Effect of Plasma-rich in Growth Factors Supplementation Timing on Cryopreservation Outcomes in Normozoospermic Samples and Its Protective Role in Asthenoteratozoospermia

Abstract

Background:

Sperm cryopreservation may have detrimental effects on sperm parameters freezing and thawing.

Aim:

In this study, the effects of plasma-rich in growth factors (PRGFs) at different stages of freezing-thawing on sperm parameters in normozoospermia and asthenoteratozoospermia (AT) were evaluated.

Settings and Design:

Aprospective experimental study conducted in two phases to evaluate the timing of PRGF addition during sperm freezing and to assess the cryoprotective effects of PRGF on AT samples.

Materials and Methods:

In the first phase, 20 normal semen samples were included in the study. After preparation, the spermatozoa were divided into the following groups: A control group that was frozen and thawed without PRGF, and three groups that received 1% PRGF before freezing, during equilibration and after thawing. The method of cryopreservation was rapid freezing. Sperm motility, viability, normal morphology, deoxyribonucleic acid (DNA) fragmentation, reactive oxygen species (ROS) levels and mitochondrial membrane potential were evaluated in different groups before freezing and after thawing. In the second phase, 1% PRGF was added to 10 AT samples, and the parameters were evaluated as in the first stage and compared with the control group.

Statistical Analysis Used:

One-way analysis of variance with Tukey’s post hoc test and the Kruskal–Wallis test with Dunn’s post hoc test were applied as appropriate.

Results:

In the first phase, progressive motility in all groups was significantly reduced after thawing compared to fresh spermatozoa. Furthermore, in the group that received PRGF during equilibration, the sperm total motility and viability increased significantly compared to the control group. The rate of DNA fragmentation and ROS also increased significantly in all groups, except in the during equilibration group. In the second phase, the group treated with 1% PRGF after thawing showed no significant differences in total motility, viability, ROS or DNA integrity compared to the fresh spermatozoa.

Conclusion:

Adding 1% PRGF to the freezing medium during the equilibration process yields effective results. PRGF effectively preserves sperm motility, viability and DNA integrity in AT samples.

INTRODUCTION

Infertility, as a multifaceted social, psychological and cognitive crisis, casts a shadow over the lives of many couples.[1] Approximately half of these infertility cases are related to male factors, which can stem from structural problems, hormonal disorders or environmental influences.[2] The critical role of sperm quality in male fertility is well established, with Asthenoteratozoospermia (AT) being one of the most common abnormalities associated with reduced sperm motility and morphology from standard values. This condition accounts for about 19% of male infertility cases.[3,4] Sperm cryopreservation, even in normal samples, is consistently associated with challenges and can affect sperm parameters such as motility, viability, deoxyribonucleic acid (DNA) integrity, morphology and reactive oxygen species (ROS) levels.[5] This is of particular importance in men with AT, as their sperm often have abnormal characteristics, including additional cytoplasm and leucocytes as a source of ROS production.[6] In men with AT, ROS production is significantly increased due to a combination of factors such as inhibition of the glycolysis pathway, impaired adenosine triphosphate production, increased membrane permeability, enzyme dysfunction, decreased axonemal protein phosphorylation, inactivation of certain biochemical pathways, and disruption of cytoplasm levels.[7] Several studies have reported a decrease in viability, motility and DNA integrity after freezing and thawing of AT sperm.[8,9] Recent studies have highlighted the use of plasma-rich in growth factors (PRGF) as a method for improving sperm quality in the freeze-thaw process. PRGF contains numerous growth factors that, by binding to sperm cell receptors and activating signalling pathways, reduce oxidative stress, improve sperm survival and motility and reduce DNA damage.[10,11] It was shown that adding 1% PRGF after 1 h of incubation improved the progressive motility of sperm with teratozoospermia.[12] Mirzaei et al. showed that adding PRGF before sperm freezing meliorates detrimental freezing effects on sperm parameters.[10] The optimal timing for antioxidant supplementation remains to be determined. Previous studies suggest that administering antioxidants both before freezing and after thawing may exert protective effects on sperm parameters.[13,14] In addition, the impact of PRGF on sperm with low motility and normal morphology is also unclear. In the first part of this study, we aimed to determine the optimal timing of PRGF supplementation in normozoospermic samples. In the second part, we investigated the effect of PRGF on the quality of AT samples during the cryopreservation process.

