|Year : 2017 | Volume
| Issue : 4 | Page : 228-232
The influence of sperm DNA damage and semen homocysteine on male infertility
Kang-Sheng Liu1, Feng Pan2, Ya-Jun Chen1, Xiao-Dong Mao3
1 Department of Clinical Laboratory, Nanjing Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu 210029, China
2 Department of Andrology, State Key Laboratory of Reproductive Medicine, Nanjing Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu 210029, China
3 Department of Endocrinology, Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, China
|Date of Submission||04-Oct-2017|
|Date of Web Publication||7-Feb-2018|
Department of Endocrinology, Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028
Source of Support: None, Conflict of Interest: None
Background: To explore the relationship of sperm DNA fragmentation index (DFI) , serum and seminal plasma homocysteine (Hcy), and semen parameters in patients with severe spermatogenetic dysfunction.
Methods: A total of 77 infertile males treated in our hospital for severe spermatogenetic dysfunction from January 2016 to November 2017 were recruited. The involved patients were divided into two groups: oligozoospermia (SOM group, 35 cases) and asthenozoospermia (OAT group, 42 cases). The control group (NM group) contained 31 healthy males without reproductive dysfunctions. All the participants involved were tested in the items below: spermatozoa parameters, spermatozoa DFI, serum Hcy level and seminal plasma Hcy level, concentration of seminal plasma malondialdehyde (MDA), and total antioxidant capacity (TAC).
Results: Between the SOM group and NM group, there were significantly difference in sperm concentration, motility and vitality, concentration of MDA, and TAC. The spermatozoa DFI and Hcy levels in SOM group were significantly higher than those of the NM group. Sperm DFI was positively correlated with serum Hcy level (r = 0.083, P < 0.05). Serum Hcy level was negatively correlated with sperm concentration (r = −0.186, P < 0.05) and sperm vitality (r = −0.216, P < 0.05). The serum Hcy level was not correlated with sperm Hcy level (r = 0.103, P > 0.05).
Conclusions: The elevated Hcy level and spermatozoa DFI may be important factors of the severe spermatogenetic dysfunction, which can be used as semen index to evaluate sperm quality and male fertility.
Keywords: DNA Fragmentation Index; Homocysteine; Male Infertility; Routine Spermatozoa Parameters
|How to cite this article:|
Liu KS, Pan F, Chen YJ, Mao XD. The influence of sperm DNA damage and semen homocysteine on male infertility. Reprod Dev Med 2017;1:228-32
| Introduction|| |
With the aggravation of environmental problems, food safety concerns, and the exposure of electromagnetic radiation, the incidence of infertility is on the rise. Male factors are responsible for half of these cases. DNA damage is largely attributable to male infertility. The normal reproductive processes, including sperm-egg fusion, membrane fusion, chromosome combination, and embryogenesis, are all dependent on the intactness of DNA. Many factors damage DNA such as pollutants (organic phosphorus, organochlorine insecticide, plasticizer, heavy metals [lead], carcinogenic polycyclic aromatic hydrocarbon, and zearalenone), male reproductive diseases such as varicocele, infection, tumor, as well as drugs, long-time driving, hot bath, and other high-temperature environment. All these factors can raise the ratio of histones or other proteins and aggravate DNA damage of sperm. Apart from the DNA damage, the abnormal spermatogenesis and spermatozoa maturation may be the most insidious factor for male infertility. However, traditional semen examination indexes can not fully reflect the sperm function. Zhao et al. found that the concentration of homocysteine (Hcy) in seminal plasma is significantly and negatively correlated with the spermatozoa motility, which can be used as an indicator for sperm motility. Crha et al. found that the concentration of Hcy in the seminal plasma of azoospermic patients is signifcantly higher than that of healthy male. In recent years, sperm DFI detection is widely used to evaluate the quality and function of sperm.,
Hcy is one of the three kinds of sulfur-containing amino acids in the body, and is an important intermediate product of methionine cycle and cysteine metabolism. Hcy does not take part in protein synthesis. More and more evidence indicates that Hcy metabolic pathway plays an important role in fertility.
