Supplementation of cryoprotectant with Pinus massoniana bark extract improves human sperm vitality and fertility potential

Abstract

AbstractBackgroundSperm cryopreservation is commonly used to preserve fertility but often decreases sperm quality by inducing oxidative stress. Pinus massoniana bark extract (Pinus massoniana, PMBE) exhibits strong antioxidant activity and has been used in traditional Chinese medicine.ObjectivesThe objective was to determine whether adding PMBE to cryoprotectant could improve human sperm quality after cryo‐resuscitation and to investigate the potential regulatory mechanisms.MethodsPMBE was used as a cryoprotectant supplement. Hydrogen peroxide was applied to generate oxidative stress. The changes in sperm quality upon cryo‐resuscitation and hydrogen peroxide treatment were measured by computer‐aided sperm analysis and fluorescein isothiocyanate‐labeled peanut (Pisum sativum) agglutinin staining. Oxidative stress was evaluated by dichlorofluorescein diacetate staining and the malondialdehyde content. The antioxidant capacity was determined by the glutathione peroxidase activity and the reduced glutathione content. Mass spectrometry was utilized to screen factors potentially regulated by PMBE, and the screened factors were evaluated by Western blot. Mouse in vitro fertilization was used to assess the effectiveness and safety of PMBE on the fertilization outcome of damaged spermatozoa.ResultsPMBE addition to cryoprotectant could effectively promote post‐thaw vitality and acrosome reaction rates, reduce intracellular reactive oxygen species, and preserve antioxidant capacity and mitochondrial function in the presence of cryo‐resuscitation and hydrogen peroxide treatment. Decreased in the expression of critical proteins such as inositol‐3‐phosphate synthase 1 (ISYNA1) and AKT1 upon resuscitation was rescued by PMBE supplementation, further reducing the cytoplasmic Nrf2 protein level and markedly increasing the nuclear level. PMBE dramatically enhanced the mRNA and protein expression of superoxide dismutase (SOD2). PMBE effectively alleviated the hydrogen peroxide‐induced decline in the two‐cell embryo rates.ConclusionSupplementing cryoprotectant with PMBE improves human sperm quality after cryo‐resuscitation by reducing oxidative stress and stabilizing the functional proteins ISYNA1 and AKT1. PMBE addition improves the fertilization outcome of oxidative stress‐damaged spermatozoa.