Ceratonia siliqua L improved cryodamage of asthenozoospermic specimens: An experimental study

Abstract Background Sperm freezing is an important procedure in assisted reproductive technology. Freezing results in physical and chemical changes in the sperm. Ceratonia siliqua L (C.siliqua) is a tree that has antioxidant properties. Objective This study aimed to investigate the effect of different concentrations of C.siliqua in a freezing medium on semen parameters, and some biochemical parameters in asthenozoospermic specimens. Materials and Methods Forty asthenozoospermic specimens (semen specimens with motility < 32%) were obtained from men aged between 20-40 yr according to the World Health Organization criteria. Each sample was divided into 6 groups: I) fresh, II) control, III) 5, IV) 10, V) 20, and VI) 30 µg/ml C.siliqua extract were added to a freezing medium respectively. Then sperm parameters, malondialdehyde, total antioxidant capacity, reactive oxygen species, and sperm DNA assay were evaluated using related protocols after thawing. Results Data analysis shows that sperm parameter, and total antioxidant capacity level increased at a concentration of 20 µg/ml of C.siliqua extract compared to the other concentrations of C.siliqua extract after cryopreservation and thawing (p < 0.001). Also, the sperm DNA fragmentation assay, reactive oxygen species, and malondialdehyde levels were significantly reduced by adding 20 µg/ml of C.siliqua extract to the sperm freezing medium compared to the other treated groups after cryopreservation (p < 0.001). Conclusion C.siliqua extract significantly improved sperm parameters after cryopreservation and thawing in asthenozoospermic specimens, and the greatest impact was observed at the 20 µg/ml C.siliqua L extract concentration (p < 0.001).


Introduction
Sperm cryopreservation has been used for fertility preservation before factors such as chemotherapy, radiotherapy, and surgery, certain diseases that may lead to testicular dysfunction, subfertility, or sterility due to gonad removal caused by adjuvant therapy (1). One of the achievements of assisted reproductive technology was the clinical use of cryopreservation for the preservation of spermatozoa in the past decade (2). The rapid cryopreservation process is based on the increasing and decreasing the temperature by immersion in liquid nitrogen. Rapid cryopreservation is a low cost, simple, and safe procedure for sperm cryopreservation (3). Despite the benefits of cryopreservation, this procedure may lead to deleterious changes in sperm structure and function (4).
The sperm preparation is done for cryopreservation, which removes the antioxidant source (5). Cryopreservation causes lipid peroxidation in the sperm membrane leading to the formation of reactive oxygen species (ROS) (6).
ROS initiates a chain of chemical reactions called lipid peroxidation that produces malondialdehyde (MDA), which leads to the destruction of the cell membrane (7). An increase in ROS can alter the sperm antioxidant defense system, leading to DNA damage of cells (8) that induces caspase activation and apoptosis (9). Antioxidant supplements such as vitamin C, vitamin E, selenium, zinc, CoQ10 (10)(11)(12), and ellagic acid (13) have shown positive effects on sperm cryopreservation as they can improve semen parameters (sperm motility, viability, and function) in infertile men.
Therefore, studies have shown that enriching media freezing with herbal extract antioxidants can reduce the harmful effects of sperm cryopreservation (14,15). Ceratonia siliqua L (C.siliqua) is an evergreen tree with a height of 12-17 m, a species of Bacchillas. C.siliqua contains high levels of fiber, minerals such as sodium, potassium, phosphorus, calcium, iron, and polyphenol, as well as vitamins such as D, C, E, B6, folic acid, and niacin (16,17). This plant has antibacterial (18), anticancer, antidiabetic (19), and antioxidant properties due to the presence of phenolic components (20).
It has also been shown that the supplementation of freezing media with C.siliqua extract improved sperm progressive motility, normal morphology, viability, and sperm DNA integrity after cryopreservation and thawing in normozoospermic aged men (21). It has been reported that using C.siliqua fruit hydroalcoholic extract improved sperm parameters (count, motility, morphology, viability), and elevated total antioxidant capacity (TAC), antioxidant enzymes activity of serum, and decreased serum MDA levels in male mice (22).
Cryopreservation caused a significant decrease in sperm parameters (motility, normal morphology, viability, and sperm DNA integrity) compared to fresh samples. To the best of our knowledge, there are no publications on the effects of C.siliqua hydroalcoholic extract as an herbal antioxidant on cryopreservation in asthenozoospermic men. Therefore, in this study, the effect of C.siliqua hydroalcoholic extract on semen parameters, sperm DNA integrity, and some biochemical parameters after freezing in fertile men with asthenozoospermic was investigated.

