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Supplementation of cryopreservation medium with TAT-Peroxiredoxin 2 fusion protein improves human sperm quality and function
Supplementation of cryopreservation medium with TAT-Peroxiredoxin 2 fusion protein improves human sperm quality and function Juan Liu, M.S.,a Wenting Wang, M.S.,a Xin Liu, Ph.D.,a Xuebo Wang, M.M.,b Jiahui Wang, Ph.D.,a Yanwei Wang, M.M.,a Ning Li, M.S.,a and Xiong Wang, M.D.c a Central Laboratory, b Department of Clinical Laboratory, and c Reproductive Medicine Center, Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, Shandong Province, People's Republic of China
Objective: To investigate the potential effects of TAT-PRDX2 protein supplementation to the cryopreservation medium on post-thaw sperm quality and function. Design: In vitro prospective study. Setting: Medical university hospital. Patient(s): Fifty normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic men undergoing semen analysis for couple infertility. Intervention(s): Each semen sample was divided into three aliquots: fresh, cryopreserved control (without additive), and cryopreserved with TAT-PRDX2 protein. Main Outcome Measure(s): Sperm motility, viability, mitochondrial potential, and DNA damage as well as reactive oxygen species (ROS) levels and lipid peroxidation were analyzed. Acrosome reaction and zona-free hamster oocyte penetration tests were performed to assess the fertilization ability of cryopreserved spermatozoa. Result(s): In normozoospermic and asthenozoospermic groups, the addition of 150 mg/mL TAT-PRDX2 significantly reduced intracellular ROS and malondialdehyde levels and enhanced post-thaw sperm motility and viability when compared with the cryopreserved control of the respective groups but did not produce any significant protective effect in the oligoasthenozoospermic group. Mitochondrial potential was significantly increased, whereas DNA fragmentation was significantly decreased, after TAT-PRDX2 supplementation only in the asthenozoospermic group when compared with the cryopreserved control. Although the penetration rate and the penetration index were not markedly improved, TAT-PRDX2 supplementation obviously reduced spontaneous acrosome reaction and increased calcium ionophore–induced acrosome reaction in the normozoospermic and asthenozoospermic groups. Conclusion(s): TAT-PRDX2 protein effectively exerted cryoprotective effects on spermatozoa by reducing intracellular ROS level and thereby improved post-thaw sperm quality and function, especially for asthenozoospermic samples. TAT-PRDX2 protein is a promising additive for developing a new and highly efficient semen cryoprotectant. (Fertil SterilÒ 2018;110:1058–66. Ó2018 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo. Key Words: TAT-peroxiredoxin 2, semen cryopreservation, oxidative stress, sperm quality, function Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertilityand-sterility/posts/36352-25677
Received January 25, 2018; revised July 2, 2018; accepted July 11, 2018. J.L. has nothing to disclose. W.W. has nothing to disclose. X.L. has nothing to disclose. X.W. has nothing to disclose. J.W. has nothing to disclose. Y.W. has nothing to disclose. N.L. has nothing to disclose. X.W. has nothing to disclose. The first three authors are considered similar in author order. This study was supported by the Key Research and Development Program of Shandong Province (grant no. 2017GSF18163) and the National Nature Science Foundation of China (grant nos. 81501313, 81571490, 81701453). Reprint requests: Juan Liu, M.S., Central Laboratory, Affiliated Yantai Yuhuangding Hospital of Qingdao University, No. 20, Yuhuangding Dong Road, Yantai 264000, Shandong Province, People's Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. 110, No. 6, November 2018 0015-0282/$36.00 Copyright ©2018 American Society for Reproductive Medicine, Published by Elsevier Inc. https://doi.org/10.1016/j.fertnstert.2018.07.008 1058
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emen cryopreservation is a vital procedure with widespread application in male fertility preservation and infertility management. However, the deleterious effects of cryopreservation on sperm quality and function are commonly observed after thawing, including reduction of motility and viability, loss of mitochondrial function, and damage of DNA, all of which ultimately impair fertilization potential (1, 2). Considerable efforts VOL. 110 NO. 6 / NOVEMBER 2018
Fertility and Sterility® have been made to prevent cryodamage, but damage control is still at an unsatisfactory level, especially for abnormal semen samples (3, 4). Excessive production of reactive oxygen species (ROS) has been suggested as a major contributing factor for cryodamage to spermatozoa. Spermatozoa usually undergo peroxidative damage during the freeze-thaw process when ROS levels exceed the antioxidant scavenging capacity (5). Particularly, semen samples from subfertile men with lower antioxidant defense show increased susceptibility to cryoinjury (6, 7). Therefore, supplementing the freezing media with antioxidants might be a useful approach for minimizing oxidative damage and improving the recovery rate of cryopreserved spermatozoa. Peroxiredoxin (PRDX) is a novel family of antioxidative proteins comprising six members (PRDX1 to 6) in mammals, which act not only as ROS scavengers to provide cytoprotection from oxidative stress (OS) but also as modulators of ROS-dependent signaling (8). PRDX1, PRDX4, PRDX5, and PRDX6 are found in abundance in human spermatozoa and seminal plasma. They can react actively with H2O2, which suggests their important roles as major protectors against OS damage in spermatozoa (9). Moreover, our previous study showed that supplementation of the TAT (transactivating transduction)-peroxiredoxin (PRDX) 2 protein in the sperm suspension from asthenozoospermic men effectively improved sperm motility and DNA integrity by reducing ROS levels (10). We speculate that the PRDX2 protein may exert antioxidant protection against cryoinjury. However, there has been no systematic investigation into the application of PRDX2 in sperm cryopreservation to date. In the present study, we systematically evaluated how the addition of TAT-PRDX2 to the freezing medium affected the quality and function of spermatozoa from normozoospermic, asthenozoospermic, and oligoasthenozoospermic men. This research should be useful for the development of a novel and efficient semen cryoprotectant.
Semen Analysis Semen samples were collected by masturbation into sterile containers after 3–5 days of sexual abstinence and left to liquefy at 37 C. Basic semen analysis including semen volume and sperm concentration, motility, viability, and morphology was performed according to 2010 World Health Organization (WHO) guidelines. To exclude observer bias, all assays for initial diagnosis and subsequent evaluation after freeze/thaw were performed by a single person with extensive training and substantial experience who was blinded to the experiment.
Sperm Cryopreservation and Thawing Each liquefied semen sample was equally divided into three aliquots: fresh semen, cryopreserved control (without additive), and cryopreserved semen with TAT-PRDX2 protein. The semen sample was mixed (2:1) with 15% glycerol-based cryoprotectant (glycerol-egg yolk-citrate medium) supplemented with or without 150 mg/mL effective concentration of the TAT-PRDX2 fusion protein prepared as described elsewhere (10). This concentration of TAT-PRDX2 was confirmed to maintain the highest sperm viability and motility after cryopreservation based on preliminary experiments (data not shown). After 10 minutes of equilibration at room temperature, the mixture was transferred to cryovials (Nunc), which were kept at 4 C for 1 hour, frozen at –20 C for 30 minutes, suspended in liquid nitrogen vapor for 15 minutes, and plunged into liquid nitrogen according to 2010 WHO guidelines with some modifications. After 1 month, the samples were left to thaw at room temperature for 10–15 minutes and washed twice with BiggersWhitten-Whittingham (BWW) buffer (35 mg/mL bovine serum albumin and 25 mM bicarbonate) by centrifugation at 500 g for 5 minutes to remove any traces of cryoprotectant. The samples were subsequently analyzed for sperm quality and function.
