Automatic detection and clinical application of semen biochemical markers / 中华男科学杂志

Human seminal plasma is rich in potential biological markers for male infertility and male reproductive system diseases, which have an application value in the diagnosis and treatment of male infertility. The methods for the detection of semen biochemical markers have been developed from the manual, semi-automatic to the present automatic means. The automatic detection of semen biochemical markers is known for its advantages of simple reagent composition and small amount of reagents for each test, simple setting of parameters, whole automatic procedure with few errors, short detection time contributive to batch detection and reduction of manpower cost, simple calibration and quality control procedure to ensure accurate and reliable results, output of results in the order of the samples in favor of clinical diagnosis and treatment, and open reagents applicable to various automatic biochemistry analyzers. At present, the automatic method is applied in the detection of such semen biochemical markers as seminal plasma total and neutral alpha-glucosidase, acid phosphatase, fructose, γ-glutamyl transpeptidase, zinc, citric acid, uric acid, superoxide dismutase and carnitine, sperm acrosin and lactate dehydrogenase C4, and semen free elastase, which can be used to evaluate the secretory functions of the epididymis, seminal vesicle and prostate, sperm acrosome and energy metabolism function, seminal plasma antioxidative function, and infection or silent infection in the male genital tract.

Association of abnormal length of Y chromosome with semen quality and outcome of assisted reproductive technology in humans / 中华男科学杂志

Objective@#To investigate the association of the abnormal length of human Y chromosome with semen quality and the outcome of assisted reproductive technology (ART).@*METHODS@#Based on the karyotype, we assigned the patients undergoing ART to a normal control, a long Y chromosome (Y>18), and a short Y chromosome group (Y18 group showed a significantly lower incidence rate of asthenozoospermia (31.03% vs 8.33%, P 18 and Y0.05).@*CONCLUSIONS@#Short Y chromosome may affect spermatogenesis, but the length of Y chromosome does not negatively influence the outcome of ART.

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

Animal models of autoimmune prostatitis and their evaluation criteria / 中华男科学杂志

Chronic prostatitis is a highly prevalent disease of unclear etiology. Researches show that autoimmune reaction is one cause of the problem. An effective animal model may help a lot to understand the pathogenesis and find proper diagnostic and therapeutic strategies of the disease. Currently used autoimmune prostatitis-related animal models include those of age-dependent spontaneous prostatitis, autoimmune regulator-dependent spontaneous prostatitis, self antigen-induced prostatitis, and steroid-induced prostatitis. Whether an animal model of autoimmune prostatitis is successfully established can be evaluated mainly from the five aspects: histology, morphology, specific antigens, inflammatory factors, and pain intensity.

Related factors of sperm DNA damage: Advances in studies / 中华男科学杂志

The detection of sperm DNA damage, as an important supplement to semen routine examination strategies, has been applied in some clinical andrology laboratories. What factors may lead to sperm DNA damage remains one of the concerns among many andrologists. Present studies show a variety of factors of sperm DNA damage, including age, environmental pollutants such as organophosphorus and organochloride pesticides, plasticizer, heavy metals such as lead, carcinogens such as polycyclic aromatic hydrocarbons (c-PAHs) and zearalenone (ZEA), male reproductive system diseases or systemic diseases such as varicocele, infection, tumor, spermatogenesis and maturation dysfunction, spinal cord injury and endocrine disorders, seasons and temperature, lifestyle, abstinence time, semen refrigeration, semen handling in vitro, and certain medications. Among them, spermatogenesis and sperm maturation dysfunction may be the most secretive factors, which are involved in the molecular mechanisms of sperm chromatin packaging and restructuring, such as the transformation of histone to protamine, single nucleotide polymorphism of genes, and the role of telomere, which may be one of the hotspots in the future studies of sperm DNA damage. Relevant researches in the future are expected to focus on the prevention of sperm DNA damage and clarification of its specific pathogenic mechanisms so as to provide some evidence for its treatment.

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>

Analysis of sperm morphology: yes or no? / 中华男科学杂志

The analysis of sperm morphology can be used to evaluate sperm fertilizing ability and spontaneous conception status, and especially the overall analysis of the sperm head, neck and tail, along with the patient's living habits, occupation and clinical manifestations, may contribute to the primary diagnosis of the patients potentia generandi. It can also be employed to assess the effects of the treatment of semen samples. Although oocyte fertilization can be achieved by the technologies of intracytoplasmic sperm injection (ICSI), motile sperm organelle morphology examination (MSOME) and intracytoplasmic morphologically selected sperm injection (IMSI) regardless of sperm morphology and / or motility, which may somewhat weaken the clinical application of sperm morphology analysis, the standardized procedure and the practice of quality control for the analysis of sperm morphology can significantly improve the accuracy of its results and largely promote its clinical application. Therefore, it is of positive necessity as well as clinical application value to perform sperm morphology analysis in andrology laboratories, reproductive centers, sperm banks and the department of laboratory medicine.

