Seminal Plasma Markers Research Articles

P10-24 The impact of zinc oxide nanoparticles on various biochemical markers in seminal plasma

P10-24 The impact of zinc oxide nanoparticles on various biochemical markers in seminal plasma

Correlation between seminal plasma biochemical markers and semen parameters in idiopathic oligoasthenoteratospermia: identification of biomarkers for L-carnitine therapy.

L-carnitine therapy for idiopathic sperm abnormalities exhibits variable effectiveness, and currently, there are no established criteria to predict patient response. This study investigated correlations between seminal plasma markers and semen parameters to identify biomarkers that can guide indications for L-carnitine therapy indications in patients with idiopathic sperm abnormalities. A retrospective review was conducted on 223 male patients with idiopathic oligoasthenoteratospermia, who sought medical attention at our clinic between January 2020 and October 2022. These patients underwent a pretreatment seminal plasma biochemical analysis, followed by a three-month continuous L-carnitine treatment. The correlation between seminal plasma biochemical parameters and pretreatment semen parameters was analyzed. Semen quality was compared between cases with normal and abnormal seminal plasma biochemical parameters, both pretreatment and posttreatment. The correlation between the changes in semen parameters after treatment and seminal plasma biochemical parameters were investigated. Correlation analyses revealed significant associations between all pretreatment semen parameters and seminal plasma biochemical markers, except for liquefying time and the ratio of normal morphology. Subgroup analysis, stratified by seminal fructose, zinc, citric acid, and neutral glycosidase levels, demonstrated that abnormal groups exhibited significantly different levels of semen parameters compared with the normal groups. The changing difference and changing ratio in the ratio of forward motile sperm showed a negative correlation with seminal fructose levels (r=-0.165 and -0.144). The changing difference in semen volume was negatively correlated with the level of seminal neutral glycosidase (r=-0.158). The changing ratio in semen volume, sperm concentration, total sperm count, and count of forward motile sperm all exhibited negative correlations with the levels of seminal neutral glycosidase (range from -0.178 to -0.224). Seminal plasma biochemical markers, particularly fructose and neutral glycosidase, may serve as valuable indicators for determining the eligibility of patients with idiopathic sperm abnormalities for L-carnitine therapy.

Analyzing and Examining Smoking Effects and Oxidative Stress Markers in Seminal Plasma in Iraqi Infertility Men

The oxidative stress (OS) and sperm DNA fragmentation have been associated with sub-infertility in males and infertility due to a lack of means of protection and defense (antioxidants) for spermatozoa and an increase in reactive oxygen spices (ROS) related to wrong lifestyle (cigarettes and hookah) or what also known as Shisha. The study included (90) men between the ages of (23-40) years, (60), infertile men who smoked cigarettes and hookahs. They were divided into two groups, the first oligozoospermia and the second asthenozoospermia, according to the guidelines of the World Health Organization for seminal fluid analysis (SFA), 5 th edition, WHO(2010). Each group included (30) smoking infertile males, with a control group included (30) non-smoking fertile males. The study aimed to estimate the concentration level of Ascorbic Acid (AA) in the seminal plasma of two groups of infertile men smoking cigarettes and hookahs using enzyme-linked immunosorbent assay (ELISA) method, with calculating the concentration of Malondialdehyde (MDA) as well as estimating the percentage of sperm DNA fragmentation (SDF) using sperm chromatin dispersion (SCD), Halo view. Compared with healthy fertile men of non-smokers of cigarettes and hookahs as one of the biomarkers of oxidative stress in infertile men in Nineveh Governorate-Iraq. The results indicated that there were significant differences at the probability level (p<0.01) for the studied parameters (AA, MDA, SDF) compared to the control group (fertile men). The results showed an increase in the percentage of sperm DNA fragmentation with increasing the concentration of MDA in seminal plasma and decreasing the concentration of AA. Cigarette and hookah smoking increased the generation of ROS with a decrease in the water-soluble non-enzymatic antioxidant Ascorbic acid (AA) and thus increased the oxidative processes of the plasma membranes of spermatozoa and the occurrence of oxidative stress and impaired fertility and caused infertility. Keywords: male infertility, oxidative stress, sperm DNA fragmentation, Ascorbic acid, MDA. Bionatura http://dx.doi.org/10.21931/RB/CSS/2023

A Comparative Analysis of the Altered Levels of Human Seminal Plasma Constituents as Contributing Factors in Different Types of Male Infertility.