Sperm Mitochondrial Potential
MATERIALS AND METHODS Subject Selection The study was approved by the Ethics Committee of Yantai Yuhuangding Hospital, and informed consent was obtained from each participant. The study included 50 normozoospermic men 26–35 years old, 50 asthenozoospermic men 28–37 years old, and 50 oligoasthenozoospermic men 27–38 years old. Three separate semen analyses were carried out at 1-month intervals for the initial screening of each participant. On the basis of medical history and physical and seminal examination findings, men with varicocele, cryptorchidism, vasectomy, leukocytospermia (greater than 1 106/mL), genital infection, chronic illness, or systemic diseases were excluded from the analysis. Men receiving any medication or antioxidant supplementation during the 3 months before the study and those with chemical or radiation exposure, smokers, and alcoholics were also excluded. VOL. 110 NO. 6 / NOVEMBER 2018
The sperm mitochondrial function was assessed using the method described by Johnson et al. (11) with minor modifications. Briefly, the fresh and frozen-thawed semen samples were washed twice by centrifugation and then resuspended in BWW buffer containing 10 mg/mL of rhodamine 123 (Sigma-Aldrich, Cat. No. R8004), followed by incubation at 37 C for 20 minutes. After washing, a drop of sperm suspension was placed on a glass slide and examined with a fluorescent microscope (Imager A1, Carl Zeiss) to assess mitochondrial integrity. Spermatozoa exhibiting bright fluorescence only at the midpiece region were considered to have functional mitochondria. At least 500 spermatozoa were scored for each sample to determine functional or nonfunctional mitochondria.
Sperm DNA Fragmentation An aliquot of washed fresh or frozen-thawed semen sample was smeared on 1% (w/v) gelatin-coated slides, air dried,
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ORIGINAL ARTICLE: ANDROLOGY fixed with ice-cold methanol for 10 minutes, and then stored at –20 C until further processing. Sperm DNA fragmentation was evaluated by TUNEL assay using the In Situ Cell Death Detection Kit, Fluorescein (No. 11684795910-1, Roche Diagnostics) as described by the manufacturer. On each slide, a minimum of 500 spermatozoa were counted. The level of DNA breaks was expressed as the DNA fragmentation index (DFI) in percentage.
Assessment of Acrosome Reaction The spontaneous and calcium ionophore-induced acrosome reactions (ARs) were measured before freezing and after thawing by using fluorescein isothiocyanate-labelled Pisum sativum agglutinin (Sigma-Aldrich). Viable spermatozoa with damaged acrosomes were interpreted as spontaneous acrosome-reacted spermatozoa, which were measured according to the method of Esteves et al. (12). The induced AR was assessed following 2010 WHO guidelines. Briefly, an aliquot of washed spermatozoa was diluted to 3 107/mL in BWW and incubated for 3 hours at 37 C in an atmosphere of 5% (v/v) CO2 in air to allow capacitation. A total of 10 mmol/L calcium ionophore A23187 (Sigma-Aldrich) was then added to the sperm suspension, followed by incubation for 15 minutes at 37 C to induce AR, and equal volume of dimethyl sulfoxide was added to another sperm suspension instead of the calcium ionophore A23187 as control. Subsequently, the samples were fixed on a glass slide with absolute methanol, air dried, and incubated for 1 hour with fluorescein isothiocyanate-labelled Pisum sativum agglutinin (working solution, 25 mg/mL) at 4 C. After washing and mounting, at least 500 spermatozoa per slide were assessed by a fluorescence microscope, and the percentage of AR after ionophore challenge was presented as the test %AR minus the control %AR.
Zona-Free Hamster Oocyte Penetration Test The sperm penetration assay was performed according to 2010 WHO guidelines with minor modifications. Briefly, the frozen-thawed semen samples were washed first by centrifugation to remove any traces of cryoprotectant. Then the highly motile spermatozoa for fresh and frozen-thawed samples were pooled by the 80% density-gradient centrifugation. The collected spermatozoa were washed and diluted to 5 106 motile spermatozoa per milliliter of fresh BWW. A total of 1.5 mmol/L of calcium ionophore A23187 was added to the sperm suspension for 3 hours at 37 C to achieve capacitation. After washing and assessing the percentage of motile spermatozoa, the sperm suspension was diluted to 3.5 106 motile spermatozoa per milliliter of fresh BWW and then dispersed under mineral oil. Zona-free hamster oocytes were obtained according to the WHO criteria and coincubated with spermatozoa for 3 hours at 37 C under 5% (v/v) CO2 in air. Sperm samples were collected from 30 normozoospermic, 30 asthenozoospermic, and 30 oligoasthenozoospermic men, and each sample from the fresh, cryopreserved control, or cryopreserved with TAT-PRDX2 group coincubated with 20 freshly prepared zona-free eggs (five oocytes per sperm 1060
drop). The oocytes were then washed three times in BWW and observed under a microscope using phase-contrast optics (100 objective). The outcome of this procedure was described in terms of both the proportion of oocytes penetrated (penetration rate, %) and the mean number of spermatozoa penetrating each oocyte (penetration index [PI]).
ROS and Lipid Peroxidation Detection Sperm ROS levels were measured with the S0033 ROS assay kit (Beyotime Institute of Biotechnology) following the manufacturer's protocol. Lipid peroxidation occurring in spermatozoa was estimated by determining the malondialdehyde (MDA) content with the A003-1 kit (Jiancheng Bioengineering) according to the manufacturer's protocol. Values are presented as nanomoles of MDA/108 cells.
Statistical Analysis All data in this study are expressed as mean SD. Mean values between two groups were compared using the unpaired t-test. Correlations between ROS and MDA, viability, progressive motility, and mitochondrial integrity as well as DNA fragmentation after cryopreservation were analyzed by the Pearson correlation test. All statistical analysis was performed with SPSS, version 18.0. P< .05 was considered statistically significant.
RESULTS Semen Parameters Semen parameters of the samples used in this study are presented in Supplemental Table 1. Samples were classified as normozoospermia (normal semen parameters), asthenozoospermia (progressive motility < 32%), and oligoasthenozoospermia (total sperm number < 39 million/ejaculate and progressive motility < 32%) according to the 2010 WHO criteria.
Effect of TAT-PRDX2 Supplementation on Sperm Motility and Viability As shown in Figure 1A, the percentage of sperm progressive motility, total motility, and viability from normozoospermic, asthenozoospermic, and oligoasthenozoospermic men decreased significantly after cryopreservation compared with those of the fresh ejaculates of the respective groups (P< .05). In both the normozoospermic and asthenozoospermic groups, the addition of the TAT-PRDX2 protein (150 mg/mL) to the cryopreservation medium significantly enhanced the post-thaw sperm progressive motility, total motility, and viability compared with those of the cryopreserved control (without additive) of the respective groups (P< .05). Notably, the progressive motility from asthenozoospermic men increased by 76.5% after adding TAT-PRDX2 compared with that of cryopreserved control (P< .05). However, in the oligoasthenozoospermic group, TAT-PRDX2 supplementation did not result in any significant ameliorative effect on sperm motility or viability. VOL. 110 NO. 6 / NOVEMBER 2018
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FIGURE 1
(A) Sperm motility and viability and (B) mitochondrial function and DNA fragmentation in 50 normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic samples cryopreserved with or without the TAT-PRDX2 protein (150 mg/mL). Spermatozoa with mitochondrial integrity and exhibiting bright fluorescence only at the midpiece region were considered to have complete mitochondrial function. The letter ‘‘a’’ indicates a value of P<.05 compared with fresh ejaculate of the respective groups; ‘‘b’’, a value of P<.05 compared with cryopreserved control (without additive) of the respective groups; ‘‘c’’, a value of P<.05 compared with fresh ejaculate of normozoospermic subjects. Liu. TAT-PRDX2 protects sperm from cryodamage. Fertil Steril 2018.
Effect of TAT-PRDX2 Supplementation on Sperm Mitochondrial Function and DNA Fragmentation
Effect of TAT-PRDX2 Supplementation on Sperm Function
As shown in Figure 1B, in both the asthenozoospermic and oligoasthenozoospermic groups, loss of mitochondrial potential was observed in spermatozoa subjected to the freeze-thaw process (P< .05). However, TAT-PRDX2 protein supplementation was found to significantly reduce the mitochondrial damage only in the asthenozoospermic group (P< .05). Moreover, the fresh ejaculates from asthenozoospermic and oligoasthenozoospermic men had a significantly higher percentage of DFI than the ejaculate from the normozoospermic samples (P< .05), and cryopreservation resulted in a significant increase in DFI in both groups compared with that in the fresh ejaculates of respective groups (P< .05). Addition of the TAT-PRDX2 protein to the cryopreservation medium significantly reduced DNA fragmentation in the asthenozoospermic group (P< .05) but did not result in any significant ameliorative effect in the oligoasthenozoospermic group.
