Micronutrients in Support to The Carbon Cycle Activate Antioxidant Defences and Reduce Sperm DNA Damage in Infertile Men Attending Assisted Reproductive Technology Programs: Clinical Trial Study

Abstract

Background: Micronutrients in support to the carbon cycle were shown to reduce sperm DNA damage both in animal models and infertile men. Besides supporting DNA methylation, their positive effect may be mediated by an improved performance of the endogenous antioxidant system but this has not yet been proven in clinical settings. The present study aimed at evaluating the effects of micronutrient supplementation in infertile male partners of assisted reproductive technology (ART) resistant couples.

Materials and methods: In this experimental clinical trial study, infertile male partners of couples resistant to at least one ART cycle, with a sperm fragmentation rate >20% (TUNEL), underwent a 4-month oral supplementation with micronutrients in support to the carbon cycle including folates, B vitamins, zinc and cysteines. Semen, sperm DNA fragmentation (TUNEL), nuclear maturation (CMA3 and blue aniline staining) and lipid peroxidation (BODIPY) were assessed before and after treatment. The couples were followed-up to record clinical outcomes.

Results: Forty-three patients completed the program but full data of pre- and post-treatment were available only for 25 patients. The treatment did not modify sperm concentration or motility but improved morphology. Nuclear maturation, DNA fragmentation and lipid peroxidation significantly improved after the treatment. Overall, 10 clinical pregnancies (23.3%) and 4 live births (9.3%) were recorded during the follow-up following expectant management (25 couples) or a new intracytoplasmic sperm injection (ICSI) cycle (18 couples).

Conclusion: The micronutrients appeared to induce both DNA methylation, resulting in improved sperm nuclear maturation, and antioxidant defences, resulting in less DNA fragmentation and lipid peroxidation. The clinical outcomes were aligned with a possible positive effect on reproductive function. Micronutrients could be regarded as an alternative to antioxidants in correcting oxidative damage in infertile men; however, to confirm such findings, further clinical investigations are warranted (Registration number: IRCT201510207223N6).

Keywords: Antioxidant; DNA Methylation; Male Infertility; Micronutrients; Sperm.

Fig 1

Concept model of micronutrients administration in support to DNA methylation (blue pathway) and antioxidant defences (grey pathway). Micronutrients: small amounts of folates, vitamins B2, B3 and B12 and zinc are needed daily to feed the carbon cycle and the methylations (SAMe). The same applies to vitamin B6, zinc and cysteine to feed glutathione (GSH) synthesis. Cross activation: SAMe acts on the enzyme CBS to increase GSH synthesis. Reducing power from GSH synthesis in turn activates the carbon cycle. Homeostatic effects: DNA methylation and antioxidant defences synergize in keeping a healthy DNA status.

Fig 2

Sperm concentration (P=0.8), total motility (P=0.4), normal morphology (P=0.001), and volume (P=0.06) before and after a 4 month exposure to micronutrients, mean values ± SE.

Fig 3

Sperm protamine deficiency (P<0.05) and nuclear maturation (P<0.001) before and after a 4 month exposure to micronutrients, mean values ± SE (n=24). CMA3 reports on sperm protamine deficiency, Aniline blue reports on sperm nuclear maturation.

Fig 4

Sperm DNA fragmentation (P=0.001) and lipid peroxidation (P<0.001) before and after a 4 month exposure to micronutrients, mean values ± SE (n=24). TUNEL reports on sperm DNA fragmentation, BODIPY reports on lipid peroxidation.

Fig 5

Outcomes from the ART cycles and couples disposition. Fertilization, cleavage (P<0.05) and top quality embryo rates were compared to those achieved by the same couples in their previous ICSI cycle. ART; Assisted reproductive technology and ICSI; Intracytoplasmic sperm injection