Introduction
With advancements in assisted reproductive techniques (ART), fertility preservation has become an increasingly salient concern, particularly for patients with cancer [1]. Sperm cryopreservation represents a crucial step of ART and is considered the standard medical approach for preserving fertility in adolescents and adults [2,3]. This technique employs extremely low temperatures to maintain the structural integrity and functionality of living cells and tissues [4]. In cases of azoospermia or oligozoospermia, sperm cryopreservation can avoid the need for repeated invasive procedures such as biopsy or aspiration [3]. Cryopreservation ensures the availability of sperm for subsequent fertility treatments, significantly increasing the chances of successful pregnancy [5,6].
Despite major advances in cryopreservation technology, the freeze-thaw process continues to negatively impact sperm structure and function. Prominent adverse effects include reduced sperm motility and viability, compromised acrosome integrity, disrupted plasma membrane function, increased generation of reactive oxygen species (ROS), mitochondrial damage, and sperm DNA fragmentation (SDF) [7-9]. Additionally, the freeze-thaw process subjects sperm to various stresses, potentially reducing fertilization potential [10,11]. Current sperm preservation methods have notable limitations, including reliance on liquid nitrogen and storage tanks; these aspects pose safety concerns for personnel, complicate the transport of frozen samples [12-14], and introduce the potential for cross-contamination in shared storage facilities [15]. Consequently, a growing demand exists for alternative preservation techniques that offer fewer drawbacks and improved cost-effectiveness. Freeze-drying, also known as lyophilization, is an innovative method for the long-term preservation of sperm, enabling storage at refrigerator temperatures and transportation at room temperature [16]. This technique involves an initial freezing stage followed by direct sublimation of water from a solid (ice) to a gaseous phase, effectively removing water from cells [17]. Freeze-drying may also inactivate viruses through the drying process [18]. Studies involving various animal species have demonstrated the feasibility of freeze-drying, resulting in live births following intracytoplasmic sperm injection (ICSI) [19-21], although success rates vary and require further optimization. Advantages of this method include minimal storage requirements and lower storage costs, ease of use, and simpler transportation [22]. Despite these benefits, freeze-drying presents challenges, such as membrane injury that results in reduced sperm viability and DNA damage due to mechanical and oxidative stresses [23,24]. Additionally, the method can induce oxidative damage due to increased accumulation of ROS and weakened antioxidant defense systems with low water content [25].
To mitigate undesirable effects associated with sperm storage, various compounds are added to semen to compensate for reductions in endogenous antioxidants in seminal plasma, combat oxidative stress, and minimize the production of free radicals [26]. Herbal medicines are recognized as effective, inexpensive, and natural alternatives for treating certain male fertility disorders. Over the past two decades, plant extracts have emerged as a promising and cost-effective source of antioxidants for semen protection [27]. Green tea (Camellia sinensis [L.] Kuntze) is a well-known plant with biological and medicinal activities [28]. Among the biologically active compounds in Camellia sinensis, catechins are the primary antioxidants [29]. Specifically, catechins derived from green tea, such as epigallocatechin gallate (EGCG), exert potent antioxidant effects by neutralizing free radicals and enhancing enzyme-mediated detoxification [30]. Studies indicate that these compounds may improve reproductive health and positively affect the functional parameters of fresh and frozen sperm [31-34]. Given the antioxidant properties of green tea catechins and their potential benefits for sperm health, this study aimed to evaluate the in vitro effects of green tea aqueous extract (GTE) on oxidative stress markers and SDF in freeze-dried human sperm.
Methods
1. Ethical approval
This study was approved by the ethics committee of the School of Medicine at Shahid Beheshti University of Medical Sciences (Approval Identifier: IR.SBMU.MSP.REC.1402.329).
2. Plant material and extraction
Dried leaves of organic green tea were purchased from a local store (Deylaman Heights) and identified at the Herbarium of the Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences (No. HMS-568). An aqueous extract was prepared by boiling the leaves for 15 minutes at a plant-to-solvent ratio of 1:10. Following extraction, the solution was dried and converted into powder form.
3. Determination of total phenolic content of the extract
The total phenolic content of the GTE was measured using Folin-Ciocalteu reagent (Sigma-Aldrich), with pyrogallol (Sigma-Aldrich) as the standard [35]. The phenolic content was determined from a calibration curve of pyrogallol with concentrations ranging from 0.0078 to 0.125 mg/mL.