As shown in Figure 2A, in the normozoospermic, asthenozoospermic, and oligoasthenozoospermic groups, the freeze-thaw process significantly increased the percentage of spontaneous acrosome-reacted spermatozoa and decreased the percentage of calcium ionophore–induced acrosome-reacted spermatozoa compared with those in the fresh ejaculates of the respective groups (P< .05). However, TAT-PRDX2 protein supplementation was found to significantly reduce the percentage of spontaneous acrosome-reacted spermatozoa and increase the percentage of induced acrosome-reacted spermatozoa only in the normozoospermic and asthenozoospermic groups compared with those of the respective cryopreserved control (without additive; P< .05). The sperm penetration assay showed that the penetration rate and PI were significantly lower in the fresh ejaculates from asthenozoospermic and oligoasthenozoospermic men than those of the normozoospermic group (P< .05), particularly in the
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FIGURE 2
Effect of TAT-PRDX2 protein supplementation on sperm function. (A) Spontaneous and calcium ionophore–induced AR in 50 normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic samples cryopreserved with or without the TAT-PRDX2 protein (150 mg/mL). (B) Penetration rate and PI in 30 normozoospermic, 30 asthenozoospermic, and 30 oligoasthenozoospermic samples cryopreserved with or without the TATPRDX2 protein. Each sample for fresh, cryopreserved control, or cryopreserved with TAT-PRDX2 was coincubated with 20 oocytes. The letter ‘‘a’’ indicates a value of P<.05 compared with fresh ejaculate of the respective groups; ‘‘b’’, a value of P<.05 compared with cryopreserved control of the respective groups; ‘‘c’’, a value of P<.05 compared with fresh ejaculate of normozoospermic subjects. Liu. TAT-PRDX2 protects sperm from cryodamage. Fertil Steril 2018.
oligoasthenozoospermic group. Cryopreservation resulted in a significant decrease in the penetration rate and PI in all three groups compared with those of the respective fresh ejaculates (P< .05), and TAT-PRDX2 protein supplementation did not markedly improve the penetration rate or PI (Fig. 2B).
tively correlated with both MDA (R ¼ 0.527–0.660, P< .05) and DNA fragmentation (R ¼ 0.403–0.647, P< .05), and higher coefficients were observed in spermatozoa of the asthenozoospermic and oligoasthenozoospermic groups (Table 1).
ROS and MDA
DISCUSSION
To investigate the possible mechanism underlying cryodamage, we monitored ROS and MDA levels in prefreeze and post-thaw spermatozoa. Figure 3 shows that in the three groups, the level of ROS and MDA after cryopreservation increased significantly compared with those of the fresh ejaculates of the respective groups (P< .05), particularly in the asthenozoospermic and oligoasthenozoospermic groups, where it increased by nearly 150%. Following the addition of TATPRDX2 protein to the freezing medium, ROS and MDA levels in both the normozoospermic and asthenozoospermic groups were significantly reduced compared with the respective cryopreserved control (without additive; P< .05), but no significant difference was found in the oligoasthenozoospermic group. Correlation analysis showed that the ROS levels in the post-thaw spermatozoa from the three groups were negatively correlated with sperm viability (R ¼ –0.475 to approximately –0.553, P< .05), progressive motility (R ¼ –0.502 to approximately –0.587, P< .05), and mitochondrial integrity (R ¼ –0.455 to approximately –0.548, P< .05), but were posi-
To analyze the mechanism for cryodamage to human spermatozoa, we examined the amount of ROS production and OS in frozen-thawed spermatozoa. MDA, the final product of lipid oxidation, was used as the marker of OS. Our results showed that ROS and MDA levels after cryopreservation increased significantly compared with those of the fresh ejaculates, and correlation analysis showed that the ROS levels in the post-thaw spermatozoa were negatively correlated with the viability, progressive motility, and mitochondrial integrity but positively correlated with both MDA and DNA fragmentation. These findings indicate that the excessive ROS production during freezing and thawing is a major contributing factor for sperm cryodamage. Because of the low content of cytoplasm in spermatozoa, which is eliminated during the final stages of spermatogenesis, seminal plasma provides the major defense against ROS through some enzymatic and nonenzymatic antioxidants (13). Nevertheless, dilution of the extender before cryopreservation diminishes the concentration of these constituents present in seminal plasma, thereby leaving
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FIGURE 3
MDA and ROS level in 50 normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic samples cryopreserved with or without the TAT-PRDX2 protein (150 mg/mL). The letter ‘‘a’’ indicates a value of P<.05 compared with fresh ejaculate of the respective groups; ‘‘b’’, a value of P<.05 compared with cryopreserved control of the respective groups; ‘‘c’’, a value of P<.05 compared with fresh ejaculate of normozoospermic subjects. Liu. TAT-PRDX2 protects sperm from cryodamage. Fertil Steril 2018.
spermatozoa vulnerable to OS. Thus, adding antioxidants to the cryopreservation medium might be a useful approach for minimizing sperm cryoinjury. Many antioxidants have been used for improving semen freezing protocols. Despite some promising data, the protective effect during cryopreservation is partial and limited. For example, supplementation with resveratrol (14) and zinc oxide nanoparticles (15) was shown to reduce sperm lipid peroxidation or DNA damage but did not significantly improve motility. On the contrary, vitamin E was shown to improve sperm motility and reduce ROS levels but did not reduce DNA damage (16). Addition of zinc before cryopreservation was found to reduce DNA and mitochondria damage in both normozoospermic and abnormal semen samples, but the protective effect for motility was statistically significant only in the normozoospermic group (17). Moreover, Rossi et al. (18) found that the supplementation of superoxide dismutase or catalase, if used separately, did not modify the sperm parameter recoveries. However, a significant improvement in both sperm motility and viability was found in combined superoxide dismutase/ catalase supplementation. Therefore, a more efficient cryoprotectant is needed, especially for abnormal semen samples. PRDXs are a novel family of antioxidative proteins with high activity and wide distribution in spermatozoa. PRDXs
are direct targets for H2O2 due to their sulfhydryl group and thus are readily oxidized in cells exposed to low H2O2 levels (19, 20). PRDXs react with H2O2 as fast as GPXs, and PRDX2 is the fastest regenerating redox protein among the six members of the PRDX family (21, 22). The six members of the PRDX family are differentially localized in the subcellular compartments of human spermatozoa depending on circumstances that allow local control of ROS levels (9). Moreover, the protective effect of PRDXs in spermatozoa is often altered in subfertile patients. Gong et al. (23) found a lower amount along with higher thiol oxidation of PRDX1 and PRDX6 in spermatozoa from patients with varicocele or idiopathic infertility compared with the spermatozoa from healthy donors. A previous study found significantly lower levels of the PRDX2 protein in spermatozoa and seminal plasma from asthenozoospermic men than in spermatozoa and seminal plasma from normozoospermic men (10). Together, these data highlight the importance of sufficient PRDXs in ensuring the normal function of spermatozoa and demonstrate the high sensitivity of PRDXs to OS. Cryopreservation requires protection of intracellular structures and biomolecules and hence preferentially requires protective agents that enable passing though the cellular membrane. TAT protein from HIV-1 has been found to have the ability to deliver heterologous proteins in vivo as well
TABLE 1 Correlation between ROS and sperm parameters in post-thaw samples. MDA Parameter (n [ 50) Normozoospermia Asthenozoospermia Oligoasthenozoospermia
Vitality
Progressive motility
R
P
R
P
R
0.527 0.653 0.660
* ** **
–0.475 –0.553 –0.551
* ** **
–0.502 –0.583 –0.587
P * ** **
Mitochondrial integrity R –0.455 –0.548 –0.546
P * ** **
DNA fragmentation R 0.403 0.645 0.647
P * ** **
Note: R ¼ regression correlation coefficient. Statistical significance of coefficients: **P< .01, * P< .05. Liu. TAT-PRDX2 protects sperm from cryodamage. Fertil Steril 2018.