MATERIALS AND METHODS

Plasma-rich in growth factors preparation

PRGF was prepared from blood samples obtained from healthy donors aged 20–35 years who visited the local blood bank. Due to inter-individual variability in platelet concentration, a standardized concentration of 300,000 platelets/μL was selected. For every 900 mL of whole blood, 400 μL of sodium citrate was added as an anticoagulant. The samples were then centrifuged at 580 × g for 8 min.[10] The resulting plasma, designated as PRGF, was carefully aspirated and incubated with a PRGF activator solution (calcium chloride) at a final concentration of 456 mM per 1 mL of plasma for 1 h at 37°C. Finally, the activated PRGF was centrifuged at 3000 × g for 15 min at 4°C, and the supernatant was collected, filtered and stored at −80°C until use.[10]

Semen collection and preparation

In the first phase of the study, 20 men aged 20–40 years with normozoospermic semen profiles were recruited based on World Health Organization (WHO) criteria. These criteria included a semen volume of >1.4 mL, sperm concentration >16 million/mL, progressive motility >30%, total motility >42% and normal morphology >4%.[15] In the second phase, 10 men aged 20–40 years with AT were selected from patients attending infertility clinics. According to WHO criteria, these individuals exhibited progressive motility <30%, total motility <42% and normal morphology <4%.[15] Semen samples were collected at the infertility centre in sterile, dedicated containers following 2–7 days of ejaculatory abstinence. Participants with underlying medical conditions, infectious diseases, substance use or current medication were excluded from the study. Normozoospermic samples were processed using the direct swim-up method, while AT samples were prepared using the simple wash method.[15] Informed consent was obtained from all participants, and the study protocol was approved by the ethics committee of the authors’ institution (Research Ethic Committee of Tarbiat Modares University, 2024/3/5, Approval ID: IR. MODARES. REC.1402.260) and conducted in accordance with the Declaration of Helsinki.

Experimental groups and sperm freezing and thawing

In the first phase, 20 normozoospermic sperm samples were cryopreserved using a rapid freezing protocol. Following sperm preparation, a glycerol-egg yolk-citrate cryoprotective agent (CPA) was gently added to each sample at a 2:1 ratio (CPA to sperm suspension). The mixtures were equilibrated at room temperature for 10 min.[15] The samples were then divided into four experimental groups: Control group; spermatozoa were mixed with CPA without PRGF and then frozen-thawed, Before freezing group; spermatozoa were incubated with 1% PRGF for 30 min prior to cryopreservation; CPA was then added and the samples were frozen-thawed, during equilibration group: 1% PRGF was added during the equilibration period with CPA before freezing, After thawing group; 1% PRGF was added to the spermatozoa immediately after thawing. Across the three intervention time points – before freeze, during equilibration and after thawing, sperm parameters including motility, viability, mitochondrial membrane potential (MMP), ROS levels and DNA fragmentation were assessed.

Based on the outcomes of the first phase, the second phase involved 10 AT samples. In this phase, PRGF was added at the optimal time point identified previously. Each AT sample was divided into two equal parts: one served as the control group, and the other was treated with PRGF at the optimal time point identified in the first phase. For rapid freezing, after equilibration, the samples were placed in microtubes and exposed to nitrogen vapour at a height of 15–20 cm above liquid nitrogen for 15 min, followed by direct immersion into liquid nitrogen. For thawing, the microtubes were removed and placed in a 37°C water bath for 5 min. Subsequently, the samples were diluted with Ham’s F-10 medium supplemented with human serum albumin (10%) and centrifuged at 300–350 g for 5 min.[15] All of the sperm evaluations and freezing–thawing process were performed by the same operator.

