Document Type : Original Article
1 Department of Anatomical Sciences, Medical Faculty, Jundishapur University of Medical Science, Ahvaz, Iran
2 Shiraz- Human Assisted Reproductive Center, Shiraz, Iran
Ketamine, a non-competitive antagonist at the glutamatergic N-methyl-D-aspartate (NMDA) receptor is currently used in human and animal medicine as an injectable anesthetic. Ketamine is also a controlled substance, illegally used as a recreational drug (
Some of the effects of ketamine on the nervous system have been identified. Earlier studies have reported ketamine use to have a significant detrimental effect on memory function (
Based on ketamine effects on the urinary system, we hypothesize that the genital system may also be affected; therefore we have investigated the effects of different doses of ketamine on quality of normal human ejaculated sperm DNA and membrane integrity. Recently, failure of the conventional semen parameters to predict fertilization show that hidden anomalies lie at the sperm membrane level or at the chromatin level. Plasma membrane function activity is one of the most important aspects of sperm biology that involves metabolic exchanges with the surrounding medium which play an important role in several events during fertilization, capacitation, acrosomal reaction and sperm-oocyte fusion (
Investigation of membrane integrity seems to offer more information about sperm fertility potential than sperm parameters and probably there is relationship between sperm membrane integrity and sperm parameters (
In this prospective study, 40 males were enrolled at Shiraz-Human Assisted Reproductive Center during one month (August 2012). The semen samples were collected by masturbation after 2-5 days of sexual abstinence. After complete liquefaction, the semen were mixed with 2 or 3 Equal of Hams F-10 medium supplemented with 10% human serum albumin and then washed twice by centrifugation at 2000 rpm for 10 minutes until sperm separated from the semen. After obtaining the normal spermogram, normal samples were selected according to World Health Organization guidelines for the next survey. In the next step, 20 million motile sperm in 1 mL of medium were adjusted to different concentrations of ketamine (
Aliquots of 0.2 mL of fresh semen samples were diluted in medium to obtain sperm concentrations that ranged from 5 to 10 million/mL. The suspensions were mixed with 1% low-melting-point aqueous agarose to obtain a 0.7% final agarose concentration at 37˚C. Aliquots of 50 μL of the mixture were pipetted onto slides which were carefully covered with coverslips. The slides were immediately immersed horizontally in a tray with freshly prepared acid denaturation solution (0.08 N HCl) for 7 minutes. A glass slide precoated with 0.65% standard agarose dried at 80˚C, covered with a coverslip (24 x 60 mm), and left to solidify at 4˚C for 4 minutes. The agarose matrix allows keeping the sample at 22˚C is resulted into generation of restricted single- stranded DNA (ss DNA) from DNA breaks. The denaturation was then stopped and proteins were removed by transferring the slides to a tray with neutralizing and lysing solution (0.4 M Tris, 0.8 M DTT, 1% SDS, 2 M NaCl, and 0.05 M Triplex) for 25 minutes at room temperature. Removal of nuclear proteins results in nucleoids with a central core and a peripheral halo of dispersed DNA loop. Slides were thoroughly washed twice in water for 5 minutes, dehydrated in sequential 70%, 90% and 100% ethanol baths (2 minutes each), and air dried. Cells were finally stained with Wright and PBS (1:1) for 10 minutes. After they were air dried, the degree of DNA dispersion was assessed by bright field microscopy. A minimum of 200 spermatozoa were evaluated by two different observers (
This staining was performed by mixing 10 ml of sperm sample with 10 ml of dye (0.5% w/v; Merck Chemical CO., Germany) on a microscope slide which was then covered with a coverslip. A total of 200 sperm cells were counted within a few minutes after the addition of the dye (
In this study, all human research was approved by Shiraz-Human Assisted Reproductive Center (Shiraz, Iran) with the help of Ahvaz Jundishapur Medical University.
The results were analyzed by ANOVA and repeated analysis for repeated measurement with p≤0.05 considered statistically significant. The mean and standard deviation (SD) was also calculated for each value.
There were control and three case groups of 10 patients per group. The results of sperm parameters at different doses of ketamine are reported in table 1. Total sperm progressive motility (fast and slow) in all case groups decreased significantly compared with the control group (p≤0.05). In case group III, there was a significant difference in progressive compared with case group II (p≤0.05). The mean percentage of sperm morphology decreased slightly in all case groups compared with the control group, but it was not significant.
