P-052 Targeted metabolomics (amino acids) profiling of seminal plasma distinguished teratozoospermia from normozoospermic samples
Authors: Hosseini E , Amirjannati N , Bazrafkan M , Moghaddasfar H , Gilany K 
Published: June 2022

Abstract

Study question
Is there a difference in metabolic composition between teratozoospermia and normozoospermic seminal samples?

Summary answer
Teratozoospermia and normozoospermia in humans are distinguished by seminal fluid metabolome markers especially amino acid content.

What is known already
Metabolomic profiling is the study of metabolites as downstream gene expression products in physiological and pathological circumstances. Identifying the metabolome of human seminal plasma (HSP) is a new research area to screen putative (in)fertility biomarkers. Knowing the semen metabolic signature and whether there are differences in metabolic composition between the teratozoospermia sample and semen with normal morphology could enhance our understanding of aspects associated with poor sperm quality and consequent male infertility.

Study design, size, duration
The Ethics Committee of Islamic Azad Tehran Medical Sciences University-Pharmacy and Pharmaceutical Branches approved the case-control study. Teratozoospermia was defined based on 5th WHO (2010) as normal morphology&lt;4%, count&gt;15*106/ml, progressive and non-progressive motility≥40%. In contrast, in normozoospermic samples from fertile men (who had at least one child), normal morphology was greater than 4%. HSP specimens were obtained from 15 infertile patients with teratozoospermia, and 12 confirmed fertile men as the control group.

Participants/materials, setting, methods
Fresh semen samples were obtained through masturbation following 3–4 days of abstention. A portion of each sample was used for routine semen analysis. Remained seminal plasma was isolated by centrifugation and stored; following post-thaw, HSPs were subjected to LC-MS-MSanalysis. MetaboAnalyst 5.0 software(www.metaboanalyst.ca) was utilized to identify fundamental metabolism alterations related to the morphology status of spermatozoa. In addition, different uni/multivariate analysis was performed to ascertain the group of metabolites responsible for the overall discrimination ability.

Main results and the role of chance
Seventeen significantly differentially metabolites (14 decreased and three increased) were observed in teratozoospermia samples vs. fertile control. Down-regulated metabolites include Cysteine, Gamma-aminobutyric acid, Glycine, Histidine, Hydroxylysine, Hydroxyproline, Methionine, Ornithine, Proline, Tryptophan, Aspartic acid, Argininosuccinic acid, Alpha-aminoadipic acid, and Beta-aminoisobutyric acid; whereas up. In contrast, up-regulatedlutamine, Asparagine, and Glycylproline.
The PLS-DA score plot demonstrated the clear separation obtained according to the sperm morphological status. The two distinct groups were well-clustered, with distinct metabolic profiles for each group.
The random forest (RF) algorithm was utilized to select relevant variables for teratozoospermia status classification by estimating the importance of each metabolite to teratozoospermia. The RF analysis defined a set of 15 metabolites that constitute the best predictors of teratozoospermia. In particular, increased Glutamine, Asparagine, and decreased Cysteine, Tryptophan, Glycine, and Valine were strong predictors of teratozoospermia. Interestingly, pathway enrichment analysis revealed that the most affected metabolic pathways in teratozoospermia men were the Aminoacyle-tRANA, arginine, valine-leucine, and isoleucine biosynthesis.
Among 34 metabolites, Asparagine, Glutamine, and Glycylproline negatively correlated with sperm morphology status; whereas 21 metabolites were positively correlated with morphology, including Alanine, Argininesuccinic, Aspartic, Cystine, Gamma-aminobutyric acid, Glutamic, Glycine, Histidine, Homocitrulline, Hydroxylysine, Hydroxyproline, Isoleucine, Leucine, Methionine, Ornithine, Phenylalanine, Proline, Threonine, Tryptophan, Tyrosine, and Valine.

Limitations, reasons for caution
Since the strict criteria were considered for teratozoospermia patients, these results are preliminary and need to be confirmed with more sample size.

Wider implications of the findings
The possible cause of metabolite difference in seminal plasma of teratozoospermia patients comes from the disability of spermatozoa for selectively absorption or secretion of specific amino acids into/out of the cell, which is critical for sperm function and maturation and reflects the potential of fertility.

Trial registration number
Not Applicable