Effective English writing for tables and figures in andrological papers / 中华男科学杂志

Tables and figures play a critical role in enhancing the quality of an andrological paper. As visual elements, well-presented tables and figures can reduce the length of the manuscript and meanwhile efficiently capture a large amount of information by clear and concise display of detailed results and complex relationships, patterns and trends, offer explicit statistical or graphical support to important ideas, and add to the value of the whole paper. The effectiveness of a table or figure lies not only in its well-crafted structural design, but even more in its language presentation. This article presents some essential and practical guidelines to effective English writing of the title, body and footnotes of tables and figures in andrological papers.

Diagnosis and treatment of idiopathic semen quality abnormalities / 中华男科学杂志

Idiopathic semen quality abnormalities include idiopathic oligozoospermia, asthenospermia, teratospermia, azoospermia and idiopathic abnormal semen liquefaction. The possible causes of idiopathic semen quality abnormality include age, non-inflammatory function changes of subsidiary gonadal organs, infection, genetic abnormalities, sperm mitochondrial changes, effects of environmental pollutants, and subtle hormonal changes. The diagnosis of idiopathic oligozoospermia, asthenospermia, teratospermia and azoospermia require detailed inquiry of the case history, physical examination, semen analysis, determination of reproductive hormones, genetic and immunological examinations, and so on, to exclude possible known causes. The treatment of idiopathic oligozoospermia, asthenospermia, and teratospermia may involve the use of Western medicines, such as clomiphene citrate, tamoxifen, recombinant FSH, Andriol, compound zinc and selenium, L-carnitine, recombinant growth hormone and pentoxifylline, the application of traditional Chinese drugs, or the combination of traditional Chinese and Western medicine. Idiopathic azoospermia can be treated by assisted reproductive technology based on the medication of spermatogenesis-promoting drugs, and idiopathic abnormal semen liquefaction can be managed with traditional Chinese drugs, integrated traditional Chinese and Western medicine, or in vitro semen processing technology. With the development of diagnostic technology, it is expected that more specific therapeutic methods will be established for idiopathic semen quality abnormalities and their incidence will be reduced.

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>

Effects of semen analysis on human sperm movement parameters at different times after semen collection / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effects of the computer-assisted semen analysis (CASA) on human sperm movement parameters at different times after semen collection.</p><p><b>METHODS</b>Ninety-two semen samples with sperm density > or = 20 x 10(6)/ml and sperm liquefaction time < 20 min were placed in a incubation box at the temperature of 37 degrees C. Then the seminal parameters were analyzed with the computer-assisted semen analysis (CASA) system at 20, 30, 60 and 90 min after semen collection.</p><p><b>RESULTS</b>The percentages of grade a and b sperm were significantly lower at 30, 60 and 90 min than at 20 min (P < 0.05), so were the percentages of grade c sperm at 60 and 90 min than at 20 and 30 min (P < 0.05), but there were no significant differences in the percentage of grade c sperm between the 20-min and 30-min groups (P > 0.05). The percentages of grade a + b and a + b + c sperm were also significantly lower at 30, 60 and 90 min than at 20 min (P < 0.05). The beat cross frequency (BCF) was significantly higher at 30 min than at 20 min (P < 0.05), while the lateral head amplitude (ALH) significantly lower at 90 min than at 30 min (P < 0.05). The sperm wobbliness (WOB) was significantly higher while the curvilinear velocity (VCL) significantly lower at 90 min than at 20 and 30 min (P < 0.05). Straightness (STR) at 30, 60 and 90 min, and average path velocity (VAP) and straight line velocity (VSL) at 90 min were significantly lower than at 20 min (P < 0.05). There were no significant differences in sperm density, average motion degree (MAD) and linearity (LIN) among the four groups (P > 0.05).</p><p><b>CONCLUSION</b>The interval between semen collection and sperm routine analysis needs to be standardized. The results of this study suggest that sperm movement parameters of normal liquefied semen samples are relatively constant at 30 -60 min after semen collection.</p>

WHO Laboratory Manual for the Examination and Processing of Human Semen: its applicability to andrology laboratories in China / 中华男科学杂志

The 5th edition of WHO Laboratory Manual for the Examination and Processing of Human Semen (2010) represents a comprehensive revision. This article aims to explore the applicability of this manual to andrology laboratories in China mainland in view of sperm count analysis, sperm motility analysis, sperm morphology analysis, sperm function analysis, anti-sperm antibody and seminal plasma biochemical marker analysis, and quality assurance and quality control of semen analysis. The authors deem that its recommendation to the analysis method and lower reference limit of sperm concentration may be a little arbitrary and lack of evidence-based support, that the revised grading sperm motility, the strict criteria and the very low cut-off value of 4% morphologically normal spermatozoa for the evaluation of sperm morphology are not applicable to andrology laboratories in China mainland, that the sperm function markers need to be supplemented, and that the determination methods of anti-sperm antibody and seminal plasma biochemical markers are incompatible with the status of Chinese andrology laboratories. However, its recommended methods for quality assurance and quality control of semen analysis have a significant directive role in China mainland. It is worth to point out that the WHO manual ignored the data obtained from Chinese which accounts for approximate 20% of the world population. Thus, given the importance of the WHO manual, its general applicability should be evaluated in China.

