Optimize human sperm cryopreservation: influence of vial sealing methods and automated freezing protocols on preserved sperm quality

Introduction

Cryopreservation is a well-established and indispensable technique in human reproductive medicine. It plays a crucial role in fertility preservation for oncological patients, whose germ cells may be compromised by the underlying disease or gonadotoxic therapies, and constitutes a central component of assisted reproductive technologies by enabling long-term storage of male fertility. The objective of sperm cryopreservation is to preserve cellular integrity and functionality during long-term storage at ultra-low temperatures by preventing irreversible cryodamage (1-3). Central to this process is the controlled regulation of water movement and ice crystal formation during cooling and thawing. During cooling, extracellular ice formation occurs first in the aqueous environment, increasing solute concentration in the remaining liquid phase and inducing osmotic water efflux from the cell (4). This dehydration process helps to prevent intracellular ice crystal formation, which represents a major source of cryodamage (5). These protective mechanisms are further supported by cryoprotectants such as glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol, which inhibit ice crystal formation, increase solution viscosity, regulate osmolarity, and reduce the fraction of freezable water, thereby limiting cellular injury during both freezing and thawing (2,6). One of the most critical determinants of cryopreservation success is the cooling rate (1). If cooling is too slow, excessive water efflux leads to pronounced cellular dehydration, while extracellular ice formation concentrates solutes in the residual unfrozen fraction, creating osmotic stress and potential damage to cytoplasmic components (7). Conversely, overly rapid cooling prevents sufficient water efflux, resulting in intracellular ice formation that can disrupt organelle integrity and cellular function (8). Thus, both excessively slow and excessively fast cooling rates can induce cryodamage. Therefore, optimized cryopreservation requires a carefully balanced cooling protocol to minimize cellular injury (9). Currently, several freezing techniques are applied in clinical practice, including slow freezing, rapid freezing, and vitrification (1,7). Rapid freezing is time-efficient, as samples are transferred into liquid nitrogen immediately after a brief pre-cooling step. However, this approach requires strict control of cryoprotectant conditions to avoid uncontrolled ice formation (10,11). Slow freezing, in contrast, uses gradual temperature reduction to promote controlled dehydration and remains the most widely used approach due to its reliability and adaptability (12). While semi-automated devices, such as the Air Liquide Nicool LM 10, rely on user-controlled nitrogen-based cooling, fully automated systems like the IceCube system (SY-LAB), offer programmable temperature profiles and standardized documentation, potentially improving reproducibility.

Despite existing legal and medical guidelines, detailed technical standards for sperm cryopreservation are still lacking, leading to substantial methodological variability across laboratories and potentially affecting post-thaw sperm quality (13-15). Therefore, the present study aims to optimize human sperm cryopreservation by developing standardized and reproducible protocols suitable for routine clinical application.

Methods

Study population

The study was conducted on semen samples obtained from 30 healthy male volunteers aged 18–35 years recruited from the student population of the Eberhard-Karls-University Tuebingen, Germany. Participants provided written informed consent, and individuals with known fertility impairment or nicotine use were excluded. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The present study protocol was reviewed and approved by the Ethics Committee of Medical Faculty of Eberhard-Karls-University Tuebingen (ethics application No. 609/2018B02).

Semen analysis and cryopreservation

Ejaculate samples were collected by masturbation following a minimum abstinence period of three days. After standardized liquefaction of 30 min in an incubator at a constant temperature of 37 ℃, semen analysis was performed at room temperature according to World Health Organization (WHO) guidelines (16), assessing macroscopic (volume, pH, color, and viscosity), and microscopic parameters (sperm concentration, total sperm count, motility, and the presence of leukocytes and erythrocytes). For determination of sperm concentration, 100 µL of liquefied ejaculate was diluted with 2 mL of formalin solution (1:21 dilution), thoroughly vortexed to ensure homogeneous distribution and immobilization of the spermatozoa, and subsequently transferred to a Neubauer-improved hemocytometer. Spermatozoa were counted in five squares of the central grid in duplicate (two independently loaded counting chambers per sample) at ×400 magnification using a transmitted-light microscope (Carl Zeiss, product number 473011 9901). Sperm concentration and total sperm count were calculated according to WHO guidelines (15).