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ORIGINAL ARTICLE: ANDROLOGY as in vitro (24–26). In the present study, we prepared TAT-PRDX2 fusion protein and confirmed its efficient transportation into spermatozoa according to the method reported elsewhere (10). Addition of TAT-PRDX2 to the freezing medium was found to significantly enhance the post-thaw sperm progressive motility, total motility, and viability in both normozoospermic and asthenozoospermic groups, but the protective effect for DNA and mitochondrial integrity was statistically significant only in the asthenozoospermic group. High DFI and ROS levels as well as decreased protection by the PRDX2 protein in fresh ejaculates from asthenozoospermic men make them more susceptible to cryoinjury, and they might benefit more from TAT-PRDX2 addition than the spermatozoa from normozoospermic men. Similar to our observations, Zhang et al. (27) found that cryopreservation medium supplemented with L-carnitine showed better protective effects on DFI and viability for asthenozoospermic samples than it did for normozoospermic semen samples. Likewise, Branco et al. (28) reported that the addition of ascorbic acid before cryopreservation reduced DNA damage only in infertile men. Poor-quality spermatozoa are more susceptible to freeze-thaw-induced DNA damage compared with normozoospermic samples, possibly because of their poor chromatin organization. In addition, our results showed that TATPRDX2 supplementation did not result in any significant ameliorative effect on sperm quality and function in the oligoasthenozoospermic group. This might be because the protective capacity of TAT-PRDX2 protein is not enough to overcome the severe cryodamage for oligoasthenozoospermic samples with higher ROS and lipid peroxidation levels. Future study will focus on improving the stability and efficacy of the TAT-PRDX2 fusion protein after administration in spermatozoa. Before fertilization, the spermatozoa should be stored within the reproductive tract to prolong their viability and motility and inhibit premature capacitation and AR until the released oocyte matures (29). In the current study, we found that the freeze-thaw process significantly increased the percentage of spontaneous acrosome-reacted spermatozoa and decreased the percentage of calcium ionophore– induced acrosome-reacted spermatozoa. This finding coincided well with the results of Cormier et al., Clarke and Johnson, and Marshburn et al., who reported that cryopreservation could induce sperm premature capacitation and spontaneous AR, resulting in a lower fertility rate (30–32). Several antioxidants for supplementation in the cryopreservation medium have been designed in an attempt to preserve sperm function. Supplementation with zinc (17) and L-carnitine (33) was shown to improve the ability of spermatozoa to undergo capacitation and AR as well as protect acrosome integrity. However, supplementation with zinc oxide nanoparticles did not alter the spermatozoa's ability to undergo AR (15). In the present study, TATPRDX2 protein supplementation was shown to significantly inhibit spontaneous AR and improve the ability of spermatozoa to undergo acrosome reaction in vitro in normozoospermic and asthenozoospermic samples, which will hopefully lead to improvement of their fertility.
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CONCLUSIONS The results of this study suggest the significance of OS as a cause for cryoinjury to spermatozoa. Supplementation of the TAT-PRDX2 protein to the cryopreservation medium effectively improved sperm quality and function by counteracting intracellular ROS, especially in asthenozoospermic ejaculates. Our results provide a promising additive for the development of a new and highly efficient semen cryoprotectant.
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Amidi F, Pazhohan A, Shabani Nashtaei M, Khodarahmian M, Nekoonam S. The role of antioxidants in sperm freezing: a review. Cell Tissue Bank 2016; 17:745–56. Zribi N, Feki Chakroun N, El Euch H, Gargouri J, Bahloul A, Ammar Keskes L. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil Steril 2010;93:159–66. Oehninger S, Duru NK, Srisombut C, Morshedi M. Assessment of sperm cryodamage and strategies to improve outcome. Mol Cell Endocrinol 2000;169:3–10. Brugnon F, Ouchchane L, Pons-Rejraji H, Artonne C, Farigoule M, Janny L. Density gradient centrifugation prior to cryopreservation and hypotaurine supplementation improve post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia. Hum Reprod 2013;28:2045–57. Aitken RJ, De Iuliis GN, Finnie JM, Hedges A, McLachlan RI. Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria. Hum Reprod 2010;25:2415–26. Agarwal A, Said TM. Oxidative stress, DNA damage and apoptosis in male infertility: a clinical approach. BJU Int 2005;95:503–7. Kalthur G, Adiga SK, Upadhya D, Rao S, Kumar P. Effect of cryopreservation on sperm DNA integrity in patients with teratospermia. Fertil Steril 2008;89:1723–7. Wood ZA, Schroder E, Robin Harris J, Poole LB. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 2003;28:32–40. O'Flaherty C, de Souza AR. Hydrogen peroxide modifies human sperm peroxiredoxins in a dose-dependent manner. Biol Reprod 2011;84:238–47. Liu J, Zhu P, Wang WT, Li N, Liu X, Shen XF, et al. TAT-peroxiredoxin 2 fusion protein supplementation improves sperm motility and DNA integrity in sperm samples from asthenozoospermic men. J Urol 2016;195:706–12. Johnson LV, Walsh ML, Chen LB. Localization of mitochondria in living cells with rhodamine 123. Proc Nat Acad Sci U S A 1980;77:990–4. Esteves SC, Sharma RK, Thomas AJ Jr, Agarwal A. Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing. Hum Reprod 2000;15:2173–9. Ben Abdallah F, Dammak I, Attia H, Hentati B, Ammar-Keskes L. Lipid peroxidation and antioxidant enzyme activities in infertile men: correlation with semen parameter. J Clin Lab Anal 2009;23:99–104. Garcez ME, dos Santos Branco C, Lara LV, Pasqualotto FF, Salvador M. Effects of resveratrol supplementation on cryopreservation medium of human semen. Fertil Steril 2010;94:2118–21. Isaac AV, Kumari S, Nair R, Urs DR, Salian SR, Kalthur G, et al. Supplementing zinc oxide nanoparticles to cryopreservation medium minimizes the freezethaw-induced damage to spermatozoa. Biochem Biophys Res Commun 2017;494:656–62. Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reprod Biomed Online 2009;18:184–9. Kotdawala AP, Kumar S, Salian SR, Thankachan P, Govindraj K, Kumar P, et al. Addition of zinc to human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm function. J Assist Reprod Genet 2012;29:1447–53. Rossi T, Mazzilli F, Delfino M, Dondero F. Improved human sperm recovery using superoxide dismutase and catalase supplementation in semen cryopreservation procedure. Cell Tissue Bank 2001;2:9–13.
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Baty JW, Hampton MB, Winterbourn CC. Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells. Biochem J 2005;389:785–95. Low FM, Hampton MB, Peskin AV, Winterbourn CC. Peroxiredoxin 2 functions as a noncatalytic scavenger of low-level hydrogen peroxide in the erythrocyte. Blood 2007;109:2611–7. Peskin AV, Low FM, Paton LN, Maghzal GJ, Hampton MB, Winterbourn CC. The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents. J Biol Chem 2007;282:11885–92. Winterbourn CC, Hampton MB. Thiol chemistry and specificity in redox signaling. Free Radic Biol Med 2008;45:549–61. Gong S, San Gabriel MC, Zini A, Chan P, O'Flaherty C. Low amounts and high thiol oxidation of peroxiredoxins in spermatozoa from infertile men. J Androl 2012;33:1342–51. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med 1998;4:1449–52. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999; 285:1569–72. Kubo E, Fatma N, Akagi Y, Beier DR, Singh SP, Singh DP. TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity. Am J Physiol Cell Physiol 2008;294:C842–55.
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Zhang W, Li F, Cao H, Li C, Du C, Yao L, et al. Protective effects of l-carnitine on astheno- and normozoospermic human semen samples during cryopreservation. Zygote 2016;24:293–300. Branco CS, Garcez ME, Pasqualotto FF, Erdtman B, Salvador M. Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen. Cryobiology 2010;60:235–7. Kawakami E, Kashiwagi C, Hori T, Tsutsui T. Effects of canine oviduct epithelial cells on movement and capacitation of homologous spermatozoa in vitro. Anim Reprod Sci 2001;68:121–31. Cormier N, Sirard MA, Bailey JL. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J Androl 1997;18:461–8. Clarke RN, Johnson LA. Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro. Gamete Res 1987;16:193–204. Marshburn PB, McIntire D, Carr BR, Byrd W. Spermatozoal characteristics from fresh and frozen donor semen and their correlation with fertility outcome after intrauterine insemination. Fertil Steril 1992;58: 179–86. Aliabadi E, Jahanshahi S, Talaei-Khozani T, Banaei M. Comparison and evaluation of capacitation and acrosomal reaction in freeze-thawed human ejaculated spermatozoa treated with L-carnitine and pentoxifylline. Andrologia 2018;50 https://doi.org/10.1111/and.12845.