So, whether the sperm DFI and Hcy can be used together as indexes to evaluate the quality and functions of sperm? There have been fewer reports to address this question. This study aimed to explore the relationship of sperm DFI, serum Hcy and seminal plasma Hcy, and semen parameters in patients with spermatogenetic dysfunction, providing a reference for its application in clinical detection.
| Methods|| |
According to the definition on male infertility by the WHO, seminal samples of 116 cases were recruited from the Andrology Department and the Urology Surgery Department at Nanjing Obstetrics and Gynecology Hospital affiliated with Nanjing Medical University from January 2016 to November 2017. The inclusion criteria are as follows: (1) participants did not take drugs that can change sperm parameters in the past several months (e.g., neomycin or gentamicin, which may degrade the sperm quality); (2) those do not have genetic diseases that can severely harm spermatogenesis (e.g., chromosome abnormality and Y chromosome microdeletion); and (3) those do not have bad habits (e.g., addiction to alcohol, cigarette, and drugs) and clinical pathological abnormality (e.g., varicocele and cryptorchidism). The research was approved by the Ethical Committee of Nanjing Medical University, and all participants signed up the written consents.
Five semen samples were excluded because of their nonconformity to the inclusion criteria; one semen sample was excluded for drug taking; and the biochemical data were missing for another two samples. The flowchart of baseline characteristics is presented in [Figure 1]. Therefore, we evaluated the semen analyses and biochemical data of 108 men.
All participants were divided into three groups: group of oligozoospermia (named group SOM, <15 × 105 mL, n = 35, aged 31.0 ± 8.2 years), group of asthenozoospermia (named group OAT, PR <32%, n = 42, aged 36.0 ± 5.8 years), and healthy group (named group NM, ≥15 × 106 mL, n = 31, aged 30.0 ± 5.5 years). The age, obesity, and other basic conditions of the three groups were measured, which was no statistically significant difference between the three groups (P > 0.05).
For all participants, sexual abstinence lasted for 3–5 days before their semen was collected. The duration of abstinence was recorded. Each semen sample was directed into a sterile plastic cup and was liquefied in an incubator at 37°C. After the semen was completely liquefed, at least 2.5 mL of it was used for the test, including 1.5 mL for the routine test and the rest 1.0 mL for detecting DFI, and seminal plasma Hcy, malondialdehyde (MDA), and total antioxidant capacity (TAC).
Routine semen analysis
According to the WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction (5th edition) and the Manual for the Standardized Investigation, Diagnosis and Management of the Infertile Male, the routine semen analysis was performed with a semen quality detection system and related kits (WLJY-9000, China). Parameters, including sperm concentration, vitality, and motility, were recorded. The sperm morphology was examined by Pap staining. To reveal the multiple defects of sperms, sperm deformity index was calculated for each sample.
Sperm chromatin dispersion (SCD) was carried out with sperm fragmentation analysis kits (BRED, Shenzhen, China). Sperms were suspended and mixed with agarose and smeared on the slides. After acidification, the sperms were lysed to remove nucleoproteins and stained for microscopic examination. Normal spermatic DNA showed radiated halos while damaged spermatic DNA showed no or small halos. For each semen sample, 500 sperms in multiple visual fields were selected to test the percentage of sperms with different halos. DFI was calculated.
Sperm fragmentation definition: the sperm head had a small or no halo. The thickness of the halo on one side was <1/3 diameter of the head's thinnest part. DFI(%) = number of sperms with fragmented DNA/the total number of observed sperms × 100%. Moreover, fragmentation rate <25% was considered normal.
Seminal malondialdehyde and total antioxidant capacity measurement
The sample was centrifuged (1,000 g, 15 min). The seminal plasma was extracted according to the instructions of the kits (Jiangcheng Bioengineering Institute, Nanjing, China). Spectrophotometry was used to test the level of MDA (μmol/L) and TAC (U/L).
