Abstract
Male infertility caused by idiopathic oligoasthenospermia (OAT) is known as idiopathic male infertility. Glutathione S-transferase (GST) and fluoride may play important roles in idiopathic male infertility, but their effects are still unknown. Our study examined the relationship between GST polymorphisms and fluoride-induced toxicity in idiopathic male infertility and determined the underlying mechanism. Sperm, blood, and urine samples were collected from 560 males. Fluoride levels were measured by a highly selective electrode method, and GST genotypes were identified using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Semen parameters, DNA fragmentation index (DFI), mitochondrial membrane potential (MMP), and oxidative stress (OS) biomarkers were statistically assessed at the P < 0.05 level. Compared with healthy fertile group, semen parameters, fluoride levels, OS biomarkers, sex hormone levels, and MMP and DFI levels were lower in the idiopathic male infertility group. For glutathione S-transferase M1 (GSTM1[-]) and glutathione S-transferase T1 (GSTT1[-]) or glutathione S-transferase P1 (GSTP1) mutant genotypes, levels of semen fluoride, OS, MMP, and DFI were considerably higher, and the mean levels of sperm parameters and testosterone were statistically significant in GSTM1(+), GSTT1(+), and GSTP1 wild-type genotypes. Both semen and blood fluoride levels were associated with oxidative stress in idiopathic male infertility patients. Elevated fluoride in semen with the genotypes listed above was linked to reproductive quality in idiopathic male infertility patients. In conclusion, GST polymorphisms and fluorine may have an indicative relationship between reproductive quality and sex hormone levels, and OS participates in the development of idiopathic male infertility.
Keywords: fluorine, glutathione S-transferase, idiopathic male infertility, oxidative stress, polymorphism
INTRODUCTION
According to the World Health Organization (WHO), male infertility is defined when a couple has had at least 12 months of regular and unprotected sexual intercourse and the female partner is unable to conceive due to male partner factors.1 Male infertility has been a global health issue for many years, affecting 8%–12% of childbearing couples worldwide.2 Recent research has shown that the quantity and quality of sperm have decreased significantly globally over the last 20 years.3 Male infertility is increasingly being recognized as a substantial psychological and social distress throughout the world, resulting in a significant economic burden on healthcare systems.4
Some etiological factors influencing male infertility have been explored, including urogenital tract infections, ductal blockage or malfunction, endocrine disorders, immunological factors, varicocele, idiopathic semen anomaly, and some genetic abnormalities.5 However, 30%–40% of cases of male infertility are considered idiopathic, and the etiology of male infertility remains unknown.6 In general, early diagnosis and investigation of unknown factors are crucial for a broad understanding of the causes of male infertility.
Our research has shown that glutathione S-transferase (GST) may play an important role in idiopathic male infertility, and we continue to focus on genetic and environmental predictors. The GST supergene family comprises related isozymes thought to be responsible for electrophile detoxification via glutathione conjugation with a wide range of chemical structures, including endogenous reactive oxygen species and exogenous polycyclic aromatic hydrocarbons.7 The enzyme activity of human cytosolic glutathione S-transferase M1 (GSTM1) and glutathione S-transferase T1 (GSTT1) is reduced by two homozygous deletions.8 Glutathione S-transferase P1 (GSTP1) variants can be stratified as the wild-type (A/A), homozygous mutation genotype (G/G), and heterozygous mutation genotype (A/G). GST polymorphisms may interfere with essential metabolic processes associated with oxidative stress (OS), leading to an increase in the rate of structural and functional sperm damage.9,10 It has also been noted that identification of certain genetic variants is an important pathogenic factor.11 Fluoride is a naturally existing contaminant that is widely distributed in the environment. Fluorosis caused by coal-burning pollution is common in Guizhou Province of southwest China. Fluoride has also been shown to affect reproductive function and male reproductive endocrine changes in mice.12 Excessive fluoride intake over time may harm various organs and tissues in the human body, particularly the male reproductive system.12,13 However, GST gene polymorphisms and fluoride-induced male reproductive issues have not yet been explored.
Therefore, this study aimed to explore interaction between GST polymorphisms and fluoride in idiopathic male infertility in an effort to provide better patient management and eliminate regional adverse factors. The relationship between GST polymorphisms and individual fluoride level as well as the underlying mechanisms for idiopathic male infertility was investigated in this study.