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ORIGINAL ARTICLE: ANDROLOGY La suplementacion del medio de criopreservacion con la proteína de fusi on TAT-Peroxiredoxin 2 mejora la calidad y la funci on del esperma humano Objetivo: Investigar los posibles efectos de la suplementaci on del medio de criopreservaci on con la proteína TAT-PRDX2 sobre la calidad y funci on del esperma despues de la descongelaci on. ~o: Estudio prospectivo in vitro. Disen Lugar: Hospital universitario medico. Pacientes: Cincuenta hombres normozoospermicos, 50 astenozoospermicos y 50 oligoastenozoospermicos sometidos a analisis de semen debido a infertilidad en la pareja. Intervenciones: Cada muestra de semen se dividi o en tres alícuotas: fresco, control criopreservado (sin aditivos) y criopreservado con la proteína TAT-PRDX2. Medida de variables principales: Se analiz o la movilidad de los espermatozoides, la viabilidad, el potencial mitocondrial y el da~ no en el ADN, así como los niveles de especies reactivas de oxígeno (ROS) y la peroxidaci on lipídica. Se realizaron las pruebas de reacci on acros omica y penetraci on en ovocitos de hamster libres de zona pel ucida para evaluar la capacidad de fecundaci on de los espermatozoides criopreservados. Resultados: En los grupos normozoospermicos y astenozoospermicos, la suplementaci on con 150 mg / ml de TAT-PRDX2, redujo significativamente los niveles intracelulares de ROS y de malondialdehído y mejor o la movilidad y viabilidad del esperma tras descongelacion en comparacion con el control criopreservado para cada uno de los grupos analizados, pero no se produjo ning un efecto protector significativo en el grupo oligoastenozoospermicos. El potencial mitocondrial aument o significativamente, mientras que la fragmentaci on del ADN disminuy o significativamente, despues de la suplementaci on con TATPRDX2 solo en el grupo astenozoospermico en comparaci on con su control criopreservado. Aunque la tasa y el índice de penetraci on no mejoraron notablemente, la suplementacion con TAT-PRDX2 redujo claramente la reacci on acrosomal espontanea y aument o la reacci on acros omica inducida por el ion oforo de calcio en los grupos normozoospermicos y astenozoospermicos. Conclusiones: La proteína TAT-PRDX2 ejerci o eficazmente efectos crioprotectores en los espermatozoides al reducir el nivel de ROS intracelular y, por lo tanto, mejor o la calidad y funci on del esperma tras la descongelaci on, especialmente en muestras astenozoospermicas. La proteína TAT-PRDX2 es un aditivo prometedor para el desarrollo de un nuevo y eficiente crioprotector de semen.
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Objective: To investigate the potential effects of TAT-PRDX2 protein supplementation to the cryopreservation medium on post-thaw sperm quality and function. Design: In vitro prospective study. Setting: Medical university hospital. Patient(s): Fifty normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic men undergoing semen analysis for couple infertility. Intervention(s): Each semen sample was divided into three aliquots: fresh, cryopreserved control (without additive), and cryopreserved with TAT-PRDX2 protein. Main Outcome Measure(s): Sperm motility, viability, mitochondrial potential, and DNA damage as well as reactive oxygen species (ROS) levels and lipid peroxidation were analyzed. Acrosome reaction and zona-free hamster oocyte penetration tests were performed to assess the fertilization ability of cryopreserved spermatozoa. Result(s): In normozoospermic and asthenozoospermic groups, the addition of 150 mg/mL TAT-PRDX2 significantly reduced intracellular ROS and malondialdehyde levels and enhanced post-thaw sperm motility and viability when compared with the cryopreserved control of the respective groups but did not produce any significant protective effect in the oligoasthenozoospermic group. Mitochondrial potential was significantly increased, whereas DNA fragmentation was significantly decreased, after TAT-PRDX2 supplementation only in the asthenozoospermic group when compared with the cryopreserved control. Although the penetration rate and the penetration index were not markedly improved, TAT-PRDX2 supplementation obviously reduced spontaneous acrosome reaction and increased calcium ionophore–induced acrosome reaction in the normozoospermic and asthenozoospermic groups. Conclusion(s): TAT-PRDX2 protein effectively exerted cryoprotective effects on spermatozoa by reducing intracellular ROS level and thereby improved post-thaw sperm quality and function, especially for asthenozoospermic samples. TAT-PRDX2 protein is a promising additive for developing a new and highly efficient semen cryoprotectant. (Fertil SterilÒ 2018;110:1058–66. Ó2018 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo. Key Words: TAT-peroxiredoxin 2, semen cryopreservation, oxidative stress, sperm quality, function Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertilityand-sterility/posts/36352-25677
Received January 25, 2018; revised July 2, 2018; accepted July 11, 2018. J.L. has nothing to disclose. W.W. has nothing to disclose. X.L. has nothing to disclose. X.W. has nothing to disclose. J.W. has nothing to disclose. Y.W. has nothing to disclose. N.L. has nothing to disclose. X.W. has nothing to disclose. The first three authors are considered similar in author order. This study was supported by the Key Research and Development Program of Shandong Province (grant no. 2017GSF18163) and the National Nature Science Foundation of China (grant nos. 81501313, 81571490, 81701453). Reprint requests: Juan Liu, M.S., Central Laboratory, Affiliated Yantai Yuhuangding Hospital of Qingdao University, No. 20, Yuhuangding Dong Road, Yantai 264000, Shandong Province, People's Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. 110, No. 6, November 2018 0015-0282/$36.00 Copyright ©2018 American Society for Reproductive Medicine, Published by Elsevier Inc. https://doi.org/10.1016/j.fertnstert.2018.07.008 1058
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emen cryopreservation is a vital procedure with widespread application in male fertility preservation and infertility management. However, the deleterious effects of cryopreservation on sperm quality and function are commonly observed after thawing, including reduction of motility and viability, loss of mitochondrial function, and damage of DNA, all of which ultimately impair fertilization potential (1, 2). Considerable efforts VOL. 110 NO. 6 / NOVEMBER 2018
Fertility and Sterility® have been made to prevent cryodamage, but damage control is still at an unsatisfactory level, especially for abnormal semen samples (3, 4). Excessive production of reactive oxygen species (ROS) has been suggested as a major contributing factor for cryodamage to spermatozoa. Spermatozoa usually undergo peroxidative damage during the freeze-thaw process when ROS levels exceed the antioxidant scavenging capacity (5). Particularly, semen samples from subfertile men with lower antioxidant defense show increased susceptibility to cryoinjury (6, 7). Therefore, supplementing the freezing media with antioxidants might be a useful approach for minimizing oxidative damage and improving the recovery rate of cryopreserved spermatozoa. Peroxiredoxin (PRDX) is a novel family of antioxidative proteins comprising six members (PRDX1 to 6) in mammals, which act not only as ROS scavengers to provide cytoprotection from oxidative stress (OS) but also as modulators of ROS-dependent signaling (8). PRDX1, PRDX4, PRDX5, and PRDX6 are found in abundance in human spermatozoa and seminal plasma. They can react actively with H2O2, which suggests their important roles as major protectors against OS damage in spermatozoa (9). Moreover, our previous study showed that supplementation of the TAT (transactivating transduction)-peroxiredoxin (PRDX) 2 protein in the sperm suspension from asthenozoospermic men effectively improved sperm motility and DNA integrity by reducing ROS levels (10). We speculate that the PRDX2 protein may exert antioxidant protection against cryoinjury. However, there has been no systematic investigation into the application of PRDX2 in sperm cryopreservation to date. In the present study, we systematically evaluated how the addition of TAT-PRDX2 to the freezing medium affected the quality and function of spermatozoa from normozoospermic, asthenozoospermic, and oligoasthenozoospermic men. This research should be useful for the development of a novel and efficient semen cryoprotectant.
