The GGC Medium Reduces the DNA Fragmentation of Human Spermatozoa via in vitro Activation

1. Introduction

Male chromosomal abnormalities, characterized by degraded deoxyribonucleic acid (DNA), suggest male infertility regardless of typical sperm features ( 1 ). Several studies have found that sperm destruction concentrations can predict pregnancy outcomes and the likelihood of multiple pregnancy losses when utilizing assisted reproductive technology ( 2 - 4 ). Therefore, the existence of DNA damage would not be a reliable predictor of reproductive potential ( 5 ). Despite the fact that DNA damage is not detectable in ordinary sperm concentration, these tests are an important part of the evaluation of an infertile man ( 5 , 6 ).

There are several methods for the assessment of sperm DNA damage ( 7 ), including disinfection with acridine orange (AO), terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate-nick-end labeling assay, single cell gel electrophoresis (COMET) assay, the sperm chromatin structure assay (SCSA), flow cytometer, and the DNA fragmentation index to determine the degree of DNA fragmentation index (DFI) ( 8 , 9 ). Sperm nucleus abnormalities include DNA strand breaks, statistical and physical genetic disorders, Y chromosome microdeletions, and alterations in the epigenetic regulation of the paternal genome, all of which affect reproductive success. Sperm DNA breakage studies are increasingly used as a sperm quality measure ( 10 ).

Single and double DNA strand breaking, metabolic alteration of a base, such as oxidation or alkylation, inter- or intra-strand cross-linkage, and DNA-protein covalent interactions are the most prevalent kinds of sperm DNA damage discovered ( 11 ). Damage to the DNA of a single sperm that fertilizes a female egg can significantly impact the growth of the embryo ( 12 ). The chromatin state of mammalian sperm is crucial for proper embryo development, and the integrity of mammalian sperm DNA is crucial for the paternal genetic contribution to a normal progeny ( 13 ).

By comparison of DFI (percentage of cells showing denatured DNA) and HDS (cells with defective chromatin condensation) in spermatozoa with threshold values, the SCSA enables the fast identification of DFI and HDS percentages. The findings of SCSA can be used to predict pregnancy success ( 13 , 14 ). Several ideas have been postulated about the molecular mechanism of sperm DNA damage. Apoptosis, oxidative stress, and aberrant chromatin packing are the most critical ( 15 ).

Tejada, Mitchell ( 16 ) were the first researchers to discuss this issue (1984). The AO technique quantifies the metachromatic shift of AO fluorescence from green (native or double-stranded DNA) to red (acid-induced denaturation) in situ to determine the susceptibility of sperm nuclear DNA to acid-induced denaturation (denatured or single-stranded DNA). The AO test is affordable and accessible; however, the color fades quickly which necessitates rapid assessment ( 17 ).

According to Zwamel and Kadhim ( 18 ), GGC medium contains antioxidants (10 mM/ml green tea extract, 10 mM/ml glutathione, 60 mM/ml vitamin C, sodium pyruvate 0.01g/L, and 10% human serum albumin applied to 1L Ringer’s solution). It decreases sperm stickiness and lipid peroxidation, allowing the sperm to flow more freely ( 19 ). Green tea has a high polyphenol content, and polyphenols have antioxidant properties that include scavenging and removing free radicals. Due to its high cell division and metabolic rates, low oxygen pressure, impaired arteries, and higher levels of unsaturated fatty acids, testicular tissue is well-prepared to fight the impacts of free radicals and oxidative stress.

Furthermore, anti-inflammatory effects have been found in green tea. In addition, glutathione protects the sperm membrane from oxidative damage, while sulfhydryl groups are important in sperm motility and metabolism. Sulfhydryl-containing compounds, such as cysteine and glutathione, are found in normal sperm ( 20 ). In addition, vitamin C is considered to combat free radicals and minimize oxidative damage due to its complete chain-breaking antioxidant potential, making it a possible treatment for unexplained male infertility ( 16 ). In this regard, the present study aimed to evaluate the quality of the human sperm DNA after in vitro activation using a GGC medium.

