Molecular screening of unculturable bacteria present in men with reproductive failure

Objective:  To determine the presence of unculturable bacteria using polymerase chain reaction (PCR) in infertile men with pyosperrmia. Design:  Perspective clinical study. Setting:  The study took place at the Department of Reproductive Medicine, Owaisi Hospital and Research Center; In vitro Fertilization Unit, Mahavir Hospital and Research Center; Center for Cellular and Molecular Biology; and Bharat Biotech Foundation, Hyderabad, India. Patients:  A total of 68 infertile men and 15 donors, all with no symptoms of genito-urinary tract infections and sterile semen cultures were included in the study. Interventions:  None. Main outcome measures:  Screening bacteria using routine bacterial cultures and PCR based screening with universal eubacterial primers. Results:  The statistical analysis of all the semen parameters in asthenazoospermic, azoospermic, ceyptazoospermic, severe oligospermic and mild oligospermic patients were found to be significant compared with the controls. All the groups were found to be significant compared with the controls (P < 0.05) except for volume and pus cells in the cryptozoospermia group. The Student's t-test also was significant for the seminal parameters before and after treatment of 68 selected individuals with pyospermia and sterile cultures. A total of 44.11% (30/68) samples were collected from the negative culture of pyospermic infertile men have shown the presence of bacteria on amplification using PCR with universal eubacterial primers. The DNA was purified and sequenced. The sequences were checked for homology using DNASTAR and Ribosomal DataBase Project II. A total of 90% of the samples have shown the nearest evolutionary relation to Pantoea P102 (AF394539) and 10% of samples have shown close relation with Burkholderia cepacia (AF042161). Conclusion:  The routine bacteriological cultures were unable to detect certain bacterial species particularly with members of enterobacteriaceae family (Pantoea species). Polymerase chain reaction, when used for screening bacteria, can detect the unculturable form of bacteria in infertile men. No amplification for bacterial DNA was obtained in control samples (fertile men with sterile semen cultures.) (Reprod Med Biol 2004; 3: 77- 84).