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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 4  |  Page : 216-221

Severity and prevalence of sperm DNA damage among infertile males at a tertiary hospital, north central, Nigeria


1 Department of Chemical Pathology, Federal Medical Centre, Abeokuta, Ogun State, Nigeria
2 Department of Chemical Pathology, University of Ilorin, Kwara State, Nigeria
3 Department of Obstetrics and Gynaecology, Federal Medical Centre, Abeokuta, Ogun State, Nigeria
4 Department of Chemical Pathology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria

Date of Submission14-Feb-2021
Date of Acceptance08-Apr-2021
Date of Web Publication28-Oct-2021

Correspondence Address:
Dr. Waliu Olatunbosun Oladosu
Department of Chemical Pathology, Federal Medical Centre, Abeokuta, Ogun State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/injms.injms_23_21

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  Abstract 


Context: Seminal fluid analysis (SFA) is the most important investigative tool for evaluation of male infertility. However, it is limited in detecting causes of sperm abnormalities while some individuals with normal SFA are still considered infertile. Evaluating sperm deoxyribonucleic acid (DNA) integrity as an ancillary investigative tool to SFA will enhance the investigation of infertility. Aims: The aim is to assess the prevalence and severity of sperm DNA damage based on semen level of 8-hydroxydeoxyguanosine (8-OHdG) in males with and without abnormal SFA. Settings and Design: This was a descriptive cross-sectional study of 120 males with at least one SFA parameter abnormalities as test controls and 120 normal controls. Subjects and Methods: Seminal 8-OHDG was assayed as a marker of sperm DNA damage using ELISA method. Statistical Analysis Used: Normally distributed data were expressed as mean ± standard deviation otherwise expressed as median and interquartile range. Results: The mean ages of subjects and controls were 35.84 ± 6.0 vs. 36.22 ± 7.56 years. The mean seminal 8-OHDG was significantly higher among subjects than among controls (15.21 ± 3.80 ng/ml vs. 12.45 ± 4.0 ng/ml, P = 0.015). The reference value of seminal 8-OHDG obtained in this study was 4.45–20.45 ng/ml and the prevalence of sperm DNA damage among subjects compared to controls was 10.8% versus 3.3%, P = 0.024. Severe DNA damage corresponding to sperm DNA fragmentation index of >30% was 3.3% in subjects and was not present in any male partner in the control group. Significant sperm DNA damage was also associated with reduced sperm count (P = 0.043), while its association with reduced sperm motility was not statistically significant in this study. Conclusions: The prevalence and severity of sperm DNA damage were more significant among males with, than those without, abnormal SFA parameters.

Keywords: Deoxyribonucleic acid damage, infertility, male, spermatozoa


How to cite this article:
Oladosu WO, Biliaminu SA, Abdulazeez MI, Jimoh SO, Ajose OA, Okesina AB. Severity and prevalence of sperm DNA damage among infertile males at a tertiary hospital, north central, Nigeria. Indian J Med Spec 2021;12:216-21

How to cite this URL:
Oladosu WO, Biliaminu SA, Abdulazeez MI, Jimoh SO, Ajose OA, Okesina AB. Severity and prevalence of sperm DNA damage among infertile males at a tertiary hospital, north central, Nigeria. Indian J Med Spec [serial online] 2021 [cited 2022 Aug 11];12:216-21. Available from: http://www.ijms.in/text.asp?2021/12/4/216/329523




  Introduction Top


The functions and replication of every cell is dependent on its deoxyribonucleic acid (DNA). DNA is responsible for the transfer of genetic information from parent cells to their progenies. However, when DNA becomes damaged, it may result in cell death (apoptosis) or mutations with loss of functions.[1] In the spermatozoa, DNA damage may transfer mutations during fertilizations.[2],[3],[4] This may lead to failed conception or early termination of pregnancy, depending on the degree of DNA damage.[5]

Sperm DNA integrity is ensured normally by compact organization of the nuclear chromatin into a tightly packaged dense and insoluble protamine-bound structure, stable enough to withstand the rigors of all the stages between deposition of semen in female genital tract postcopulation and immediate postfertilization stage.[1] The delicate intricate arrangement of the chromatin makes DNA within it susceptible to damage.

