged into liquid nitrogen at -196°C, achieving an ultra-rapid cooling rate exceeding 20,000°C per minute. This process prevented the formation of intracellular ice, converting the water within sperm cells into a glass-like solid state.

1. Thawing Procedure

Thawing was carried out in a standardized manner for both cryopreservation techniques. Slow-frozen samples were removed from liquid nitrogen and thawed in a 37°C water bath for 5 minutes. The cryoprotectant was then gradually removed through stepwise dilution with a sperm washing medium. Similarly, vitrified samples were thawed at 37°C for 5 minutes, followed by cryoprotectant removal through stepwise dilution with a sperm washing medium containing decreasing concentrations of sucrose to prevent osmotic shock.

1. Post-Thaw Sperm Analysis

Post-thaw sperm quality was assessed through various standardized tests:

• Motility Assessment: Computer-assisted sperm analysis (CASA) was employed to classify sperm motility into progressive motility (active forward movement), non-progressive motility (limited or erratic movement), and immotile sperm (no movement).
• Viability Testing: Eosin-nigrosine staining was used to distinguish viable sperm from non-viable ones, with at least 200 sperm counted per sample to determine the percentage of viable cells.
• Morphology Evaluation: Sperm morphology was examined using Papanicolaou staining, wherein 200 sperm per sample were assessed under a light microscope, and normal and abnormal forms were recorded based on WHO guidelines.
• DNA Fragmentation Test: Sperm DNA integrity was analysed using the Sperm Chromatin Structure Assay (SCSA), which measures DNA susceptibility to denaturation under acidic conditions. The DNA fragmentation index (DFI) was quantified using flow cytometry, with higher DFI values indicating greater DNA damage.

1. Statistical Analysis

All statistical analyses were performed using SPSS (Statistical Package for the Social Sciences) version 25. Descriptive statistics, including mean values and standard deviations, were calculated for motility, viability, morphology, and DNA fragmentation across both cryopreservation methods. Paired t-tests were employed to compare pre- and post-thaw values within each method, while an Analysis of Variance (ANOVA) was conducted to determine differences between the two techniques. A p-value of less than 0.05 was considered statistically significant.

III. RESULT

1. Overview Of Sample Collection

A total of 30 semen samples were collected from healthy male participants aged between 25 and 40 years. Each sample was divided into two aliquots for cryopreservation: one for slow freezing and the other for vitrification. Prior to freezing, baseline semen analyses were performed to ensure all samples met the WHO (2010) criteria for normal semen parameters.

Baseline Characteristics Of Semen Samples(Who, 2010)

1. Post Thaw Sperm Quality Assessment

After thawing, sperm samples were assessed for motility, viability, morphology, and DNA integrity. The findings from these evaluations are detailed below.

Sperm Motility

Post-thaw motility was significantly affected by the cryopreservation method.

Comparison Of Sperm Motility Post- Thawing

The results indicated that sperm motility was significantly higher in the vitrification group compared to the slow freezing group (p < 0.001). These findings suggest that vitrification is more effective in preserving sperm motility after thawing

Sperm Viability

Sperm viability was assessed using eosin-nigrosine staining.

Comparison Of Sperm Viability Post- Thawing

Sperm viability was also significantly higher in the vitrification group compared to the slow freezing group (p < 0.001). These results highlight the superior effectiveness of vitrification in maintaining sperm viability post-thaw.

Sperm Morphology

Morphological evaluation revealed differences in sperm structure between the two methods.

Comparison Of Sperm Morphology Post- Thawing

The morphological assessment indicated that a greater percentage of sperm exhibited normal morphology in the vitrification group compared to the slow freezing group (p < 0.01), underscoring the benefits of vitrification in preserving sperm morphology.

1. DNA Fragmentation  Assessment

DNA integrity was evaluated using the SCSA technique.

                                       COMPARISON OF DNA FRAGMENTATION INDEX  POST- THAWING

The SCSA results showed a significantly lower DNA fragmentation index in the vitrification group compared to the slow freezing group (p < 0.001), indicating that vitrification not only preserves motility and viability but also maintains sperm DNA integrity more effectively.

Summary of the Finding

The results of this study indicate that vitrification is superior to slow freezing in preserving post-thaw sperm quality. All evaluated parameters—motility, viability, morphology, and DNA integrity—showed significantly better outcomes with vitrification. These findings provide strong evidence for the adoption of vitrification as a preferred method for sperm cryopreservation in clinical settings.

IV. discussion

1. Overview Of Key Findings

This study highlights the superior efficacy of vitrification over slow freezing in preserving post-thaw human sperm quality. Vitrification significantly outperformed slow freezing across all key parameters: motility, viability, morphology, and DNA integrity. Post-thaw total motility was markedly higher in vitrified sperm (55.1%) compared to slow freezing (36.5%). Similarly, sperm viability was significantly greater in the vitrification group (70.9% vs. 48.3%), and normal morphology was better preserved (7.0% vs. 4.5%). Furthermore, DNA fragmentation was notably lower in vitrified samples (18.5%) compared to slow-frozen samples (34.6%).

These findings align with existing literature supporting vitrification as a superior cryopreservation method due to its ability to mitigate cryoinjury and ice crystal formation, which are common drawbacks of slow freezing.

1. Interpretation Of Results

Sperm Motility

The significantly higher post-thaw motility in vitrified sperm corroborates previous studies. Gonzalez et al. (2016) demonstrated that vitrification minimizes ice crystal formation, a key factor in preserving cell viability. The rapid cooling process prevents cellular dehydration and intracellular ice formation, maintaining sperm membrane integrity—crucial for motility.