4. Extract preparation
A stock solution of GTE was prepared by dissolving 4 mg of dried extract in 10 mL of phosphate-buffered saline (PBS; Genocell). The solution was sterilized using a 0.22-μm syringe filter and diluted with sterile PBS to achieve a final concentration of 40 µg/mL [34]. The GTE was stored at 4 °C until use.
5. Participant criteria and sample collection
Ten semen samples were obtained from men aged 20 to 45 years attending the Andrology Laboratory at the Infertility and IVF Treatment Center, Taleghani Hospital (Shahid Beheshti University of Medical Sciences), between July and September 2023. After informed consent was obtained, samples were collected through masturbation after 2 to 7 days of sexual abstinence. Macroscopic and microscopic characteristics of the samples were assessed according to the 2021 World Health Organization (WHO) guidelines [36]. Samples of at least 3 mL with normal parameters (total sperm number >39×106/mL, total motility >42%, and progressive motility >30%) were selected for the study. Exclusion criteria included liquefaction time >30 minutes; history of cancer treatment; consumption of dietary supplements, vitamins, or antioxidants within 3 months prior to the study; history of varicocele or diabetes; smoking; and alcohol consumption.
6. Processing of semen samples
Semen samples were processed according to WHO guidelines [36]. Each sample was divided into three groups (n=10 per group): conventionally cryopreserved (group A), freeze-dried (group B), and freeze-dried with pre-incubation in GTE (group C). For each group, seminal plasma was separated by centrifugation at 2,500 ×g for 5 minutes, and the pellet was washed twice with human tubal fluid (HTF) medium (Genocell) containing 10% human serum albumin (Kedrion Biopharma). The pellet was then resuspended in 1 mL of the same medium.
7. Freezing/thawing procedures
Rapid freezing was used for cryopreservation [37]. A 1:1 mixture of sperm suspension and CryoSperm freezing medium (Origio) was equilibrated at room temperature according to the manufacturer’s instructions. During equilibration (cryoprotectant exposure), samples were kept at 20 °C for 10 minutes. Subsequently, they were loaded into cryovials, exposed to N2 vapor for 30 minutes while positioned horizontally 3 cm above liquid N2, and then completely immersed in liquid N2 for storage. After 1 month, the samples were thawed by transferring from the liquid nitrogen to a 37 °C water bath for 5 minutes. They were then moved to test tubes, and the volume was gently adjusted with 1 mL of albumin-supplemented HTF medium. The samples were washed twice by centrifugation at 2,500 ×g for 5 minutes to remove the cryoprotectant. All procedures were performed swiftly under controlled and uniform lighting conditions. Finally, the samples were assessed.
8. Freeze-drying/rehydration method
Washed sperm samples were resuspended in 1 mL of freeze-drying solution (10 mM Tris-HCl [Sigma-Aldrich], 50 mM NaCl [Kian Kaveh Azma], and 50 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid [EGTA; Sigma-Aldrich]; pH 7.5) at room temperature [38]. The samples were subsequently transferred to sterile culture plates, exposed to liquid nitrogen vapor for 15 minutes, and then freeze-dried for 6 hours at −80 °C and 0.03 mbar pressure using a freeze dryer (Zirbus Technology GmbH). The plates containing dried samples were sealed with parafilm, wrapped in aluminum foil, and stored at 4 °C for 1 month. After storage, each dried sample was rehydrated with 1 mL of warm (37 °C) Milli-Q water and evaluated. Samples in group C were incubated for 30 minutes in 1 mL of 40 µg/mL GTE at 37 °C before freeze-drying.
9. SDF assessment
SDF was assessed using the sperm chromatin dispersion test with a commercially available sperm chromatin dispersion assay kit (IVFCo.). First, 50 µL of the sperm suspension was added to liquid agarose gel, and 25 µL of this mixture was placed onto a slide and incubated at 4 °C for 5 minutes. The slide was sequentially covered with a denaturing solution for 7 minutes and a lysis solution for 15 minutes. Then, the slide was washed with distilled water and dehydrated sequentially in 70%, 90%, and 100% alcohol for 2 minutes each. After staining, the slide was washed, dried, and examined under a light microscope (Nikon) at ×100 magnification with immersion oil. Spermatozoa displaying large and medium halos were considered normal (indicating no DNA fragmentation), while those with small or no halos were identified as abnormal (indicating DNA fragmentation).
