Genetic Diversity of Pluripotent Stem Cell Marker OCT4, NANOG and SOX9 Gene Expression with Functional Impact on Non-Obstructive Azoospermic Patients

Stem cells, an early transcription factors play an important role in reproductive biology during differentiation of primary spermatocytes and further development of gonads. During fertilization, embryonic stem cells have ability to differentiate in three cell-lineages - ectoderm, endoderm, and mesoderm. Male gonads shows dual in origin i.e ectodermal and mesoderm, where NANOG, OCT4 and SOX9 are responsible for maintaining pluripotency during normal spermatogenesis. The differentiation of testes till normal functioning is purely a genetic event and are highly sensitive to environmental factors. During spermatogenesis, germ cells divides in synchronize fashion to maintain “genetic pool” till development of mature sperm. These stem cell markers NANOG or OCT4 or SOX9 are early transcription factors to maintain pluripotency during spermatogenesis. Present study has been designed with the aims to assess the functional role of NANOG, OCT4 and SOX9 genes in clinically diagnosed cases of Non-Obstructive Azoospermia (NOA). Genomic DNA was isolated from peripheral blood under sterile conditions, quantified and RT-PCR techniques were used for characterization of gene-expression (either over or under expression) using specific primers. The frequency of DNA Copy Number Variations (DNACNV) were also evaluated to assess the genomic instability. Interestingly, findings reveals that NANOG showing highest frequency (42.0 %) of over-expression with the values of odd ratio (0.07) with confidence intervals (C.I) at 95% were varies between 0.029 - 1.430. The over-expression of SOX9 observed in 33% cases with odd ratio (0.11) and calculated value of C.I at 95% varies between 0.040 - 0.070. The frequency of complete disappearance (mutation) band of OCT4 was observed in 9.0% and SOX9 (20.8%) cases, while, NANOG (28.8%) showing significant (p<0.01) differences between cases and controls. OCT4 showing higher frequency of DNACNVs (blue bar) as compare to controls (yellow bar) having calculated values of O.R. (0.12) and C.I. at 95% interval varies between 0.014 to 1.089. The present study concludes that 1) NANOG play an important role to regulate for germ- cells proliferation, 2) Over-expression of SOX9 inhibit the  proliferation of differentiating cells, and 3) OCT4- gene-expression showing increase genetic susceptibility due to poor signalling. Hence, early detection of these stem cells may be use as “biomarker” and these stem cells in future becomes relevant for stem-cell therapy as a source of regenerative medicine.