INTRODUCTION

Infertility is defined by the World Health Organization as a disease of the reproductive system characterized by the inability to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse¹. This condition affects nearly 20% of couples of reproductive age, with the male factor contributing to 50% of the cases. The first analysis performed on a man visiting an assisted reproduction center to predict his fertile potential is the semen analysis, which generally includes macroscopic and microscopic examination of the seminal fluid. Additionally, new complementary tests are now considered, evaluating other aspects of sperm such as the integrity of their genetic material².

The importance of analyzing sperm DNA lies in the fact that various studies have shown that the integrity of sperm DNA affects clinical outcomes in assisted reproduction treatments. Despite the information provided by the semen analysis to evaluate sperm quality, approximately 10% to 15% of men diagnosed with infertility present semen parameters within normal ranges but may have defects in sperm DNA. Sperm DNA strand breaks are attributed to several causes, including excessive production of free radicals in the ejaculate, as well as exposure to environmental, occupational factors, and toxic habits³. High sperm DNA damage has been correlated with infertility, defective embryonic development, implantation failure, and an increase in recurrent miscarriages⁴.

Cyclophosphamide (CP) [N, N-bis(2-chloroethyl) tetrahydro-2H-1,3,2-oxazaphosphorin-2-amino 2-oxide] is an alkylating agent commonly used as an antineoplastic and immunosuppressive drug. CP's cytotoxicity is mediated by DNA alkylation at the N7 position of guanine and the formation of DNA-DNA and DNA-protein cross-links, as well as single-strand DNA breaks^5-7. Cyclophosphamide induces infertility by interrupting meiosis before the pachytene stage, causing genotoxic damage to the germline and impairing Leydig cells^8-10. Eukaryotic cells, to maintain genetic stability, halt their cell cycle, which allows for the activation of DNA repair mechanisms¹¹. However, when the damage is severe, cell death pathways such as apoptosis are activated¹². Apoptosis describes a unique morphological pattern of cell death characterized by chromatin condensation, membrane blebbing, and DNA fragmentation; this mechanism plays an important role in the homeostasis of multicellular organisms. Abnormal apoptosis function has been associated with several human diseases, including neurodegenerative disorders and cancers.

"Camu camu" (Myrciaria dubia) is a fruit that grows in the Amazon and is notable for its high vitamin C or ascorbic acid content¹³. Ascorbic acid has been reported to have a protective role in spermatogenesis due to its excellent reducing action, making it a good antioxidant^14-16. Reports have shown that the aqueous extract of Myrciaria dubia H. B. K. Mc Vaugh "camu camu" has an antimutagenic effect against damage caused by fluoride salts, demonstrated through the in vivo micronucleus assay in mouse bone marrow. When administered beforehand, it also has a cytoprotective effect on the same cell line¹⁷. Another study conducted in our laboratory confirmed the protective effect of camu camu on three cell lines previously treated in vivo with potassium bromate¹⁸. Based on the reported scientific evidence, the objective of this research is to determine whether "camu camu" can reverse the negative effect of CP on the male germline in mice, focusing this evaluation on the DNA fragmentation index of sperm. The achieved results could be extrapolated for human use, thereby restoring fertility in patients who have required the use of this drug in their cancer treatment.

The Halomax test is a technique used to identify and evaluate sperm with damaged DNA. It identifies sperm with damaged genetic material (DNA) and differentiates them from those without damage. This test establishes the proportion of sperm with fragmented DNA in the total analyzed sample. It is estimated that, using normal reproduction methods, a percentage of sperm with fragmented DNA above 30% reduces or, in some cases, eliminates the possibility of achieving a full-term pregnancy.

The purpose of this research was to evaluate in vivo the cytoprotective capacity of the aqueous extract of Myrciaria dubia (Kunth) McVaugh "camu camu" fruit against the mutagenic damage caused by the antineoplastic cyclophosphamide (CP) on the male germline in mice.

METHODS

RESULTS

No significant differences were observed in the increase of body weight, weight of the testes, epididymis, and prostate (p>0.05) (Table I); as well as in sperm morphology (not included in the tables), among the groups analyzed during the experiment. Similarly, the results of vitality, motility, membrane integrity, and sperm count are detailed in Table II.

The cytological differences between fragmented and non-fragmented sperm are shown in Figures 1 and 2. In Figure 1, representing the positive control group (cyclophosphamide only, 50 mgKg-1 BW), large halos are observed, indicating sperm DNA damage in a value higher than those in Figure 2, where sperm from group 4 (cyclophosphamide only, 50 mgkg-1 BW) + camu camu extract (100 mgkg-1 BW) show few sperm with large halos. It is important to note that the presence of the flagellum differentiates sperm from other possible cells involved.

Figura 1 Resultados del Test Halomax en espermatozoides de ratón tratados con ciclofosfamida (50 mgKg^-1PC). La presencia de halos grandes, evidencia daño en el ADN, signo de fragmentación 400x 

Figura 2 Resultados del Test Halomax en espermatozoides de ratón tratados con ciclofosfamida (50 mgKg^-1PC) y extracto acuoso de camu camu (100mgKg^-1PC). La presencia de opocos espermatozoides fragmentados sugiere el efecto protector del fruto. 400x 

DISCUSSION

Reproduction and fertility are the foundation of species continuity. However, when referring to our own species, this premise goes beyond simply fulfilling our biological purpose. While infertility as a disease does not cause death, it often leads to situations that can be deemed as lacking psychological and social well-being¹. From this perspective, any effort to generate knowledge that helps individuals achieve conception should be considered a priority from both biological and clinical viewpoints.

The protective effect of antioxidant substances against genotoxicity can occur in three ways: by decreasing the assimilation of pro-oxidant genotoxicants, preventing their formation within the diet itself; as a reducing agent at the sites of pro-oxidant action, and by inducing detoxifying enzymes capable of reducing active oxygen intermediates^3-16.

Analyzing the protective effect of the aqueous extract of Myrciaria dubia fruit through the sperm DNA integrity test showed no significant differences between NC and the treatment groups. This result suggests a protective effect of the aqueous extract of camu camu fruit against CP oxidative damage. Additionally, it was determined that oral supplementation with vitamin C in humans reduces DNA damage induced by hydrogen peroxide (H2O2). In vivo studies in human cells and in vivo studies in rodents have demonstrated that high intracellular concentrations of ascorbic acid reduce mutations caused by oxidative stress from KBrO3(20). It is likely that the high content of ascorbic acid (vitamin C) in camu camu fruit is responsible for the protective effect observed in the results, as a similar number of grade 0 cells were found between T1 and T4.

It is known that CP induces permanent alterations due to different types of damage, which can be detected in a micronucleus test by blocking cytokinesis^22,23. These findings indicate that CP induces DNA damage through various mechanisms besides oxidative stress.

CONCLUSIONS

It is concluded that the oral administration of aqueous extract of camu camu can counteract, modulate, and neutralize the effects of CP, as evidenced by the reduced rate of sperm with nuclear DNA damage in treated samples.

LIMITATIONS OF THE STUDY

The study's limitations were logistical in nature at the institution where the work was carried out, primarily due to unplanned suspensions of activities during weekends (sometimes starting on Friday), which disrupted the dosing and continuous monitoring of the animals. Additionally, the inability to access the research pavilion on Sundays due to a lack of security personnel caused delays and repetition of the experimental design.