(1) Background: The relationships between the biochemical and immunological components in seminal plasma and their physiological effects on male reproductive system have been underreported. In this study, we evaluated the potential of several seminal plasma biochemical and immunological markers in the pathophysiological developments of the infertile male patients. The study was designed to identify and assess different markers that may be associated with semen functions in different types of male infertility. (2) Methods: A total of 50 infertile male patients who underwent checkup for fertility assessment and 50 fertile controls were included in this study. The complete medical history of each recruited participant was reviewed. The infertile sub-groups (non-obstructive azoospermia (NOA), asthenozoospermia (AS), normozoospermic infertile (NI), and oligozoospermia (OZ)) were characterized based on sperm motility and concentration, while NI patients were included after a thorough check up of their female partners as well. We investigated each sample for 21 different analytes, enzymes, trace elements, and immunological markers to find crucial markers posing as contributing factors to a specific type of male infertility. (3) Results: The levels of 15 out of 21 markers, assayed from the seminal plasma of infertile males, were significantly altered in comparison to fertile controls (p < 0.05). For the first time, microprotein levels were also analyzed. The presence of monocytes, lymphocytes, and granulocytes was limited to semen from NOA patients, while a significant increase in the level of platelets was observed in AS. Hierarchical clustering and ROC-AUC analysis identified the three most significant markers (zinc, LDH, and TG) for the healthy control group and asthenozoospermic group (AUC, of 0.92 and 0.81, respectively). (4) Conclusions: The altered levels of biochemical and immunological markers in seminal plasma might be associated with the different male infertility profiles and could be required for the sperm metabolism and maintenance. However, a larger sample size and follow up analysis is required for establishing the hypothesized panel of markers as biomarkers at clinical stage.

Stable bull fertility protein markers in seminal plasma

Bull fertility is an important trait in breeding as the semen of one bull can, potentially, be used to perform thousands of inseminations. The high number of inseminations needed to obtain reliable measures from Non-Return Rates to oestrus creates difficulties in assessing fertility accurately. Improving molecular knowledge of seminal properties may provide ways to facilitate selection of bulls with good semen quality. In this study, liquid chromatography mass spectrometry (LC-MS/MS) was used to analyze the protein content from the seminal plasma of 20 bulls with Non-Return Rates between 35 and 60%, sampled across three seasons. Overall, 1343 proteins were identified and proteins with consistent correlation to fertility across multiple seasons found. From these, nine protein groups had a significant Pearson correlation (p < 0.1) with fertility in all three seasons and 34 protein groups had a similar correlation in at least two seasons. Among notable proteins showing a high and consistent correlation across seasons were Osteopontin, a lipase (LIPA) and N-acetylglucosamine-1phosphotransferase subunit gamma. Three proteins were combined in a multiple linear regression to predict fertility (r = 0.81). These sets of proteins represent potential markers, which could be used by the breeding industry to phenotype bull fertility. SignificanceThe ability of bull spermatozoa to fertilize oocytes is crucial for breeding efficiency. However, the reliability of this trait from field measures is relatively low and the prediction of fertility given by conventional methods to evaluate sperm quality is currently not very accurate. In this work, we identify sets of proteins in bull seminal plasma from repeated samples collected at different times of the year that correlate to fertility in a consistent way. We combined these individual proteins to build a molecular signature predictive of fertility. This study provides an overview of proteins linked to fertility in seminal plasma, thereby increasing knowledge of the bull seminal plasma proteome. Protein signatures from the latter, potentially related to fertility, may be of use to predict fertility for individual bulls.

