Critical evaluation of two models of flow cytometers for the assessment of sperm DNA fragmentation:an appeal for performance verification

Lack of standardized,reproducible protocols and referencO values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation(SDF)is a unique example.Any change in the setting of the standard instrument,including upgrades of hardware or software,can lead to different results and may affect clinicians'decision for treatment.Therefore,we compared TUNEL results of SDF obtained from a standard(C6)flow cytometer with a newer version of the same instrument(C6 Plus)and examined the cutoff,sensitivity,and specificity without calibration(adjustment)and after adjustment.Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers.The strength of agreement of the results between the two observers was also assessed.After adjustment of the settings,overall concordance became high and the two cytometers showed 100%positive and negative predictive value with 100%area under the curve.The overall correlation coefficient observed between C6 and C6 Plus was highly significant(P<0.0001;r=0.992;95%confidence interval[Cl]:0.982-0.997).After adjustment,the two cytometers showed very high precisi on of 98%and accuracy of>99%.The in terobserver agreeme nt on C6 flow cytometer for the two observers was 0.801±0.062 and 0.746±0.044 for C6 Plus.We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.

Spermatozoa protein alterations in infertile men with bilateral varicocele

Among infertile men, a diagnosis of unilateral varicocele is made in 90% of varicocele cases and bilateral in the remaining varicocele cases. However, there are reports of under-diagnosis of bilateral varicocele among infertile men and that its prevalence is greater than 10%. In this prospective study, we aimed to examine the differentially expressed proteins （DEP） extracted from spermatozoa cells of patients with bilateral varicocele and fertile donors. Subjects consisted of 17 men diagnosed with bilateral varicocele and 10 proven fertile men as healthy controls. Using the LTQ-orbitrap elite hybrid mass spectrometry system, proteomic analysis was done on pooled samples from 3 patients with bilateral varicocele and 5 fertile men. From these samples, 73 DEP were identified of which 58 proteins were differentially expressed, with 7 proteins unique to the bilateral varicocele group and 8 proteins to the fertile control group. Majority of the DEPs were observed to be associated with metabolic processes, stress responses, oxidoreductase activity, enzyme regulation, and immune system processes. Seven DEP were involved in sperm function such as capacitation, motility, and sperm-zona binding. Proteins TEKT3 and TCP11 were validated by Western blot analysis and may serve as potential biomarkers for bilateral varicocele. In this study, we have demonstrated for the first time the presence of DEP and identified proteins with distinct reproductive functions which are altered in infertile men with bilateral varicocele. Functional proteomic profiling provides insight into the mechanistic implications of bilateral varicocele-associated male infertility.