O-185 Cryopreservation of Human Spermatozoa: Utilization of L-Proline as a Novel Additive to Improve Sperm Quality Following Freezing-Thawing Process
Authors: Moradi B , Moradi M , Faramarzi A 
Published: June 2022

Abstract

Study question
Does supplementation of human sperm freezing media with L-Proline improve sperm quality and DNA Integrity following the freezing-thawing process?

Summary answer
The inclusion of L-proline as a novel additive to human sperm cryopreservation media improves sperm parameters and DNA integrity via mitigating oxidative stress.

What is known already
Sperm cryopreservation is an essential aspect of assisted reproductive technique (ART) and male fertility preservation. Although frozen-thawed semen has great practical benefits for reproduction, it is widely reported that the cryopreservation process induces physical and chemical detrimental changes in sperm functions. Indeed, freezing induces nitro-oxidative stress, lipid peroxidation, DNA fragmentation, and apoptosis. L-proline plays versatile roles in osmotic protection and oxidative stress, cell signaling, programmed cell death, and nutrient adaptation. This multifunctional amino acid is a natural osmoprotectant and the protective effect of L-proline against freezing-thawing-induced damages in stallion and donkey spermatozoa have been reported.

Study design, size, duration
Thirty normozoospermic semen samples were collected by masturbation after 3–5 days of sexual abstinence from men who were referred to the IVF clinic of Kermanshah Motazedi Hospital from December 2020 to June 2021. Each prepared semen sample was aliquoted to 4. In aliquots 1 to 4, experimental concentrations of L-proline (0, 1, 2, and 4 mmol/L), were included in the freezing medium.

Participants/materials, setting, methods
30 normozoospermic, healthy, non-smoker men were enrolled. Sperm parameters (progressive motility, viability, and morphology) were assessed. Sperm chromatin quality was measured by Aniline blue (AB), Toluidine blue (TB), and Chromomycin A3 (CMA3) staining. DNA integrity was evaluated by the Sperm chromatin dispersion (SCD) test. Furthermore, the levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and total antioxidant capacity (TAC) were determined in sperm freezing media. All assessments were conducted before cryopreservation and after thawing.

Main results and the role of chance
Our findings showed that sperm progressive motility and viability were significantly higher in the 4 mmol/L L-proline treated groups compared to the control groups (p = 0.003, p = 0.0200) respectively. The percentage of normal morphology was improved in L-Proline-treated groups. However, this improvement was not considerable (p &gt; 0.5). Moreover, the level of ROS production significantly diminished in the 4 mmol/L L-proline group compared to the control group (p &lt; 0.001). Besides, the level of TAC was significantly enhanced in the 4 mmol/L L-proline group by comparison to the control group (p &lt; 0.001). Furthermore, the results of AB and TB tests demonstrated that chromatin packaging after supplementation with 4 mmol/L L-proline significantly improved compared to the control group (p &lt; .0001). In this regard, there was a significant improvement between the 4 mmol/L   L-proline group and the control group in terms of CMA3 evaluation (p &lt; .0001). Also, there was a difference between the 4 mmol/L L-proline group and the control group in reducing the level of fragmented DNA. Although 4 mmol/L of Praline could diminish the SCD level compared to the control group, this amelioration was not statically significant (p = 0.053).

Limitations, reasons for caution
We were not able to perform further studies to verify the effects of L-proline on mitochondrial activity and membrane potential, and sperm apoptosis as well as to confirm the efficiency and safety of L-proline in terms of more indicators such as the in vitro embryonic development and live birth rate.

Wider implications of the findings
These findings can draw attention to the potential role of L-proline as a novel additive and antioxidant to human freezing medium in preserving sperm quality and protecting spermatozoa against ROS attack during sperm cryopreservation in infertility clinics.

Trial registration number
N/A