Hcy measurement in semen and serum
Instruments and kits: HITACHI (a fully automatic biochemical analyzer) was used to test the level of Hcy by means of colorimetry. The standard code of the kits was YZB/0072-2005 (Registration No.: 3400016 by SFDA, 2006). Due to the high content and viscosity of seminal protein, influence factors were strictly controlled during the examination. The normal range of serum Hcy level was 5–15 μmol/L for those aged 1–50 years and 5–20 μmol/L for those aged >50 years. The Hcy reference range of normal seminal plasma (aged: 24–36 years) was 6.02–21.96 μmol/L.
SPSS19.0 statistical software (SPSS Inc., Chicago, IL, USA) was used to compare the data in the form of mean ± standard deviation. Nonparametric tests of Mann–Whitney and Kruskal–Wallis were used to analyze the sperm parameters, Hcy level, and sperm DFI of each group. Data correlation analysis was performed by Passing–Bablok linear regression. P < 0.05 (two tail) was considered statistically significant.
| Results|| |
Sperm concentration, motility, and vitality in the SOM, OAT, and NM groups
Sperm concentration and motility had a significant difference between the three groups (P < 0.05). Comparison between NM group and SOM group, vitality and motility levels had a statistical difference, while there was no statistical difference between SOM group and OAT group (t = 0.71, P = 0.63) [Table 1].
Rate of deformed sperms, sperm DNA fragmentation index, and Hcy in the SOM, OAT, and NM groups
The DFI and Hcy levels had no statistical difference between SOM group and OAT group. The DFI and Hcy levels were higher in SOM group and OAT group than those in NM group. Rate of deformed sperms in NM group was obviously lower than that in the other two groups, and the difference was statistically significant [Table 2].
|Table 2: Comparison of rate of deformed sperm, DFI, serum Hcy, and seminal plasma Hcy|
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Level of MDA and TAC in the SOM, OAT, and NM groups
The mean of MDA in NM group was obviously lower than that in the other two groups [Table 3], whereas the mean of TAC in NM group was obviously higher than that in the other two groups. The difference was statistically significant [Table 3]. The above results indicated that more MDA was produced during the seminal lipid peroxidation and that the drop of TAC level triggered oxidative stress reaction and destroyed the spermatic membranes.
Correlations between serum Hcy and semen parameters
According to the results of Spearman analysis, there was a positive correlation between serum Hcy and DFI levels (r = 0.083, P < 0.05) and a negative correlation between serum Hcy level and spermatozoa concentration (r = −0.186, P < 0.05), and serum Hcy level and sperm vitality (r = −0.216, P < 0.05). As the sperm concentration and sperm motility rise, the serum Hcy level drops. The correlation between serum Hcy level and seminal plasma Hcy level was not significant (r = 0.103, P = 0.601) [Table 4].
|Table 4: Correlation of serum Hcy with DFI, spermatozoa concentration, seminal plasma Hcy, and sperm vitality|
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| Discussion|| |
Recent evidence from literatures has proved the importance of Hcy metabolic pathways in human reproduction. Hcy does not participate in the protein synthesis. Hcy is metabolized through three pathways: (a) with the help of MS (a methionine synthase and Vitamin B12 (a coenzyme), Hcy and N5-methyl-tetrahydrofolic acid (reduced from MTHFR) are synthesized into methionine and tetrahydrofolic acid; (b) with the help of CBS (a cystathionine synthase) and Vitamin B6 (a coenzyme), Hcy and serine are synthesized into cystathionine that is further broken down into cystine and ketobutyric acid; (c) once formed in the cell, Hcy is released into extracellular fluid. In some conditions, Hcy accumulation brings with hyperhomocysteinemia, triggering harmful systematic influence on the body.,
Human sperm DNA is the carrier of hereditary information of human beings. The integrity of sperm chromatin is critical to the fertilization and embryonic development. Environmental factors, genetic mutation, and chromosome abnormality can all damage sperm DNA and deform sperm nucleus. Studies have unveiled the mechanism of sperm DNA damage: cell apoptosis during spermatogenesis and influence of oxygen free radicals on sperm migration. Studies on the abnormal package of chromatin have proved that the sperm DNA damage could negatively affect reproduction, either in natural or artificial means, and was associated with recurrent abortion.,,,,
Sperm DNA fragmentation can be examined in direct or indirect methods. The direct methods involve terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, SCD, and comet assay. Indirect methods mainly involve sperm chromatin structure assay and acridine orange flowcytometry. SCD, first invented by Fernández et al. in 2003, serves to analyze sperm integrity through testing the susceptibility of sperm DNA to acid denaturation. Sperms without fragmented DNA display characteristic halos, while those with fragmented DNA does not, so the sperm integrity can be evaluated within a short period.