In contrast, slow freezing exposes sperm to osmotic shock and cellular damage, leading to reduced motility. Morris et al. (2018) noted that gradual cooling allows ice formation within cells, impairing post-thaw movement.

Sperm Viability

Higher viability in vitrified samples mirrors the motility trends observed. Liu et al. (2020) reported similar findings, attributing this to the protective effects of cryoprotectants like glycerol and sucrose, which create a favourable osmotic environment and preserve membrane integrity.

The lower viability in the slow freezing group underscores the risks of cryoinjury, where ice formation disrupts sperm function. These findings emphasize the importance of optimizing cryopreservation techniques to minimize cell damage.

Sperm Morphology

The increased proportion of morphologically normal sperm in vitrified samples supports prior research by Zhang et al. (2019), which found that vitrification reduces mechanical stress on sperm cells. The absence of ice crystal formation prevents structural deformation, preserving sperm integrity.

Conversely, slow freezing introduces mechanical trauma due to ice crystallization, resulting in morphological defects. This is clinically significant, as sperm morphology directly influences fertilization potential.

1. DNA Fragmentation

Lower DNA fragmentation in vitrified sperm is particularly noteworthy, given the critical role of DNA integrity in fertilization and embryo development. Kumar et al. (2020) established a strong correlation between high DNA fragmentation and poor reproductive outcomes, highlighting the necessity of preservation methods that minimize genetic damage.

The superior DNA integrity observed in the vitrification group suggests that this method offers enhanced protection against oxidative stress and cryoinjury, potentially improving ART success rates.

1. Discussion of Anomalies and Limitations

Despite the strong overall results favouring vitrification, some anomalies were noted. A subset of samples in both groups exhibited lower-than-expected motility and viability, likely due to biological variability or undetected subclinical conditions. Further research is needed to explore these factors.

Additionally, this study was limited by a relatively small sample size and a geographically homogeneous participant pool. Expanding the cohort and incorporating diverse populations in future studies would enhance the generalizability of these findings.

1. Clinical Implications

The findings of this study have significant implications for assisted reproductive technologies (ART). Given the clear advantages of vitrification in preserving sperm quality, ART clinics should consider adopting this method as the preferred standard for cryopreservation.

Vitrification not only improves post-thaw sperm motility and viability but also maintains morphological and genetic integrity, thereby enhancing fertilization potential and embryo quality. By minimizing cryoinjury, this approach may improve overall ART success rates.

Furthermore, ongoing research into advanced cryopreservation techniques is essential to optimize fertility preservation strategies, particularly for individuals undergoing medical treatments that may impact reproductive health, such as chemotherapy. Continuous innovation in this field will contribute to improved reproductive outcomes and broader accessibility to effective fertility preservation methods.

Conclusion

A. Summary Of Findings This study compared the effectiveness of slow freezing and vitrification in sperm cryopreservation, evaluating key parameters such as motility, viability, morphology, and DNA integrity. The results clearly demonstrated that vitrification outperforms slow freezing across all metrics. Post-thaw motility was significantly higher in vitrified samples (55.1%) compared to those frozen slowly (36.5%). Similarly, sperm viability was better preserved in the vitrification group (70.9% vs. 48.3%), and normal morphology was maintained at a higher rate (7.0% vs. 4.5%). Additionally, DNA fragmentation was markedly lower in vitrified sperm (18.5%) than in slow-frozen samples (34.6%). These findings reinforce vitrification as a superior method for sperm preservation, minimizing cryoinjury and ensuring better post-thaw sperm function, which is crucial for reproductive success. B. Significance Of The Study The implications of these findings extend beyond laboratory analysis, emphasizing the clinical importance of vitrification in assisted reproductive technologies (ART). Given its ability to better maintain sperm integrity, this method has the potential to enhance fertility treatment outcomes, offering improved chances of successful conception for individuals and couples undergoing ART. Furthermore, this study contributes to the broader understanding of cryopreservation techniques, reinforcing the need for ongoing advancements in the field. By highlighting the advantages of vitrification, these findings advocate for a shift in clinical practice toward more effective sperm preservation methods that maximize reproductive potential. C. Future Research Directions While this study provides compelling evidence supporting vitrification, further research is needed to explore its long-term implications. Future studies should focus on assessing the reproductive outcomes of offspring conceived using vitrified sperm, ensuring that the method does not introduce unforeseen risks. Expanding research to include more diverse populations will also help determine the generalizability of these findings. Additionally, comparative studies involving emerging cryopreservation techniques could provide insights into optimizing sperm preservation further. Investigating the influence of individual factors such as lifestyle, environmental exposure, and underlying health conditions on post-thaw sperm quality could refine cryopreservation strategies and lead to more personalized fertility preservation approaches. Exploring how vitrification interacts with other ART techniques, such as intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing (PGT), may reveal additional benefits that enhance overall treatment success rates. D. Final Thoughts In conclusion, this study establishes vitrification as a more effective sperm cryopreservation method than slow freezing, demonstrating its clear advantages in maintaining sperm quality. By minimizing cryoinjury and preserving crucial functional parameters, vitrification offers significant potential to improve fertility treatment success. These findings not only lay the groundwork for future advancements in sperm preservation but also reinforce the importance of continued innovation in reproductive medicine. As the field evolves, optimizing cryopreservation methods will remain essential in addressing infertility challenges and improving reproductive outcomes for individuals worldwide.

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Copyright

Copyright © 2025 Ms. Tithi Karmakar, Dr Sunil Kumar, Dr TK Karmakar. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.