# Bicarbonate buffer enhances functional sperm selection compared to Zwitterionic buffers in sperm preparation

## TL;DR

Bicarbonate buffer improves sperm quality more than zwitterionic buffers like HEPES and MOPS during sperm preparation for fertility treatments.

## Contribution

This study demonstrates that bicarbonate buffer outperforms zwitterionic buffers in selecting functionally superior spermatozoa for ART.

## Key findings

- Bicarbonate-buffered media significantly increased sperm motility and mitochondrial activity in both normozoospermic and non-normozoospermic samples.
- Zwitterionic buffers like HEPES and MOPS were linked to reduced motility, poor kinematic parameters, and lower mitochondrial potential.
- Bicarbonate buffer supports better sperm selection compared to zwitterionic buffers in assisted reproductive technology.

## Abstract

Sperm preparation media used in assisted reproductive technology (ART) laboratories typically contain single or combined buffers to maintain pH and support spermatozoa homeostasis. Concerns exist that zwitterionic buffers, such as 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) and 3-(N-morpholino) propane sulfonic acid (MOPS), may negatively affect intrinsic cellular functions. Hence, this prospective study evaluated the impact of various buffers on sperm selection by swim up from fifty-four sub-fertile men, which were then divided into normozoospermic samples (n = 27) and non-normozoospermic (n = 27) cohorts. Each ejaculate was split into six aliquots, and swim-up was performed with media buffered by bicarbonate, HEPES, MOPS, bicarbonate + HEPES, or bicarbonate + MOPS. Post-swim-up spermatozoa were assessed for motility, morphology, kinematics, acrosome reaction, mitochondrial integrity, and DNA damage. Bicarbonate-buffered medium yielded spermatozoa with significantly higher motility and mitochondrial activity in both normozoospermic and non-normozoospermic samples (p < 0.001). In contrast, media containing HEPES or MOPS were associated with reduced motility (p < 0.001), compromised sperm kinematic parameters (p < 0.05), and diminished mitochondrial potential (p < 0.05). The study findings highlight the importance of bicarbonate buffer over zwitterionic buffers in sperm preparation media for the selection of better spermatozoa during ART procedures.

The online version contains supplementary material available at 10.1038/s41598-026-44733-9.