[Establishment of a method for detection of thiols in seminal plasma and its application in patients with hyperuricemia].

OBJECTIVE: To establish a method of reversed-phase ultra-performance liquid chromatography with fluorescence (UPLC-FL) detection for simultaneous determination of homocysteine (Hcy), cysteine (Cys), cysteine glycine (CysGly) and glutathione (GSH) and analysis of the contents of the four thiols in the seminal plasma of normal men and patients with hyperuricemia (HUA). METHODS: Seminal plasma samples were collected from 30 normal sperm donors and 30 HUA patients and reduced with tris (2-carboxyethyl) phosphine hydrochloride. Then, the samples were subjected to protein precipitation with trichloroacetic acid solution, derivative reaction with 7-fluorobenzofuran-4-sulfate and isolation with a C18 column, with 0.025 mol/L KH2PO4 (pH 3.0) for mobile phase A and pure methanol for B, followed by equal gradient elution with an excitation wavelength of 380 nm and an emission wavelength of 510 nm. RESULTS: The correlation coefficients (R2) of the four thiols all exceeded 0.999, with an average recovery rate of 94.23－107.87%. Compared with the normal sperm donors, the HUA patients showed significantly increased contents of Cys (ï¼»108.01 ± 48.03ï¼½ vs ï¼»250.10 ± 55.87ï¼½ µmol/L, P < 0.05), Hcy (ï¼»113.97 ± 6.32ï¼½ vs ï¼»48.35 ± 15.07ï¼½ µmol/L, P < 0.05), and GSH (ï¼»3.15 ± 1.48ï¼½ vs ï¼»4.63 ± 1.17ï¼½ µmol/L, P < 0.05), but a decreased level of CysGly (ï¼»12.79 ± 3.18ï¼½ vs ï¼»5.94 ± 0.99ï¼½ µmol/L, P < 0.05). CONCLUSIONS: The method of reversed-phase UPLC-FL detection established in this study has made it possible simultaneous detection of Hcy, Cys, CysGly and GSH in the seminal plasma, which is applicable to laboratory research and clinical routine examination. Patients with hyperuricemia may incur oxidative damage to the reproductive system.

[Ultra-performance liquid chromatography for determination of the contents of biogenic amines in the seminal plasma].

OBJECTIVE: To establish an ultra-performance liquid chromatography (UPLC) method for determination of the contents of the three biogenic amines putacine, spermidine and spermine in human seminal plasma. METHODS: Seminal plasma samples were extracted with 5% trichloroacetic acid and processed by pre-column derivatization with dansyl chloride. Chromatographic separation was performed with a C18 (2.1×50 mm,1.7 µm) chromatographic column using water and acetonitrile for mobile-phase gradient elution at a flow rate of 0.3 ml/min, a detection wavelength of 245 nm, a column temperature of 35℃ and an injection volume was 3.0 µl. The contents of putacine, spermidine and spermine in the seminal plasma of 52 healthy sperm donors (the normal group) and 23 azoospermia patients (the AS group) were measured, and their correlation with routine semen parameters were analyzed. RESULTS: The three biogenic amines showed a good linearity (r ≥ 0.999), with a lower detection limit of 0.03－0.08 µg/ml. The relative standard deviation (RSD) of precision was ≤ 0.72% and the average recovery rate was 79.74%－108.87%. The normal group, compared with the AS patients, showed significantly higher contents of putrescine (ï¼»8.19 ± 7.85ï¼½ vs ï¼»2.43 ± 1.38ï¼½ mg/ml, P < 0.05), spermidine (ï¼»77.30 ± 32.58ï¼½ vs ï¼»31.99 ± 16.21ï¼½ mg/ml, P < 0.05) and spermine (ï¼»246.44 ± 83.99ï¼½ vs ï¼»166.15 ± 79.28ï¼½ mg/ml, P < 0.05). However, the contents of the three biogenic amines in the seminal plasma exhibited no significant correlation with the routine semen parameters in the normal group. CONCLUSIONS: The ultra-performance liquid chromatography method we established, with the advantages of high sensitivity and reproducibility and short peak-time, can quickly and accurately determine the contents of biogenic amines in the seminal plasma.

[Correlation of seminal plasma biochemical indexes with routine semen parameters in sperm donors].

