Accuracy evaluation of the depth of six kinds of sperm counting chambers for both manual and computer-aided semen analyses

Background: Although the depth of the counting chamber is an important factor influ-encing sperm counting, no research has yet been reported on the measurement and comparison of the depth of the chamber. We measured the exact depths of six kinds of sperm counting chambers and evaluated their accuracy

Materials and Methods: In this prospective study, the depths of six kinds of sperm counting chambers for both manual and computer-aided semen analyses, including Makler [n=24], Macro [n=32], Geoffrey [n=34], GoldCyto [n=20], Leja [n=20] and Cell-VU [n=20], were measured with the Filmetrics F20 Spectral Reflectance Thin-Film Measurement System, then the mean depth, the range and the coefficient of variation [CV] of each chamber, and the mean depth, relative deviation and acceptability of each kind of chamber were calculated by the closeness to the nominal value. Among the 24 Makler chambers, 5 were new and 19 were used, and the other five kinds were all new chambers

Results: The depths [mean +/- SD, ?m] of Makler [new], Macro and Geoffrey chambers were 11.07 +/- 0.41, 10.19 +/- 0.48 and 10.00 +/- 0.28, respectively, while those of GoldCyto, Leja and Cell-VU chambers were 23.76 +/- 2.15, 20.49 +/- 0.22 and 24.22 +/- 2.58, respectively. The acceptability of Geoffrey chambers was the highest [94.12%], followed by Macro [65.63%], Leja [35%] and Makler [20%], while that of the other two kinds and the used Makler chamber was zero

Conclusion: There existed some difference between the actual depth and the corresponding nominal value for sperm counting chambers, and the overall acceptability was very low. Moreover, the abrasion caused by the long use, as of Makler chamber, for example, may result in unacceptability of the chamber. In order to ensure the accuracy and repeatability of sperm concentration results, the depth of the sperm counting chamber must be checked regularly

Correlations of 24 biochemical markers in seminal plasma with routine semen parameters / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the correlations of 24 biochemical markers in the seminal plasma with routine semen parameters.</p><p><b>METHODS</b>According to the WHO5 standards, we analyzed the routine semen parameters of 66 subfertile men, including the semen volume, sperm concentration, total sperm count, sperm motility, and the percentage of progressively motile sperm (PR). Based on the calibration and quality control measures and using an automatic biochemistry analyzer or electrolyte analyzer, we detected 24 biochemical markers in the seminal plasma of the patients, including total protein (TP), albumin (Alb), globulin (Glb), uric acid (UA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), γ-glutamyltransferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK), alpha hydroxybutyrate dehydrogenase (αHBDH), adenosine deaminase (ADA), glucose (Glu), triglyeride (TG), total cholesterol (TC), urea nitrogen (UN), creatinine (Cr), high-sensitive C-reactive protein (hsCRP), K+, Na+, Cl- , Ca, Mg, and phosphorus (P). Then we analyzed the correlations of the 24 biochemical markers with routine semen parameters.</p><p><b>RESULTS</b>The levels of the TP, Alb, and Glb proteins in the seminal plasma were positively correlated with sperm concentration, so was that of Alb with the total sperm count, and the AST and LDH activities with sperm concentration and total sperm count. The AKP activity in the seminal plasma was correlated negatively with the semen volume, but positively with sperm motility. The αHBDH activity exhibited a positive correlation with both sperm concentration and total sperm count, with a coefficient of correlation (r) above 0.7. The UN level was correlated negatively with the semen volume, so was the Cr level with the semen volume, sperm concentration, and total sperm count, and the Glu level with sperm concentration and total sperm count. The TG level was correlated positively with the semen volume, but negatively with sperm motility. The levels of seminal plasma ALT, GGT, ADA, UA, TC, CK, and hsCRP showed no correlation with the above-mentioned semen parameters. None of the seminal plasma K+, Na+, Ca, Mg, and P levels was found correlated with semen parameters except the Cl- level, which was negatively correlated with the semen volume.</p><p><b>CONCLUSION</b>Many biochemical markers in the seminal plasma are closely related to routine semen parameters, indicating that these biochemical components may play roles in spermatogenesis, sperm maturation, and physiological metabolism.</p>

Influence of the depth of the sperm counting chamber on sperm motility / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the influence of the depth of the sperm counting chamber on sperm motility.</p><p><b>METHODS</b>We measured the depths of sperm counting chambers using the Filmetrics F20 Spectral Reflectance Thin-Film Measurement System. Then, according to the WHO5 manual, we analyzed 36 semen samples for the percentages of progressively motile sperm (PR) and non-progressively motile sperm (NP) and sperm motility (PR + NP) with the Ruiqi CFT-9201 computer-aided sperm analysis system, and compared the results of analysis.</p><p><b>RESULTS</b>The depths of the 4 sperm counting chambers were 9.8, 12.7, 15.7 and 19.9 microm, respectively, and the obtained PR were (44.00 +/- 11.63), (41.96 +/- 12.62), (40.86 +/- 11.71) and (37.78 +/- 11.38)%, NP (13.54 +/- 3.01), (14.13 +/- 2.94), (14.91 +/- 3.02) and (16.53 +/- 2.77)%, and sperm motility (57.53 +/- 11.06), (56.08 +/- 11.97), (55.78 +/- 11.55) and (54.31 +/- 12.11)% from the 4 chambers, respectively. The depth of the sperm counting chamber was correlated negatively with PR (r = -0.993, P < 0.05) and sperm motility (r = -0.978, P < 0.05), but positively with NP (r = 0.989, P < 0.05). There were statistically significant differences between the 9.8 microm and 19.9 microm deep chambers in PR and NP (P < 0.05) though not in sperm motility among the 4 chambers of different depths.</p><p><b>CONCLUSION</b>The impact of the depth of the sperm counting chamber on sperm motility should not be ignored, for the deviation of the results from the chambers of different depths may lead clinicians to incorrect diagnosis and consequently inappropriate therapeutic approaches. Different reference ranges of sperm motility need to be normalized in correspondence to the depths of sperm counting chambers.</p>

