One-carbon cycle support rescues sperm damage in experimentally induced varicocoele in rats Parisa Mohammadi* † , Hassan Hassani-Bafrani †‡ , Marziyeh Tavalaee*, Maurizio Dattilo § and Mohammad H. Nasr-Esfahani* ¶ *Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, Academic Center for Education, Culture and Research (ACECR), ¶ Isfahan Fertility and Infertility Center, Isfahan, † Anatomical Sciences Research Center, ‡ Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran, and § Parthenogen, Lugano, Switzerland Objectives To investigate whether micronutrients in support of the one- carbon cycle and glutathione synthesis are effective in improving sperm damage after surgical varicocoele induction in rats and whether any effect is achieved without a rebound reductive stress as seen with oral antioxidants. Materials and Methods Surgical varicocoele was induced in adult male Wistar rats and resulted in significant damage to the testis and sperm cells measured at 2 and 4 months after surgery. At 2 months after surgery, rats received a 2-month oral supplementation in support of the one-carbon cycle containing B vitamins (B2, B3, B6, folic acid and B12), N-acetyl-cysteine, zinc, small amounts of vitamin E, and a natural source of betalains and quercetine (Condensyl â ; Parthenogen SAGL, Lugano, Switzerland and Nurilia SARL, Lyon, France). Results One-carbon cycle supplementation, compared to untreated controls, significantly improved the morphometric characteristics of testis (P < 0.05), sperm concentration, motility and abnormal morphology (P < 0.001), sperm chromatin condensation (aniline blue staining, P < 0.05), sperm DNA damage (acridine orange staining, P < 0.05) and sperm lipid peroxidation (BODIPY C11, P < 0.001). The improvement in both nuclear condensation and DNA damage and the lack of excessive inhibition of lipid peroxidation confirmed that no reductive stress had occurred. Conclusions Micronutrients in support of the one-carbon cycle are effective in the treatment of surgically induced varicocoele in rats, probably by activating natural antioxidant defences and epigenetics. These results support the idea that essential micronutrients including B vitamins may also have a positive influence in clinical varicocoele, which should be tested in prospective clinical trials. Keywords Condensyl, DNA damage, lipid peroxidation, nuclear condensation, varicocoele Introduction Varicocoele, an enlargement of the veins within the scrotum causing retrograde blood flow from the internal spermatic and cremasteric veins into the pampiniform plexus, is the main surgically reversible cause of male infertility. According to clinical reports, the prevalence of varicocoele is 15% in the male population, 19–41% in men with primary infertility, and 45–81% in men with secondary infertility [1]. The lack of normal thermoregulation of the scrotum causing testicular hyperthermia is considered as the main cause of spermatogenic abnormality associated with varicocoele. Several studies have shown that overproduction of reactive oxygen species (ROS) is one of the pathological hallmarks in varicocoele [2,3] and is thought to be the consequence of heat stress [4–6]. At the cellular level, oxidative stress reflects an imbalance between the load of ROS and cellular defences aimed to readily detoxify the reactive intermediates or pro-oxidants and to repair the resulting damage. ROS may induce male fertility problems in two ways: first, by impairing cell membrane integrity through lipid peroxidation, consequently diminishing sperm motility; second, by altering cellular components, especially DNA integrity. Thus, although the correlation between oxidative stress and infertility in varicocoele is not definitely proven, the administration of antioxidants is considered as one of the therapeutic options [1,7,8]. Many clinical trials investigating the effects of daily supplementation (a combination of antioxidants and © 2018 The Authors BJU Int 2018; 122: 480–489 BJU International © 2018 BJU International | doi:10.1111/bju.14385 wileyonlinelibrary.com Published by John Wiley & Sons Ltd. www.bjui.org Andrology