Evaluation of sperm quality in male fertility is commonly determined by seminal fluid analysis (SFA). This procedure basically does not access the sperm DNA integrity, therefore assumption of viability of spermatozoa based on light microscopy assessment of semen, may not be accurate. This perhaps is the cause of increasing number of infertile males, who though have adequate sperm parameters based on SFA, are unable to achieve fertility.[2],[4] This may also be the picture in most Artificial Reproductive Technology (ART) centers, where eggs are harvested from fertile surrogate mothers and fertilized by sperm obtained from men with assumed fertility based on the adequacy of their SFA parameters and subjected to techniques such as intrauterine insemination (IUI) and in vitro fertilization (IVF). Yet the failure rate is very high despite the huge financial commitment of patients, emotional burden of anticipatory parenthood, and huge amount of manpower and facility deployed by the experts. For men found to have high levels of DNA damage or fragmentation, their best chance of achieving conception is through intra-cytoplasmic sperm injection (ICSI) rather than IVF or IUI.[6]

There are various methods of assessing DNA damage. These include Comet assay, fluorescence in-situ hybridization technique, sperm chromatin structure assay, terminal transferase dUTP nick end labeling assay, the Sperm Chromatin Dispersion (Halo) test, and the DNA damage quantitation using ELISA method.[3] The most feasible of all these methods is DNA quantitation using the ELISA method. This is because of its affordability in developing countries with limited resources.


  Subjects and Methods Top


The study was conducted at the Department of Chemical Pathology and Immunology, University of Ilorin Teaching Hospital, Ilorin, Kwara State. The hospital receives referrals of infertile male patients from Kwara State and other neighboring states. The Hospital has a General Outpatient Department (GOPD) which serves as a referral point for infertile couples and Specialist Gynaecological and Urology clinics attending to female and male infertility cases, respectively, as well as an Artificial Reproductive Technology Unit (ARTU). The Department of Microbiology and ARTU receives the request for the SFA of about 400/year from Gynaecology, Urology, and GOPD Clinics in the hospital as part of the evaluation of infertile couples.

The study population included male partners of infertile couples who were requested to carry out SFA at the Microbiology Laboratory in University of Ilorin Teaching Hospital, Ilorin, Kwara State and at the ARTU.

This was a descriptive cross-sectional study of consecutive infertile male partners of infertile couples.

The study was conducted using the consecutive sampling method.

The minimum sample size required for the study was estimated using the Fisher formula.[7]

Given as:



n = the desired minimum sample size.

z = the standard normal deviation usually set at 1.96 which corresponds to 95% confidence interval.

p = the prevalence of male infertility in the target population from the previous study. This was estimated to be 8.45%.52

q = the proportion in the target population who do not have a particular characteristic, i.e.

q = 1 – P = 1–0.0845 = 0.9155,

d = tolerable margin of error, an observed difference of 5% was taken as being significant.

Therefore,



The sample size was 120

120 participants with normal semen parameters according to the WHO guidelines served as controls.

Study participants were consenting male patients who were referred from Gynaecology, Urology and GOPD clinics to the Medical Microbiology laboratory or ARTU for SFA. Patients found to have at least one defective semen parameter were recruited as subjects while patients with normal semen parameters were recruited as controls.

The normal SFA parameters were based on current WHO criteria as stated below:[8]

Semen Volume (ml):1.5 ml, Concentration (Mill/cc):15 Mill/cc, Motility (%): 40%.

Recruitment into the study was preceded by obtaining an informed consent. The process was repeated for consecutive patients until the required sample size was achieved.

Included in this study were all consenting male partners of infertile couples. Those with at least one defective semen parameter after SFA were recruited as subjects while those with normal SFA parameter were regarded as controls.

The following individuals were excluded from this study:

  1. Patients with abnormalities like cryptorchidism (undescended testes) or atrophic testes
  2. Patients with Azoospermia (absence of spermatozoa in the semen)
  3. Patients with Aspermia, those who are unable to produce semen.


Written consent for inclusion into the study was obtained after explanation of the study and the procedure before samples were taken from the patients. Written permission was sought and obtained from the Heads of Chemical Pathology and immunology and Medical Microbiology of the hospital. Clearance was obtained from the Ethical Committee of the University of Ilorin before commencing the study.