Sperm motility, viability and morphology

To evaluate sperm motility, 10 μL of the semen sample was aspirated with a micropipette and placed on a clean glass slide. A coverslip was gently positioned over the sample. Motility assessment was conducted using a light microscope equipped with a ×40 objective lens. Sperm motility patterns – including progressive (rapid or slow), nonprogressive and immotile spermatozoa – were classified according to WHO guidelines. For enumeration, a counting chamber was used, and 200 spermatozoa were assessed across four different microscopic fields. The average percentage of each motility category was then calculated.[15,16] Sperm cell viability was evaluated using the eosin–nigrosin staining method. In this technique, spermatozoa with compromised plasma membranes absorb the eosin dye and appear pink to red, while those with intact membranes exclude the dye and remain unstained (white). A minimum of 200 sperm cells were assessed per sample by light microscope (Labomed, USA, ×1000).[17] Sperm morphology was assessed by preparing smear slides stained with a diff-quick sperm staining kit (in vitro fertilisation [IVF], Iran). Morphological abnormalities in the head, midpiece and tail were evaluated and classified based on WHO criteria. At least 200 spermatozoa were examined per sample by light microscope (Labomed, USA, ×1000).[12]

Reactive oxygen species evaluation

To evaluate ROS levels, two fluorescent dyes – Dihydroethidium (DHE) and 2’, 7’-Dichlorodihydrofluorescein (DCFH) – were utilised. Both dyes are membrane-permeable and capable of entering cells. To allow complete reaction with superoxide anion and hydrogen peroxide, incubation times of 20 min for DHE and 40 min for DCFH were applied, respectively. During incubation, samples were maintained in test tubes under dark conditions at room temperature. Following incubation, fluorescence emissions were measured using flow cytometry, and the emission wavelengths were analysed using a Facscalibur analyser (BD Biosciences, USA).[18]

Deoxyribonucleic acid integrity

Sperm DNA fragmentation was evaluated using the sperm chromatin dispersion kit (IVF, Iran). Sperm samples were first washed and adjusted to a concentration of 10–15 million spermatozoa per millilitre. Dehydration with an alcohol-based solution and subsequent staining were performed according to the manufacturer’s protocol. In this assay, spermatozoa exhibiting no halo or a small halo were classified as having DNA fragmentation, whereas those with medium to large halos were considered intact and free of fragmentation [Figure 1]. A minimum of 200 spermatozoa were assessed by light microscope (Labomed, USA, ×1000).[19]

Figure 1.

Sperm chromatin dispersion staining to assess the sperm deoxyribonucleic acid (DNA) fragmentation. (A) Large halo sperm, (B) Medium halo sperm with intact, unfragmented DNA, (C) Small halo sperm, (D) Nonhalo sperm with fragmented DNA (×1000)

Mitochondrial membrane potential

MMP was analysed using the JC-1 staining kit in combination with flow cytometry. At low MMP, JC-1 remains in its monomeric form and emits green fluorescence (530 ± 15 nm), whereas at high MMP, it forms aggregates that emit red-to-orange fluorescence (590 ± 17.5 nm). Thus, a decrease in the red-to-green fluorescence intensity ratio indicates mitochondrial depolarisation, while an increase reflects mitochondrial polarisation. The samples were assessed by the Faxcalibur flow cytometry system (BD Biosciences, USA).[20]

Statistical analysis

The normality of quantitative data distribution was evaluated using the Shapiro–Wilk test. Based on the distribution of the data, for comparisons between groups, one-way analysis of variance (One-way ANOVA) followed by Tukey’s post hoc test was used for normally distributed data, whereas the Kruskal–Wallis test followed by Dunn’s post hoc test was used for nonnormally distributed data. All tests were one-tailed, and a significance level of <0.05 was considered.

RESULTS

First phase

In the first phase of the study, the mean progressive motility of normozoospermic spermatozoa significantly declined in all experimental groups after thawing compared to fresh spermatozoa, except in the during equilibration group, where no significant change was observed. Both total motility and viability were significantly reduced in all groups following freezing and thawing; however, the during equilibration group demonstrated a statistically significant improvement in these parameters compared to the control group. Sperm morphology was noticeably affected by the cryopreservation process, with a marked reduction in the percentage of normal forms observed across all groups compared to the fresh spermatozoa, although no significant differences were found between the groups.