Semen Analysis Semen samples were collected by masturbation into sterile containers after 3–5 days of sexual abstinence and left to liquefy at 37 C. Basic semen analysis including semen volume and sperm concentration, motility, viability, and morphology was performed according to 2010 World Health Organization (WHO) guidelines. To exclude observer bias, all assays for initial diagnosis and subsequent evaluation after freeze/thaw were performed by a single person with extensive training and substantial experience who was blinded to the experiment.
Sperm Cryopreservation and Thawing Each liquefied semen sample was equally divided into three aliquots: fresh semen, cryopreserved control (without additive), and cryopreserved semen with TAT-PRDX2 protein. The semen sample was mixed (2:1) with 15% glycerol-based cryoprotectant (glycerol-egg yolk-citrate medium) supplemented with or without 150 mg/mL effective concentration of the TAT-PRDX2 fusion protein prepared as described elsewhere (10). This concentration of TAT-PRDX2 was confirmed to maintain the highest sperm viability and motility after cryopreservation based on preliminary experiments (data not shown). After 10 minutes of equilibration at room temperature, the mixture was transferred to cryovials (Nunc), which were kept at 4 C for 1 hour, frozen at –20 C for 30 minutes, suspended in liquid nitrogen vapor for 15 minutes, and plunged into liquid nitrogen according to 2010 WHO guidelines with some modifications. After 1 month, the samples were left to thaw at room temperature for 10–15 minutes and washed twice with BiggersWhitten-Whittingham (BWW) buffer (35 mg/mL bovine serum albumin and 25 mM bicarbonate) by centrifugation at 500 g for 5 minutes to remove any traces of cryoprotectant. The samples were subsequently analyzed for sperm quality and function.
Sperm Mitochondrial Potential
MATERIALS AND METHODS Subject Selection The study was approved by the Ethics Committee of Yantai Yuhuangding Hospital, and informed consent was obtained from each participant. The study included 50 normozoospermic men 26–35 years old, 50 asthenozoospermic men 28–37 years old, and 50 oligoasthenozoospermic men 27–38 years old. Three separate semen analyses were carried out at 1-month intervals for the initial screening of each participant. On the basis of medical history and physical and seminal examination findings, men with varicocele, cryptorchidism, vasectomy, leukocytospermia (greater than 1 106/mL), genital infection, chronic illness, or systemic diseases were excluded from the analysis. Men receiving any medication or antioxidant supplementation during the 3 months before the study and those with chemical or radiation exposure, smokers, and alcoholics were also excluded. VOL. 110 NO. 6 / NOVEMBER 2018
The sperm mitochondrial function was assessed using the method described by Johnson et al. (11) with minor modifications. Briefly, the fresh and frozen-thawed semen samples were washed twice by centrifugation and then resuspended in BWW buffer containing 10 mg/mL of rhodamine 123 (Sigma-Aldrich, Cat. No. R8004), followed by incubation at 37 C for 20 minutes. After washing, a drop of sperm suspension was placed on a glass slide and examined with a fluorescent microscope (Imager A1, Carl Zeiss) to assess mitochondrial integrity. Spermatozoa exhibiting bright fluorescence only at the midpiece region were considered to have functional mitochondria. At least 500 spermatozoa were scored for each sample to determine functional or nonfunctional mitochondria.
Sperm DNA Fragmentation An aliquot of washed fresh or frozen-thawed semen sample was smeared on 1% (w/v) gelatin-coated slides, air dried,
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ORIGINAL ARTICLE: ANDROLOGY fixed with ice-cold methanol for 10 minutes, and then stored at –20 C until further processing. Sperm DNA fragmentation was evaluated by TUNEL assay using the In Situ Cell Death Detection Kit, Fluorescein (No. 11684795910-1, Roche Diagnostics) as described by the manufacturer. On each slide, a minimum of 500 spermatozoa were counted. The level of DNA breaks was expressed as the DNA fragmentation index (DFI) in percentage.
Assessment of Acrosome Reaction The spontaneous and calcium ionophore-induced acrosome reactions (ARs) were measured before freezing and after thawing by using fluorescein isothiocyanate-labelled Pisum sativum agglutinin (Sigma-Aldrich). Viable spermatozoa with damaged acrosomes were interpreted as spontaneous acrosome-reacted spermatozoa, which were measured according to the method of Esteves et al. (12). The induced AR was assessed following 2010 WHO guidelines. Briefly, an aliquot of washed spermatozoa was diluted to 3 107/mL in BWW and incubated for 3 hours at 37 C in an atmosphere of 5% (v/v) CO2 in air to allow capacitation. A total of 10 mmol/L calcium ionophore A23187 (Sigma-Aldrich) was then added to the sperm suspension, followed by incubation for 15 minutes at 37 C to induce AR, and equal volume of dimethyl sulfoxide was added to another sperm suspension instead of the calcium ionophore A23187 as control. Subsequently, the samples were fixed on a glass slide with absolute methanol, air dried, and incubated for 1 hour with fluorescein isothiocyanate-labelled Pisum sativum agglutinin (working solution, 25 mg/mL) at 4 C. After washing and mounting, at least 500 spermatozoa per slide were assessed by a fluorescence microscope, and the percentage of AR after ionophore challenge was presented as the test %AR minus the control %AR.
Zona-Free Hamster Oocyte Penetration Test The sperm penetration assay was performed according to 2010 WHO guidelines with minor modifications. Briefly, the frozen-thawed semen samples were washed first by centrifugation to remove any traces of cryoprotectant. Then the highly motile spermatozoa for fresh and frozen-thawed samples were pooled by the 80% density-gradient centrifugation. The collected spermatozoa were washed and diluted to 5 106 motile spermatozoa per milliliter of fresh BWW. A total of 1.5 mmol/L of calcium ionophore A23187 was added to the sperm suspension for 3 hours at 37 C to achieve capacitation. After washing and assessing the percentage of motile spermatozoa, the sperm suspension was diluted to 3.5 106 motile spermatozoa per milliliter of fresh BWW and then dispersed under mineral oil. Zona-free hamster oocytes were obtained according to the WHO criteria and coincubated with spermatozoa for 3 hours at 37 C under 5% (v/v) CO2 in air. Sperm samples were collected from 30 normozoospermic, 30 asthenozoospermic, and 30 oligoasthenozoospermic men, and each sample from the fresh, cryopreserved control, or cryopreserved with TAT-PRDX2 group coincubated with 20 freshly prepared zona-free eggs (five oocytes per sperm 1060
drop). The oocytes were then washed three times in BWW and observed under a microscope using phase-contrast optics (100 objective). The outcome of this procedure was described in terms of both the proportion of oocytes penetrated (penetration rate, %) and the mean number of spermatozoa penetrating each oocyte (penetration index [PI]).
ROS and Lipid Peroxidation Detection Sperm ROS levels were measured with the S0033 ROS assay kit (Beyotime Institute of Biotechnology) following the manufacturer's protocol. Lipid peroxidation occurring in spermatozoa was estimated by determining the malondialdehyde (MDA) content with the A003-1 kit (Jiancheng Bioengineering) according to the manufacturer's protocol. Values are presented as nanomoles of MDA/108 cells.
Statistical Analysis All data in this study are expressed as mean SD. Mean values between two groups were compared using the unpaired t-test. Correlations between ROS and MDA, viability, progressive motility, and mitochondrial integrity as well as DNA fragmentation after cryopreservation were analyzed by the Pearson correlation test. All statistical analysis was performed with SPSS, version 18.0. P< .05 was considered statistically significant.
RESULTS Semen Parameters Semen parameters of the samples used in this study are presented in Supplemental Table 1. Samples were classified as normozoospermia (normal semen parameters), asthenozoospermia (progressive motility < 32%), and oligoasthenozoospermia (total sperm number < 39 million/ejaculate and progressive motility < 32%) according to the 2010 WHO criteria.