2. Materials and Methods

2.1. Sample Collection and Semen Analysis

The present study was performed on 200 semen samples. The semen samples were collected from individuals within the age range of 18-45 years old after 3-5 days of sexual abstinence. The samples were held at 37 °C for up to 40 min to allow for liquefaction, and then the seminal fluid analysis was performed according to the standard criteria of WHO (2010). The semen samples were divided into three groups before activation by swim-up technique. The control group (G1) received no GGC medium, while G2 and G3 were activated with Ferticult flushing medium and GGC medium, respectively. Afterward, the sperm DFI was assessed at the pre- and post-swim-up activation.

2.2. Preparation of the Acridine Orange

The AO stain was prepared according to the instructions provided by Tejada, Mitchell ( 16 ). As a stock solution, 1 g of AO was soaked in 1,000 mL of distilled water and kept at 4 °C in the dark. Moreover, 40 mL of 0.1 M citric acid, 2.5 mL of 0.3 M Na₂HPO₄.7H₂O, and 10 mL of stock suspension were pipetted. It should be mentioned that during dyeing, the pH was adjusted to 2.5. Except for the AO, which was stored in the refrigerator, the other stock solutions were stored at ambient temperature.

2.3. Preparation of Tyrode’s Solution

For the preparation of Tyrode's solution, a small portion of warmed distilled water (0.24 g) was applied to MgCl₂, which was followed by all of the NaCl components (7.054 mg). The KCl is a salt of potassium chloride (0.439 mg), CaCl₂.2H₂O is a salt of calcium chloride (0.24 gm), and Na₂HPO₄.2H₂O is a salt with the formula Na₂HPO₄.2H₂O (0.187 mg). Moreover, NaHCO₃ is a salt of sodium chloride (1.302 mg). When the volume reached 1 L, it was dialyzed to 7.3 using the method provided by Tejada, Mitchell ( 16 ).

2.4. Preparation of Fixative Solution (Carnoy’s Solution)

This mixture was produced using three main parts methanol and one part glacial acetic acid, as reported by Tejada, Mitchell ( 16 ).

2.5. Preparation of GGC Medium

The GGC medium was prepared by adding 10 mM/ml green tea extract, 10 mM/ml glutathione, 60 mM/ml Vitamin C, sodium pyruvate 0.01 g/L, and 10% human serum albumin to 1 L Ringer’s solution ( 18 ).

2.6. Assessment of DNA Fragmentation Index

The AO technique was used to evaluate the chromatin stability of sperm. A smear was collected from each specimen and placed on a transparent microscope slide on the same day of the inspection. Afterward, it was mixed with a freshly prepared Carnoy's solution. The next day, the slides were inserted into the AO solution for 5 min. Afterward, they were thoroughly washed with tap water before being stored in a cool and dark place until the evaluation was finished. The fraction of spermatozoa with normal DNA was determined by measuring at least 200 spermatozoa under a fluorescence microscope at a magnification of 400. Spermatozoa with normal, undamaged, and double-stranded DNA fluoresced green at a wavelength of 450-490 nm, while those with denatured DNA fluoresced red or orange. Genomic instability was measured using the DFI, which measures the amount of transient transfection as a percentage of total DNA ( 27 ).

3. Results

Based on the comparison of pre- and post-activation stages, the findings of genomic instability indicated a significant reduction at the post-activation stage. Figure 1 shows that the samples activated with GGC medium (G3) had the lowest DFI post-activation, compared to other groups. Despite the fact that the level of DFI was lower in G3, compared to that in G2, the difference between them was non-significant. Normal sperm DNA and sperm DNA fragmented in the AO test are represented in figures 2 and 3.

Figure 1. DNA fragmentation of pre- and post-in vitro sperm activation using GGC for subjects.Means with different superscripts within each column are significantly different (P<0.05).Means with similar superscripts within each column are non-significantly different (P>0.05).

Figure 2. Sperm head under magnification power of (1000) of oil immersion displaying green fluorescence as sperms with intact DNA

Figure 3. Sperms head under (☓40) HPF displaying intact and abnormal fragmentation DNA of human sperm. The green fluorescence refers to sperms with intact DNA, while the orange and yellow fluorescence considers sperms with fragmented DNA

4. Discussion

Sperm DNA fragmentation was a helpful technique for the evaluation of male fertility and infertility ( 21 ). Peroxidation is the most frequent source of DNA fragmentation in spermatozoa ( 22 ). The high content of unsaturated fatty acids in the plasma membrane of human sperm renders them more susceptible to free radicals. These unsaturated fatty acids enhance extracellular matrix protein processes (e.g., acrosome activation and sperm-oocyte interaction) and sperm motility. Due to their unsaturated nature, many components are also vulnerable to free radical assault and lipid peroxidation of the sperm plasma membrane ( 23 ). Lipid peroxide in human spermatozoa has been associated with a significant decrease in motility ( 24 ). In the present study, the increase in sperm motility might be related to the GGC medium stopping oxidative damage pathways in human spermatozoa when effective free radical hunters play a protective role.