PATIENTS AND METHODS
Study population
This study protocol and informed consent form were approved by The Affiliated Hospital of Guizhou Medical University (Guiyang, China; approval No. 2013023). Written and verbal informed consent was obtained from all participants before participation in the study. From March 2017 to June 2020, 596 unrelated men were recruited; 36 patients withdrew from the study, and the remaining 560 patients completed the study, including 345 patients with idiopathic male infertility and 215 fertile volunteers. The WHO’s 5th edition criterion was used to identify eligible men who matched the included patients.14 Semen parameters were evaluated in at least two different analyses to identify abnormal semen. The primary inclusion criterion for the healthy fertile male participants was a childbirth history in the past 1 year; their indicators, such as sex hormone levels and semen parameters, were all within normal limits. The exclusion criteria for all participants were as follows: (1) abnormal sexual function; (2) chromosome abnormality; (3) ejaculation disorder; (4) hypogonadism; (5) varicocele; (6) use of immunosuppressants or cytotoxic drugs; (7) serious primary disease, metabolic disease, or mental disease; (8) long-term smoking (a pack of cigarettes per day for more than 20 years) or alcoholism (one drink per day for more than 10 years); or (9) unhealthy sexual partner(s). The same exclusion criteria were applied to the healthy men as to the patients with idiopathic male infertility.
Sample collection and analysis
According to the WHO’s recommendation of 5–7 days of sexual abstinence, all semen specimens were voluntarily obtained by masturbation in our quiet room, involving ejaculation into a collection vessel. The precise collection time was noted, and the semen specimens were subsequently incubated at 37°C for liquefaction. Sperm parameters were measured by computer-assisted semen analysis (CASA; WLJY-9000, Weili New Century, Beijing, China). The ejaculate volume, sperm concentration, mass activity, motility percentage, viability, and morphology of spermatozoa were all analyzed. Peripheral venous blood (6 ml) was drawn from all patients, of which 3 ml was mixed with ethylenediaminetetraacetic acid (EDTA) anticoagulant and stored at −80°C as a reserve. The other 3 ml of blood was placed in a rapid coagulating tube and centrifuged for 10 min at 1000g (TGL-16 GB, Anting Medical Equipment Co., Ltd., Shanghai, China) to separate the plasma and blood cells, which were then frozen at −80°C for future use. The AxyPrep genomic DNA miniprep kit (Axygen Biosciences, Union City, CA, USA) was used to extract genomic DNA from the blood according to the manufacturer’s instructions. Urine samples with particular collection time were labeled and maintained at room temperature. Fluoride levels were quantified using the highly selective electrode method once sufficient samples had been obtained.
Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay
The TUNEL assay was used to investigate DNA damage (Roche Diagnostics GmbH, Mannheim, Germany). The test and negative control samples were fixed with 4% paraformaldehyde for 30 min at room temperature and washed once with phosphate buffer solution (PBS). The suspension was mixed with 0.1% Triton X-100 and placed in an ice bath for 2 min. The spermatozoa were then stained with 3, 3-diaminobenzidine (DAB; Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence intensity and the proportion of TUNEL-positive sperm were used to determine DNA fragmentation index (DFI) levels.
Mitochondrial membrane potential assay
Mitochondrial function in spermatozoa specimens was assessed using JC-1 (Sigma‒Aldrich Chemical Co., St. Louis, MO, USA) staining and flow cytometry (CytoFLEX, Beckman Coulter, Indianapolis, IN, USA). JC-1 exhibits red fluorescence at a high mitochondrial membrane potential (MMP), which indicates normal mitochondrial functionality and energy status. Green fluorescence is seen at a low MMP.
