P-063 Multivariate analysis identifies oxidative stress levels and acrosome status of motile spermatozoa as independent predictors of motile sperm hyaluronan binding ability
Authors: Raad G , Serdarogullari M , Yarkiner Z , Bazzi M , Mourad Y , Fakih F , Fakih C 
Published: June 2022

Abstract

Study question
Is it possible to predict motile sperm hyaluronan binding ability (HBA) from motile sperm functional parameters?

Summary answer
Using multivariate analysis, oxidative stress levels and acrosome status of motile spermatozoa, derived from semen swim-up, were identified as predictors of sperm HBA.

What is known already
Semen analysis is a gold standard practice for fertility evaluation. However, motility and morphology of spermatozoa are not always indicative of the molecular makeup of sperm cells. In this context, several sperm functional tests were developed to enhance our understanding of spermatozoa biology. Interestingly, several studies have shown that the plasma membrane of mature sperm cells contain hyaluronan binding sites. HBA was associated with intact acrosome, decreased DNA damage, and high viability. Nevertheless, it has not been demonstrated yet if hyaluronan binding ability can be predicted solely by swim-up derived- motile spermatozoa functional parameters.

Study design, size, duration
A prospective study was performed at Al-Hadi Medical Center, beirut, Lebanon, between January and July 2020. Freshly ejaculated semen samples obtained from infertile men with concentration of ≥ 20 x 106/ml and with total sperm motility of ≥ 30% were included. The primary outcome was to assess if the motile sperm functional parameters may influence HBA. A sample size of 54 was calculated using power = 85% and alpha = 5% for multiple linear regression.

Participants/materials, setting, methods
54 semen samples were assessed according to WHO guidelines (2010). Thereafter, samples were processed using the semen swim-up technique. Supernatants containing motile spermatozoa were collected for further analysis. In motile spermatozoa-enriched fractions, chromatin integrity was assessed using toluidine blue (TB) staining. Oxidative stress levels were detected using a nitroblue tetrazolium (NBT) test. Acrosome status was determined using triple staining technique. HBA was evaluated via a commercially available hyaluronan binding assay (HBA® Assay).

Main results and the role of chance
In the motile spermatozoa-enriched fractions, mean ± standard deviations for concentration, progressive motility, non-progressive motility and normal morphology were 26.17±11.48, 76±18.97, 20.94±12.61 and 0.75 ± 1.4, respectively. In addition, mean ± standard deviations for TB + cells, NBT + cells, living spermatozoa with intact acrosome, and HBA were 16.42 ± 13.4, 57.4 ± 9.4, 93.9 ±3.2, and 48.98±11.95, respectively. The values of the HBA had a non-normal distribution; therefore, these values were first linearized by taking the logarithm of the outcome. Afterwards, multiple linear regression (MLR) was used to predict the outcome. The overall regression of the final model was statistically significant (R2 = 0.346), F(4.43) =5.962, p = 0.001. The results of the final MLR model indicated that a unit increase in non-progressive motility (%) (p = 0.002) and NBT + cells (%) (p = 0.009) increased the HBA by 1.505 units and 1.409 units, respectively. However, a unit increase in dead spermatozoa with unreacted acrosome (%) (p = 0.032) and living spermatozoa with reacted acrosome (%) (p = 0.026) decreased the HBA by 1.317 units and 1.342 units, respectively.

Limitations, reasons for caution
This is an interim study and these results should be taken as preliminary. In order to confirm these findings, further studies including larger sample size and motile spermatozoa derived from semen samples with abnormal parameters (such as severe asthenozoospermia, leukocytospermia, severe oligozoospermia) are required.

Wider implications of the findings
Several studies indicated that using damaged spermatozoa may affect the intracytoplasmic sperm injection (ICSI) outcomes. In addition, using damaged spermatozoa during fertilization may predispose the offspring to various diseases later in life. These results may be considered as an impetus to reconsider sperm selection during ICSI.

Trial registration number
Not applicable