P-024 Revitalizing male fertility: Crocin’s dual role in antioxidant mastery and mitochondrial activation to enhance sperm motility
Authors: Boussabbeh M , Haddar M , Amira S , Abir C , Rania K , Salwa A  E , Meriem M 
Published: July 2024

Abstract

Study question
The study aims to investigate the potential effect of crocin, a major constituent of Crocus sativus (saffron), on improving sperm motility in patients with asthenozoospermia.

Summary answer
In vitro crocin treatment enhances sperm motility, reduces ROS, improves mitochondrial membrane potential, and boosts mitochondrial activity, suggesting the potential role of enhancing sperm motility.

What is known already
Male infertility can result from various factors, with excessive production of reactive oxygen species (ROS) adversely affecting sperm motility. Crocin, known for its antioxidant properties in scavenging ROS, has led to increasing interest in natural compounds to enhance sperm quality.

Study design, size, duration
The current study involved the evaluation of 95 patients at the Service of Reproductive Biology at the Maternity and Neonatology Center of Monastir. Sperm samples were collected and incubated for 3 hours at 37 °C with increasing concentrations of Crocin (0, 0.2, 0.5, 1, 1.5 mM).

Participants/materials, setting, methods
This study included asthenozoospermic patients, categorized into two groups: G1 with progressive motility (PM) between 20% and 30%, and G2 with PM between 10% and 15%. Sperm samples underwent treatment with Crocin. Evaluation encompassed sperm motility, considering the distance covered and velocity via video-microscopy. Additionally, intracellular ROS levels were assessed using DCFH-DA fluorochrome, mitochondrial membrane potential was measured with rh123, and mitochondrial activity was determined through succinate dehydrogenase activity via the MTT assay.

Main results and the role of chance
The results demonstrate that sperm motility is significantly enhanced after in vitro treatment with 1mM Crocin for 3 hours, with 1.9-fold (p &lt; 0.001) and 1.7-fold (p &lt; 0.001) increases in sperm velocity and distance traveled, respectively. Additionally, Crocin reduces intracellular ROS levels, exhibiting a reduction of 1.5-fold (p &lt; 0.001) in G1 and 2.4-fold (p &lt; 0.001) in G2. Fluorescence microscopy of spermatozoa from a patient with severe asthenozoospermia showed intense ROS-induced fluorescence, which was attenuated after incubation with 1 mM Crocin for 3 hours. Furthermore, Crocin demonstrates a noteworthy improvement in mitochondrial membrane potential, with 1.7-fold (p &lt; 0.001) for G1 and 1.9-fold (p &lt; 0.001) for G2, suggesting its capability to prevent the decline of mitochondrial potential. Microscopic fluorescence revealed a marked increase in fluorescence, particularly in the midpiece after Crocin treatment, confirming the protective effect of Crocin on mitochondrial membrane potential.
Moreover, there is a substantial increase in mitochondrial activity with Crocin, particularly in G1, showing a 2.14-fold (p &lt; 0.001) increase compared to the G2 group’s 1.85-fold (p &lt; 0.001) increase. These findings collectively highlight the multifaceted benefits of Crocin, encompassing enhanced sperm motility, reduced ROS levels, preserved mitochondrial membrane potential, and heightened mitochondrial activity, underscoring its potential as a therapeutic agent for male infertility, particularly in cases of asthenozoospermia.

Limitations, reasons for caution
While promising, this study’s limitations include the in vitro treatment, and not fully replicating complex in vivo conditions. Focused on asthenozoospermia, generalizing findings to other male infertility forms necessitates further investigation. Validation of Crocin’s therapeutic potential in male infertility requires additional clinical trials.

Wider implications of the findings
Our findings suggest that in vitro enhancement of sperm quality with crocin may serve as a promising solution to improve assisted reproductive techniques, mitigating exogenous oxidative stress during laboratory procedures. This insight holds potential for advancing reproductive interventions and warrants further exploration in clinical settings.

Trial registration number
not applicable