Measurement of malondialdehyde (MDA), total antioxidant capacity (TAC), nitric oxide (NO), and 8-hydroxydesoxyguanosine (8-OHdG)
Commercial assay kits (Jiancheng, Nanjing, China) were used to identify MDA, NO, and TAC in seminal plasma. The liquefied sperm samples were centrifuged for 5 min at 12 000g at room temperature (Thermo Fisher Scientific, Osterode, Germany). The semen was separated and the seminal plasma was extracted, and seminal plasma MDA, NO, and TAC levels were measured as optical density by spectroscopic analysis (BioTek Epoch Company, Winooski, VT, USA) according to the manufacturer’s protocols. An enzyme-linked immunosorbent method was used to measure the concentrations of 8-OHdG in seminal plasma (ELISA kit, Thermo Fisher Scientific, Waltman, MA, USA). The color reaction product was detected by an ELISA reader when exposed to 450 nm laser light.
Measurement of serum sex hormones
All participants were advised not to take hormone medicines for 72 days and to attend to the measurement visit with an empty stomach. Blood samples were collected form all patients, and the levels of sex hormones in the serum were measured within 1 h. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were measured and assessed based on the manufacturer’s recommendations.
GST gene polymorphisms
GSTM1, GSTT1, and GSTP1 genotypes were identified by multiplex polymerase chain reaction (PCR) using published primer sequences as follows: GSTM1 gene, 5′-GAACTCCCTGAAAAGCTAAAG C-3′ (forward) and 5′-GTTGGGCTCAAATATACGGTGG-3′ (reverse); GSTT1 gene, 5′-TTCCTTACTGGTCCTCACATCTC-3′ (forward) and 5′-TCACCGGATCATGGCCAGCA-3′ (reverse). The GSTP1 genotype was identified by PCR-restriction fragment length polymorphism (RFLP) to detect three common mutations. The GSTP1 gene primer sequences were as follows: 5′-ACCCCAGGGCTCTATGGGAA-3′ (forward) and 5′-TGAGGGCACAAGAACCCCT-3′ (reverse). β-actin was selected as an internal control in our study; the forward primer was 5′-ACTCCCCATCCCAAGACC-3′, and the reverse primer was 5′-CCTTAATGTCACGCACGAT-3′. Agarose gel electrophoresis was used to detect the amplification products: GSTM1 219 base pairs (bp) and GSTT1 480 bp. Homozygous Ile-Ile (A/A) patients showed a single 177-bp fragment, and homozygous Val-Val (G/G) patients showed two fragments: 85 bp and 92 bp; heterozygous Ile-Val (A/G) individuals showed three fragments: 85 bp, 92 bp, and 177 bp.
Statistical analyses
Statistical analyses were performed with SPSS 24.0 software (SPSS Inc., Chicago, IL, USA). Some variables were expressed as the median and interquartile range. For continuous variables, the data were reported as the mean ± standard deviation. When the data followed a normal distribution, an independent t-test with two-tailed values was employed to compare two categorical variables. A nonparametric test (Wilcoxon rank test) was performed when the data were nonnormally distributed. The spearman test was applied to analyze the correlation. Differences in frequencies of GST genotypes between the two groups were checked using the Chi-squared test. Multivariable analyses were performed and corrected to control for confounders. In all cases, P < 0.05 was considered statistically significant.
This study was registered in the Chinese Clinical Trial Registry Center (Chi CTR-IPR-14005580). All data supporting the conclusion of this article are included.
RESULTS
The detailed characteristics of all participants enrolled in our study are shown in Table 1. In terms of demographic characteristics, there was no substantial difference between the two groups. The frequency of the GSTM1 null genotype was 39.1% in the idiopathic male infertility group and 59.1% in the healthy fertile group. Infertile men had a GSTT1 null genotype frequency of 47.4% and healthy fertile men had a frequency of 58.8%. A significant difference between the two groups was found. In addition, the frequency of the GSTP1 A/G + G/G genotype was 37.2% in the healthy fertile group and 35.1% in the infertile group.
Table 1.