Semen samples were collected by the patients from their respective homes using masturbation into a sterile, wide-mouthed container, after at least 72 h (3–4 days) of sexual abstinence. The samples were transported from their respective homes to the laboratory within 1 h of collection, to preserve the viability of the spermatozoa. During transportation to the laboratory samples were kept as much as possible close to the body temperature, this is best achieved by placing the container inside a plastic or polythene bag and transporting it in the front pocket in the person's clothing.

Samples were allowed to liquefy at room temperature (25°C–30°C) monitored by thermometer fitted on the wall inside the laboratory, for at least 45 min. After liquefaction, samples were analyzed for volume, pH, sperm count, motility, morphology, and viability (where necessary). The basic laboratory procedures for SFA was according to the WHO guideline.[9]

Samples were centrifuged for 20 min at 1000 × g (validated by a timed stopwatch). The sediments were collected and divided into aliquots and stored at − 80°C inside a freezer (Thermofisher, UK, located in the Rotavirus Research Laboratory of the Hospital) monitored by 12 hourly temperature chart of automatic factory fitted thermometer, for not >6 months to avoid the loss of bioactivity and contamination. Semen samples that are found to be normospermic, according to the WHO criteria, were taken as controls.

Laboratory procedures

Laboratory procedures were in two phases, which include: (1) Sperm DNA isolation and (2) Sperm DNA damage quantification

  1. Protocol for sperm DNA Extraction using the Zymo kit by BioTechniques:[10]


  • Add 200 μl Trizol reagent to 200 μl of sperm cells in an Eppendorf tube
  • Add 20 μl of Proteinase K to the mixture
  • Incubate the mixture to 60°C and vortex the mixture intermittently to disrupt and solubilize the material for an hour
  • Add 500 μl of ethanol (95%–100%) to the homogenized mixture and vortex for 30 s
  • Transfer the mixture into a Zymo Spin™ IICR Column in a Collection Tube and centrifuge at 8000 rpm for 2 min
  • Transfer the column into a new collection tube and discard the flow-through and reassemble the column into the collection tube
  • Add 700 μl Direct-zol™ DNA Wash 1 to the column and centrifuge for 2 min. Discard the flow-through and reassemble the column into the collection tube
  • Add 700 μl Direct-zol™ DNA Wash 1 to the column and centrifuge for 2 min. Discard the flow-through and reassemble the column into the collection tube
  • To ensure complete removal of the wash buffer centrifuge the reassembled tubes for 3 min
  • Carefully, transfer the column into a nuclease-free tube
  • To elute DNA, add 50ul of Direct zol™ Elution Buffer directly to the column matrix and centrifuge for 1 min.


Sperm deoxyribonucleic acid damage quantification

The OxiSelect™ Oxidative DNA Damage ELISA kit (produced by Cell Biolabs, Incorporated, 7758 Arjons Drive San Diego, CA 92126) was used.[11] It is a competitive enzyme immunoassay developed for rapid detection and quantitation of 8-hydroxydeoxyguanosine (8-OHdG) in urine, serum, or other cell or tissue DNA samples.

Sperm deoxyribonucleic acid samples preparation

  1. DNA was extracted from sperm cell samples as described above
  2. The extracted DNA was then dissolved in water at 1–5 mg/mL
  3. The DNA samples were then converted to single-stranded DNA by incubating the samples at 95°C for 5 min and rapidly chilling on ice
  4. The DNA samples were then digested to nucleosides by incubating the denatured DNA with 5–20 units of nuclease P1 (previously reconstituted in the manufacturer's recommended buffer) for 2 h at 37°C in a final concentration of 20 mM Sodium Acetate, pH 5.2
  5. Then 5–10 units of alkaline phosphatase (previously reconstituted in the manufacturer's recommended buffer) was added, plus sufficient Tris buffer to a final concentration of 100 mM Tris, pH 7.5, and incubated for 1 h at 37°C
  6. The reaction mixture was then centrifuged for 5 min at 6000 × g and collected the supernatant for use in the ELISA.


