Numerous biological processes, such as cell division and differentiation, depend significantly on miRNAs. They are highly conserved across species, extremely stable, and prevalent in seminal plasma and various body secretions [3, 15]. miRNAs were found to be highly abundant in semen; however, it is important to note that different methods of investigations may produce different results [16,17,18,19].

In the current study, two miRNA markers (miR-20b and miR-197) were selected, and their relative expression levels were applied to distinguish between semen and vaginal secretion as well as to study their accuracy to differentiate between fertile and infertile semen (oligospermia and azoospermia) clinical samples. The expression levels of miR-20b and miR-197 showed significant expression differences between all body fluid samples with a P-value < 0.001, but the expression levels of miR-197 in azoospermia samples were relatively similar to those in vaginal fluid without significant differences.

We designed a model based on Fisher’s discriminant function to differentiate various bodily fluid samples using miR-197 and miR-20b markers. The findings can be directly derived using expression data and a mathematical formula, avoiding some subjectivity and producing reliable and accurate results. With three novel equations, we were able to accurately distinguish between fertile semen & vaginal fluid, fertile & infertile semen and between oligospermia & azoospermia samples with validation accuracy of 81.3%, 100% and 100%, respectively.

Weber et al [20] studied the expression level of miRNAs in 12 human body fluids and stated that miR-197 and miR-20b are among the top 20 most prevalent miRNAs in semen samples, while the miRNA species that are uniquely detected in semen fluids are miR-197, miR-20b, miR-20b miR-380, miR-29b-2, miR-508-5p, miR-340, miR- 644, miR-17, miR-588, miR-617, and miR-1. They also demonstrated that miR-10a, miR-135b, miR-135a, and miR-10b have co-expression with semen in different body fluids.

MiR-891a and miR-888 were discovered by other researchers to have a uniform and focused distribution. These markers are able to successfully distinguish semen from a variety of forensically relevant body fluids with great semen specificity [16,17,18, 21]. However, according to the findings of Tong et al. [19] and Hanson et al. [22], there was overlapping in the expression of miR-891a regarding normal semen and various bodily fluids.

He et al. [6] developed a statistical model for classifying five human body fluids on the basis of the different expression levels of 10 miRNAs (miR-451a, miR-214-3p, miR-144-3p, miR-205-5p, miR-144-5p, miR-654-5p, miR-888-5p, miR-891a-5p, miR-203-3p, and miR-124a-3p) in semen, saliva, vaginal secretions, saliva, peripheral blood, and menstrual blood. A discriminant function has been created using stepwise discriminant analysis, and the model’s accuracy in self-validation, cross-validation, identification validation set accuracy, and blind test result accuracy were all 100%.

As regards identification of infertile semen samples, in the research done by Tian et al. [10], the level of expression of different sets of miRNA markers (miR-10a, miR-10b, miR-135b, miR-888, miR-135a and miR-891a) has been assessed in normal semen as well as four different types of infertile semen samples: (asthenospermia, oligospermia, azoospermia, and asthenospermia) using real-time quantitative PCR. They stated that these markers have significant high expression in normal semen and the model’s self-validation accuracy was 100%.

Tian et al [23] investigated the expression levels of two miRNAs (10b and 135b) in various types of semen from infertile males, including asthenospermia and azoospermia and the results revealed that fertile semen expressed substantially more of the two miRNAs than did the asthenospermia and azoospermia samples. Additionally, Haas et al. [24] studied the expression of the semen miRNA markers (PRM2 and PRM1) in fertile and azoospermic semen and they stated that it was negative in azoospermic samples.

Our results were sufficient to demonstrate the existence of a difference in the expression of the studied markers between semen and vaginal fluid, also between fertile and infertile semen and between oligospermia and azospermia semen clinical samples. Moreover, this study is the first to examine the efficacy of miR-197 and miR-20b to distinguish between fertile & infertile semen and between azoospermia & oligospermia semen samples and few studies, to our knowledge, have examined the significant effect of semen fertility problems on other miRNAs markers. We could therefore introduce the use of these miRNAs markers in the field of body fluid differentiation. However, this study is based on clinical samples, thus, further studies are needed using real forensic samples from crime scenes to investigate if these markers expression can provide significant forensic body fluid identification when such samples are found in different crime scenes as in sexual assault cases.