Effect of Heat Stress on Sperm Production, Oxidative Markers and their Association in Native Breeding Bulls

The objective of the study was to evaluate the effect of climatic conditions on fortnightly semen quality and its oxidative markers, and to evaluate the correlation between them in zebu cattle and buffalo breeding bulls under weekly twice semen collection schedule. The macro- (observatory; daily; n=330) and micro- (bull shed; hourly; n=8540) climatic parameters, viz., ambient temperature (AT °C) and relative humidity (RH %) were measured during Jan to Dec 2018 and temperature humidity index (THI) was generated. There were high associations between micro and macro AT (r=0.982), RH (r=0.897) and THI (r=0.985). Overall, micro-AT was 1.20°C higher, micro-RH was 5.42% lower and the micro-THI was 0.50 higher than the corresponding macro-climatic parameters. Therefore effect of micro-climatic AT, RH and THI was evaluated on biweekly collected 26 ejaculates of three bulls each of Surti, Murrah buffalo and Gir cattle breeds. The macro and microscopic parameters of semen and oxidative markers in seminal plasma, viz., catalase, lipid peroxidation (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined. During the day 72.6% of hours, bulls experienced stress (THI &gt; 72). No impact of season was observed on seminal attributes and oxidative markers of cattle and buffalo bulls, however correlation coefficients were achieved. The results of our study warrants further investigation on more number of bulls including their semen freezability and fertility.

Oxidative stress markers in seminal plasma of idiopathic infertile men may be associated with glutathione S-transferase M1 and T1 null genotypes.

This study aimed to investigate the association between glutathione S-transferase (GST) M1 and T1 null genotypes and thiobarbituric acid reactive substances (TBARS), total antioxidant capacity (TAC) and nitric oxide (NO) levels in male infertility. For this purpose, semen samples were collected from fertile and infertile subjects, and then they were genotyped for GSTT1 and GSTM1 genes using multiplex-PCR. The TBARS, TAC and NO levels in seminal plasma were then measured via the ferric-reducing ability of plasma (FRAP). A significant association was observed between GSTT1 null genotype and oligozoospermia, asthenozoospermia and teratozoospermia. But, the GSTM1 null genotype was merely associated with teratozoospermia. Moreover, the GSTT1-/GSTM1+ combined genotype was associated with all subgroups of male infertility. Besides, an association was observed between GSTT1-/GSTM1- genotype and asthenozoospermia and teratozoospermia. Further analysis showed that the GSTT1 null genotype was associated with increased NO in asthenozoospermia. Also, the GSTT1 null genotype was associated with increased TBARS in oligozoospermia and asthenozoospermia. As well, GSTM1 null genotype was associated with decreased TAC and increased NO in asthenozoospermia respectively. As a preliminary conclusion, the GSTM1 and GSTT1 null genotypes could be considered as genetic risk factors for male infertility, interfering with some oxidative stress markers in infertile men.

Correlations Amongst Functional and Morphological Attributes and Oxidative Markers of Fresh and Cryopreserved Semen of Gir and Murrah Bulls

This study was undertaken during the winter season on healthy mature Gir cattle and Murrah buffalo bulls (n=3 each). The semen samples (6 ejaculates/bull, total 36 ejaculates) collected in the morning using artificial vagina were evaluated for routine seminal attributes, including acrosomal and plasma membrane integrity. The samples were then diluted @ 100 million sperm/ml with tris fructose yolk glycerol extender without and with sericin @ 0.1, 0.25, 0.5 and 1.0% (w/v), filled in French mini-straws, and frozen in LN2 using biofreezer as per standard freezing protocol. Straws were thawed in water bath at 37°C for 30 sec and evaluated for post-thaw quality, viz., motility, viability, morphology, acrosome integrity and plasma membrane integrity (HOST). Lipid peroxidation (malondialdehyde - MDA production) and activities of enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) were assessed as oxidative markers in seminal plasma of freshly diluted and frozen-thawed semen samples. Sericn at 0.5% level significantly (p less than 0.01) improved the post-thaw sperm quality with reduced oxidative stress in both the species. The breed-wise correlation coefficients (r) among sperm quality attributes and oxidative markers were studied in fresh and frozen-thawed semen of each species, and also for fresh with frozen-thawed semen. The findings revealed significant interrelationships amongst most of the attributes of fresh as well as post-thawed semen and also of fresh semen attributes with those of cryopreserved semen including oxidative markers in both the species. Sperm motility estimation in fresh, pre-freeze and post-thawed semen was a legitimately good indicator of quality of spermatozoa at various steps of semen processing/freezing, and its fertilizing potential. Thus, the sperm motility, HOS test and either MDA or SOD/GPx activity alone may be used as valuable and practical tools for routine assessment of bovine semen quality considering significant correlations found between them.