Distribution of glutathione S-transferase genotypes among 560 patients in Guizhou province from 2017 to 2020
| GST genotypes | Idiopathic man infertility group (total=215), n (%) | Healthy fertile group (total=345), n (%) | χ2 | P | OR (95% CI) |
|---|---|---|---|---|---|
| GSTM1(+) | 131 (60.9) | 141 (40.9) | 21.34 | <0.001 | 2.256 (1.593–3.196) |
| GSTM1(-) | 84 (39.1) | 204 (59.1) | |||
| GSTT1(+) | 113 (52.6) | 142 (41.1) | 6.94 | <0.001 | 1.584 (1.124–2.232) |
| GSTT1(-) | 102 (47.4) | 203 (58.8) | |||
| GSTP1 | |||||
| A/A | 135 (62.8) | 224 (64.9) | 0.263 | 0.608 | 0.912 (0.640–1.299) |
| A/G + G/G | 80 (37.2) | 121 (35.1) |
(+): present genotype; (-): null genotype. GST: glutathione S-transferase; GSTM1: glutathione S-transferase M1; GSTT1: glutathione S-transferase T1; GSTP1: glutathione S-transferase P1; OR: odds ratio; CI: confidence interval; A/A: wild-type genotype; G/G: homozygous mutation genotype; A/G: heterozygous mutation genotype
Table 2 illustrates the clinical parameters of the two studied groups. The difference between the idiopathic male infertility group and the healthy fertile group in terms of clinical demographic features such as age and body mass index (BMI) was not significant. Comparison of sperm parameters revealed that sperm concentration, motility, and morphological integrity were significantly higher in the healthy fertile group than those in the idiopathic male infertility group, though semen volume did not show a statistically significant difference based on the independent t-test (all P < 0.05). A statistical trend for higher semen and blood fluoride levels was found in the idiopathic infertility patients (both P < 0.05), but the mean values of urine fluoride appeared to be unaffected. Overall, an increase in OS biomarkers was detected in the healthy fertile group. These results, such as 8-OHdG, NO, TAC, and MDA (Table 2), indicated that the difference between the two groups was significant (all P < 0.05). Further analysis of the data revealed significantly lower T levels in the idiopathic infertile group (P < 0.05), while there was no significant difference in the mean values of follicle-stimulating hormone (FSH) and LH between the two groups. The mean MMP values were also considerably lower in the idiopathic male infertility group, and mean DFI values differed significantly as well (both P < 0.05).
Table 2.
Clinical parameters related to the idiopathic male infertility group and healthy fertile group
| Clinical parameter | Idiopathic male infertility group (n=215) | Healthy fertile group (n=345) | P |
|---|---|---|---|
| Age (year) | 28.1±1.5 | 28.0±1.6 | 0.302 |
| BMI (kg m−2) | 22.53±0.60 | 22.47±0.54 | 0.221 |
| Semen parameters | |||
| Semen volume (ml) | 2.98±0.67 | 3.03±0.66 | 0.386 |
| Sperm concentration (×106 ml−1) | 16.55±5.42 | 72.81±13.48 | <0.01 |
| Sperm motility (%) | 37.86±12.64 | 64.31±6.23 | <0.01 |
| Sperm morphology (%) | 6.78±3.39 | 19.78±4.54 | <0.01 |
| Fluoride levels (mg l−1) | |||
| Semen fluoride | 0.78±0.24 | 0.18±0.12 | <0.01 |
| Blood fluoride | 1.21±0.42 | 0.24±0.16 | <0.01 |
| Urine fluoride | 0.65±0.38 | 0.28±0.14 | 0.124 |
| OS biomarkers | |||
| 8-OHdG (pg ml−1) | 368.66±110.19 | 284.12±94.92 | <0.01 |
| NO (nmol ml−1) | 31.08±6.07 | 34.92±8.01 | <0.01 |
| TAC (mmol l−1) | 1.51±0.32 | 1.82±0.41 | <0.01 |
| MDA (nmol ml−1) | 23.48±5.38 | 21.03±4.76 | <0.01 |
| Sex hormone levels | |||
| FSH (IU l−1) | 8.54±0.64 | 8.48±0.66 | 0.290 |
| T (nmol l−1) | 13.72±3.43 | 15.01±2.98 | <0.01 |
| LH (IU l−1) | 7.29±0.31 | 7.34±0.30 | 0.059 |
| MMP (%) | 24.18±3.28 | 40.96±5.64 | <0.01 |
| DFI (%) | 30.92±3.88 | 24.06±3.68 | <0.01 |