10. Mitochondrial membrane potential assessment
Mitochondrial membrane potential (MMP) was assessed using JC-1 staining (Sigma-Aldrich). Specifically, 1 mL of sperm suspension diluted in PBS (Genocell) at a concentration of 1×106 cells/mL was incubated with 2 μM JC-1 for 30 minutes at 37 °C in the dark. After washing with PBS, samples were analyzed by flow cytometry (BD FACSLyric; BD Biosciences) using a 488-nm excitation laser. The ratio of red (585 nm) to green (530 nm) fluorescence was used as an indicator of MMP; a higher ratio indicates a higher MMP [39].
11. Intracellular ROS assessment
Intracellular ROS levels in sperm cells were evaluated using 2',7'-dichlorofluorescein diacetate (DCFH-DA), as previously described by Mahfouz et al. [40], with modifications. Initially, the sperm sample was centrifuged at 1,200 rpm for 5 minutes at 4 °C. Then, 100 µL of 20 µM DCFH-DA (CAS: 2044-85-1) was added, and the sample was incubated for 45 minutes at 37 °C in the dark. Subsequently, 900 µL of PBS was added, and the sample was centrifuged again at 1,200 rpm for 5 minutes at 4 °C. Finally, the sample was analyzed by flow cytometry (BD FACSLyric; BD Biosciences) with excitation at 485 nm and emission at 530 nm.
12. Lipid peroxidation assessment
Lipid peroxidation in sperm samples was assessed by measuring malondialdehyde (MDA) levels using an MDA assay kit (ZellBio GmbH), following the manufacturer’s instructions. The lysed sperm samples were incubated with the kit reagents to form an MDA-thiobarbituric acid adduct. After incubation, samples were cooled, and absorbance was measured spectrophotometrically at 532 nm. MDA concentrations were calculated using a standard curve.
13. Glutathione level assessment
Intracellular glutathione (GSH) levels in sperm samples were assessed using a GSH assay kit (ZellBio GmbH) according to the manufacturer’s instructions. Briefly, sperm samples were lysed to release intracellular GSH. After adding assay buffer and chromogenic reagent, samples were incubated at room temperature. Absorbance was measured spectrophotometrically at 412 nm, and GSH concentrations were calculated by comparison with a standard curve.
14. Statistical analysis
Statistical analysis was performed using SPSS ver. 26.0 (IBM Corp.). The Shapiro-Wilk test was used to confirm the normality of the data distribution. For normally distributed datasets, repeated measures analysis of variance and the Tukey test were applied, with the results reported as mean±standard deviation. The Friedman test, followed by the Wilcoxon test, was utilized for non-normally distributed data, with the results presented as median and interquartile range. p-values of less than 0.05 were considered to indicate statistical significance.
Results
1. GTE production and standardization
The GTE was brown, with an extraction yield of 20.6%. The total phenolic content, determined using pyrogallol as a standard, was 20.26%±0.16%.
2. Baseline semen sample evaluation
Table 1 presents baseline sperm parameters for the men participating in the study, who had an average age of 34.30±6.03 years.
3. Effects of freeze-drying and GTE
Incubating sperm samples with GTE for 30 minutes before freeze-drying significantly improved both oxidative stress markers and SDF. Freeze-drying alone (group B) caused significantly less DNA damage compared to the cryopreservation control (group A; p=0.005) (Table 2, Figure 1). However, freeze-drying alone significantly increased intracellular ROS levels (p<0.0001) and MDA concentration (p<0.0001) and significantly decreased the percentage of sperm with high MMP (p<0.0015) and the GSH concentration (p<0.0001) compared to group A (Table 2, Figures 2 and 3). Freeze-drying resulted in loss of sperm motility in both groups B and C; based on our results, GTE did not significantly affect the motility of freeze-dried human sperm (Table 2). Furthermore, GTE pretreatment (group C) significantly mitigated the detrimental effects of freeze-drying. Compared to both control groups (A and B), group C displayed lower DNA fragmentation (p=0.005 vs. A; p=0.026 vs. B) (Figure 1), lower intracellular ROS levels (p<0.0001 vs. A; p<0.0001 vs. B), a lower MDA concentration (p<0.0001 vs. A; p<0.0001 vs. B), a higher percentage of sperm with high MMP (p<0.0001 vs. A; p<0.0001 vs. B), and a higher GSH concentration (p<0.0001 vs. A; p<0.0001 vs. B) (Table 2, Figures 2 and 3).