Establishment and evaluation of an automatic method for seminal plasma gamma-L-glutamyl transpeptidase detection / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish an automatic method for seminal plasma gamma-L-glutamyl transpeptidase (GGT) detection and evaluate its accuracy, repeatability and linear range.</p><p><b>METHODS</b>We detected the GGT activity in the seminal plasma by rate assay, and established the detection parameters on an automatic biochemical analyzer. Then, we evaluated the reagent blank absorbance, accuracy, repeatability and linear range of the automatic method, and compared the results obtained from the method and the seminal plasma GGT detection kit (Xindi Biological Pharmaceutical Engineering Co., Ltd, Nanjing, China) commonly used in clinical laboratories.</p><p><b>RESULTS</b>The average absorbance of reagent blank was 0.0476, and the average change rate of blank absorbance (deltaA/min) was 0.000168. The coefficients of variation (CV) for 3 seminal plasma samples with high, middle and low GGT activity detected for 10 times, respectively, were 0.26%, 4.83% and 1.60%. The accuracy of the automatic method was evaluated by a comparison test, and the relative deviation for each concentration point of 40 seminal plasma samples ranged from 13.38% to 11.05%, which met the requirement of < 15%. There was a good linear relationship (r > 0.99) when the seminal plasma GGT activity was between 299 and 1 833 U/L. A significant positive correlation was found between the seminal plasma GGT detection kit (a colorimetric method) as the control and the automatic method as the test reagent in the results of 115 seminal plasma samples (r = 0.981, P < 0.01), with a Kappa value of 0.776 (P < 0.05) and a coincidence rate of 90.43%.</p><p><b>CONCLUSION</b>The established automatic method to detect seminal plasma GGT activity has a low reagent blank, good repeatability and accuracy, and fine concordance with the colorimetric method commonly used in clinical laboratories. It is simple, rapid and suitable for screening large numbers of samples, avoids the necessity of diluting the seminal plasma sample, and saves a lot of manpower and reagents.</p>

Advances in researches on polymorphonuclear neutrophil elastase in semen / 中华男科学杂志

Reproductive tract infection is one of the important factors of male reproduction. Polymorphonuclear neutrophil elastase (PMNE) in semen, as a marker of male reproductive tract inflammation, especially recessive infection, potentially affects male fertility. The concentration of PMNE in semen is correlated significantly not only with semen white blood cell count and seminal plasma ROS level, but also with the levels of other inflammation related cytokines, such as IL-6, IL-8, and TNF-alpha. Furthermore, PMNE has a negative impact on sperm quality by decreasing sperm motility, increasing the percentage of morphologically abnormal sperm and interfering with DNA integrity. PMNE inhibitors in semen can form a compound with PMNE, and the imbalanced proportions of the two may promote the development of chronic inflammation, and consequently lead to male infertility. At present, PMNE in semen is detected mainly by enzyme immunoassay, but this method still needs to be standardized, and the diagnostic standards to be unified.

Preparation and identification of recombinant human neutrophil elastase / 中华男科学杂志

<p><b>OBJECTIVE</b>To prepare a purified recombinant human neutrophil elastase (HNE) using genetic engineering technology, and pave the way for the preparation of the antibody to HNE and establishment of semen HNE detection methods.</p><p><b>METHODS</b>HNE mRNA was obtained from human peripheral blood granulocytes with specific HNE primers, and the cDNA of HNE was cloned into the plasmid pGEX-2T to derive a recombinant plasmid pGEX-2T/HNE. After PCR identification, double-enzyme digestion and gene sequencing, the recombinant plasmid was transferred into competent Escherichia coli DH5alpha and further induced to express the recombinant fusion protein GST/HNE by isopropyl beta-D-thiosulfate galactosidase (IPTG). The recombinant fusion protein was cleaved by thrombin and further purified with glutathione agarose beads to obtain purified recombinant HNE.</p><p><b>RESULTS</b>The recombinant plasmid pGEX-2T/HNE was successfully prepared and transferred into E. coli DH5o; the expression of the recombinant fusion protein GST/ HNE was successfully induced by IPTG at 18 degrees C overnight; and the purified recombinant protein HNE was successfully obtained by thrombin cleavage and purification of glutathione agarose beads.</p><p><b>CONCLUSION</b>The acquirement of purified recombinant HNE has prepared the ground for the preparation of the antibody to HNE and establishment of semen HNE detection methods.</p>