P<0.05 was considered statistically significant. The data are reported as mean±s.d. BMI: body mass index; OS: oxidative stress; 8-OHdG: 8-hydroxydesoxyguanosine; NO: nitric oxide; TAC: total antioxidant capacity; FSH: follicle-stimulating hormone; LH: luteinizing hormone; T: testosterone; MMP: mitochondrial membrane potential; DFI: DNA fragmentation index; MDA: malondialdehyde; s.d.: standard deviation
The results obtained for clinical parameters associated with GSTM1, GSTT1, and GSTP1 genotypes for the men with idiopathic infertility are presented in Table 3. The mean fluoride values in GSTM1 and GSTT1 null genotype groups were significantly higher than those in the idiopathic infertility patients, and the mean fluoride values also showed a significant difference between the wild-type group (A/A) and the mutation group (A/G + G/G) with all P < 0.05. No significant difference in sperm volumes was found when comparing the GSTM1 and GSTT1 genotype groups with the wild-type group. In terms of the other three semen parameters, a significant difference was detected (all P < 0.05). The present genotype group and wild-type group had significantly higher sperm concentration and motility than the null genotype or mutation genotype group (all P < 0.05). OS indicators, in particular, revealed that the GSTM1 and GSTT1 present genotype groups differed significantly from the two null genotype groups. For idiopathic male infertility, the mean levels of 8-OHdG, NO, TAC, and MDA in the wild-type group were significantly higher compared with the mutation group (all P < 0.05). Furthermore, an independent t-test found significant differences in mean T and LH levels in males with the GSTM1 present genotype for idiopathic infertility (both P < 0.05). The mean FSH and T levels in the GSTT1 present genotype, on the other hand, were significant (both P < 0.05). A significant difference in T and LH levels was also observed (both P < 0.05), but no significant difference in FSH was found compared to the mutant genotype group. Based on the MMP and DFI data in Table 3, the GSTM1 and GSTT1 present genotype groups had significantly higher levels than the GSTM1 and GSTT1 null genotype groups (both P < 0.05). The mean values of MMP and DFI in the wild-type group were significantly higher than those in the mutation group (both P < 0.05).
Table 3.
Clinical parameters of GSTM1, GSTT1 and GSTP1 genotypes for idiopathic infertility patients
| Genotype | Fluoride | Semen parameters | |||||||
|---|---|---|---|---|---|---|---|---|---|
|
|
|
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| Semen (µg l−1) | Blood (µg l−1) | Urine (µg l−1) | Semen volume (ml) | Sperm concentration (×106 ml−1) | Sperm motility (%) | Sperm morphology (%) | |||
| GSTM1(+) (n=131) | 0.32±0.09* | 0.67±0.26* | 0.59±0.21* | 3.56±0.37 | 14.23±1.39* | 36.11±3.46* | 8.82±1.28* | ||
| GSTM1(-) (n=84) | 0.65±0.08 | 0.96±0.28 | 0.82±0.24 | 3.61±0.41 | 9.52±0.79 | 19.52±1.87 | 6.43±1.47 | ||
| GSTT1(+) (n=113) | 0.36±0.08* | 0.73±0.23* | 0.62±0.19* | 3.82±0.64 | 17.38±2.13* | 35.24±4.82* | 8.06±1.32* | ||
| GSTT1(-) (n=102) | 0.71±0.10 | 1.02±0.21 | 0.85±0.18 | 3.78±0.60 | 11.30±1.62 | 17.66±2.74 | 6.10±1.14 | ||
| GSTP1 A/A (n=135) | 0.42±0.18* | 0.69±0.41* | 0.49±0.19* | 3.61±0.52 | 14.96±2.35* | 30.74±11.35* | 5.47±1.27* | ||
| GSTP1 A/G+G/G (n=80) | 0.92±0.21 | 0.93±0.32 | 0.47±0.21 | 3.42±0.49 | 9.89±2.02 | 15.28±5.48 | 4.85±1.06 | ||
|
| |||||||||
| Genotype | OS biomarkers | Sex hormone levels | MMP (%) | DFI (%) | |||||
|
|
|
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| 8-OHdG (ng ml−1) | NO (nmol ml−1) | TAC (µmol l−1) | MDA (nmol ml−1) | FSH (IU l−1) | T (nmol l−1) | LH (IU l−1) | |||
|
| |||||||||
| GSTM1(+) (n=131) | 298.66±60.19* | 35.74±5.88* | 1.76±0.30* | 22.48±3.56* | 8.54±0.64 | 15.24±2.92* | 7.65±0.79* | 28.86±4.11* | 25.12±4.88* |