Sperm motility was assessed microscopically at ×400 magnification by analyzing at least five randomly selected fields per sample and classifying spermatozoa as progressively motile, non-progressively motile, or immotile according to WHO criteria. Motility evaluation was supported by an electronic counting device (Assistent Differenziergerät Counter AC-8, Karl Hecht KG). Leukocytes and erythrocytes were detected by peroxidase staining and evaluated microscopically at ×400 magnification; leukocytes were identified as peroxidase-positive cells, while erythrocytes were identified based on their characteristic morphology. All parameters were documented according to WHO reference standards for further analysis.

Inclusion criteria for the cryopreservation analyses required normozoospermia according to the reference ranges defined in the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition, 2010) (16). A sample was classified as normozoospermic when all assessed macroscopic and microscopic parameters, including semen volume (≥1.5 mL), pH, sperm concentration (≥15×10⁶/mL), total sperm count (≥39×10⁶ per ejaculate), and progressive as well as total motility, fell within the WHO reference ranges. Samples exhibiting any pathological finding (e.g., oligozoospermia, asthenozoospermia, or azoospermia) were excluded from the subsequent freezing and thawing comparisons.

Following semen analysis, samples were processed under sterile conditions and mixed 1:1 with a commercially available cryoprotective freezing medium (Quinn’s Advantage^TM Sperm Freezing Medium, ART-8022; SAGE In Vitro Fertilization, CooperSurgical Company, Trumbull, USA) containing human serum albumin, glycerol as a cryoprotectant, and gentamicin for antimicrobial stabilization. No additional dilution was performed to adjust sperm concentration prior to cryopreservation; the standardized 1:1 mixing ratio was applied uniformly across all samples to ensure a consistent final cryoprotectant concentration. The prepared samples were aliquoted into 250 µL plastic straws (Minitüb GmbH, Tiefenbach, Germany) and mounted in standardized storage cassettes.

Straw sealing methods

To evaluate sealing methods, straws were sealed either by ultrasonic heat sealing (CryoSealer; Ref. 13135/0000, Minitüb GmbH) or by the conventional metal ball closure technique from the human sperm packaging set (article No. 13490/0036; Minitüb GmbH). For the conventional method, filled straws were manually sealed by inserting a metal closure ball into the open end and applying pressure to ensure a secure closure. Ultrasonic sealing was performed at the manufacturer-recommended duration of 4.9 seconds (s), as well as a shortened duration of 2.5 s, to evaluate potential time savings and protection from ultrasound damage in routine clinical use. Straw integrity, airtightness, and post-thaw sperm vitality and motility were assessed and compared between sealing durations and against the conventional ball-sealing method as a reference standard.

Comparison of straw sealing methods

Each ejaculate was divided into three equal aliquots, with three straws per aliquot assigned to one of three sealing methods. Post-thaw sperm vitality and survival rates—calculated as the percentage of motility relative to the fresh sample—were assessed, including total motility [progressive motility (PR) + non-progressive motility (NP)] and PR.

Formula for survival rate (%):

Survivalrate=MotilityafterthawingMotilityinnativeejaculate×100%[1]

All straws underwent standardized cryopreservation using the conventional Nicool freezing device (LM10, Air Liquide Deutschland GmbH, Duesseldorf). Primary endpoints were post-thaw sperm vitality and motility immediately after thawing.

Leakage test of the CryoSealer

Straw integrity after ultrasonic sealing with the CryoSealer was evaluated using methylene blue–stained samples. Four straws were filled with methylene blue samples and sealed with the CryoSealer for 2.5 s (n=2) or 4.9 s (n=2) and submerged completely overnight in water. Leakage was assessed visually to confirm complete and reliable sealing.

Development of a cryopreservation protocol using the IceCube 11XS System

To establish a standardized protocol for clinical sperm cryopreservation, ejaculates with physiological reference values were processed using the fully automated IceCube 11XS freezing device (SY-LAB Geräte GmbH, Neupurkersdorf, Austria). This computer-controlled system enables programmable cooling with uniform temperature distribution, continuous chamber monitoring, and real-time internal sample temperature measurement. The integrated microcontroller ensures precise control of cooling and heat transfer through program-controlled liquid nitrogen supply, allowing reproducible cryopreservation. Optimization of the freezing process was conducted based on systematically testing different cooling rates, hold times, and temperature profiles.