DNA integrity analysis revealed a significant post-thaw decrease in all groups except the during equilibration group. Similarly, ROS levels measured by the DCFH assay were significantly elevated after thawing in all groups except the during equilibration group, where the increase was not statistically significant. In contrast, ROS levels assessed using the DHE assay did not show significant differences between the groups. Furthermore, a decrease in MMP was observed in all groups post-thawing compared to the pre-freezing, although this reduction did not reach statistical significance [Table 1].

Table 1.

Comparison of sperm parameters between different groups

Parameters (%)	Fresh	Control	Before freezing	During equilibration	After thawing
Progressive motility	70±11.87	55.08±10.93a	49.83±12.22a	60.25±16.16	43.83±12.33a,b
Total motility	85.29±6.35	58.8±7.28a,b	56±8.01a,b	69.07±9.86a	51.5±10.32a,b
Viability	91.06±5.69	64.07±9.08a,b	62.5±9.09a,b	73.12±10.22a	62.47±8.53a,b
Normal morphology*	7.2±3.32 5.5 (5–14)	3.4±2.22 3.5 (1–7)	3.1±1.44 2 (1–12)	4±3.8 2 (1–11)	4.2±3.76 2 (1–12)
DNA integrity	94.2±1.64	87±4.06a	85.5±3.69a	91±1.87	83.4±5.17a,b
High MMP*	58.83±10.61 54.7 (49.8–78.8)	27.84±41.31 5.67 (2.35–75.5)	23.45±35.48 3.86 (2.08–47.9)	20.66±23.68 9.15 (4.94–47.9)	19.13±21.65 8.81 (4.57–44)
DCFH positive cells*	0.44±0.24 0.41 (0.14–0.79)	2.8±0.6a 2.7 (2.2–3.42)	3.32±1.08a 2.82 (2.58–4.56)	2±1.5 2.05 (0.47–3.48)	4.56±0.92a,b 4.95 (3.51–5.23)
DHE positive cells*	0.21±0.16 14 (0.05–0.47)	43.7±17.27a 45.5 (10.8–46.5)	29.53±17.92 31.3 (10.8–46.5)	19.15±22.35 9.53 (3.23–44.7)	32.51±27.43 34.3 (4.23–59)

*Data are presented as mean±SD, median (minimum–maximum), ^aP˂0.05 versus fresh, ^bP˂0.05 versus During equilibration, Data are presented as mean±SD. SD=Standard deviation, MMP=Mitochondrial membrane potential, DCFH=2’,7’-Dichlorodihydrofluorescein, DHE=Dihydroethidium

Second phase

In the second phase of the study, which involved AT samples, progressive motility declined after freezing and thawing in both the control and PRGF1% groups; however, the reduction was not statistically different between the groups. In the control group, total motility and viability were significantly reduced following cryopreservation, whereas these reductions were not significant in the 1% PRGF group. Cryopreservation adversely affected sperm morphology across all groups, leading to a decreased proportion of normal forms, although the difference between the control and PRGF1% groups was not statistically significant. A significant increase in the sperm DNA fragmentation was observed in the control group after thawing, while no such increase was detected in the 1% PRGF group. ROS levels, measured using both the DCFH and DHE assays, did not differ significantly between the groups. Likewise, no significant differences in MMP were observed among the groups in this phase of the study [Figure 2].

Figure 2.