Assay principle

The OxiSelect™ Oxidative DNA Damage ELISA kit is a competitive ELISA method for the quantitative measurement of 8-OHdG. The unknown 8-OHdG samples or 8-OHdG standards were first added to an 8-OHdG/BSA conjugate preabsorbed microplate. After a brief incubation, an anti-8-OHdG monoclonal antibody is added, followed by an HRP conjugated secondary antibody. The 8-OHdG content in unknown samples was determined by comparison with the predetermined 8-OHdG standard curve.

Quality control

The performance of the equipment, reagents, analytical method and analyst was validated by running control with each batch of samples.

Statistical analysis

Statistical analysis was done with the Statistical Package for the Social Sciences (SPSS) version 20.0 (SPSS Inc., Chicago, Ill, USA). Normally distributed data were expressed as mean ± standard deviation (SD), while nonnormally distributed data were expressed as median and interquartile range; categorical variables will be reported as percentages. For nonnormally distributed data, the comparison will be performed employing Mann–Whitney U or Kruskal–Wallis tests when appropriate. Comparison of normally distributed data was performed using ANOVA. Parameters displaying P < 0.05 will be considered statistically significant.


  Results Top


A total of 120 males who had abnormal SFA results, involving sperm count, motility or both were recruited for this study as test controls and 120 males with no sperm count or motility abnormality were recruited as controls. The mean age of the test controls was 36.22 ± 7.56 years while the mean age of control was 35.84 ± 6.27 years as shown in [Table 1]. The test subjects and controls were mainly civil servants, constituting 56% and 50%, respectively and were educated to tertiary level. The age and profession of male partners of both groups were matched and comparable. The sociodemographic distribution is shown in [Table 1].
Table 1: Distribution of the sociodemographic characteristics of participants

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The mean seminal 8-OHDG level among test controls (15.21 ± 3.80 ng/ml) is significantly higher than among controls (12.45 ± 4.00 ng/ml), P 0.024, as shown in [Table 2].
Table 2: Assessment of the effects of significant sperm DNA damage on seminal fluid analysis parameters

Click here to view


The mean seminal 8-OHDG level among the 120 fertile controls is 12.45 ± 4.00 ng/ml. Therefore, taking the reference value as mean ± 2S. D based on the clinical and laboratory standard institute (CLSI) recommendation, reference value of seminal 8-OHDG was 4.45–20.45 ng/ml.55 Values of Seminal 8-OHDG >20.45 ng/ml, the upper limit of the reference interval, were considered as suggestive of significant sperm DNA damage. This was used as the threshold value for estimation of the prevalence of sperm DNA damage among subjects and controls. Based on this, the prevalence of sperm DNA damage among subjects in this study was 10.8% compared to 3.3% among fertile controls.

Sperm count and motility were compared with significant sperm DNA damage, sperm count was significantly lower among participants with significant sperm DNA damage compared to those without significant sperm DNA damage (P = 0.043). However, significant sperm DNA damage was not associated with sperm motility, though percentage motility was lower among subjects than controls.

A previous study established a relationship between sperm DNA damage estimation using sperm DNA fragmentation index (DFI %) and seminal 8-OHDG concentration (in ng/ml), where sperm DFI of 21.73% ± 9.49% was equivalent to seminal 8-OHDG of 19.27 ± 5.01 ng/ml.56 Sperm DFI of >30% which is will be equivalent to the seminal 8-OHDG concentration of >26.6 ng/ml in this study is established as severe sperm DNA damage. 3.3% of individuals in this study had severe sperm DNA damage compared to 0% among controls.


  Discussion Top


The mean ages of individuals and controls in this study were 36.22 ± 7.56 years and 35.84 ± 6.27 years, respectively. Majority of the research participants were civil servants, constituting 56% and 50% among individuals and controls, respectively, and mostly educated to tertiary level. This is likely because the study site is located in the state capital where the majority of the civil servants reside and the fact that literate individuals tend to seek medical care more than the less educated.

