To evaluate the
Semen samples (n=13) were collected from healthy donors and a semen analysis was performed according to World Health Organization. Density gradient centrifugation was performed to isolate motile sperm. Samples were incubated with different concentrations (0, 1, 10, 100, and 1,000 ng/mL) of RA. The non-exposed group (0 ng/mL) was defined as the control group. Samples were analyzed for motility at different time points (0, 60, 150, 240, and 300 minutes) and for vitality and oxidation reduction potential (ORP) (at 0, 240, and 300 minutes). Sperm motility was assessed manually and motion kinetic parameters were recorded by computer aided semen analysis.
RA at any tested concentration significantly increased sperm motility compared to the control in a time-dependent manner with a maximum increase after 240 minutes. Motion kinetic parameters were not comparable. For sperm vitality, supplementation with RA significantly maintained survival at higher levels, while non-treated sperm gradually died. These higher levels of vitality were maintained with rising RA concentrations of up to 1,000 ng/mL. A non-significant trend of increased ORP was observed in all study groups.
RA increases sperm motility and maintains vitality at any concentration tested. Therefore, RA might be utilized to improve sperm quality in asthenozoospermic specimens. However, further investigation is ongoing to evaluate the effect of RA on other sperm parameters.
Attention-deficit hyperactivity disorder (ADHD) is a frequent neurodevelopmental disorder with an increasing prevalence over the last 2 decades [
The most commonly used first line pharmacologic treatment for ADHD is methylphenidate (MPH). The mechanism of action of MPH in ADHD is still a matter of debate, but several studies indicated that MPH inhibits the presynaptic dopamine transporter [
Ritalinic acid (RA), the major inactive metabolite of MPH, is one of the isomers of amphetamine. The circulating concentrations of RA greatly exceed that of the parent drug [
Due to the wide use of MPH to treat ADHD, several studies have been conducted to evaluate its impact on various body biological systems in general and the reproductive system in particular; however, most of them are animal studies. Chronic use of MPH decreases the body weights of male mice as compared to a control group. In addition, the weight of the testes and seminal vesicles decreased significantly [
Previous studies, mainly animal ones, investigated the impact of orally administrated MPH on biological systems. However, no study assessed the effect of MPH or its metabolites on human sperm. This lack of information might be related to the young age of the patients, mostly prepubertal boys, to whom MPH was prescribed at the time the drug was approved for clinical use in 1955. Nonetheless, the growing use of MPH in adolescents and adults who are in their reproductive age, and the growing rate of MPH abuse, requires an evaluation of the impact of MPH on male reproduction. The aim of the present study is to investigate the direct effect of RA on human semen parameters
Following the approval of the study by the Institutional Review Board (IRB) of the Cleveland Clinic Foundation Hospital (Reg. No. 15-435) according to the Declaration of Helsinki, a total of 13 semen samples were collected from healthy donors by masturbation after 2 to 3 days of abstinence. An informed, written consent has been obtained from each donor.
All samples were allowed to liquefy completely for 20 minutes at 37℃ before further processing. The initial semen analysis was performed according to the World Health Organization (WHO) 5th edition [
The following inclusion criteria were applied for samples to be eligible for enrollment into the study: generally healthy males over 18 years of age who were willing to abstain from sexual activities for 2 to 3 days prior to semen collection, and normal semen analysis as per WHO 2010 guidelines. Subjects presenting with any of the following were excluded: sample of high viscosity semen, non-compliance with protocol requirements, and any abnormal semen parameter according to the WHO 2010 criteria.
Following liquefaction, after the sample has been in the incubator for 20 minutes at 37℃, the sample is examined for viscosity by the ease of pipetting in a graduated serological pipette. Viscosity is graded as follows: Normal viscosity: the sample can easily pass in the pipette and expelled easily, slight viscosity: the sample is not expelled easily out of the pipette, moderate: difficult to pipette and expel, high viscosity: the sample is a coagulum and cannot be aspirated. In the current study, none of our donors presented with moderate or high viscosity. Hence none of the samples were excluded. Semen specimens were evaluated for volume, liquefaction, color, pH, and volume as well as sperm concentration, motility, and vitality as per WHO 2010 guidelines. Motile sperm were obtained by pooling of semen samples prepared by double density gradient separation. The motile sperm were tested for the effect of different concentrations of RA (1, 10, 100, and 1,000 ng/mL) on spermatozoa.
In order to evaluate the better fraction of motile sperm, spermatozoa were prepared by separation on a double density gradient by layering 2 mL each of upper (45%) and lower phase (90%) layer (Vitrolife, San Diego, CA, USA). Two mL of liquefied semen were layered on top. Samples were centrifuged at 300 ×
A human sperm motility assay was used to test the toxicity of the RA at different concentrations [
Sperm vitality was tested using the eosin-nigrosine stain. An amount of 0.67 g eosin Y (color index 45,380) and 0.9 g sodium chloride (NaCl) were dissolved in 100 mL of purified water. Ten grams of nigrosin (color index 50,420) were added to the 100 mL of eosin Y solution. Semen samples were mixed with an equal volume of eosin-nigrosin suspension in a porcelain spot plate well or test-tube. A smear on a glass slide was made from each suspension and was allowed to air dry. Immediately after drying, each slide was examined at ×1,000 magnification under oil immersion. The number of stained (dead) or unstained (vital) cells was evaluated and the percentage of vital sperm was calculated.