Development of a new freezing protocol

The primary goal was to develop an optimized, cell-preserving freezing protocol for the IceCube 11XS system following WHO guidelines [2010] and manufacturer instructions. A major focus was preventing uncontrolled temperature spikes at the crystallization point, where the exothermic transition of the processed ejaculate-cryoprotectant solution from liquid to solid can negatively affect sperm viability and motility. Without countermeasures, the released crystallization heat can cause brief temperature fluctuations that may negatively affect sperm vitality and motility. The aim was therefore to compensate for this heat release by a precisely adapted freezing curve, ensuring uniform and controlled cooling. Optimization was performed through iterative testing of staggered cooling rates to identify the crystallization point, observed at approximately −18 ℃ during a linear cooling rate of −2 ℃/min. The freezing program was then adjusted to counteract crystallization heat via an early rapid-cooling phase followed by a holding phase. The final protocol (‘freezing program 27’, see Table 1) implemented staged cooling rates: −1.5 ℃/min before the crystallization complex, −6 ℃/min immediately after, and −10 ℃/min from −130 ℃ onward. Figure 1 illustrates the optimized temperature profile resulting from freezing program 27. This approach successfully compensated for crystallization heat, ensured controlled cooling, and provided optimal conditions for post-thaw sperm viability and motility, forming the basis for subsequent validation.

Figure 1 Freezing curve: after reaching the starting temperature of 20 ℃, continuous cooling began at a rate of −1.5 ℃/min (Segment 1). At −12 ℃, rapid cooling was initiated with a rate of −40 ℃/min (Segment 2), followed by a warming phase lasting 18 seconds with a heating rate of 10 ℃/min (Segment 3) and a holding phase of 50 seconds (Segment 4). Subsequently, continuous cooling resumed at a rate of −6 ℃/min (Segment 6), and from −130 ℃ onwards, the cooling rate was set to −40 ℃/min.

Validation of the IceCube 11XS freezing protocol

After establishing this optimized IceCube 11XS protocol, its performance was directly compared with the conventional semi-automated Nicool LM 10 freezing device. In the Nicool system, cryopreservation is achieved via indirect cooling using evaporating nitrogen. Sealed straws are placed in a rack above a liquid nitrogen container (−196 ℃), and an integrated fan ensures uniform nitrogen distribution. The cooling rate can be adjusted across nine fan settings, applied sequentially for 3 min each, with the highest setting maintained until the chamber reaches −130 ℃. After reaching the target temperature, straws are transferred to liquid nitrogen (−196 ℃) for storage. Chamber temperatures were monitored, while exact sample temperatures were not directly measured. For protocol validation, 11 ejaculates were split into two aliquots. One aliquot was frozen using the Nicool LM 10 standard protocol, and the other using the IceCube 11XS protocol. All samples were stored in liquid nitrogen at −196 ℃ for at least 24 hours. Thawing controls were then performed to compare post-thaw sperm vitality and motility as mentioned.

Thawing and quality control

Two straws per aliquot were thawed following the in-house protocol based on WHO guidelines: 3 min at room temperature, followed by 20 min in a 35–37 ℃ water bath. Each straw was opened, and a drop of semen was placed on a microscope slide for analysis using the same methodology as native semen assessment. Sperm vitality was additionally assessed via eosin staining. At least five randomly selected fields of view were analyzed per sample, evaluating ≥200 spermatozoa. Mean values per aliquot were calculated, and percentages of motility and vitality were determined.

Statistical analyses

Data were analyzed using GraphPad Prism 10.4.2. Descriptive statistics included mean ± standard deviation (SD), median, and interquartile range (IQR). Owing to the limited sample size, no formal test for normal distribution was performed; non-parametric tests were applied a priori, representing the methodologically more conservative approach: the Friedman test for the three straw sealing methods, and the Wilcoxon signed-rank test for paired cryopreservation comparisons. A P value ≤0.05 was considered statistically significant.