Effects of plasma-rich in growth factors on sperm parameters after freezing-thawing in asthenoteratozoospermia, (a) Progressive motility, (b) Total motility, (c) Viability, (d) Normal morphology, (e) deoxyribonucleic acid integrity, (f) 2’,7’-Dichlorodihydrofluorescein-positive sperm, (g) Dihydroethidium-positive sperm, (h) Sperm with high mitochondrial membrane potential. Data are presented as mean ± standard deviation

DISCUSSION

A variety of endogenous compounds have been proposed as candidates for supplementation to protect cellular structures from the damaging effects of ROS and other free radicals, which are further exacerbated by cryopreservation. PRFG is a great example of endogenous biological sample enriched with different growth factors. PRGF is regarded as the first form of autologous platelet-rich plasma (PRP). PRGF is widely recognised for its pivotal role in tissue regeneration and cellular differentiation. Moreover, recent evidence suggests that PRGF possesses significant therapeutic potential for improving sperm parameters under freezing and thawing conditions.[21] It was shown that growth factors can mitigate oxidative damage, with pronounced benefits for sperm motility.[11] Oxidative stress induced by the freeze–thaw process primarily results from mitochondrial injury and subsequent decreases in adenosine triphosphate (ATP) production, which are essential for optimal sperm motility.[5] Previous findings confirm that the addition of PRGF can effectively enhance motility and viability in normozoospermic samples during cryopreservation.[10] Furthermore, the beneficial effects of fibroblast growth factor (FGF) on human sperm and the increased phosphorylation of fibroblast growth factor receptors in the acrosomal region underscore the high therapeutic potential of growth factors in improving sperm function.[22]

Our data showed that adding PRGF during equilibration has more beneficial effects when compared to before freezing or after thawing. To our knowledge, there are few studies that evaluate the effect of timing of antioxidant supplementation in the literature. Kafi et al. showed that the addition of cysteine either before freezing or post-thawing had the same results regarding the ROS level in ram sperm. Furthermore, they reported that supplementing cysteine both before freezing and after thawing increases sperm motility and viability after cryopreservation.[14] Another study by Najafi et al. showed that supplementation of either pre-freezing or post-thawing medium with brain-derived neurotrophic factor (BDNF) could increase human sperm total and progressive motility compared to control, and no significant differences were seen between pre-freezing and post-thawing. They showed that the hydrogen peroxide was also significantly decreased, and the rate of viable spermatozoa with intact plasmalemma was higher in both groups of pre-freezing and post-thawing compared to control. However, the rate of apoptotic altered plasma lemma spermatozoa showed an increased level in post-thawing group compared to pre-freezing group, and the rate of dead spermatozoa was also significantly lower in post-thawing group compared to pre-freezing group.[13] Damages to the membrane and subcellular organelles could be occurred during thawing procedure. In this phase, recrystallisation may be occurred.[23] It was shown that the highest velocity of DNA damage is occurred during the first 4 h (8.3% per hour) of incubation after thawing.[24] It seems adding antioxidant before freezing or during equilibration may integrate to CPA medium and mitigate ROS production during both freezing and thawing. An important consideration is that the timing of antioxidant supplementation may depend on the specific type of antioxidant used. It should be noted that in our study, spermatozoa were not incubated with PRGF post-thawing, whereas Najafi et al. reported improved outcomes following a 60-min post-thaw incubation.[13] Furthermore, Kafi et al. demonstrated that the highest sperm motility and viability occurred immediately (0 min) after thawing, underscoring the potential benefits of immediate evaluation or use following thawing.[14] It is possible that post-thaw incubation may have altered our results, and this warrants further investigation.

