P-071 L-proline enhances sperm quality and chromatin condensation of normozoospermic men during in vitro incubation
Authors: Moradi M , Moradi B , Peysokhan M , Rashidi S , Faramarzi A 
Published: June 2023

Abstract

Study question
Does supplementation of human sperm media with L-Proline improve sperm function and chromatin quality during in Vitro Incubation?

Summary answer
The inclusion of L-proline as an antioxidant in human sperm media improves sperm function and chromatin integrity probably by modulating oxidative stress

What is known already
Infertility affects millions of people of reproductive age globally. The quality of gametes is one of the most critical aspects that could affect the success rate of infertility therapy. The storage of spermatozoa at the incubator to achieve capacitation could rise sperm DNA fragmentation as a result of prolonged incubation. Although mild and low levels of Reactive Oxygen Species (ROS) could boost fertilization capacity by improving hyperactivation, and capacitation, oxidative stress is one of the major contributors to low sperm quality. L-Proline plays a pivotal role in many aspects of cell metabolism and physiology, including osmotic protection and oxidative stress

Study design, size, duration
The study was conducted on thirty-five healthy men attending the Motazedi Hospital, Kermanshah of the Division of Andrology, from December 2020 to June 2021. Each semen sample was equally divided into 4 groups: one group as control and three as treatment groups. The control group contained only sperm media (Hams, F10 medium supplemented with 5 mg/ml HSA). In treatment groups, different concentrations of Proline (1, 2, and 4 mmol/L) were also added.

Participants/materials, setting, methods
35 normozoospermic men with healthy lifestyles were included. Sperm parameters including motility, viability, and morphology were evaluated. The levels of ROS, lipid peroxidation (LPO), and total antioxidant capacity (TAC) were determined in sperm media. Sperm chromatin integrity was analyzed by Aniline blue (AB), Toluidine blue (TB), and Chromomycin A3 (CMA3) staining tests. DNA fragmentation was measured by the Sperm chromatin dispersion (SCD) test. All assessments were conducted after 1, 4, and 24 hours of incubation.

Main results and the role of chance
The inclusion of Proline (2 mmol/L) resulted in noticeably improved maintenance of motility and viability after 24 hr of incubation compared to the control group (P &lt; 0.05). Also, 2 mmol/L of Proline was able to significantly maintain normal morphology compared to other groups (P &lt; 0.01). However, the level of ROS production non-significantly diminished in the L-proline groups compared to the control group (P &gt; 0.05). In terms of lipid peroxidation, the presence of 2 mmol/L of Proline kept the MDA production of spermatozoa at lower levels after 4 hr of incubation compared with the control group (P &lt; 0.05). The supplementation of sperm media with 2 mmol/L of Proline led to improved maintenance of TAC level, compared to any other group (P &lt; 0.01). Additionally, the results of AB and TB tests indicated that chromatin quality after supplementation with 2 mmol/L L-proline significantly improved compared to the control group (P &lt; 0.05). In terms of CMA3 assessment, however, there was a minor improvement between the 2 mmol/L Proline group and the control group. Moreover, 2 mmol/L Proline significantly reduced the level of fragmented DNA by comparison to the control group after 24 hr of incubation (P &lt; 0.05).

Limitations, reasons for caution
Our team was unable to carry out additional studies to verify the impact of L-proline on mitochondrial activity, membrane potential, and apoptosis and to validate the efficiency and safety of L-proline using additional parameters such as the in vitro embryonic development and live birth rate.

Wider implications of the findings
While further studies are required to uncover the mechanisms underlying the antioxidant properties of Proline, these findings extend our knowledge of human male fertility and pave the way to improving the human sperm culture and in vitro procedure routinely used in infertility clinics for sperm incubation.

Trial registration number
IR.KUMS.REC.1399.625