Oxidation reduction potential (ORP) levels were measured in sperm suspension by using the MiOXSYS analyzer (Aytu Bioscience, Eaglewood, CO, USA) at 0 and 240 and 300 minutes parallel to the vitality and motility tests. ORP measures the transfer of electrons from a reductant (or antioxidant) to an oxidant. Briefly, 30 mL of liquefied semen at room temperature were applied to the MiOXSYS sensor. The sensor was pre-inserted into the MiOXSYS analyzer; measurements begin automatically. ORP is measured in milli-volts (mV) and is the integrated measure of the existing balance between total oxidants and reductants in a biological system.
Each sample was measured in triplicate, and the average values for ORP were recorded. Data were then normalized to sperm concentration to control for differences in cell numbers. Thus, data are presented as mV/106 sperm/mL. To assess the effects of time on ORP measures, samples were incubated at room temperature for 0 and 120 minutes before being measured.
For statistical analysis, MedCalc Statistical Software, ver. 17.4 (MedCalc Software bvba, Ostend, Belgium) was used. After testing for normal distribution of the data by means of the Kolmogorov—Smirnov test, the parametric (paired-samples t-test) and non-parametric tests (Wilcoxon test and Kruskal-Wallis test with Jonckheere-Terpstra trend), respectively, were employed. Data are presented as mean±standard error of mean. A p<0.05 was considered significant.
All semen samples underwent an initial semen analysis according to the WHO 5th edition. All samples were found eligible and no sample was required to be excluded.
A total of 10 samples were evaluated for sperm motility. Samples were analyzed at different time points (0, 60, 150, 240, and 300 minutes) based on the half-life of RA in plasma. All concentrations of RA used in this study significantly (p<0.05) increased sperm motility with a maximum after 240 minutes of incubation (
A total of 13 samples were evaluated for sperm vitality (
A total of 8 samples were evaluated for ORP before and after exposure to RA. Samples were analyzed at 3 time points (0, 240, and 300 minutes) after the observed increase in sperm motility was observed in 60 and 150 minutes (
MPH in its various administration forms is widely used for treatment of ADHD in the general population, including those patients at reproductive age. Increasing age of the treated patients and increased rates of MPH abuse have been reported as well [
Results of the current study clearly show that MPH main metabolite, RA, was able to increase human sperm motility, while sperm kinematic parameters, such as LIN, ALH, and VCL were not affected. In addition, vitality was maintained over time at significantly higher levels as compared to the control. It is also important to note that despite increased oxidative stress (OS) levels as measured as ORP after 240 and 300 minutes, respectively, these increases were not significant. Moreover, the impact of RA on sperm functions was comparable in all studied RA concentrations including the subclinical dose of 1 ng/mL, and the abuse equivalent doses of 100 and 1,000 ng/mL. However, due to the fact that the actual RA concentrations in semen have never been reported before, concentrations used in this study were only based on plasma levels and has to be considered as a limitation of our study.
The exact mechanism of action of MPH in ADHD treatment is still debatable. It is hypothesized that MPH acts as a catecholamine reuptake inhibitor impacting mainly dopamine and norepinephrine levels. MPH blocks the dopamine transporter in the central nervous system, thus causing an increased dopamine concentration in the synaptic cleft [
The observed increased motility cannot be attributed to decreased OS levels as measured by ORP since these slightly, but not significantly, increased. At this stage, it can also only be speculated whether this increased ROS activity is due to increased metabolic activity of the spermatozoa or simply caused by the elimination of protective antioxidants present in the seminal plasma. Excessive ROS production that impairs the fine redox balance between oxidants and anti-oxidants (enzymatic and non-enzymatic) yield OS. In turn, OS has a well-established deteriorating effect on sperm function including reduced sperm motility, decreased ability of spermatozoa to adhere to the oocyte [
On the other hand, naturally generated low ROS levels are major role players in male reproductive physiologic processes such as spermatogenesis and the triggering of sperm capacitation. Ultimately, the molecular processes involved in sperm binding to the zona pellucida are also mediated by hydrogen peroxide. Likewise, ROS in low levels are important for proper sperm motility [
A potential application of the observed impact of RA (or any other dopamine reuptake inhibitor) on sperm cells may be as a motility enhancer. This would be of particular interest in patients with asthenozoospermia or during various fertility treatment procedures such as intra-uterine insemination (IUI). IUI is often a first line treatment in patients with adequate sperm numbers since it is minimally invasive and far less expensive when compared to
The current study has some limitations. The first is the small study sample of the evaluated parameters. Another limitation is the fact that only selected sperm parameters including vitality, total motility, VCL, ALH, LIN, and ORP were measured. However, the fact that despite the small sample significant differences were observed between the study groups reflects the substantial effect of RA on sperm function parameters.
In conclusion, RA significantly increased sperm motility and maintained vitality over a long incubation period. This may allow its
The study was supported by funds from the American Center for Reproductive Medicine. Andrology lab technologists helped with scheduling of subjects. Figures were drawn by graphic artists from the Cleveland Clinic Arts department.
Special thanks to: Reva Prabha Peer, Ibrahim Altamimi, Mohamed Iesar Mohamed, Muna Abuayash, Abdullah Alzaaqi, Fares Bamajbuor.
The study was presented in part at the American Society for Reproductive Medicine (ASRM) 2017 Scientific Congress, San Antonio, TX, USA.
Supplementary materials can be found
The impact of ritalinic acid on sperm vitality.
The impact of ritalinic acid on oxidation reduction potential.