Semen parameters
Twenty-nine normozoospermic volunteers as controls and 51 asthenozoospermic patients were enrolled in this study for free fatty acid analysis using gas chromatography and a flame-ionization detector GC-FID. Semen samples of the asthenozoospermic patients were analyzed before and after oral antioxidant supplementation (vitamin E 400 IU/day + selenium 60 mg/day + folic acid 5 mg/day) for three months (ASTAntiOxSupp).
The demographic data, clinical features, and spermiogram parameters of all 80 participants are summarized in Table 1. The control (normozoospermic) and asthenozoospermic groups showed significant differences in age and BMI. The three groups did not differ in terms of abstention time, ejaculate volume, and pH. Significant differences were observed in terms of the sperm concentration, total sperm count, and motility between the normozoospermic, asthenozoospermic and ASTAntiOxSupp groups (P<0.0001). However, there were no significant changes in the semen parameters between groups 2 (asthenozoospermic before treatment) and group 3 (asthenozoospermic after treatment; ASTAntiOxSupp). Furthermore, normal sperm morphology in the three groups was in normal range and the differences were not significant.
Table 1. Demographic and spermiogram and pregnancy data of partners of the volunteers.
Variable
|
Group 1 (n=29)
|
Group 2
(n=51)
|
Group 3
(n=51)
|
1vs.2vs.3
|
1 vs. 2
|
1 vs. 3
|
2 vs. 3
|
Age (years)
|
39.4±5.6
|
36.1 ± 6.1
|
|
<0.05a
|
|
|
BMI (kg/m2)
|
28.5±3.8
|
26.7 ± 3.2
|
|
0.03
|
|
|
Abstention time (day)
|
4±2
|
3.4±1
|
3.6 ± 2
|
|
|
|
|
Semen Parameters
|
|
|
|
|
|
|
|
Volume (ml)
|
3.8±1.9
|
3.2 ± 1.2
|
3.3 ± 1.3
|
|
|
|
|
pH
|
7.5±0.1
|
7.5 ± 0.1
|
7.5 ± 0.1
|
|
|
|
|
Sperm concentration (106/ml)
|
45±2.9
|
32 ± 2.8
|
35 ± 2.4
|
0.0001b
|
0.0001
|
0.002
|
n.s.
|
Total sperm count
(106 /ejaculate)
|
170±5.5
|
96 ± 3.4
|
105 ± 3.1
|
0.0001
|
0.0004
|
0.003
|
n.s.
|
Progressive (%)
|
41±2
|
25±2
|
28 ± 2
|
0.0001
|
0.0001
|
0.0001
|
n.s.
|
Morphology (%)
|
5.8±0.4
|
4.1 ±0.4
|
4.3 ± 0.3
|
|
|
|
|
|
|
|
|
|
|
|
|
Pregnancy data
|
|
|
|
|
|
|
|
Total pregnancy
|
-
|
0/51
|
9/51
|
|
|
|
|
Clinical pregnancy rate
|
|
0/51
|
9/51
|
|
|
|
|
Miscarriage rate
|
|
0/51
|
3/9
|
|
|
|
|
The values are expressed as the means (±SEMs). Group 1, normozoospermic; Group 2, asthenozoospermic before antioxidant therapy; Group 3, asthenozoospermic after antioxidant therapy. (a) Student's t test was performed for two groups. (b) ANOVA and post hoc Tukey’s tests were performed for statistical analyses. BMI: body mass index.
Total antioxidant capacity
Figure 1 shows the TAC of the seminal plasma from normozoospermic, and asthenozoospermic patients before and after antioxidant therapy.
Figure 1. Total antioxidant capacity of seminal plasma following antioxidant supplementation. The values are expressed as the normalized means±SEMs. AST: asthenozoospermic, ASTAntiOxSupp: asthenozoospermic antioxidant supplement.
The concentration of TAC was significantly higher in the normozoospermic controls compared to asthenozoospermic patients before and after antioxidant therapy (P=0.0001). However, although the mean value of normalized TAC of the asthenozoospermic patients before the antioxidant treatment was slightly higher than after the treatment, there was no significant difference (P=0.4).
Fatty acid analysis
Preliminary injection of 28 fatty acids by GC-FID allowed the identification and quantification of 5 FFAs in the panel using 1 µL of seminal plasma. The most abundant identified FFAs in the seminal plasma were palmitic acid, vaccenic acid, eicosatrienoic acid, stearic acid, and myristoleic acid (Figure 2).
Figure 2. The pattern of palmitic acid (a), eicosatrienoic acid (b), myristoleic acid (c), stearic acid (d), and vaccenic acid (e) levels in normozoospermic patients (n=29) and following antioxidant therapy in asthenozoospermic patients (n=51). The values are expressed as the normalized mean (±SEM) area percentage. AST, asthenozoospermic; ASTAntiOxSupp, asthenozoospermic after antioxidant supplementation. * Significant difference between normozoospermia and AST. £ Significant difference between AST and ASTAntiOxSupp. † Significant difference between normozoospermia and ASTAntiOxSupp.
Figure 2a shows the levels of palmitic acid in the studied groups. Palmitic acid had a significantly (P=0.0001) lower concentration in SP of asthenozoospermic patients than in the controls. After antioxidant treatment its levels were significantly (P= 0.0001) restored to near-control levels.
Figure 2b shows that the level of eicosatrienoic acid is significantly lower in asthenozoospermic patients before (P=0.0001) and after (P=0.01) antioxidant therapy compared to normozoospermic subjects. Additionally, after antioxidant therapy, the level of eicosatrienoic acid increased. However, this increase is not significant (P=0.2).
Figure 2c shows the level of myristoleic acid in the studied groups. The myristoleic acid level is higher in asthenozoospermic men than in normozoospermic samples. Following oral antioxidant supplementation, the levels of myristoleic acid decreased to levels between those of normozoospermic and asthenozoospermic men. Yet, these changes are not statistically significant.
Figures 2d and 2e show the levels of stearic acid and vaccenic acid in the normozoospermic and asthenozoospermic before and after antioxidant supplementation. Levels of both fatty acids are lower in asthenozoospermic men compared to normozoospermic subjects. After antioxidant treatment, the levels of these fatty acids returned to values comparable to near-control levels. However, these changes are not statistically significant.
Correlations of semen parameters with FFA levels
In this study, we conducted a correlation analysis between semen parameters, including concentration, count, motility, and morphology, and identified FFAs levels. There were no statistically significant correlations with sperm parameters and identified FFAs.
Clinical outcome
As indicated in Table 1, over the six-month follow-up period post-antioxidant treatment, 9 out of 51 female partners achieved pregnancy (pregnancy rate = 17.6%), in contrast to the pre-treatment phase (0/51).