L‐proline as a novel additive to cryopreservation media improved post‐thaw quality of human spermatozoon via reducing oxidative stress

Abstract

Sperm cryopreservation as a routine technique in assisted reproductive technique (ART) laboratories has detrimental effects on spermatozoa. Various methods have been introduced to improve it. The aim of this research was to evaluate the effects of L‐proline supplementation in cryopreservation medium on normozoospermic semen samples. A total of 30 semen samples were collected from normozoospermic men. Cryopreservation media were supplemented with different concentrations of L‐proline (0, 1, 2 and 4 mmol/L). The semen samples were cryopreserved. After thawing, sperm parameters and chromatin integrity (aniline blue (AB), toluidine blue (TB), sperm chromatin dispersion test (SCD) and chromomycin A3 (CMA3)), reactive oxygen species (ROS), and total antioxidant capacity (TAC) and malondialdehyde (MDA) levels were evaluated. A total of 4 mmol/L L‐proline significantly improved progressive motility and viability (p < 0.05). MDA and ROS levels significantly diminished in samples were cryopreserved by 4 mmol/L L‐proline supplemented cryopreservation media (p < 0.001). Also, it significantly increased TAC level. Also, chromatin damages (AB, TB and CMA3) significantly improved in samples were cryopreserved by 4 mmol/L L‐proline supplemented cryopreservation media (p < 0.05). The results support that the usage of L‐proline supplemented cryopreservation media to improve sperm quality after cryopreservation.

Beneficial effect of L‐ Proline supplementation on the quality of human spermatozoa

“…In treatment groups, similar volumes of different concentrations of Proline (1, 2 and 4 mmol/L) were added. The Proline concentrations were chosen based on the pre‐experiments we conducted and the limited available data (Moradi, Faramarzi, et al, 2021; Moradi, Goodarzi, et al, 2021). All groups were incubated for 1, 4 and 24 h at room temperature (25°C) (Figure 1).…”

“…To assess sperm morphology, sperm were stained according to the Papanicolaou staining protocol based on guidelines of the WHO (World Health Organization, 2010). Then, phase‐contrast microscopy (Olympus Co., Tokyo, Japan) (×1000) was used to examine at least 200 spermatozoa per sample (Moradi, Faramarzi, et al, 2021; Moradi, Goodarzi, et al, 2021).…”

“…ROS levels were measured using a fluorimeter by a spectrometer‐based ELISA reader. Finally, the fluorescence intensity at 485 nm and 520 nm was reported as a relative fluorescence unit (RFU) (Moradi, Faramarzi, et al, 2021; Moradi, Goodarzi, et al, 2021).…”

“…These microtubes were placed in a boiling water bath and ice bath for 1 h in the next step. Finally, supernatant absorbance was assessed using a microplate reader/ELISA reader at 535 nm (Moradi, Faramarzi, et al, 2021; Moradi, Goodarzi, et al, 2021).…”

“…Then, microplates were incubated for 2 min at room temperature. Finally, a plate ELISA reader at 490 nm was used to evaluate and read the absorbance (Moradi, Faramarzi, et al, 2021; Moradi, Goodarzi, et al, 2021).…”