International Journal of

Introduction

Infertlity is a major problem for 15-20% of young couples and 50% of cases are related to male factors (1). The causes of some male infertility have been identified such as gene mutant, aneuploidies, infections, diseases, varicocele, radiation, drugs and so on. On the other hand, in many cases the etiology of male infertility is not clear (2), so the study about life styles and several habits, which can give rise to male infertility are important. The quality of sperm DNA is very important in the mammalian reproductive potential (3). The sperm cell transmits the paternal genetic codes to the embryo (4). So, the men with normal sperm motility and morphology may be still infertile, due to the owning abnormal sperm DNA (3). In fact, the accurate transfer of genome depends on sperm chromatin structure (2), as a result of facing with chemical and physical agents during transfer in both sexes genital tracts (5). So, sperm cell DNA needs to be denser and more organized than somatic one (6). In this structure, histones are replaced by basic protamines, which are smaller and have more positive charge than histones.
In addition, the genetic material in male sperm has a critical role in embryonic development specially during the first days (7) and it is an important factor in successful assisted reproductive treatment (ART). Tesarik et al. suggested sperm DNA fragmentation can cause Intra-cytoplasmic Sperm Injection failure (8). Moreover, Borini et al. have shown that the paternal genome not only has strong effect on fertilization and embryo quality, but also affects embryo viability (9).
There are several factors that menace sperm DNA sperm integrity. Pathological and environmental factors like varicocele, alcohol, narcotics and some drugs such as ibuprofen could affect sperm DNA structure and chromatin condensation (10). These painkiller drugs are available without prescription in several parts of the world and many people use them real vast without paying attention to their dosage and probable side effects (11).
Ibuprofen is a propionic acid and one of the non-steroidal anti-inflammatory drugs (NSAD_S) that has both analgesic and antipyretic effects (12). Ibuprofen and other NSADs are ingested for many reasons like fevers, cold, flu, general pains, and rheumatoid arthritis. These drugs inhibit the activity of cyclooxygenases (COX1 and COX2), which synthesize and release prostaglandins (PGs) (13, 14). Although, the PGs have many diverse roles in the most tissues, but, their roles in spermatogenesis are not well known. Templeton et al. have shown that prostaglandin E, 19-hydroxy prostaglandin E, prostaglandin F, and 19-hydroxy prostaglandin are present in human semen, but they did not explain their roles. It was shown that PGs come from prostate (15). However, the study of Fang et al. has indicated that the prostaglandin D synthase has a key role in the regulation of spermatogenesis in Chines mitten crab Eriocheirsinensis (16).
The aim of present study was to investigate the effects of ibuprofen on sperm parameters, chromatin condensation, and DNA integrity using cytochemical based (Aniline blue and Chromomycin A3) and molecular based (sperm chromatin dispersion) assays in mice.
 
Materials and methods
 
Animals
This experimental study was done in Research and Clinical Center for Infertility, Yazd, Iran from September to December of 2014. Totally 36 adult male NMRI mice with average weight of 37 g and 11 weeks old were obtained from the mouse stock of Shahid Sadoughi University of Medical Sciences. These mice were divided into three groups; control (group I, n=12), normal dosage (group II, n=12) and high dosage (group III, n=12). In groups II and III, 30 and 57 mg/kg body weight ibuprofen were dissolved in daily drink water, respectively (10), but, in the control group, we did not have any medication. During experiment, animals were kept in standard conditions with a temperature range of 25±3^oC and mean relative humidity of 50±5% in the animal house. During the course of study, it was followed the recommendations by our Institutional Animal Care and Use Committee for the handling, maintenance, treatment, and killing of the animals. This experimental project was approved by the Ethical Committee of Yazd Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences.
 
Epididymal sperm preparation
The duration of spermatogenesis in mouse is about 35 days (17). We assessed the spermatozoa of drug-treated mice after 1, 2 and 3 durations of spermatogenesis in each group. In other word, the sperm samples were analyzed on 35, 70 and 105 days post treatments. The mice in each groups were killed by cervical dislocated and the cauda epididymis of each animal was cut and placed in 1 ml Ham’s F10 medium. The dishes were incubated at 37 and 5% CO₂ for 10 min to make spermatozoa swim out (18).
 
Sperm parameters analysis
The sperm motility, normal morphology, viability and sperm count were evaluated for at least 200 spermatozoa of each animal. Sperm movement analysis was done by Makler chamber and light microscopy at x 40 eyepiece magnification (Olympus Co., Tokyo, Japan). Motility was expressed as the percentages of progressive motility, including progressive (Rapid and Slow), non-progressive and immotile spermatozoa. The morphologically normal spermatozoa and the percentage of viable sperm cells were assessed by Papanicolaou staining and Eosin test, respectively (19). The light microscope was set at × 100 eyepiece magnification.
 