The mean seminal 8-OHDG levels among individuals were significantly higher than among controls in this study (15.21 ± 3.80 ng/ml vs. 12.45 ± 4.0 ng/ml), P < 0.05. This showed higher levels of spermatozoan DNA damage by reactive oxygen species (ROS) in subjects than controls since 8-OHDG formation results from oxidation of DNA, specifically guanine base by ROS. This is similar to findings in many other studies.[12],[13],[14] The effects of excessive free radicals/ROS attack on DNA in the sperm nucleus include nucleotide modifications, DNA strand breaks, and chromatin cross-linking and may result in male infertility.[15] There are about 20 oxidized nucleic acids found in the human body and the most abundant of these is 8-OHDG.[16]

The mean seminal 8-OHDG level of 12.45 ± 4.0 ng/ml obtained among the 120 controls was used to estimate the reference values for seminal 8-OHDG, using mean ±2SD in line with the CLSI recommendation for a normally distributed measurement,[17] since there was no known established reference value for seminal 8-OHDG level. The reference values of 4.45–20.45 ng/ml were obtained for seminal 8-OHDG and values >20.45 ng/ml, the upper limit of the reference interval, were taken as suggestive of significant sperm DNA damage. This is equivalent to 23.06% sperm DNA damage by DFI using 21.73% DFI equivalent to 19.27 ng/ml obtained in a previous study.[18] Based on this, the prevalence of sperm DNA damage was 10.8%, which was statistically significantly >3.3% among controls. This is similar to findings of 16.8% and 19.25% obtained among infertile men in previous studies.[19],[20] The higher values obtained in those studies may be due to differences in their method of DNA damage evaluation. Their evaluations were based on sperm DNA fragmentation which would have detected sperm DNA damages from other causes aside ROS while the method in this study detected sperm DNA damage from ROS only.

The finding of significant sperm DNA in only about 10.8% of males with abnormal SFA parameters suggests that there are numerous other etiological causes of abnormal SFA parameters. Endocrinological causes of abnormal SFA parameters include hypoandrogenism, hyperprolactinemia, and thyroid disorders, while metabolic causes include diabetes mellitus and obesity. Moreover, ROS can also cause sperm damage by lipid peroxidation of its membranes and also negatively impact SFA parameters, not necessarily through sperm DNA damage. Furthermore, the observation of significant sperm DNA in about 3.3% of the controls, who had normal SFA parameters suggests that normal SFA does not confer fertility in all cases and consistent with the finding in a previous study that up to 15% of males with normal SFA are infertile and classified as male infertility of idiopathic origin, which may in fact be due to sperm DNA damage when properly evaluated.[21],[22]

Seminal 8-OHDG >26.6 ng/ml equivalent to sperm DFI of >30% was taken as the threshold of severe sperm DNA damage and 3.3% of the subjects in this study had severe sperm DNA damage and was not present in any male partner in the control group. Only a few studies have been done on assessment of the severity of sperm DNA damage, however, the severity in this study is lower than 8.4% severity of sperm DNA among infertile males obtained from a similar study, though subjects selected for the study were men with unexplained infertility.[23] Infertile males with >30% sperm DNA damage, classified as severe sperm DNA damage will benefit from ICSI as a means of assisted reproductive technique rather than the conventional IVF. Sperm DNA damage evaluation, therefore, becomes very important for infertile males selected for ART before choosing the specific ART method, to minimize the failure rate which is very common with such procedures.

Significant sperm DNA damage was found to be associated with reduced sperm count among research participants compared with those without significant sperm DNA damage (P < 0.05), while sperm motility was also lower among participants with significant sperm DNA damage but the difference was not statistically significant in this study (P > 0.05). The common denominator in sperm DNA damage, reduction of sperm concentration and sperm motility among infertile males, is excessive ROS Sperm DNA damage, as demonstrated in numerous other studies.[24],[25] Proposed mechanism includes lipid peroxidation of sperm DNA, membranes, and mitochondria. Others include activation of caspases-mediated sperm cell apoptosis.[26]

To buttress the importance of ROS as a major cause of male infertility, several recent studies have proved that oral anti-oxidant therapy including Vitamins E and C, carnitines, lycopene, folic acid, selenium and Zinc, etc., can improve male fertility by enhancing sperm quality, including counts and motility, improving the outcomes of ART and the number of life births, by reversing the damaging consequences of ROS on sperm DNA and membranes.[27]


  Conclusions Top


Significant and severe sperm DNA damage are more prevalent among male partners with abnormal SFA parameters than those without abnormal SFA parameters. Therefore, including sperm DNA damage estimation in the evaluation of male partners of infertile couples, using seminal 8-OHDG level especially in low resource settings will enhance the investigation and treatment of males with infertility.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2]



 

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