Discussion
This study investigated the in vitro effects of GTE on oxidative stress markers and SDF following freeze-drying in normozoospermic men. Freeze-drying poses several challenges, including increased oxidative stress levels and reduced sperm motility and viability [23-25]. Previous studies have aimed to mitigate these effects, demonstrating improvements in sperm parameters during freeze-drying through the application of cryoprotectants such as trehalose and antioxidants [16,24]. To our knowledge, no previous research has evaluated the effects of GTE on human sperm function following freeze-drying; this study thus represents a first step in this field. Our findings indicate that although freeze-drying increases intracellular ROS levels and MDA concentration and decreases the percentage of sperm with high MMP and GSH concentration in normal human sperm samples compared to conventional cryopreservation, pretreatment with GTE significantly mitigates these adverse effects.
In the present study, sperm motility was compromised after freeze-drying and subsequent rehydration, similar to findings from previous studies in this field [16,41,42]. However, viable offspring have successfully been produced through ICSI using freeze-dried sperm in various animal species [19-21]. This suggests that freeze-drying may offer a viable alternative to conventional sperm cryopreservation, potentially influencing the future of reproductive technology [16]. Our findings indicated that SDF was significantly lower in the freeze-drying group compared to the cryopreservation group. Additionally, pretreatment with GTE further decreased DNA fragmentation compared to both control groups, to a significant extent. These results align with the findings of Gianaroli et al. [41], who reported better preservation of DNA integrity during freeze-drying than during cryopreservation of human sperm. Similarly, two other studies found that freeze-drying preserved sperm DNA integrity, indicating that although motility is affected, DNA integrity remains intact [42,43]. In contrast, Octarina et al. [44] examined the impact of alpha-lipoic acid (ALA) supplementation on DNA fragmentation index (DFI) in freeze-dried human sperm. They found that, while freeze-drying significantly increased DFI across all groups (control, supplemented with 1.25 mg ALA, and supplemented with 2.5 mg ALA), the use of ALA at either concentration did not result in significantly less damage. As acknowledged by the researchers, the absence of protective agents during freeze-drying likely explains the increased DNA fragmentation observed.
EGTA is essential for preserving sperm during the freeze-drying process, as it stabilizes the environment and maintains DNA integrity. As a chelating agent, EGTA binds calcium and magnesium ions, preventing the activation of nucleases that can damage DNA, a function that is crucial for successful fertilization and viability of offspring. Studies indicate that incorporating EGTA into the freeze-drying buffer significantly increases sperm DNA integrity, highlighting the importance of effective chelating agents in optimizing sperm quality and improving outcomes of reproductive technologies [14,45,46]. Our findings on the positive effect of GTE in reducing SDF are consistent with recent studies on fresh and cryopreserved human sperm [33,34]. The strong antioxidant properties of green tea likely help reduce oxidative stress and associated DNA damage by neutralizing free radicals [30]. Moreover, the catechins in green tea may indirectly modulate inflammatory responses, suggesting that green tea could also reduce oxidative stress mediated by inflammation [47]. Comparable findings were reported by Wang and Zhu [16] regarding the effects of rosmarinic acid (RA) on DNA integrity in freeze-dried human sperm. Their study demonstrated that RA did not enhance DNA integrity after 1 week but significantly preserved it relative to the control group after 6 months of storage. Like GTE, RA exhibits antioxidant and anti-inflammatory properties that can reduce oxidative stress and support sperm DNA integrity, especially during long-term freeze-drying. It also helps maintain stable methylation levels of key genes, which may indirectly contribute to overall sperm health [16,48]. However, more research is needed to fully understand the direct effects of RA on DFI compared to well-established antioxidants such as GTE.
Furthermore, another study reported that adding 0.2 M trehalose to freeze-dried human sperm improved acrosomal and DNA integrity, indicating that trehalose helps protect sperm during freeze-drying and enhances long-term preservation prospects [24]. Trehalose effectively improves human sperm preservation by reducing oxidative and osmotic stresses, thereby protecting DNA integrity and sperm function [49,50]. While both trehalose and GTE demonstrate significant benefits for sperm quality, their mechanisms differ: trehalose primarily acts as a cryoprotectant, whereas GTE functions as an antioxidant. This distinction may influence their application in clinical settings, suggesting potential combined use to maximize sperm preservation outcomes.