Effect of TAT-PRDX2 Supplementation on Sperm Motility and Viability As shown in Figure 1A, the percentage of sperm progressive motility, total motility, and viability from normozoospermic, asthenozoospermic, and oligoasthenozoospermic men decreased significantly after cryopreservation compared with those of the fresh ejaculates of the respective groups (P< .05). In both the normozoospermic and asthenozoospermic groups, the addition of the TAT-PRDX2 protein (150 mg/mL) to the cryopreservation medium significantly enhanced the post-thaw sperm progressive motility, total motility, and viability compared with those of the cryopreserved control (without additive) of the respective groups (P< .05). Notably, the progressive motility from asthenozoospermic men increased by 76.5% after adding TAT-PRDX2 compared with that of cryopreserved control (P< .05). However, in the oligoasthenozoospermic group, TAT-PRDX2 supplementation did not result in any significant ameliorative effect on sperm motility or viability. VOL. 110 NO. 6 / NOVEMBER 2018
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FIGURE 1
(A) Sperm motility and viability and (B) mitochondrial function and DNA fragmentation in 50 normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic samples cryopreserved with or without the TAT-PRDX2 protein (150 mg/mL). Spermatozoa with mitochondrial integrity and exhibiting bright fluorescence only at the midpiece region were considered to have complete mitochondrial function. The letter ‘‘a’’ indicates a value of P<.05 compared with fresh ejaculate of the respective groups; ‘‘b’’, a value of P<.05 compared with cryopreserved control (without additive) of the respective groups; ‘‘c’’, a value of P<.05 compared with fresh ejaculate of normozoospermic subjects. Liu. TAT-PRDX2 protects sperm from cryodamage. Fertil Steril 2018.
Effect of TAT-PRDX2 Supplementation on Sperm Mitochondrial Function and DNA Fragmentation
Effect of TAT-PRDX2 Supplementation on Sperm Function
As shown in Figure 1B, in both the asthenozoospermic and oligoasthenozoospermic groups, loss of mitochondrial potential was observed in spermatozoa subjected to the freeze-thaw process (P< .05). However, TAT-PRDX2 protein supplementation was found to significantly reduce the mitochondrial damage only in the asthenozoospermic group (P< .05). Moreover, the fresh ejaculates from asthenozoospermic and oligoasthenozoospermic men had a significantly higher percentage of DFI than the ejaculate from the normozoospermic samples (P< .05), and cryopreservation resulted in a significant increase in DFI in both groups compared with that in the fresh ejaculates of respective groups (P< .05). Addition of the TAT-PRDX2 protein to the cryopreservation medium significantly reduced DNA fragmentation in the asthenozoospermic group (P< .05) but did not result in any significant ameliorative effect in the oligoasthenozoospermic group.
As shown in Figure 2A, in the normozoospermic, asthenozoospermic, and oligoasthenozoospermic groups, the freeze-thaw process significantly increased the percentage of spontaneous acrosome-reacted spermatozoa and decreased the percentage of calcium ionophore–induced acrosome-reacted spermatozoa compared with those in the fresh ejaculates of the respective groups (P< .05). However, TAT-PRDX2 protein supplementation was found to significantly reduce the percentage of spontaneous acrosome-reacted spermatozoa and increase the percentage of induced acrosome-reacted spermatozoa only in the normozoospermic and asthenozoospermic groups compared with those of the respective cryopreserved control (without additive; P< .05). The sperm penetration assay showed that the penetration rate and PI were significantly lower in the fresh ejaculates from asthenozoospermic and oligoasthenozoospermic men than those of the normozoospermic group (P< .05), particularly in the
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FIGURE 2
Effect of TAT-PRDX2 protein supplementation on sperm function. (A) Spontaneous and calcium ionophore–induced AR in 50 normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic samples cryopreserved with or without the TAT-PRDX2 protein (150 mg/mL). (B) Penetration rate and PI in 30 normozoospermic, 30 asthenozoospermic, and 30 oligoasthenozoospermic samples cryopreserved with or without the TATPRDX2 protein. Each sample for fresh, cryopreserved control, or cryopreserved with TAT-PRDX2 was coincubated with 20 oocytes. The letter ‘‘a’’ indicates a value of P<.05 compared with fresh ejaculate of the respective groups; ‘‘b’’, a value of P<.05 compared with cryopreserved control of the respective groups; ‘‘c’’, a value of P<.05 compared with fresh ejaculate of normozoospermic subjects. Liu. TAT-PRDX2 protects sperm from cryodamage. Fertil Steril 2018.
oligoasthenozoospermic group. Cryopreservation resulted in a significant decrease in the penetration rate and PI in all three groups compared with those of the respective fresh ejaculates (P< .05), and TAT-PRDX2 protein supplementation did not markedly improve the penetration rate or PI (Fig. 2B).
tively correlated with both MDA (R ¼ 0.527–0.660, P< .05) and DNA fragmentation (R ¼ 0.403–0.647, P< .05), and higher coefficients were observed in spermatozoa of the asthenozoospermic and oligoasthenozoospermic groups (Table 1).
ROS and MDA
DISCUSSION
To investigate the possible mechanism underlying cryodamage, we monitored ROS and MDA levels in prefreeze and post-thaw spermatozoa. Figure 3 shows that in the three groups, the level of ROS and MDA after cryopreservation increased significantly compared with those of the fresh ejaculates of the respective groups (P< .05), particularly in the asthenozoospermic and oligoasthenozoospermic groups, where it increased by nearly 150%. Following the addition of TATPRDX2 protein to the freezing medium, ROS and MDA levels in both the normozoospermic and asthenozoospermic groups were significantly reduced compared with the respective cryopreserved control (without additive; P< .05), but no significant difference was found in the oligoasthenozoospermic group. Correlation analysis showed that the ROS levels in the post-thaw spermatozoa from the three groups were negatively correlated with sperm viability (R ¼ –0.475 to approximately –0.553, P< .05), progressive motility (R ¼ –0.502 to approximately –0.587, P< .05), and mitochondrial integrity (R ¼ –0.455 to approximately –0.548, P< .05), but were posi-
To analyze the mechanism for cryodamage to human spermatozoa, we examined the amount of ROS production and OS in frozen-thawed spermatozoa. MDA, the final product of lipid oxidation, was used as the marker of OS. Our results showed that ROS and MDA levels after cryopreservation increased significantly compared with those of the fresh ejaculates, and correlation analysis showed that the ROS levels in the post-thaw spermatozoa were negatively correlated with the viability, progressive motility, and mitochondrial integrity but positively correlated with both MDA and DNA fragmentation. These findings indicate that the excessive ROS production during freezing and thawing is a major contributing factor for sperm cryodamage. Because of the low content of cytoplasm in spermatozoa, which is eliminated during the final stages of spermatogenesis, seminal plasma provides the major defense against ROS through some enzymatic and nonenzymatic antioxidants (13). Nevertheless, dilution of the extender before cryopreservation diminishes the concentration of these constituents present in seminal plasma, thereby leaving
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FIGURE 3
MDA and ROS level in 50 normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic samples cryopreserved with or without the TAT-PRDX2 protein (150 mg/mL). The letter ‘‘a’’ indicates a value of P<.05 compared with fresh ejaculate of the respective groups; ‘‘b’’, a value of P<.05 compared with cryopreserved control of the respective groups; ‘‘c’’, a value of P<.05 compared with fresh ejaculate of normozoospermic subjects. Liu. TAT-PRDX2 protects sperm from cryodamage. Fertil Steril 2018.