OBJECTIVE: To investigate the relationship of seminal plasma biochemical indexes with routine semen parameters and that between seminal plasma biochemical indexes. METHODS: Using the automatic biochemical analyzer, we measured the contents of neutral α-glucosidase (NAG), fructose hexokinase (Fru) , citric acid (CA), acid phosphatase (ACP), (zinc) Zn, uric acid (UA), lactase dehydrogenase (LDH) and α-hydroxybutyrate dehydrogenase (α-HBDH) in the seminal plasma of 84 sperm donors in the Human Sperm Bank of Hebei Province. We analyzed the correlation between these indexes and that between routine semen parameters and these indexes. RESULTS: Sperm concentration and total sperm count were correlated positively with the contents of seminal plasma NAG, ACP, Zn, CA, LDH and α-HBDH (P<0.05) but negatively with Fru (P<0.05), the percentage of progressively motile sperm positively with seminal plasma Zn (P<0.05), and CA positively with NAG, Zn, LDH, α-HBDH and ACP (P<0.01) but negatively with Fru (P<0.01), NAG positively with Zn, LDH, α-HBDH and ACP (P<0.05) but Fru negatively with ACP (P<0.01), Zn positively with LDH, α-HBDH and ACP (P<0.01), and LDH positively with α-HBDH and ACP (P<0.01) but UA negatively with ACP (P<0.05). CONCLUSIONS: Biochemical indexes in the seminal plasma of healthy men are not only closely related to each other, but also to some routine semen parameters.

[Correlation of reproductive hormone levels and seminal plasma oxidative stress with semen quality in obese males].

OBJECTIVE: To investigate the correlation of the levels of reproductive hormones and oxidative stress in the seminal plasma with semen parameters in obese males. METHODS: Based on the body mass index (BMI), we divided 138 infertile men into three groups: normal (BMI <24 kg/m2, n = 48), overweight (24 kg/m2≤BMI<28 kg/m2, n = 47), and obesity (BMI ≥28 kg/m2, n = 43). We determined the concentrations of follicle-stimulating hormone (FSH), luteotropic hormone (LH), prolactin (PRL), testosterone (T) and estradiol (E2) in the serum by electrochemiluminescence and measured the levels of superoxide dismutase (SOD), glutathione-S-transferases (GSTs), reactive oxygen species (ROS) and malondialdehyde (MDA) in the seminal plasma by ELISA, compared the above indexes among the three groups, and analyzed their correlation with the semen volume, sperm concentration, total sperm count, and percentage of progressively motile sperm (PMS). RESULTS: The semen volume was significantly lower in the obesity than in the normal group (ï¼»2.63 ± 0.74ï¼½ vs ï¼»3.37 ± 1.00ï¼½ ml, P < 0.05), and so was the percentage of PMS in the overweight and even lower in the obesity than in the normal group (ï¼»47.91 ± 12.89ï¼½ and ï¼»41.27 ± 15.77ï¼½ vs ï¼»54.04 ± 13.29ï¼½%, P < 0.05). Compared with the normal group, both the overweight and obesity groups showed markedly decreased levels of serum T (ï¼»4.83 ± 1.42ï¼½ vs ï¼»3.71 ± 1.22ï¼½ and ï¼»3.49 ± 1.12ï¼½ ng/ml, P<0.05), T/LH ratio (1.53 ± 0.57 vs 1.19 ± 0.54 and 0.97 ± 0.51, P<0.05), SOD (ï¼»112.05 ± 10.54ï¼½ vs ï¼»105.85 ± 6.93ï¼½ and ï¼»99.33 ± 8.39ï¼½ U/ml, P<0.05), and GSTs (ï¼»31.75±6.03ï¼½ vs ï¼»29.54±5.78ï¼½ and ï¼»29.02±4.52ï¼½ U/L, P<0.05), but remarkably increased seminal plasma ROS (ï¼»549.93±82.41ï¼½ vs ï¼»620.61±96.13ï¼½ and ï¼»701.47±110.60ï¼½ IU/ml, P<0.05) and MDA (ï¼»7.46 ± 2.13ï¼½ vs ï¼»8.72 ± 1.89ï¼½ and ï¼»10.47 ± 2.10ï¼½ nmol/L, P<0.05). BMI was correlated positively with ROS and MDA, but negatively with the semen volume, PMS, T, T/LH, SOD and GSTs (P<0.05); LH negatively with sperm concentration, total sperm count and GSTs (P<0.05); PRL negatively GSTs (P<0.05); E2 positively with SOD (P<0.05); T positively with SOD (P<0.05) but negatively with MDA (P<0.05); T/LH positively with PMS and SOD (P<0.05) but negatively with ROS and MDA (P<0.05); SOD positively with semen volume, PMS and GSTs (P<0.05) but negatively with ROS and MDA (P<0.05); GSTs negatively with sperm concentration; total sperm count and MDA (P<0.05); ROS positively with MDA (P<0.01) but negatively with PMS (P<0.05); and MDA negatively with semen volume (P<0.05). Multivariate logistic regression analysis showed that the independent factors influencing the semen volume were BMI and GSTs, those influencing the total sperm count were BMI and T, and those influencing PMS were BMI and MDA. CONCLUSIONS: Increased BMI induces changes in the levels of male reproductive hormones and seminal plasma oxidative stress and affects semen quality, which may be associated with male infertility.