Influence of Antioxidant Sericin in Tris Extender on Oxidative Markers during Cryopreservation (–196ºC) of Bovine Semen

This investigation was carried out during winter season on the semen of three mature, healthy breeding bulls each of Gir cattle and Murrah buffalo breeds. The aim was to assess the effect of different concentration of antioxidant Sericin (0.0, 0.1, 0.25, 0.50 and 1.0%, w/v) in standard Tris fructose egg yolk glycerol (TFYG) extender on cryopreservability of bovine semen based on sperm motility and oxidative markers in seminal plasma of freshly diluted and cryopreserved semen. The mean sperm motility observed in freshly diluted and frozen-thawed Gir bull semen, irrespective of Sericin levels, were 76.93±0.39 and 43.47 ± 0.58 % and in Murrah bulls 78.20±0.38 and 44.10 ± 0.48 %, respectively. The values of malondialdehyde (MDA, μmol/ml) in seminal plasma of freshly diluted and frozen-thawed semen of Gir bulls, irrespective of Sericin levels, were 21.68±0.38 and 24.99 ± 0.56, and in Murrah bulls 21.49±0.57 and 25.60±0.94, respectively. The corresponding values of superoxide dismutase (SOD, U/ml) were 1.77 ± 0.06 and 1.37 ± 0.05 in Gir and 1.18 ± 0.06 and 0.85 ± 0.04 in Murrah bulls, and those of glutathione peroxidase (GPx, nmol/min/ml) 417.10 ± 12.00 and 349.76 ±11.92 in Gir and 385.71±9.21 and 320.02±9.49 in Murrah bull semen. Sperm motility and activities of all three enzymes differed highly significantly (plessthan00.01) between stages. SOD was significantly (plessthan00.05) lower in buffalo than cattle semen. Inclusion of 0.5% and/or 0.25% Sericin in TFYG extender gave better protection to spermatozoa over other levels against ROS mediated injuries as the MDA production was significantly reduced with increased sperm motility and higher levels of SOD and GPx enzymes in the seminal plasma.

Proteomic analysis of seminal plasma from bilateral varicocele patients indicates an oxidative state and increased inflammatory response.

Seminal plasma is a rich source of proteins and serves as an ideal sample for proteomic analysis of male infertility. In varicocele-associated infertility, the contributory role of seminal plasma proteins specific to unilateral and bilateral varicocele is not clear. Furthermore, there is a lack of specific protein biomarker to differentiate bilateral from unilateral varicocele. The main objective is to identify the differentially regulated molecular and cellular pathways in bilateral varicocele. Furthermore, we intend to identify seminal plasma biomarkers to differentiate bilateral and unilateral varicocele patients in comparison with fertile healthy men. Global proteomic analysis of seminal plasma proteins has identified the functionality of differentially expressed proteins (DEPs) in varicocele patients. Bioinformatic analysis has revealed response to reactive oxygen species and oxidative stress, and tissue homeostasis as top process pathways that are affected in bilateral varicocele patients compared to fertile healthy men. In comparison with unilateral varicocele patients, inflammatory response pathways were dysregulated, especially interleukin 6 (IL-6) signaling and Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathways, in bilateral varicocele patients, owing to the involvement of underexpressed DEPs. Key DEPs associated with oxidative stress (peroxiredoxin 2; PRDX2), DNA fragmentation (fatty acid synthase; FASN), and inflammatory response (fibronectin 1; FN1) validated by western blot analysis revealed differential expression of these proteins in unilateral and bilateral varicocele groups. Altered expression of DEPs and its association with key processes show that the seminal plasma homeostasis is compromised in bilateral varicocele patients. Furthermore, we propose PRDX2, FASN, and FN1 as potential noninvasive seminal plasma markers for the differentiation of unilateral and bilateral varicocele patients.