| GSTM1(-) (n=84) | 400.92±86.23 | 30.26±5.12 | 1.39±0.28 | 29.96±3.80 | 8.48±0.66 | 14.12±2.58 | 7.06±0.68 | 20.69±3.43 | 28.98±5.02 |
| GSTT1(+) (n=113) | 264.82±68.24* | 36.06±5.64* | 1.68±0.35* | 24.56±4.08* | 9.09±0.66* | 16.50±2.06* | 7.10±0.72 | 29.18±4.03* | 25.88±3.23* |
| GSTT1(-) (n=102) | 360.92±70.93 | 30.81±4.72 | 1.23±0.26 | 32.98±4.84 | 8.89±0.82 | 13.84±1.88 | 6.98±0.77 | 19.69±3.09 | 30.64±2.42 |
| GSTP1 A/A (n=135) | 352.45±98.72* | 38.68±6.35* | 1.54±0.29* | 27.46±4.71* | 10.04±0.76 | 12.68±3.45* | 6.09±0.42* | 20.38±2.56* | 23.45±3.68* |
| GSTP1 A/G+G/G (n=80) | 470.23±121.06 | 28.14±6.30 | 1.21±0.34 | 36.35±5.64 | 9.98±0.81 | 9.55±1.93 | 5.16±0.35 | 14.36±1.69 | 29.74±3.75 |
*P<0.05 was considered statistically significant. P values were calculated by the Wilcoxon rank sum test because of the skewness of the fluoride data. The data are expressed as mean±s.d. (+): present genotype; (-): null genotype. GSTM1: glutathione S-transferase M1; GSTT1: glutathione S-transferase T1; GSTP1: glutathione S-transferase P1; A/A: wild-type genotype; G/G: homozygous mutation genotype; A/G: heterozygous mutation genotype; OS: oxidative stress; 8-OHdG: 8-hydroxydesoxyguanosine; NO: nitric oxide; TAC: total antioxidant capacity; MDA: malondialdehyde; FSH: follicle-stimulating hormone; LH: luteinizing hormone; T: testosterone; MMP: mitochondrial membrane potential; DFI: DNA fragmentation index; s.d.: standard deviation; TSH: thyroid stimulating hormone
Table 4 shows the sperm parameters of idiopathic infertile men and correlation estimations for fluoride level and GST null genotype. We also found that the interaction between specimen fluoride and the GST null genotype was significant (P < 0.05). For other sperm parameters, however, the relationship between the urine fluoride null genotype or mutation genotype was not significant. GST gene deletion or mutation was linked to poor semen quality when the concentration of semen fluoride increased. The data in Table 5 show the relationship between semen fluoride and OS based on GST genetic polymorphisms. Overall, we found that the number of idiopathic male infertility individuals with the GSTM1/GSTT1 null or GSTP1 mutant genotype decreased as the fluoride level in their sample increased. Similar observations were obtained for blood and urine fluoride, which reached statistical significance when multivariate analysis was carried out (all P < 0.01).
Table 4.
Spearman correlation analysis between fluoride and the glutathione S-transferase null genotype with sperm parameters for idiopathic male infertility patients
| Parameter | GSTM1(-) (n=84) | GSTT1(-) (n=102) | GSTP1 A/G+G/G (n=80) | ||||||
|---|---|---|---|---|---|---|---|---|---|
|
|
|
|
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| Semen fluoride | Blood fluoride | Urine fluoride | Semen fluoride | Blood fluoride | Urine fluoride | Semen fluoride | Blood fluoride | Urine fluoride | |
| Sperm concentration | −0.62 (<0.05) | −0.34 (0.24) | −0.45 (0.21) | −0.60 (<0.05) | −0.52 (0.28) | −0.32 (0.26) | −0.59 (<0.05) | −0.40 (<0.05) | −0.37 (0.29) |
| Sperm motility | −0.55 (<0.05) | −0.58 (<0.05) | −0.48 (<0.05) | −0.53 (<0.05) | −0.43 (<0.05) | −0.53 (<0.05) | −0.57 (<0.05) | −0.41 (<0.05) | −0.43 (0.11) |
| Sperm morphology | −0.44 (<0.05) | −0.59 (<0.05) | −0.54 (<0.05) | −0.55 (<0.05) | −0.51 (<0.05) | −0.61 (<0.05) | −0.49 (<0.05) | −0.46 (<0.05) | −0.48 (<0.05) |
The data are expressed as correlation coefficient (P). (+): present genotype; (-): null genotype. GSTM1: glutathione S-transferase M1; GSTT1: glutathione S-transferase T1; GSTP1: glutathione S-transferase P1; G/G: homozygous mutation genotype; A/G: heterozygous mutation genotype
Table 5.