We evaluated membrane integrity using Eosin-Y staining and observed an increased rate of necrospermia in the case groups compared to the control group. However, this result was significant only in case group III (p≤0.05; Fig 1).
The difference in human sperm parameters between control and case groups (mean ± SD)
|Sperm parameters||Control group||Case group I||Case group II||Case group III|
|70.17 ± 1.12||47.48 ± 1.33 *||39.35 ± 1.4 *||29.95 ± 1.2 */**|
|71.38 ± 1.26||68.55 ± 1.33||54.44 ± 1.4||48.06 ± 1.27|
*; Significant difference between case group I and control group and **; Significant difference between case group II and control group.
Comparison of sperm membrane integrity, evaluated by eosin-Y staining between control and case groups (mean ± SD).
a; Difference between case group III and control group.
Determination of sperm DNA fragmentation by the SCD test using conventional bright-field microscopy showed the presence of four SCD patterns as seen in figure 2: 1. sperm cells with large halos whose halo width was similar or higher than the minor diameter of the core; 2. sperm cells with medium size halos whose halo size is between those with large and small halos; 3. sperm cells with small size halo where the halo width is similar or smaller than one third of the minor diameter of the core; and 4. sperm cells without halos.
Table 2 showed a substantial difference in the percentage of positive SCD stained spermatozoa. In the control group nucleoids with medium halo (63.44 ± 1.2) showed a significant difference with all case groups I (15.44 ± 0.45), II (9.05 ± 1.16) and III (10.55 ± 1.14), respectively. Between case groups, nucleoids with large and medium halos showed significant differences in case group II and III compared with case group I. Nucleoids with medium halo had a significant difference between case groups II and III (p≤0.05).
Nucleoides from human sperm cells obtained with the sperm chromatin dispersion (SCD) test. Nucleoides with large halos of DNA dispersion A. medium halos, B. small halos, C. no halos and D. degenerated.
Sperm chromatin dispersion (SCD) data (mean ± standard error of mean) from semen samples between case groups and control group
|Sperm sample||Large halo (%)||Medium halo (%)||Small halo (%)||No halo (%)|
|28.11 ± 1.22||63.44 ± 1.2||7.88 ± 1.33||10.33 ± 1.1|
|21.88 ± 1.32||15.44 ± 0.45 a||8.11 ± 1.33||10.55 ± 0.73|
|7.8 ± 1.33 a, b||9.5 ± 1.16 a, b||11.11 ± 1.25||10.66 ± 1.33|
|10.33 ± 1.33 a, b||10.55 ± 1.14 a, b, c||10.66 ± 1.2||12.66 ± 1.33|
a; Significant difference between case group I and control group, b; Significant difference between case group II and control group and c; Significant difference between case group III and control group.
Ketamine is a dissociative anesthetic developed in 1963 to replace PCP and presently used in human anesthesia and veterinary medicine. Most ketamine sold on the street has been obtained from veterinarians’ offices. Ketamine is a slightly acidic solution administered through injection whose chemical name is (±)-2-(o-chlorophenyl)-2-(methylamino) cyclohexanone hydrochloride (
In the 1990s ketamine abuse, as a new type of drug abuse, appeared in areas of Southeastern China and in high-populated cities and spread quickly into mainland China (
Until now researchers have mainly studied the neurological and urinary tract effects of ketamine. For the first time, Tan et al. (
It has been suggested that sperm concentration, motility and normality are three important parameters to evaluate infertility (
Oxidative stress (OS) represents an imbalance between the productions of ROS or free radicals and the available antioxidant system (
On the other hand, ROS has been shown to correlate with reduced male fertility by causing peroxidative damage to the sperm plasma membrane (
It has been shown that OS is a major case of infertility and is intimately related to DNA fragmentation (
Our findings of the chromatin dispersion pattern according to the SCD test after ketamine dosing have shown that abnormalities in sperm parameters reflected DNA structure.
Our results demonstrate that ketamine administration significantly decreases sperm motility, viability and normal chromatin dispersion. These results may disturb male reproductive function.