[Correlation of insulin resistance with male reproductive hormone levels and semen parameters].

OBJECTIVE: To investigate the influence of insulin resistance on male reproductive hormones and semen quality. METHODS: Using the electrochemiluminescence method, we measured the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), estradiol (E2) and testosterone (T) in the serum of 83 infertile males. We detected the levels of fasting plasma glucose (FPG) and fasting insulin (FINS) and calculated the insulin resistance index presented as homeostasis model assessment of insulin resistance (HOMA-IR). Based on HOMA-IR, we divided the patients into three tertile groups, T1 (HOMA-IR 0.36－0.55, n = 27), T2 (HOMA-IR 0.56－0.80, n = 28) and T3 (HOMA-IR 0.81－1.97, n = 28), obtained their semen parameters by computer-assisted semen analysis (CASA) and analyzed the correlation of HOMA-IR with male reproductive hormone levels and semen parameters. RESULTS: With the elevation of HOMA-IR, the patients of the T1, T2 and T3 groups showed significant decreases in the serum T level (ï¼»14,26 ± 4.27ï¼½ vs ï¼»14.75 ± 5.00ï¼½ vs ï¼»11.62 ± 3.68ï¼½ nmol/L, P <0.05) and the percentage of progressively motile sperm (PMS) (ï¼»51.04 ± 15.10ï¼½% vs ï¼»48.04 ± 16.24ï¼½% vs ï¼»37.84 ± 18.23ï¼½%, P <0.05). HOMA-IR was correlated negatively with the serum T level (r = －0.333, P = 0.002), semen volume (r = －0.23, P = 0.029) and PMS (r = －0.27, P = 0.015), and so was FINS with the serum T level (r = －0.327, P = 0.003) and PMS (r = －0.315, P = 0.004), while the semen volume was correlated positively with the levels of serum T (r = 0.221, P = 0.048) and FSH (r = 0.222, P = 0.047). Multivariate linear regression analysis showed that HOMA-IR was an independent influencing factor for PMS and the body mass index (BMI) was that for the semen volume and total sperm count. CONCLUSIONS: Insulin resistance may reduce semen quality by changing the levels of male reproductive hormones.

[Correlation of seminal plasma zinc alpha-2 glycoprotein with semen quality in obese males].

OBJECTIVE: To investigate the relationship between seminal plasma zinc alpha-2 glycoprotein (ZAG) and semen quality in obese males. METHODS: This study included 130 obese male patients with idiopathic infertility Based on the concentration of seminal plasma ZAG, we divided the patients into three tertile groups: tertile 1 (T1, 73.45－97.15 µg/ml, n = 43), T2 (97.16－115.46 µg/ml, n = 44), and T3 (115.47－220.11 µg/ml, n = 43). We measured the concentrations of seminal plasma zinc (SPZ) and ZAG of the patients by ELISA, obtained the semen parameters, and analyzed the correlation of semen quality with the levels of SPZ and ZAG and the influence of obesity on SPZ, ZAG and semen quality. RESULTS: The mean level of seminal plasma ZAG in the 130 obese male patients was (111.29 ± 26.50) µg/ml. There were statistically significant differences in sperm concentration and total sperm count among the three tertile groups (P < 0.05). The level of seminal plasma ZAG was correlated negatively with the body mass index (BMI), waist circumference (WC), sperm concentration and sperm count (P < 0.01), that of SPZ positively with BMI and WC (P < 0.05) but negatively with semen volume and the percentage of progressively motile sperm (P < 0.05). The level of serum ZAG, however, exhibited no correlation with SPZ, seminal plasma ZAG or semen quality. Obesity was found to be associated with significantly decreased concentration of seminal plasma ZAG and percentage of progressively motile sperm but remarkably increased level of SPZ (P < 0.05). CONCLUSIONS: Obesity may induce the metabolic disorder of SPZ and ZAG, change the microenvironment of seminal plasma, and consequently affect semen quality.