Results

Semen analysis of fresh samples

Microscopic analysis of fresh samples from 30 participants showed that most parameters were within reference ranges. Minor deviations were noted for ejaculate volume (6.7%), sperm concentration (10.0%), and total sperm count (6.7%), including one case each of mild oligozoospermia, moderate oligozoospermia, and azoospermia. Impairments in motility were rare, with total motility (PR + NP) below reference in 3.3% and progressive motility below reference in 13.3% of participants (Table 2).

Comparison of straw closure methods

Analysis in a subset of four participants revealed no statistically significant differences among the three closure methods regarding sperm vitality, total motility, or progressive motility, see Table 3. The CryoSealer method with 4.9 s sealing tended to show the highest vitality, while the 2.5 s sealing exhibited the highest total motility survival rate. The conventional ball-seal method yielded the highest progressive motility survival rate. Overall, the CryoSealer, independent of sealing duration, produced results comparable to the ball-seal method, with minor, non-significant differences (Figure 2). No methylene blue leakage was observed in any sample, confirming complete airtightness and reliability of the CryoSealer method for both sealing durations.

Figure 2 Representation of vitality and survival rate of all motile as well as progressively motile sperms after the freezing and thawing process by a Nicool system, depending on the closure method used.

Sperm motility after cryopreservation

Cryopreservation markedly reduced sperm motility in both freezing systems. Total motility (PR + NP) and progressive motility (PR) were significantly lower after thawing compared to fresh samples (both P=0.001, Tables 4,5, Figure 3A), demonstrating the impact of freezing independent of the system. Direct comparison between the Nicool and IceCube systems revealed no statistically significant differences in total motility (P=0.75) or progressive motility (P=0.40). Slightly higher mean and median values were observed for both parameters with the IceCube system, as well as for survival rates of total (P=0.83) and progressive motility (P=0.52), indicating a non-significant trend toward improved performance. Table 6 and Figure 3B present a comparison of sperm parameters after the freeze-thaw process for both freezing systems (Nicool, IceCube). A statistically significant difference was observed for sperm vitality, which was higher in the IceCube group compared to Nicool (P=0.02), suggesting better preservation of cellular integrity. Graphical demonstration illustrated the overall decrease in motility after cryopreservation for both systems and especially highlighted substantial inter-individual variability. For most participants, total and progressive motility tended to be higher with IceCube, although differences did not reach significance except for vitality (Table 6, Figure 3C,3D).

Figure 3 Comparison of sperm quality parameters before and after the freeze-thaw process using two different cryopreservation systems (Nicool and IceCube). (A) Depiction of total motility and progressive motility before freezing (native) and after the freeze-thaw process depending on the freezing system used (Nicool, IceCube). (B) Depiction of survival rates for total and progressive motility after the freeze-thaw process depending on the freezing system used (Nicool, IceCube). (C) Depiction of total and progressive motility in fresh samples and after the freeze-thaw process for each freezing system (Nicool and IceCube); individual participant values shown. (D) Depiction of survival rates for total and progressive motility after the freeze-thaw process for each freezing system (Nicool and IceCube); individual participant values shown. ns; ***.

Discussion

The increasing importance of fertility preservation in oncology reflects improved survival rates due to modern cancer therapies (17,18). As chemo- and radiotherapy often cause irreversible germ cell damage, sperm cryopreservation prior to treatment remains the primary, and often only, option for safeguarding future fertility (19). Optimizing cryopreservation protocols is therefore critical, particularly as sperm quality may already be compromised before therapy.

Secure sealing of cryostraws is a key factor for the safety and quality of cryopreserved samples. Even minor leaks can increase the risk of microbial contamination or process-related damage (20-23). Conventional metal ball sealing is user-dependent and shows limited reproducibility, partially failing to meet current recommendations for fully closed vessel systems (15,24). In this study, standardized thermal sealing using the CryoSealer was evaluated as an alternative. Both the manufacturer-recommended 4.9 s and a shortened 2.5 s sealing application produced airtight straws, with no leakage observed during methylene blue testing. Post-thaw sperm vitality, total motility, and progressive motility were comparable, or slightly improved relative to the conventional ball-seal method. The shorter sealing time (2.5 s) demonstrated practical advantages by reducing processing time without compromising sample quality and showed a tendency toward higher survival rates of progressively motile and total motile sperms. Overall, thermal sealing provided a reproducible, operator-independent, and hygienically reliable approach, suitable for routine clinical use in line with WHO guidelines.