Regression coefficients and 95% confidence intervals for semen fluoride levels associated with oxidative stress according to glutathione S-transferase genotype for idiopathic infertility patients
| Genotype | Unadjusted | ||
|---|---|---|---|
|
| |||
| β | 95% CI | ||
| Semen fluoride | GSTM1(+) | 36.8 | −15.6–99.8 |
| GSTM1(-) | −24.6 | −108.3–−2.2 | |
| GSTT1(+) | 30.4 | −10.8–92.6 | |
| GSTT1(-) | −20.3 | −89.2–10.5 | |
| GSTP1 A/A | 38.2 | −120.5–66.7 | |
| GSTP1 A/G+G/G | −40.6 | −100.3–−18.4 | |
Regression models based on GST genetic polymorphisms, included semen fluoride levels and oxidative stress biomarkers (8-OHdG, NO, TAC, and MDA). (+): present genotype; (-): null genotype. CI: confidence interval; GST: glutathione S-transferase; GSTM1: glutathione S-transferase M1; GSTT1: glutathione S-transferase T1; GSTP1: glutathione S-transferase P1; A/A: wild-type genotype; G/G: homozygous mutation genotype; A/G: heterozygous mutation genotype; 8-OHdG: 8-hydroxydesoxyguanosine; NO: nitric oxide; TAC: total antioxidant capacity; MDA: malondialdehyde
DISCUSSION
The prevalence of idiopathic male infertility has been progressively rising in recent years. Moreover, sperm parameters dropped by 50%–60% between 1973 and 2011, posing a threat to human reproductive health.15 Exploration of these factors is critical to better understanding both infertility and risks of genetic anomalies in idiopathic male infertility as a complicated lifestyle-related disorder.11 Based on clinical data for 560 individuals, we conducted a detailed assessment of clinical parameters in idiopathic male infertility in this study. Fluoride levels have diagnostic and prognostic value and may be of great relevance given the link between GST genotype and OS and sex hormone levels. Furthermore, we report that semen parameters of idiopathic male infertility differ greatly among various GST subtypes. These differences might be crucial determinants for idiopathic male infertility.
Semen analysis, which provides essential auxiliary information for the diagnosis and prognosis of male infertility, such as semen volume, sperm motility, sperm morphology, and sperm concentration, is the cornerstone for evaluating male infertility and reproductive potential.16 CASA was applied to analyze 560 male sperm samples in this study. We found that GSTM1 and GSTT1 genotypes are related to high sperm concentration and motility but that the GSTP1 wild-type genotype is associated with high semen volume, sperm concentration, sperm motility, and sperm morphology. These results support the 5th edition of the WHO manual released in 2010. The recommended cutoff values for semen parameters are dramatically lower than those in the 4th edition in 1999, especially regarding motility and morphology.17 A prospective study suggested that a combination of sperm parameters offers greater clinical value for predicting a man’s fertility than a single sperm parameter.18 In idiopathic male infertility, involvement of the GSTM1 and GSTT1 present genotypes, as well as the GSTP1 wild-type genotype, provides indicative predictors.
GSTs are a supergene family of related isozymes considered to detoxify electrophiles by glutathione conjugation with a wide variety of chemical structures.7 GST catalyzes direct conversion of harmful H2O2 into water and hydroperoxides.19 Changes in gene activity are caused by genetic variations in human GST. GSTM1, GSTT1, and GSTP1 in particular may be responsible for an individual’s vulnerability to OS damage.20 Increasing evidence suggests that damage to spermatozoa caused by reactive oxygen species (ROS) is a major factor in infertility.19,21 Mitochondria are the source and often also a target of oxidation.19,21 With deficient ATP, energy sources for sperm motility are low.22 Through our study, we found that GSTM1 and GSTT1 present genotypes, as well as the GSTP1 wild-type genotype, are related to high levels of 8-OHdG and MDA and low levels of NO and TAC. Comparison of our findings with those of other studies confirms a widespread diagnostic criterion for oxidative damage to spermatozoa.