Consistent with our findings, Setumo et al. [34] reported that incubating human semen samples with GTE for 1 hour resulted in a dose-dependent decrease in intracellular ROS concentrations in normozoospermic men. In contrast, Alqawasmeh et al. [33] found no significant difference in ROS levels after freeze/thawing when 1.0 ng/mL of GTE was added to the sperm-freezing medium compared to a control group. This discrepancy across studies may stem from differences in the type of extract used (aqueous, alcoholic, or commercially prepared polyphenol mixtures), the dose investigated, and the incubation time of the samples with the extract. Among the bioactive compounds in green tea, catechins are the primary antioxidant agents, demonstrating potent free radical scavenging properties both in vitro and in vivo [51,52]. Green tea catechins exert their protective effects by scavenging ROS and upregulating antioxidant enzymes such as GSH peroxidase, catalase, and GSH reductase [30,33].
Our findings also indicated that treating samples with GTE maintained intracellular GSH concentrations significantly better than both control groups. Results from another study indicated that adding green tea to the diet of male rabbits at a dose of 6 g/kg increased antioxidant enzyme activity and GSH levels in their sperm [53]. These findings suggest that the catechins present in green tea, due to their antioxidant effects, may enhance antioxidant defense mechanisms in sperm cells and help maintain GSH levels by reducing oxidative stress [54].
High levels of ROS in sperm can lead to oxidative stress by compromising antioxidant defenses, potentially resulting in apoptosis due to lipid peroxidation [55]. Moreover, studies have demonstrated that lipid peroxidation adversely impacts overall sperm quality, reducing fertilization and embryo development rates in ART [56,57]. The use of antioxidants has been proposed as a therapeutic strategy to mitigate the harmful effects of oxidative stress on sperm, with multiple studies indicating that antioxidant supplementation can improve sperm quality by reducing lipid peroxidation [58-61]. In the present study, we found that pre-incubation with GTE in the freeze-drying group significantly decreased MDA concentration. Additionally, research has shown that adding catechins to sperm preservation media can increase sperm viability and reduce markers of oxidative stress. Dias et al. [62] conducted a comparative study of white tea and green tea, focusing on the beneficial effects of catechins in sperm preservation. They demonstrated that treatment with EGCG reduced MDA levels during sperm storage at room temperature. These findings support the potential of catechins as additives in sperm preservation protocols, indicating their capacity to help maintain sperm quality by reducing oxidative damage.
MMP is a key biomarker of mitochondrial activity, essential for sperm motility and fertilization capability [63]. We observed that the percentage of sperm with high MMP increased after the freeze-drying process when normal sperm samples were incubated with GTE. Setumo et al. [34] also demonstrated that GTE significantly improves human sperm function, associated with a notable increase in the percentage of live sperm exhibiting healthy MMP and a simultaneous reduction in intracellular ROS production. Conversely, Dias et al. [64] showed that adding EGCG at a concentration of 50 µM to Sertoli cell culture medium decreased MMP, potentially due to interference with ATP production. This underscores a complex interaction in which balanced catechin levels may improve sperm quality, while excessive dosages could adversely affect MMP. The capacity of green tea to regulate MMP likely stems from the antioxidant properties of its catechins, which help mitigate oxidative stress. Future research should explore the mechanisms of action of GTE and its key components in this context.
This experimental study provides the first evidence that treatment of human sperm with GTE before freeze-drying significantly reduces oxidative stress markers (ROS, MDA, and DFI) and improves indicators of sperm health (MMP and GSH) compared to conventional cryopreservation. This protective effect likely arises from the antioxidant properties of green tea polyphenols, flavonols, and tannins [65,66]. However, further research is essential to optimize the extract concentration and incubation time use. While this study indicates that GTE improves post-rehydration sperm quality by reducing oxidative stress, several limitations should be acknowledged. First, the relatively short duration of sample storage may not accurately reflect long-term stability in clinical settings. More importantly, since our experimental design focused on in vitro parameters, we did not assess fertility outcomes such as fertilization potential or pregnancy rates. Additionally, the limited sample size in this study poses a notable challenge, as it reduces the statistical power and increases the risk of type II errors (false negatives). Thus, the findings should be interpreted cautiously. To address these gaps, future studies should use larger sample sizes to validate these preliminary results. Extended storage studies are also crucial for assessing the long-term protective effects of GTE. Furthermore, in vitro fertilization experiments using treated spermatozoa will help evaluate fertilization capacity. Ultimately, preclinical studies on embryonic development will provide further insights into the role of GTE in male reproductive health and clarify the potential of freeze-drying as an alternative to conventional cryopreservation in clinical practice.
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