Sperm chromatin and DNA evaluations
Sperm chromatin condensation and DNA integrity were assessed using Sperm Chromatin Dispersion (SCD) test, chromomycin A3 (CMA3) and Aniline blue (AB) staining.
 

1. Aniline blue

Aniline blue staining indicates the excessive histones in chromatin structure. After smearing the epididymal spermatozoa, the slides were air-dried and then fixed in 3% buffered glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 30 min at room temperature. Each smear was stained with 5% aqueous AB stain (Sigma, St Louis, MO, USA) in 4% acetic acid (pH 3.5) for 15 min (18).
 

2. Chromomycin A3 staining

CMA3 is a fluorochrome antibiotic, which competes with the protamines for binding to the minor groove of DNA (6). The air-dried smears were fixed with  Carnoy's  solution  at 4 °C for 10 min. Then, each slide was stained with 500 µl of CMA3 solution (50 µl stock stain solution + 450 µl McIlvaine buffer) (Sigma, St Louis, MO, USA) in darkroom for 20 min (20).
 

3. SCD test

The SCD test was used for the assessment of sperm DNA fragmentation. The SCD test was performed using the Halosperm® Kit (INDAS laboratories, Madrid, Spain). For the mouse sperm staining, after adding 25 μl of semen to 50 μl of low melting agarose, 10 μl of this suspension was decanted on coated slide of Halosperm® Kit. A small lamella was put on it and kept on 4°C for 5 min. The slide was immersed in Denaturant Agent (for 7min) and Lysis Solution (for 20 min). After dehydrating, the slides were stained with Staining Solution A & B for 7 min (20).
 
Statistical analysis
All statistical analyses were performed by using Statistical Package for the Social Sciences, version 20.0, SPSS Inc, Chicago, Illinois, USA (SPSS 20) software. Data were expressed in mean±SD (standard deviation). One-way ANOVA followed by Tukey-HSD and Pearsons´ correlation test was considered for comparison of the results and the term 'statistically significant' was used for p-value less than 0.05.
 
Results
 
Table I indicates the results of semen analysis for sperm parameters. Some of the parameters such as the numbers of sperm cells with normal morphology, and viability (except for group III) were changed with only one period of drug treatment (35 days). While, some other parameters like sperm count were revealed significant changes after ongoing use of the drug for two periods of spermatogenesis (70 days). Although, some of the parameters showed significant variation with a continuous use of drug such as progressive motility, but some of them did not change. The variables were perused during three times of exposure to drugs in each group. Also, the number of non-progressive motile spermatozoa in group III, which were received 35 days drug were increased compared to the same group after receiving drug in 70 and 105 days (p value=0.011 and 0.022, respectively). Total motility of sperm had significant decrease in mice that were treated 35 mg/kg/day for 70 days (p value=0.001).
Table II shows the results of sperm chromatin analysis. The results of samples that were treated for one period of spermatogenesis were positive for all three sperm chromatin tests. While these results for other samples were different. After 105 days of drug treatment, the result of DNA assessment did not show increasing in DNA fragmentation and protamine deficiency (CMA3 staining). In this study, it was used Pearsons´ correlation test to find out the correlation between the variables. The results with p value less than 0.05 of this test are as follows:
•    The correlation between progressive motility and count (p-value<0.001and Pearsons' correlation= 0.816).
•    Progressive motility and viability (p-value< 0.001 and Pearsons' correlation= 0.816).
•    Progressive motility and Aniline blue staining (p-value= 0.007 and Pearsons' correlation= -0.638).
•    Progressive motility and normal morphology (p-value= 0.002 and Pearsons' correlation= 0.400).
•    DNA fragmentation (SCD test) and abnormalities in sperm tail (p-value= 0.018 and Pearsons' correlation= 0.363).
•    Normal morphology and protamine deficiency (CMA3 staining) (p value= 0.029 and Pearson correlation= -0.337).
•    Normal morphology and DNA fragmentation (SCD test) (p-value= 0.004 and Pearson correlation= -0.436).
•    Protamine deficiency and DNA fragmentation (p-value= 0.050 and Pearsons' correlation= 0.304).

• Protamine deficiency and Aniline blue staining (p-value= 0.021 and Pearsons' correlation= -0.354).