Fluoride is a common chemical element with an effect on male fertility that has attracted much attention.23 Fluoride-induced reductions in T levels and sperm motility have been detected in male rats, as described in a literature review.24 Fluoride-induced OS has been reported to cause impaired antioxidant defense systems, mitochondrial dysfunction, and genotoxicity in animal models, and antioxidant intervention might be affected by antioxidants.24,25 However, fluorine-induced male reproductive toxicity and GST polymorphisms have not been elucidated in humans. Our findings reveal that sperm parameters and the spermatogenesis process are directly associated with male residency in fluorosis-endemic areas. Furthermore, when these individuals carry the GST null genotype, they have more ROS levels and reduced antioxidant ability, as evidenced by decreased NO and TAC levels and a significant rise in 8-OHdG and MDA. These tests assess the extent of OS and involve quantification of ROS levels. This finding might support the hypothesis that fluorosis exposure with the GST null genotype is related to poor prognosis in idiopathic male infertility and that the GST present genotype protects against OS.22 We can deduce from our findings that OS has adverse effects on sperm structural and functional integrity. Spermatozoa are known to be particularly sensitive to OS-induced DNA fragmentation, and much evidence suggests that DNA damage is caused by free radicals.26 Considerable DNA damage in spermatozoa may hasten the death of germ cells and reduce sperm count.27 As a result, early antioxidant treatments should be emphasized for male individuals with the GST null genotype who reside in fluorosis-endemic areas.
The role of T is especially compelling. Fluoride transplacental transit and assimilation into fetal tissues following passage via the placenta have been explored.28 However, the present study provides additional evidence concerning T. It is possible that the decrease in serum T concentration influences the spermatogenesis process and sperm parameters, thereby affecting the reproductive abilities of these fluoride-exposed individuals. Although no statistical significance was found in our study, analysis of FSH is part of routine investigation for male patients with infertility.29
Interestingly, we found that excessive fluoride significantly reduced mitochondrial membrane potential. Mitochondria are energy suppliers and free-radical oxidation targets. Mitochondrial DNA is particularly vulnerable, and its ability to repair DNA is very limited. Sperm cells with multiple damaged mitochondria are unable to execute apoptosis due to DNA damage, resulting in sperm cells with damaged DNA in ejaculate.30 Hypospermatogenesis may stem from mitochondrial dysfunction, which produces more free radicals and less adenosine triphosphate (ATP).22
Fluoride and genetic polymorphisms were the focus of this study, though other factors that may have an impact on male fertility cannot be excluded. Although we complied strictly with the WHO manual methods, we noticed some shortcomings and deficiencies in standard semen analysis during our research. Furthermore, CASA systems have flaws in accurately measuring sperm morphology. In addition, the single-center design in our study was a limitation, and multicenter studies are required for further clarification.
CONCLUSIONS
In the development of idiopathic male infertility, GST polymorphisms and fluorine may provide important evidence of the relationship between reproductive quality and sex hormone levels. Some OS biomarkers involved in idiopathic male infertility may be affected by a GST null or mutant genotype. These findings highlight the need for new policy guidelines to reduce fluoride exposure, particularly with GST deletion or mutation and genetic susceptibility. There are further issues that need to be researched regarding the underlying causes of male infertility and toxicity.
AUTHOR CONTRIBUTIONS
JH participated in the design and conducted the experiment, analyzed the results, and wrote the manuscript. YM and ML analyzed the results and performed the experiments. BWC, WJZ, and KHC performed the experiments. KFT supervised the project, reviewed the results, and helped draft the manuscript. All authors accept responsibility for the paper as published. All authors read and approved the final manuscript.
COMPETING INTERESTS
All authors declare no competing interests.
ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (No. 81660263) and the Science and Technology Fund Project of Guizhou Health Commission (No. gzwkj2021-211).
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