spermatozoa vulnerable to OS. Thus, adding antioxidants to the cryopreservation medium might be a useful approach for minimizing sperm cryoinjury. Many antioxidants have been used for improving semen freezing protocols. Despite some promising data, the protective effect during cryopreservation is partial and limited. For example, supplementation with resveratrol (14) and zinc oxide nanoparticles (15) was shown to reduce sperm lipid peroxidation or DNA damage but did not significantly improve motility. On the contrary, vitamin E was shown to improve sperm motility and reduce ROS levels but did not reduce DNA damage (16). Addition of zinc before cryopreservation was found to reduce DNA and mitochondria damage in both normozoospermic and abnormal semen samples, but the protective effect for motility was statistically significant only in the normozoospermic group (17). Moreover, Rossi et al. (18) found that the supplementation of superoxide dismutase or catalase, if used separately, did not modify the sperm parameter recoveries. However, a significant improvement in both sperm motility and viability was found in combined superoxide dismutase/ catalase supplementation. Therefore, a more efficient cryoprotectant is needed, especially for abnormal semen samples. PRDXs are a novel family of antioxidative proteins with high activity and wide distribution in spermatozoa. PRDXs
are direct targets for H2O2 due to their sulfhydryl group and thus are readily oxidized in cells exposed to low H2O2 levels (19, 20). PRDXs react with H2O2 as fast as GPXs, and PRDX2 is the fastest regenerating redox protein among the six members of the PRDX family (21, 22). The six members of the PRDX family are differentially localized in the subcellular compartments of human spermatozoa depending on circumstances that allow local control of ROS levels (9). Moreover, the protective effect of PRDXs in spermatozoa is often altered in subfertile patients. Gong et al. (23) found a lower amount along with higher thiol oxidation of PRDX1 and PRDX6 in spermatozoa from patients with varicocele or idiopathic infertility compared with the spermatozoa from healthy donors. A previous study found significantly lower levels of the PRDX2 protein in spermatozoa and seminal plasma from asthenozoospermic men than in spermatozoa and seminal plasma from normozoospermic men (10). Together, these data highlight the importance of sufficient PRDXs in ensuring the normal function of spermatozoa and demonstrate the high sensitivity of PRDXs to OS. Cryopreservation requires protection of intracellular structures and biomolecules and hence preferentially requires protective agents that enable passing though the cellular membrane. TAT protein from HIV-1 has been found to have the ability to deliver heterologous proteins in vivo as well
TABLE 1 Correlation between ROS and sperm parameters in post-thaw samples. MDA Parameter (n [ 50) Normozoospermia Asthenozoospermia Oligoasthenozoospermia
Vitality
Progressive motility
R
P
R
P
R
0.527 0.653 0.660
* ** **
–0.475 –0.553 –0.551
* ** **
–0.502 –0.583 –0.587
P * ** **
Mitochondrial integrity R –0.455 –0.548 –0.546
P * ** **
DNA fragmentation R 0.403 0.645 0.647
P * ** **
Note: R ¼ regression correlation coefficient. Statistical significance of coefficients: **P< .01, * P< .05. Liu. TAT-PRDX2 protects sperm from cryodamage. Fertil Steril 2018.
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ORIGINAL ARTICLE: ANDROLOGY as in vitro (24–26). In the present study, we prepared TAT-PRDX2 fusion protein and confirmed its efficient transportation into spermatozoa according to the method reported elsewhere (10). Addition of TAT-PRDX2 to the freezing medium was found to significantly enhance the post-thaw sperm progressive motility, total motility, and viability in both normozoospermic and asthenozoospermic groups, but the protective effect for DNA and mitochondrial integrity was statistically significant only in the asthenozoospermic group. High DFI and ROS levels as well as decreased protection by the PRDX2 protein in fresh ejaculates from asthenozoospermic men make them more susceptible to cryoinjury, and they might benefit more from TAT-PRDX2 addition than the spermatozoa from normozoospermic men. Similar to our observations, Zhang et al. (27) found that cryopreservation medium supplemented with L-carnitine showed better protective effects on DFI and viability for asthenozoospermic samples than it did for normozoospermic semen samples. Likewise, Branco et al. (28) reported that the addition of ascorbic acid before cryopreservation reduced DNA damage only in infertile men. Poor-quality spermatozoa are more susceptible to freeze-thaw-induced DNA damage compared with normozoospermic samples, possibly because of their poor chromatin organization. In addition, our results showed that TATPRDX2 supplementation did not result in any significant ameliorative effect on sperm quality and function in the oligoasthenozoospermic group. This might be because the protective capacity of TAT-PRDX2 protein is not enough to overcome the severe cryodamage for oligoasthenozoospermic samples with higher ROS and lipid peroxidation levels. Future study will focus on improving the stability and efficacy of the TAT-PRDX2 fusion protein after administration in spermatozoa. Before fertilization, the spermatozoa should be stored within the reproductive tract to prolong their viability and motility and inhibit premature capacitation and AR until the released oocyte matures (29). In the current study, we found that the freeze-thaw process significantly increased the percentage of spontaneous acrosome-reacted spermatozoa and decreased the percentage of calcium ionophore– induced acrosome-reacted spermatozoa. This finding coincided well with the results of Cormier et al., Clarke and Johnson, and Marshburn et al., who reported that cryopreservation could induce sperm premature capacitation and spontaneous AR, resulting in a lower fertility rate (30–32). Several antioxidants for supplementation in the cryopreservation medium have been designed in an attempt to preserve sperm function. Supplementation with zinc (17) and L-carnitine (33) was shown to improve the ability of spermatozoa to undergo capacitation and AR as well as protect acrosome integrity. However, supplementation with zinc oxide nanoparticles did not alter the spermatozoa's ability to undergo AR (15). In the present study, TATPRDX2 protein supplementation was shown to significantly inhibit spontaneous AR and improve the ability of spermatozoa to undergo acrosome reaction in vitro in normozoospermic and asthenozoospermic samples, which will hopefully lead to improvement of their fertility.
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CONCLUSIONS The results of this study suggest the significance of OS as a cause for cryoinjury to spermatozoa. Supplementation of the TAT-PRDX2 protein to the cryopreservation medium effectively improved sperm quality and function by counteracting intracellular ROS, especially in asthenozoospermic ejaculates. Our results provide a promising additive for the development of a new and highly efficient semen cryoprotectant.
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ORIGINAL ARTICLE: ANDROLOGY La suplementacion del medio de criopreservacion con la proteína de fusi on TAT-Peroxiredoxin 2 mejora la calidad y la funci on del esperma humano Objetivo: Investigar los posibles efectos de la suplementaci on del medio de criopreservaci on con la proteína TAT-PRDX2 sobre la calidad y funci on del esperma despues de la descongelaci on. ~o: Estudio prospectivo in vitro. Disen Lugar: Hospital universitario medico. Pacientes: Cincuenta hombres normozoospermicos, 50 astenozoospermicos y 50 oligoastenozoospermicos sometidos a analisis de semen debido a infertilidad en la pareja. Intervenciones: Cada muestra de semen se dividi o en tres alícuotas: fresco, control criopreservado (sin aditivos) y criopreservado con la proteína TAT-PRDX2. Medida de variables principales: Se analiz o la movilidad de los espermatozoides, la viabilidad, el potencial mitocondrial y el da~ no en el ADN, así como los niveles de especies reactivas de oxígeno (ROS) y la peroxidaci on lipídica. Se realizaron las pruebas de reacci on acros omica y penetraci on en ovocitos de hamster libres de zona pel ucida para evaluar la capacidad de fecundaci on de los espermatozoides criopreservados. Resultados: En los grupos normozoospermicos y astenozoospermicos, la suplementaci on con 150 mg / ml de TAT-PRDX2, redujo significativamente los niveles intracelulares de ROS y de malondialdehído y mejor o la movilidad y viabilidad del esperma tras descongelacion en comparacion con el control criopreservado para cada uno de los grupos analizados, pero no se produjo ning un efecto protector significativo en el grupo oligoastenozoospermicos. El potencial mitocondrial aument o significativamente, mientras que la fragmentaci on del ADN disminuy o significativamente, despues de la suplementaci on con TATPRDX2 solo en el grupo astenozoospermico en comparaci on con su control criopreservado. Aunque la tasa y el índice de penetraci on no mejoraron notablemente, la suplementacion con TAT-PRDX2 redujo claramente la reacci on acrosomal espontanea y aument o la reacci on acros omica inducida por el ion oforo de calcio en los grupos normozoospermicos y astenozoospermicos. Conclusiones: La proteína TAT-PRDX2 ejerci o eficazmente efectos crioprotectores en los espermatozoides al reducir el nivel de ROS intracelular y, por lo tanto, mejor o la calidad y funci on del esperma tras la descongelaci on, especialmente en muestras astenozoospermicas. La proteína TAT-PRDX2 es un aditivo prometedor para el desarrollo de un nuevo y eficiente crioprotector de semen.
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