Background & Aim: A large number of infertile men suffer from non-obstructive azoospermia. To evaluate their spermatogenesis status, they must undergo the invasive procedure of testicular biopsy. Analyzing the expression pattern of testis-specific genes and using them as molecular markers of testicular spermatogenesis will lead to a drastic change in therapeutic approaches. Furthermore, mRNA content of the semen gives us most valuable information about the patient's spermatogenesis level which is almost undetectable by histopathological methods. In this study, in comparison with histopathological results, the expression of testis specific genes, DAZ, AKAP4, CSNK2a2, PRM1 and PRM2 as molecular markers was monitored.
Material & Methods: Azoospermic men (203 persons) who underwent testicular biopsy at the Avesina Infertility Clinic (AIC), Tehran, Iran were included in this study. Biopsied testicular tissue was used for histologic evaluation by an expert clinical pathologist. In addition to semen analysis, the level of LH, FSH and testosterone was measured in patients' serum and was compared with the molecular results.
Total RNA was extracted from the semen pellets using RNAbee reagent. First-strand cDNA was synthesized and subsequently amplified using M-MuL V RT. PCR was carried out using sequence specific primers for above testis-specific genes and as ?-actin a house keeping gene. Testis biopsies showing normal spermatogenesis were used as positive controls.
Results: PCR for testis-specific genes was carried out on b-actin positive samples (110 patients). Molecular results for DAZ, PRM1 and PRM2 genes were in agreement with the histopathological results. However AKAP4 and CSNK2a2 genes showed insignificant agreement with the pathology. Comparing hormonal and molecular results was showed large number of men with spermatogensis failures had normal levels of LH and FSH and testosterone. Interestingly, there were 7 patients with germ cell aplasia who had positive molecular results which show the presence of focal spermatogenesis in their testis.
Conclusion: Introduction of this non-invasive molecular diagnostic tool, Using PRM1, PRM2 and AKAP4 transcripts in semen along with DAZ as a testis-specific internal control can help clinicians to predict the presence of spematogenesis in testis of NOA men and will prevent to undergoing unnecessary testicular biopsies in most patients.