Study design
Each liquefied asthenozoospermic specimen was divided into 6 groups: I) fresh (before cryopreservation), II) control (cryopreserved without C.siliqua extract), III) 5, IV) 10, V) 20, and VI) 30 µg/ml C.siliqua extract were added to a freezing medium respectively. All assessments were done before cryopreservation and after thawing. These doses were according to the previous research (15).

Rapid cryopreservation and thawing
Rapid cryopreservation and thawing processes were applied to provide semen specimen. To

Sperm DNA fragmentation assay (SDFA)
The determination of SDFA was carried out    Finally, the C.siliqua fruit extract was stored at -20°C.

Ethical considerations
In addition, this research was approved by the

Research Ethics Committees of Kermanshah
Razi University, Kermanshah, Iran (Code: IR.RAZI.REC.1400.085), and informed consent was obtained from all participants in the study.

Statistical analysis
The current study was analyzed using SPSS version 26 (SPSS Inc., Chicago), where the significance level was set at p = 0.001. The Shapiro-Wilk and Kolmogorov-Smirnov tests were utilized for normality assessment and the one way-ANOVA followed by TOKEY POSTHOC test for quantifying the significance of differences between groups.

Discussion
Human sperm cryopreservation is a step in the process of fertility treatment that maintains men's fertility for years regardless of their infertility etiology. In this study, different concentrations of C.siliqua hydroalcoholic extracts in a freezing medium were used to optimize the sperm freezing medium.
It has been shown that it is used as herbal In addition, it has been shown that C.siliqua positively affects on male fertility and spermatogenesis (15). A previous study reported that C.siliqua extract induced by alcohol in the liver has a protective effect and causes a decrease of oxidative stress and the levels of malondialdehyde (28). In addition, many authors showed that C.siliqua has protective potential against oxidative stress, and it showed a significant reduction in blood glucose and biochemical profiles in diabetic rats at various doses (19).
Also, the present study demonstrated that a freezing medium supplemented with C.siliqua extract increased TAC levels. These results are in agreement with other studies (17,22).
The use of the herbal extract in freezing and/or thawing media to improve sperm cryopreservation in treating male infertility has been the attention of many infertility clinics (29).
In agreement with our study, it showed that adding antioxidant agents to freezing and/or thawing media might be an effective approach to improve the sperm function efficiency (30). C.siliqua extract can be used dose-dependently as a supplement in the sperm cryopreservation process which is a cost-convenient technique to help infertile men increase their fertilization rate, pregnancy rate, and ultimately, live birth.
The limitation of this study is that the number of asthenozoospermic patients were low. However, we believe this is the first study to evaluate the effect of C.siliqua extract on sperm parameters, sperm DNA integrity, and biochemical parameters after cryopreservation in asthenozoospermic specimens.

Conclusion
This study showed that the supplementation of freezing media with C.siliqua extract as an antioxidant significantly improved sperm progressive motility, normal morphology, viability, and chromatin condensation after cryopreservation and thawing in asthenozoospermic specimens. Also, our results showed that the greatest impact was observed at the 20 µg/ml C.siliqua extract concentration in asthenozoospermic specimens (p < 0.001).