P-178 The interaction between spermatozoa and cumulus cells: a more physiological approach to the selection of good quality spermatozoa for assisted reproduction
Authors: Scarica C , Petrocelli P , Pontemezzo E , Haxhiu A , Perez Casasus S , Luddi A 
Published: June 2023

Abstract

Study question
Does selection by cumulus cells (CCs) improve sperm quality in terms of acrosome integrity and mitochondrial functionality compared to standard techniques?

Summary answer
The sperm-CCs interaction method appears to be effective in selecting sperm with an intact acrosome and functioning mitochondria

What is known already
In nature, the selection of the best quality sperm is very demanding and involves high morphological qualities and dynamic features. Semen preparation in ART has long been carried out using either the swim-up or density gradient method; although both methods provide motile sperm, they cannot really replicate the physiological selection processes. As a result, several methods have been proposed to mimic the natural selection that occurs in the female reproductive tract, allowing the most competent sperm to provide the paternal contribution to the zygote. However, the literature on these techniques is limited and does not provide consistent conclusions

Study design, size, duration
Proof-of-principle study conducted at the New Fertility Group (NFG) in Rome and the Department of Molecular and Developmental Medicine at the University of Siena (Italy) from July 2022 to December 2022, including 9 semen samples. Comparison of the CCs selection model with the standard swim-up by assessing mitochondrial and acrosome integrity. In addition, mitochondrial activity was assessed by oxygraphic analysis in spermatozoa exposed to immortalised human granulosa cells (hGL5) compared to standard swim-up sorted spermatozoa

Participants/materials, setting, methods
The CCs-model was prepared by running two channels of medium, one with CCs (study group) and one with medium alone (control). Spermatozoa were added to each channel and incubated at 37 °C for 1 hour. Moreover, spermatozoa were also added on hGL5 and incubated for 1.5h. The collected spermatozoa were compared with controls for acrosomal integrity, assessed by FITC-labelled PSA, and mitochondrial membrane potential, assessed by Mitotracker. Finally, sperm oxygen uptake was measured by oxygraphic analysis

Main results and the role of chance
Assessment of sperm acrosome integrity showed a higher percentage of responding acrosomes in the CCs model compared to swim-up and controls (61% ± 0.3; 39% ± 0.4; 19% ± 0.4 p &lt; 0.05). The CCs model also showed 83% ± 0.3 of spermatozoa with active mitochondria compared to 65% ± 0.3 in the control group (p &lt; 0.005). A similar pattern was observed in the swim-up group. Quantitative analysis of mitochondrial membrane potential (MMP) confirmed that CCs and swim-up groups had higher MMP compared to the control group (p &lt; 0.005). Evaluation of mitochondria in the hGL5 treated group showed active mitochondria in 78% ± 0.49 of the sperm compared to 59% ± 0.65 in the control group (p = 0.1). The same profile was detected by fluorescence quantification of MMP in the hGL5 group compared to the control group (p &lt; 0.01). Oxygraphic analysis, as an index of oxidative phosphorylation and consequently of ATP production, was performed on hGL5 exposed spermatozoa, swim-up selected spermatozoa and controls. hGL5 exposed and swim-up selected spermatozoa showed a similar O2 consumption, about 4 times higher than unselected spermatozoa (p &lt; 0.05)

Limitations, reasons for caution
The limited sample size and the heterogeneity of the samples were the main limitations of this study. Although CC and hGL5 sperm selection models appear to be more physiological, further studies on other parameters such as sperm DNA integrity are needed.

Wider implications of the findings
Both the CCs model and the hGL5 model appear to be effective in selecting good quality sperm. As these models avoid sperm centrifugation, further research is needed to understand their efficacy and reliability and whether they are a valid alternative to conventional sperm selection methods in terms of IVF outcomes.

Trial registration number
none