Sperm DNA integrity was more desirable at the post-activation stage, compared to that at the pre-activation stage. However, the GGC medium exhibited the highest recovery rate in sperm DNA, compared to the control group, which is in concordance with the method of Harrison ( 25 ). Despite a reasonable balance of oxygen reduction reactions, glutathione has a positive impact, minimizing oxidative stress and decreasing DNA strand breaking ( 26 ). It may be deduced from the data that sperm motility and DNA damage have a strong connection.

Some defensive effects of green tea used in in vitro culture medium include low water solubility, limited absorption, and decreased accessibility of polyphenols, like quercetin, which possess high in vitro antioxidant properties ( 27 ). The GGC medium also contains glutathione, which protects sperm-oocyte fusion potential by inhibiting ferrous ion-catalyzed oxidative damage in vitro. Superoxide dismutase and glutathione peroxidase/reductase strands are always considered to have two superoxides (O₂-) and H₂O₂ protective factors ( 28 ). It was also discovered that the direct addition of vitamin C to the sperm has a more therapeutic effect, as inadequate seminal vesicle activity causes low ascorbate levels in these people. A wide range of reactive oxygen species (ROS) is trapped by vitamin C, which explains its capacity to prevent free radical damage, either mediated gene damage or ROS generation ( 29 ).

In conclusion, the novel GGC medium was found to be effective in improving sperm motility and reducing sperm DNA fragmentation in asthenozoospermic individuals by in vitro sperm stimulation utilizing the straight swim-up approach.

Authors' Contribution

Study concept and design: A. H. Z. and N. K. K.

Acquisition of data: N. K. K.

Analysis and interpretation of data: A. H. Z.

Drafting of the manuscript:

Critical revision of the manuscript for important intellectual content: A. H. Z.

Statistical analysis: A. H. Z.

Administrative, technical, and material support: N. K. K.

Ethics

Informed consent was obtained from all the participants before the collection of semen samples. Besides, the local medical Ethics Committee approved this study.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