SCD was performed in our study to test DFI. DFI and Hcy levels in groups of abnormal sperms were significantly higher than those in the control group, indicating that the levels of DFI and Hcy are associated with sperm concentration. The more severe the DNA damage, the fewer the number of sperms, which is consistent with the research result of He et al. and Gong et al. The level of Hcy was negatively correlated with the sperm concentration. The lower the concentration, the higher the level of Hcy, which is consistent with the research result of Ge et al. We have also studied the correlation between Hcy and DFI levels and found that DFI level rose obviously as Hcy level crept up, which was consistent with the research results of He et al. and Sun et al. Our findings confirm the intricate correlation between the two parameters. The possible underlying reasons may be high level of Hcy leads to the direct or indirect damage of vascular endothelial cells, overproduction of reactive oxygen species (ROS), inhibition of transmethylation, and abnormal gene expression.,
ROS is the most important free radical product of oxidation reaction. The antioxidant in the seminal plasma can protect sperm DNA from being oxidized and promotes sperm capacitation and acrosomal reaction. However, high level of ROS, if not immediately eliminated by the defense system of the body, will break down the double strand of sperm DNA into single strand. The underlying mechanism is that ROS covalently binds DNA through directly oxidizing DNA bases or lipid peroxidation and breaking down DNA strands and structures. Once DNA is damaged, the sperms become more vulnerable to be attacked by ROS. All these physiological changes form a vicious cycle in which sperm DNA is gradually decomposed. Furthermore, increased number of leukocytes in the semen can stimulate sperms to produce ROS. Mediated by various pathways such as cell-to-cell interaction or production of soluble materials, DNA integrity is doomed to be broken.,
Semen is blessed with a natural resistance against oxidation. MDA is conventionally used as an index for reactive oxygen and TAC as an index for oxidation resistance. In thses studies, the control group had an obviously lower seminal MDA level and an obviously higher seminal TAC level than that the other two groups, indicating that too much MDA is produced during seminal lipid peroxidation and that the drop of TAC level triggered oxidative stress reaction and destroyed the spermatic membranes., According to Ni et al. and Fu et al., sperm DNA damage of patients with varicocele could be caused by ROS. Shang et al. and Greco et al. have reported that antioxidant can decrease the rate of DNA fragmentation, suggesting that the seminal ROS participates in the process of sperm damage.
Hcy is involved in the metabolism of one-carbon units. Hcy accumulation evokes hyperhomocysteinemia. The rise of seminal Hcy level will increase the inflammatory factors that could undermine sperm quality (evidenced by the fall of parameters) and male fertility. In addition, high level of Hcy weakens the body's ability to metabolize nitric oxide, which further imposes a bad impact on blood–testis barrier, sperm motility, sperm capacitation, acrosomal reaction, and fertilization., However, it requires more researches to confirm whether sperm DNA damage and low spermatogenesis are directly correlated with Hcy-induced ROS level rise.
The research is not without limitations. First, the sample size was comparatively small. Second, due to the different protein concentrations and viscosity of seminal plasma, errors were unavoidable in testing the level of seminal Hcy, in spite of the quality control having been done before the research.
In conclusion, for patients with severe oligozoospermia and asthenozoospermia, it might be effective to examine the sperm quality by Hcy and DFI. If these indexes are high, early intervention should be performed to promote the odds and safety of fertilization.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]