Our results showed that 1% PRGF improved the sperm parameters of AT samples, that is in line with a recent study that showed that incubation of teratozoospermic samples with PRGF may improve sperm parameters.[12] The in vitro protective effect of PRGF on sperm parameters is thought to result from its content of growth factors, antioxidant enzymes, trace elements, and bioenergetic compounds. The important roles of different growth factors such as BDNF, nerve growth factor (NGF), FGFs, vascular endothelial growth factor and insulin-like growth factor I (IGF-I) have been comprehensively reviewed elsewhere.[11] Two main mechanisms have been proposed for the mechanism of action of growth factors on decreasing cryodamage in sperm cryopreservation, including stabilising the cell membrane[25] and acting as non-enzymatic antioxidant.[26] During cryopreservation, damage from ice crystal formation further compromises sperm viability.[27] Growth factors, by control mitochondrial activity and reducing ROS levels, play a crucial role in preventing sperm DNA damage.[11] Specifically, NGF and IGF-I have proven effective in supporting mitochondrial health and reducing ROS,[28,29] suggesting the importance of including these factors during sperm cryopreservation. The cryopreservation process disrupts the plasma membrane and mitochondrial function of spermatozoa, leading to increased production of ROS and cytochrome C release, thereby activating apoptotic pathways.[30] Previous studies have demonstrated that supplementation with NGF which, along with its receptors TrKA and p75NTR, is expressed in reproductive tissues, including sperm can significantly improve sperm viability and motility while reducing ROS-induced apoptosis.[31] Similarly, the addition of IGF-I has also been shown to enhance sperm motility and viability following incubation with normal and abnormal spermatozoa.[28] While earlier research investigated the protective effects of these individual growth factors, the present study utilised PRGF, which contains a broad spectrum of synergistically acting growth factors. This more physiologically relevant approach provides a comprehensive supportive environment with enhanced protective effects for spermatozoa during cryopreservation. Our findings also suggest that the positive effects of PRGF on sperm viability may be attributed to the collective actions of its constituent factors, especially through the activation of signalling pathways such as phosphoinositide 3-kinase and extracellular signal-regulated kinase, which are pivotal for maintaining mitochondrial integrity.[32] Nevertheless, further research is warranted to clarify the optimal timing and dosage of PRGF administration for maximal benefit on sperm function. Türk et al. showed that concentrations of 4%, 8% and 10% of PRP demonstrated efficacy in mitigating oxidative stress and prevent apoptotic and ion channel (CatSper-1) damages during the short-term preservation of ram sperm at a temperature of 5°C over a duration of 96 h, while the concentration of 20% is harmful.[33] Furthermore, Bader et al. 2020 showed that incubating 2%, 5% and 10% PRP with human spermatozoa at 37°C for 24 h reduced ROS levels induced by H₂O₂.[34] Suárez-Barrio et al. showed that PRGF has neuroprotective properties on retinal epithelial cells by reducing oxidative stress through modulating the antioxidant pathway. They found that PRGF mitigates stress by increasing GSH and decreasing Nrf2 and Keap1 gene expression.[35] It was shown that the level of ROS is higher in AT samples compared to normal samples.[36] AT samples have low-quality chromatin packaging, making them more vulnerable to DNA damage caused by the freeze–thaw process compared to normal samples.[37] Therefore, the cryopreservation media need to be optimised for AT samples. Our data showed that total motility, viability and DNA fragmentation were preserved during cryopreservation in AT samples like normozoospermia. However, PRGF has no beneficial effects on progressive motility, and the rate of DCFH cells in contracts to normal samples. One cause may be that the AT samples need more concentration of antioxidant compared to normal samples due to having a high level of ROS. Our study has several limitations, including lacks of evaluating different time points of incubation, before freezing and after thawing, on cryosurvival of spermatozoa following freezing–thawing. Our findings in AT samples should be confirmed by larger studies with greater sample sizes. In addition, PRGF was prepared using donor blood; however, to advance toward personalised medicine, it would be preferable to use each individual’s own blood for PRGF preparation. The results of the present study should be interpreted with caution and that future clinical studies are needed to confirm the translational relevance of these findings.

CONCLUSION

The addition of 1% PRGF during the equilibration time of normozoospermic sperm samples with CPA had the most protective effect on sperm quality. This concentration also maintained motility, viability and DNA integrity in AT samples. These results highlight the importance of further research and clinical application of PRGF in sperm cryopreservation and underscore the need for comprehensive approaches in male infertility treatment that prioritise the preservation of sperm quality, especially in abnormal spermatozoa.

Authors’ contributions

The study was designed by I.H and M.M. Data collection and the initial drafting of the manuscript were performed by S.K. Data analysis was conducted by S.K and I.H. All authors reviewed and approved the final version of the manuscript.

Conflicts of interest

There are no conflicts of interest.

Data availability statement

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

Disclosure on use of Artificial Intelligence

In the preparation of this paper, no Artificial Intelligence tools were used.

Funding Statement

This study was supported by Tarbiat Modares University.