How to write an andrological paper: standardization, mechanics and techniques / 中华男科学杂志

Andrological research papers not only reflect the current status and academic level of andrology, but also constitute an important communication platform for researchers and clinicians engaged in this field and contribute significantly to the development of andrology. It would be made easier to write a high-quality andrological paper once the author observes the basic requirements of research papers, adheres to the use of standard scientific terminology, knows the special writing mechanics, and equips himself with some essential writing techniques. Based on the long experience of editorship, we present a detailed introduction of the standardization, mechanics and techniques of writing an andrological paper.

Status quo of the researches on HSV vaccines / 中华男科学杂志

Herpes simplex virus (HSV) infection can cause severe recurrent disease in humans and establish lifelong latency in the host. Furthermore, it can significantly increase the risk of HIV infection and bring substantial psychosexual as well as physical morbidity to the patients. Several antiviral therapies are available for the control of its symptoms and spreading, but they can neither cure nor alter the nature of HSV infection. The development of an efficacious HSV vaccine is therefore necessitated for controlling the occurrence, transmission and recurrence of the infection. Those that have undergone clinical evaluation include subunit vaccines, attenuated live virus vaccines, replication defective virus vaccine, naked DNA vaccines and viral vector vaccines. Most of the above vaccine strategies have elicited protective immunity in animal models, but none has yet been effective in human beings. For all that, scholars have never stopped their exploration for an effective vaccine against the notorious virus. The authors present an overview of a few most promising candidate vaccines for the prevention and treatment of HSV infection.

Culture of HSV-2 and cloning of specific fragment of the gG-2 gene / 中华男科学杂志

<p><b>OBJECTIVE</b>To clone the glycoprotein G gene and its specific fragment with high conservation and antigenicity by culturing and amplifying herpes simplex virus type 2 and extracting its whole genome.</p><p><b>METHODS</b>We obtained a great deal of suspension with HSV-2 virus after infecting the cultured Hela cells with HSV-2 virus, extracted the whole genome of the virus by the phenol-chloroform method, and amplified the US4 gene coding gG-2 by PCR. Then we selected the specific target fragment according to the amino acid sequence alignment of the gG-2 gene and cloned it with the designed primers with restricted endonuclease sites.</p><p><b>RESULTS</b>We successfully obtained a lot of suspension with HSV-2 virus, and cloned the gG-2 gene from the whole genuine and its specific target fragment. Sequencing showed that both the sequences were identical with those printed in the GenBank.</p><p><b>CONCLUSION</b>It is feasible to obtain the virus genome and specific fragment of the gG-2 gene from virus-infected cells, especially for HSV-2 virus with relatively stable hereditary trait. It has prepared the ground for further constructing the expression plasmid of the specific fragment, expressing related proteins and identifying their antigenicity.</p>

Construction of plasmid expression vector for specific peptide of the rubella virus E1 gene / 中华男科学杂志

<p><b>OBJECTIVE</b>To construct a recombinant plasmid vector of the RV specific fragment for expressing the specific fragment of RV E1 protein.</p><p><b>METHODS</b>RNA of the RV attenuated live vaccine Wistar RA27/3 strain was extracted and reversely transcribed. The specific fragment of the E1 gene was amplified and the PCR products cloned in the vector pGEX-2T after purification. Positive clones were selected and identified by two-enzyme digestion and sequence analysis.</p><p><b>RESULTS</b>A 330 bp target fragment was successfully cloned, and the sequence of the recombinant plasmid was consistent with the original sequence.</p><p><b>CONCLUSION</b>Successful cloning of the RV El specific fragment and the construction of the recombinant plasmid have laid a foundation for further expressing the recombinant protein.</p>

Advances in molecular biology of rubella virus structural proteins / 中华男科学杂志

Rubella virus (RV), one of pathogens in TORCH syndrome, can lead to anisotropy of the fetus, and therefore it is of great significance to screen RV infection among women at child-bearing age. At the present, the screening of RV infection is mainly based on the ELISA method, the specificity of RV antigens is very important for ELISA tests. The antigenicity of RV is closely associated with its structural proteins, including capsid protein C, and envelope glycoproteins E1 and E2, which are important surface antigens of RV. This paper reviews the advances in the studies on the structure features, immunogenicity and recombinant proteins of the three structural proteins.