1. Lombardo F, Sansone A, Romanelli F, Paoli D, Gandini L, Lenzi A. The role of antioxidant therapy in the treatment of male infertility: an overview. Asian J Androl. 2011; 13(5):690.
2. Mohan ZH, Al-Hilli NM, Selman MO. Effects of Vaginal Misoprostol after Intrauterine Insemination IUI. IJEIR. 2019; 9(2):39-54.
3. Idowu OO. Green tea extract and reproduction: A review. E3 J Med Res. 2017; 6:001-6.
4. Dadashpour Davachi N, Norouzi E, Didarkhah M, Eslampanah M, Hablolvarid MH. In vitro Production of Grivet Monkey (C hlorocebus aethiops) Embryo. Iran J Vet Med. 2022; 16(3):257-64.
5. The effects of green tea (Camellia sinensis) extract on mouse semen quality after scrotal heat. Vet Res Forum. 2011.
6. Roychoudhury S, Agarwal A, Virk G, Cho C-L. Potential role of green tea catechins in the management of oxidative stress-associated infertility. Reprod Biomed Online. 2017; 34(5):487-98.
7. Kolesnikova L, Kurashova N, Bairova T, Dolgikh M, Ershova O, Dashiev B, et al. Role of glutathione-S-transferase family genes in male infertility. Bull Exp Biol Med. 2017; 163(5):643-5.
8. Buhling K, Schumacher A, Zu Eulenburg C, Laakmann E. Influence of oral vitamin and mineral supplementation on male infertility: a meta-analysis and systematic review. Reprod Biomed Online. 2019; 39(2):269-79.
9. Rahman SU, Huang Y, Zhu L, Feng S, Khan IM, Wu J, et al. Therapeutic role of green tea polyphenols in improving fertility: a review. Nutrients. 2018; 10(7):834.
10. Dutta S, Majzoub A, Agarwal A. Oxidative stress and sperm function: A systematic review on evaluation and management. Arab J Urol. 2019; 17(2):87-97.
11. Mahmoudi R, Azizi A, Abedini S, Jahromi VH, Abidi H, Barmak MJ. Green tea improves rat sperm quality and reduced cadmium chloride damage effect in spermatogenesis cycle. J Med Life Sci. 2018; 11(4):371.
12. Fafula R, Onufrovych O, Iefremova U, Melnyk O, Nakonechnyi I, Vorobets D, et al. Glutathione content in sperm cells of infertile men.  Regul Mech Biosyst. 2017; 2(8):157-61.
13. Chávez JC, Hernández-González EO, Wertheimer E, Visconti PE, Darszon A, Treviño CL. Participation of the Cl−/HCO3− exchangers SLC26A3 and SLC26A6, the Cl− channel CFTR, and the regulatory factor SLC9A3R1 in mouse sperm capacitation. Biol Reprod. 2012; 86(1):1-14.
14. Agarwal A, Majzoub A, Esteves SC, Ko E, Ramasamy R, Zini A. Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios. Transl Androl Urol. 2016; 5(6):935.
15. Sciorio R, Thong J, Pickering S. Comparison of the development of human embryos cultured in either an EmbryoScope or benchtop incubator. J Assist Reprod Gene. 2018; 35(3):515-22.
16. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril. 1984; 42(1):87-91.
17. Custers IM. Intrauterine insemination: Fine-tuning a treatment. 2013.
18. New medium from green tea extract, glutathione and vitamin C (GGC) for activation of human spermatozoa in vitro. Human Reproduction; 2019: Oxford Univ Press Great Clarendon St, Oxford Ox2 6dp, England.
19. van der Linden M, Buckingham K, Farquhar C, Kremer JA, Metwally M. Luteal phase support for assisted reproduction cycles. Cochrane Database Syst Rev. 2015; 7
20. Barbonetti A, Calogero A, Balercia G, Garolla A, Krausz C, La Vignera S, et al. The use of follicle stimulating hormone (FSH) for the treatment of the infertile man: position statement from the Italian Society of Andrology and Sexual Medicine (SIAMS). J Endocrinol Invest. 2018; 41(9):1107-22.
21. Barati E, Nikzad H, Karimian M. Oxidative stress and male infertility: Current knowledge of pathophysiology and role of antioxidant therapy in disease management. Cell Mol Life Sci. 2020; 77(1):93-113.
22. Opuwari CS, Henkel RR. An update on oxidative damage to spermatozoa and oocytes. Biomed Res Int. 2016; 2016
23. Shirata C, Kaneko J, Inagaki Y, Kokudo T, Sato M, Kiritani S, et al. Near-infrared photothermal/photodynamic therapy with indocyanine green induces apoptosis of hepatocellular carcinoma cells through oxidative stress. Sci Rep. 2017; 7(1):1-8.
24. Samardzija M, Getz I, Lojkic M, Valpotic H, Djuricic D. Optimization of sperm for in vitro production of bovine embryos. SOJ Vet Sci. 2015; 1(2):1-7.
25. Harrison R. A highly efficient method for washing mammalian spermatozoa. Reproduction. 1976; 48(2):347-53.
26. Zhang N, Duncan FE, Que EL, O’Halloran TV, Woodruff TK. The fertilization-induced zinc spark is a novel biomarker of mouse embryo quality and early development. Sci Rep. 2016; 6(1):1-9.
27. Raimondo S, Gentile T, Gentile M, Donnarumma F, Esposito G, Morelli A, et al. Comparing different sperm separation techniques for ART, through quantitative evaluation of p53 protein. J Hum Reprod Sci. 2020; 13(2):117.
28. Agarwal A, Majzoub A, Baskaran S, Selvam MKP, Cho CL, Henkel R, et al. Sperm DNA fragmentation: a new guideline for clinicians. World J Mens Health. 2020; 38(4):412.
29. Cyrus A, Kabir A, Goodarzi D, Moghimi M. The effect of adjuvant vitamin C after varicocele surgery on sperm quality and quantity in infertile men: a double blind placebo controlled clinical trial. Int Braz J Urol. 2015; 41:230-8.