Total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs.

The aim of this study was to evaluate the total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs. The study was conducted on 10 infertile and 10 fertile dogs of various breeds. Infertility was defined as conception failure at least three matings with different bitches. Semen was collected by manual manipulation. The sperm concentration and motility parameters were evaluated using CASA Hamilton Thorne, Vers. IVOS 12.3. The morphology of spermatozoa and the percentage of live and dead sperm cells were assessed microscopically, total antioxidant capacity and the content of SH-groups in seminal plasma were determined spectrophotometrically, the contents of protein peroxidation markers in seminal plasma, bityrosine and formylokinurenine, were determined using spectrofluorimetric methods. Sperm concentration and total sperm count were significantly (p<0.05) lower in infertile dogs than in fertile dogs (99.92±3 0.05×106 /ml vs. 282.07±48.27×106 /ml; 214.19±114.74×106 vs. 747.57±210.94×106 , respectively). The percentage of spermatozoa with normal morphology and the most determined motility parameters differed significantly (p<0.05) between both groups. The mean values of total antioxidant capacity in the seminal plasma were significantly (p<0.05) lower (19.95±20.94 vs. 25.66±23.18µmol/g protein), whereas the mean contents of bityrosine and formylokinurenine in seminal plasma were significantly (p<0.05) higher in infertile dogs than in fertile dogs (3.71±4.83µg/mg protein vs. 1.55±2.00µg/mg protein and 0.37±0.45µg/mg protein vs. 0.14±0.08µg/mg protein, respectively). In conclusion, the obtained results suggest that the poor semen quality and infertility in dogs could be associated with lowered total antioxidant capacity and increased protein peroxidation in seminal plasma as a consequence of oxidative stress.

In-depth quantitative proteome analysis of seminal plasma from men with oligoasthenozoospermia and normozoospermia.

Can seminal plasma markers for oligoasthenozoospermia be identified by comparison of the human seminal plasma proteome in men with oligoasthenozoospermia and normozoospermia? An in-depth quantitative proteome analysis was conducted using a high-throughput method named isobaric tag for relative and absolute quantification. A total of 734 seminal plasma proteins were quantified by mass spectrometry. Compared with the seminal plasma from men with normozoospermia, 22 upregulated proteins and 20 downregulated proteins were identified in the oligoasthenozoospermic seminal plasma. These differential seminal plasma proteins were involved in various physiological processes, including metabolism, transport, antioxidation and immune response. The confidence of some proteome data was further verified by western blot of (prostate-specific antigen [KLK3], lactotransferrin [LTF], alpha-1-antitrypsin [SERPINA1] and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). Additionally, 38% of the seminal plasma proteins identified in this study have not been reported in previously published studies on seminal plasma proteome, and 53% of our seminal plasma proteins were shared with published studies on human plasma proteome. Our seminal plasma proteome research provides new complementary high-confidence data, and also enhances understanding of the pathogenic mechanisms in oligoasthenozoospermia.