[Correlation of the levels of seminal plasma homocysteine, folate and cobalamin with semen parameters in obese men].

OBJECTIVE: To investigate the correlation of seminal plasma homocysteine (Hcy) and its metabolic factors folate (FA) and cobalamin (VB12) with semen quality in obese men. METHODS: We randomly selected 83 male patients with idiopathic infertility for this study and, according to the body mass index (BMI), divided them into a normal BMI (n = 28), an overweight (n = 28) and an obesity group (n = 27). We determined the levels of Hcy, FA and VB12 in the seminal plasma by ELISA and analyzed their correlation with the semen parameters of the patients in different groups. RESULTS: Compared with the normal BMI group, the obese males showed significant decreases in the semen volume (ï¼»3.23 ± 0.86ï¼½ vs ï¼»2.58 ± 0.77ï¼½ ml, P < 0.05), total sperm count (ï¼»191.35 ± 103.00ï¼½ vs ï¼»121.55 ± 88.08ï¼½ ×106, P < 0.05), percentage of progressively motile sperm (ï¼»52.88 ± 15.58ï¼½ % vs ï¼»38.97 ± 16.52ï¼½ %, P < 0.05), seminal plasma Hcy (ï¼»7.41 ± 1.29ï¼½ vs ï¼»6.62 ± 0.85ï¼½ µmol/L, P < 0.05), and VB12 (ï¼»282.41 ± 30.38ï¼½ vs ï¼»230.07 ± 37.75ï¼½ pmol/L, P < 0.05). There were no statistically significant differences in the semen parameters between the overweight group and the normal BMI or the obese group (P > 0.05), or in sperm concentration or the FA level among the three groups of patients (P > 0.05). The levels of seminal plasma Hcy and VB12 were correlated positively with the semen volume (r = 0.281 and 0.242, P < 0.05) and total sperm count (r = 0.229 and 0.258, P < 0.05) but negatively with BMI (r = －0.293 and －0.238, P < 0.05). No correlation, however, was found either between sperm concentration and the percentage of progressively motile sperm (P > 0.05) or between the FA level and BMI or semen parameters (P > 0.05). CONCLUSIONS: The levels of seminal plasma Hcy and VB12 are correlated with BMI, semen volume and total sperm count, which suggests that the concentrations of seminal plasma Hcy and VB12 may be associated with the fertility of obese men.

[Hyperuricemia reduces semen parameters in infertile men].

OBJECTIVE: To explore the impact of hyperuricemia (HUA) on semen quality in infertile men. METHODS: Based on the level of fasting serum uric acid (SUA ≥420 µmol/L), 402 infertile men were divided into a normal SUA group (n = 304) and an HUA group (n = 98), and also into four age groups: 20－24, 25－29, 30－34, and 35－44 years old. Semen parameters were obtained from the patients by computer-assisted semen analysis and the levels of SUA determined by chemical colorimetry. RESULTS: The mean semen volume was significantly lower in the HUA than in the normal SUA group (2.40 vs 2.80 ml, P < 0.05), and so was the total sperm count (120.51 vs 187.21 ×106, P < 0.05). The mean semen volume was remarkably lower in the 25－29 and 30－34 years old patients with HUA than in those with normal SUA (2.40 and 2.55 ml vs 2.85 and 2.90 ml, P < 0.05), and so was the total sperm count in the 30－34 years old patients with HUA than in those with normal SUA (109.69 vs 192.91 ×106, P < 0.05). Correlation analysis showed that the level of SUA was negatively correlated with the semen volume (r = －0.193, P < 0.05) and total sperm count of the patients (r = －0.163, P < 0.05). CONCLUSIONS: Hyperuricemia significantly reduces the semen volume and total sperm count of infertile men, and the level of serum uric acid is closely related with semen quality.

[Metabonomics in the diagnosis and treatment of male infertility].

Metabonomics is an emerging branch of science for the study of endogenous small molecule metabolites in organisms, which plays an important role in evaluatingthediagnosis and treatment of male infertility by exploring the metabolites of body fluids, cells and tissues. With its advantages ofmass information, noninvasiveness, and celerity, metabonomics will be widely applied to clinical researches in the future. This review introducesmetabonomics and its analytical techniques and data processing procedures,its latest application in the studies of the etiology, diagnosis and the treatment of male infertility, and the prospect of its future application in the researches of male reproduction.

[Correlation of semen parameters with inflammatory factors in the seminal plasma of obese males].