Despite the clinical importance of sperm cryopreservation, there is a lack of standardized, validated freezing protocols. Available evidence focuses on basic methods or broad cooling ranges without providing precise, device-specific parameters for the procedure (25). Consequently, reproducible, clinically applicable reference values are scarce. Protocol development has largely relied on theoretical cryobiological principles—controlled cooling, dehydration, and minimization of ice crystal formation—which provide guidance but do not directly translate into precise temperature profiles for modern automated systems. Even the WHO Laboratory Manual provides only general guidelines and does not consider the specific characteristics of modern freezing systems (15).

This study addresses this methodological gap by developing a standardized reproducible freezing protocol for the fully automated IceCube 11XS system. Iterative adjustment of cooling rates and controlled staging around the critical crystallization phase ensured a stable temperature decline and minimized abrupt osmotic shifts, which are crucial for preserving sperm integrity.

Consistent with previous reports, cryopreservation significantly reduced total and progressive motility compared with fresh samples (both P=0.001), highlighting the inherent stress associated with freezing procedures. When comparing the Nicool and IceCube systems, no statistically significant differences in motility parameters were observed after thawing. However, descriptive trends favored the IceCube system, suggesting a potential advantage in preserving functional sperm characteristics. Notably, sperm vitality was significantly higher with the IceCube system (56.0% vs. 48.5%; P=0.02), likely reflecting the benefit of precise and continuous temperature control, particularly within the critical crystallization zone. Although the absence of statistical significance for some endpoints may be related to the limited sample size, the consistent direction of effects supports a biologically relevant advantage of the automated system. Overall, these findings indicate that fully automated, programmable freezing systems may enhance the stability and reproducibility of cryopreservation outcomes.

The IceCube protocol combines precise temperature regulation with full automation and digital documentation, thereby reducing operator dependency, minimizing procedural variability, and improving standardization and laboratory efficiency. While not all functional endpoints showed statistically significant superiority, the observed improvement in vitality highlights the potential clinical relevance of controlled and standardized freezing approaches for long-term fertility preservation.

The limitations of this study are acknowledged and should be interpreted in the context of the findings. The relatively small sample size and the use of healthy normospermic donors may limit direct extrapolation to oncological patients, who often present with impaired baseline sperm quality. However, the use of standardized donor samples was essential to ensure methodological control and to isolate the effects of the cryopreservation protocols themselves. In addition, only basic functional parameters (vitality, motility, and survival rates) were assessed, whereas functional markers such as DNA integrity, oxidative stress (ROS), or membrane stability were not included. Likewise, factors such as cryoprotectant composition, thawing conditions, and seeding were not systematically varied in this study. These aspects represent important biological and technical variables that may further influence post-thaw sperm function and should be addressed in future studies. Importantly, the present work provides a standardized methodological framework and reproducible baseline data, which can serve as a foundation for these future extensions and for further optimization of clinical cryopreservation protocols.

Conclusions

The developed IceCube 11XS-protocol provides results at least equivalent to the conventional Nicool system, with significantly higher sperm vitality and a trend towards improved motility. Fully automated, programmable freezing enables standardized, reproducible, and operator-independent cryopreservation, forming a robust basis for clinical application. Further validation in oncological patients and additional optimization of cryoprotectant use, thawing protocols, and individual sample parameters are warranted to maximize clinical outcomes.

Acknowledgments

The authors would like to thank Olga Dobler and Andrea Hohneder for excellent technical assistance.

Footnote

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0266/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0266/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0266/coif). A.S. serves as Secretary General and a member of the Board of the European Association of Urology (EAU) and NPO. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The present study protocol was reviewed and approved by the Ethics Committee of Medical Faculty of Eberhard-Karls-University Tuebingen (ethics application No. 609/2018B02). Informed consent was submitted by all subjects when they were enrolled.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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