THE STUDY OF THE LEVELS OF METALLOPROTEINASES, CYTOKINES AND LYMPHOCYTE ACTIVATION MARKERS IN SEMINAL PLASMA OF MEN, DEPENDING ON FERTILITY

The study of the concentration of metalloproteinases, pro- and anti-inflammatory cytokines, lymphocyte activation markers in seminal plasma of men with oligosymptomatic forms of chronic inflammation of the urogenital tract (CIUT), depending on the fertility rate are presented in the article. Quantitative characteristics in male sperm with different forms of pathospermia were studied for matrix metalloproteinases (MMP) -2 and MMP-9 and their inhibitors - tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2. It was shown, that during chronic inflammation of the urogenital tract of men are shifting levels of cytokine profile, reducing the concentration of metalloproteinase-2, chemokines - fractalkine and regulated by activation, expression and secretion of normal T-cells (RANTES), sharp increase in IL-8, MCP-1 and elevation of the CD25+ / CD95+, indicating that the disturbance of apoptosis of pathological forms generative cells and their accumulation in the sperm. The ratio of immunological indices IL-2/IL-4, IL-10/IL-12 was calculated and a significant increase in the IL-10/IL-12 index was noted in the group of individuals with elevated levels of hypercapitated form of sperm, and a reduced proportion of this ratio was observed in the microsomatic morphology of sperm. Prolonged inflammation in the genital area accompanied by depletion of the local immune system, resulting in the development of infertility. Immunocorrection therapy for men with CIUT should take into account the peculiarities of changes in local immunity and be differentiated depending on the prevalence of certain pathological forms of sperm and changes in the cytokine profile of the seminal plasma.

Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics

Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization–time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility.

Вплив імунокорекції на запальний процес урогенітального тракту у чоловіків з безпліддям

We studied the concentration of metalloproteinases, pro-and anti-inflammatory cytokines, lymphocyte activation markers in seminal plasma of men with oligosymptomatic forms of chronic inflammation of the urogenital tract complicated with infertility. It was shown that chronic inflammation of the urogenital tract shifts the levels of cytokine profile, reduces the concentration of metalloproteinase-2 (576.4 +/- 89.3 ng/ml, P < 0.001), chemokines - fractalkine (16.3 +/- 1.3 pg/ml, P < 0.001) and chemmmokine regulated by activation, expression and secretion of normal T-cells (RANTES (12.7 +/- 1.0 pg/ ml, P < 0.001). Additionally, we observed a sharp increase in IL-8 (215.5 +/- 7.8 pg/ml, P < 0.01), MCP-1 (926.4 +/- 30.2 pg/ml, P < 0.001) and elevation of the CD25 + / CD95 + ratio. These observations point for alterations in apoptotic mechanisms in pathological forms of the generative cells and their accumulation in the sperm. Prolonged inflammation of the genital area is accompanied by depletion of the local immune system resulting in the development of infertility. The multidirectional shifts in IL-10/IL-12 index have been established: an increase in the group of patients with elevated level of hypercapitated spermatozoa and its reduction in microsomatic morphotype of spermatozoa. These data emphasize the importance of the microenvironment in maturation of gametes. It was shown that an inclusion of alfagin and gepatomax in immunorehabilitation of men with oligosymptomatic forms of urogenital infections improves leukocyte subpopulation content of sperm and concentrations of pro-and anti-inflammatory cytokines, and therefore, results in an increase in fertilizing potential.

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability

The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80–85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0–5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9–7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8–4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability.

MRNA profiling for the identification of sperm and seminal plasma

mRNA profiling is a promising new method for the identification of body fluids from biological stains. In this study we aimed to establish a multiplex RT-PCR protocol for the detection and differentiation of sperm and seminal plasma. The Agilent Bioanalyzer and Nanodrop spectrophotometer were shown not to be suitable for assessing RNA quality and quantity of forensic stains. Semen specificity of the mRNA markers was successfully confirmed with singleplex PCR. Our data indicated that semen samples down to 0.1 μl and up to 20-year-old could be identified with mRNA profiling. With the semen multiplex, including 2 sperm markers (PRM1, PRM2) and 2 novel seminal plasma markers (SEMG1, PSA), samples from azoospermic men (absence of sperm in semen) are clearly distinguishable from those of normozoospermic men (having a normal sperm production). We think that our multiplex RT-PCR protocol is a reliable and sensitive method for the identification of semen in forensic samples.

Evaluation of seminal plasma parameters in patients with chronic prostatitis or leukocytospermia.