OBJECTIVE: To investigate the influence of inflammatory factors on semen parameters in the seminal plasma of obese men. METHODS: Based on the body mass index (BMI), 171 males were divided into a normal group (BMI < 24 kg/m2, n = 59), an overweight group (24 ≤ BMI < 28 kg/m2, n = 54), and an obesity group (BMI =≥ 28 kg/m2, n = 58). The routine semen parameters of the subjects were obtained by computer-assisted semen analysis, the levels of TNF-α, IL-6 and VEGF in the seminal plasma were measured by ELISA, and the correlation of BMI with the above indexes was analyzed. RESULTS: Sperm concentration was significantly decreased in the obesity group in comparison with the normal and overweight groups (ï¼»40.19 ± 24.05ï¼½ vs ï¼»66.54 ± 34.81ï¼½ and ï¼»57.73 ± 24.61ï¼½ ×106/ml, P <0.01), and so was the total number of sperm (ï¼»110.22 ± 75.44ï¼½ vs ï¼»200.75 ± 102.66ï¼½ and ï¼»157.46 ± 112.89ï¼½ ×106, P <0.01) and the percentage of progressively motile sperm (PMS) (ï¼»30.80 ± 15.56ï¼½ vs ï¼»50.75 ± 10.17ï¼½ and ï¼»39.71 ± 9.73ï¼½%, P <0.01). The levels of TNF-α and IL-6 in the seminal plasma were markedly elevated in the obesity group as compared with the normal and overweight groups (ï¼»76.90 ± 14.64ï¼½ vs ï¼»64.47 ± 11.92ï¼½ and ï¼»69.74 ± 12.32ï¼½ pg/ml, P <0.05; ï¼»54.17 ± 17.81ï¼½ vs ï¼»39.26 ± 9.09ï¼½ and ï¼»46.25 ± 13.66ï¼½ pg/ml, P <0.01), while that of VEGF remarkably reduced in the former group in comparison with the latter two (ï¼»154.24 ± 30.23ï¼½ vs ï¼»199.23 ± 36.28ï¼½ and ï¼»181.57 ± 34.41ï¼½ pg/ml, P <0.01). The levels of TNF-α, IL-6, and VEGF were significantly correlated with BMI (r = 0.254, 0.321 and －0.407, P <0.01), those of TNF-α and IL-6 negatively with the percentage of PMS (r =－0.163, P <0.05; r = －0.333, P <0.01). There was a positive correlation between TNF-α and IL-6 (r = 0.468, P <0.01), a negative correlation between IL-6 and VEGF (r = 0.177, P <0.05), but no correlation between TNF-α and VEGF (r = 0.058, P >0.05). CONCLUSIONS: The levels of TNF-α and IL-6 are increased and that of VEGF decreased in the seminal plasma of obese males, which may affect the semen quality.

[Correlation of serum zinc alpha 2 glycoprotein with blood lipid and reproductive hormone levels in men].

OBJECTIVE: To study the changes of the serum zinc alpha 2 glycoprotein (ZAG) level in men and its relationship with blood lipid male reproductive hormones. METHODS: We enrolled 297 men aged 25－ 65 years in this study, 152 with hyperlipemia (HL) and the other 145 with normal blood lipid (normal control). We divided them into four age groups (25－35 yr, 36－45 yr, 46－55 yr, and 56－65 yr) and three tertile groups (Q1, Q2, and Q3) according to the tertiles of the serum ZAG level, and examined their blood lipid, blood glucose, serum ZAG, and reproductive hormones. RESULTS: The serum ZAG level was decreased gradually with the increase of age in both the HL patients and normal controls, significantly in the 36－45 and 56－65 yr age groups (P <0.05), and markedly lower in the HL than in the control men in the 25－35 and 36－45 yr groups (P <0.05). The levels of follicle-stimulating hormone (FSH) and total testosterone (TT) changed significantly with the ZAG level. The level of serum ZAG was correlated negatively with age (r = －0.58, P<0.05), waist circumference (r = －0.21, P <0.05), body mass index (BMI) (r = －0.22, P <0.05), fasting blood glucose (r = －0.16, P <0.05) , and triglyceride (TG) (r = －0.27, P <0.05) but positively with TT (r = 0.36, P <0.05). Age, BMI and TG were independent factors influencing the serum ZAG level. CONCLUSIONS: The serum ZAG level is decreased with the increase of age and associated with lipid metabolism, abdominal obesity, and reproductive hormone levels in males.