Though detailed cytological and microbiological diagnostic procedures are routinely carried out in male genital tract infection, the correct diagnosis and localization of inflammation or infection is often difficult. In this prospective study, the relevance of the seminal plasma markers PMN elastase, complement C3, CRP, fructose, PSP 94, PSA, and alpha-glucosidase was investigated in 13 patients with chronic prostatitis, 31 patients with significant leukocytospermia, and 58 patients with non-inflammatory diseases (controls). Statistically relevant results were obtained for PMN elastase when comparing chronic prostatitis with controls, leukocytospermia with controls (P < 0.001) and chronic prostatitis with leukocytospermia (P < 0.05); for complement C3 chronic prostatitis and leukocytospermia vs. controls (P < 0.05) and for fructose/ejaculate leukocytospermia vs. controls (P < 0.05). No statistically relevant differences were found for C-reactive protein, alpha-glucosidase, PSA and prostatic secretory protein (PSP 94). To delimit genital tract inflammation from non-inflammatory patients, cutpoint levels for PMN elastase of 230 ng ml-1 and for C3c of 0.01 g l-1 were suggested. PMN elastase was shown to possess the strongest discriminating power. The assessment of a cutpoint for fructose to indicate seminal vesicle dysfunction is not possible as the significance level is weak (P < 0.05).

Preliminary investigations on the standardization and quality control for the determination of acid phosphatase activity in seminal plasma

Background The determination of ACP in seminal plasma was considered as an appropriate biochemical marker to evaluate prostate function, as recommended by the WHO manual. However, few reports on the standardization and quality control for the determination of biochemical markers in seminal plasma have been documented. Methods Two frozen samples of seminal plasma with or without phenylmethylsulfonyl fluoride were determined for their acid phosphatase (ACP) levels. The ACP level and sperm concentration of each of 72 samples of seminal plasma obtained at 1000× g for 10 min or 3000× g for 15 min centrifugation were assayed. ACP activity in 10 samples of seminal plasma was measured immediately or standing for 30 min after dilution. The ACP levels in seminal plasma with or without chymotrypsin were also assayed. Results There was no significant difference of ACP levels ( P = 0.166) but of sperm concentrations ( P = 0.000) in seminal plasma obtained by centrifugation at different velocity. ACP activities in seminal plasma measured when standing for 30 min after dilution were significantly lower than those measured immediately after dilution ( P = 0.001). Both chymotrypsin and freezing–thawing had no apparent effect on the determination of ACP in seminal plasma. Conclusion The results indicated that standing time after dilution and centrifugation velocity should be standardized, and frozen seminal plasma could serve as the quality control products for the determination of ACP activity among different laboratories.

Seminal plasma characteristics as indicators of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in men with obstructive azoospermia

Objective: To determine the prognostic value of seminal plasma volume, pH, fructose, and α-glucosidase for the detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations. Design: Retrospective data analysis. Setting: University infertility clinic (referral center). Patient(s): Fifty-nine men with obstructive azoospermia. Intervention(s): Semen analysis including seminal plasma volume, pH, fructose, α-glucosidase, molecular genetic diagnosis of CFTR mutations and FSH measurement. Main Outcome Measure(s): Sensitivity and specificity of seminal plasma markers for the detection of CFTR mutations. Result(s): A CFTR mutation was detected in 26 of 59 patients with obstructive azoospermia. Patients carrying a mutation had significantly lower seminal plasma volume (mean ± SEM: 1.5 ± 1.4 mL vs. 2.8 ± 2.2 mL), lower pH levels (25th percentile, median, 75th percentile: 6.5, 6.8, 7.5 vs. 7.7, 7.9, 7.9) and lower fructose content (1.0, 1.1, 3.7 vs. 5.8, 20.0, 83.0 μmol/ejaculate) than those without mutations. Diagnostic efficacy for detection of mutations was best (pH 81.4%, fructose 81.8%) at a cutoff level for pH of 7.4 and fructose of 2 μmol/ejaculate. Conclusion(s): Seminal plasma markers provide an effective, noninvasive method to predict CFTR mutations in men with obstructive azoospermia.