https://doi.org/10.5812/numonthly.101792

Effects of Chronic Use of Methylphenidate on Spermatogenesis and Sexual Hormones in Adult Male Rats

authors:

avatar Alireza Akhavan Rezayat ORCID 1 , avatar Amir Abbas Asadpour ORCID 1 , * , avatar Samaneh Boroumand Noughabi 2 , 3 , avatar Hassan Ahmadnia ORCID 1 , avatar Hamid Mohseni 4 , avatar Iman Broomand 5 , avatar Maliheh Dadgar Moghadam 6

Kidney Transplant Complications Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
Department of Pathology, Mashhad University of Medical Sciences, Mashhad, Iran
Department of Hematology and Blood Banking, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Mashhad University of Medical Sciences, Mashhad, Iran
Department of Community Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Nephro-Urology Monthly: Vol.12, issue 2; e101792
published online: May 30, 2020
article type: Research Article
received: February 23, 2020
revised: April 29, 2020
accepted: May 14, 2020

how to cite: Akhavan Rezayat A, Asadpour A A, Boroumand Noughabi S, Ahmadnia H, Mohseni H, et al. Effects of Chronic Use of Methylphenidate on Spermatogenesis and Sexual Hormones in Adult Male Rats. Nephro-Urol Mon. 2020;12(2):e101792. https://doi.org/10.5812/numonthly.101792.

Abstract

Background:

The growing prevalence of Attention Deficit Hyperactivity Disorder (ADHD) and the non-medical use of Methylphenidate (MPH) among the youth have lead male infertility to be a major health problem.

Objectives:

The present study was conducted to investigate the impacts of MPH administration on different aspects of productivity, including total body weight, testis weight, spermatogenesis, sperm motility, histopathology changes, and sex hormone serum levels in male rats.

Methods:

This study was performed with 54 eight-week-old male rats divided into one control and two experimental groups. The experimental groups were gavaged with 2 and 10 mg/kg methylphenidate daily while the control group was gavaged with normal saline (at the same dosage). After 60 days, rats were subjected to blood sampling and bilateral orchidoepididymectomy under anesthesia. Spermogram, histological, and hormonal evaluations were performed on the samples. Testes weight and total body weight were also recorded.

Results:

The results revealed significant differences between the MPH and experimental groups in terms of hormonal, spermographic, and histopathologic features, as well as weight. Luteinizing hormone and testosterone levels, sperm count and motility, Leydig cell hyperplasia, spermatogenesis, congestion and necrosis levels, total body weight, and testis weight were significantly different between the experimental and control groups. However, no difference was observed between the experimental and control groups concerning follicle-stimulating hormone, maturation arrest, and edema levels.

Conclusions:

Based on the findings, MPH exposure exerts a significant effect on the testis and total body weight, as well as hormonal, spermatographic, and histopathologic characteristics. Accordingly, the present study provided an insight into the negative impression of MPH on sexual parameters.

1. Background

Methylphenidate (MPH), which is also known as Ritalin, is a Central Nervous System (CNS) stimulant. This agent is a common effective drug for Attention Deficit Hyperactivity Disorder (ADHD). This disorder is a highly prevalent psychiatric disease among children (1, 2) with a worldwide prevalence of 5.29% (3). Based on the statistics, the prevalence of this condition is 2-18% in the United States (2). While the short-term use of this mediation is safe in children suffering from ADHD, its long-term impacts on different systems, including the reproductive system, are a matter of concern. Male factors play important roles in the infertility of many populations, imposing a notable financial burden on patients and governments (4).

Reproductive impairments are reported as the adverse effects of several medications.(5) The adverse effects of MPH on the male reproductive system have been reported in several studies. Some of these adverse effects include decreased testes weight, hormone levels, spermatogenesis rate, quality of sperms, and germinal cell function (6-11). The growing prevalence of ADHD and the non-medical use of MPH among the youth (12) have lead male infertility to be a major health problem (4).

2. Objectives

With this background in mind, the current study was performed to investigate the impacts of MPH administration on important parameters of productivity in male rats. These parameters included total body weight, testis weight, spermatogenesis, sperm motility, histopathologic changes, and sex hormone serum levels.

3. Methods

3.1. Study Population

This study was conducted on 54 Wistar male rats from November 2017 to March 2018 at the Animal Laboratory of the Medical Faculty of Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. The rats were randomly divided into three groups of 18. They aged eight weeks with an average weight of 250 g. The rats were obtained from the Animal Laboratory of the Medical Faculty of MUMS. Before and after the study, they were examined by a veterinarian, and none of them had any health issues. In all steps of this study, the animals were treated as per the protocols of animal studies. They were kept in a standard temperature and humidity condition. Hydration and nutrition were adequate, and they were all normal rats.

3.2. Procedures

For 60 days, the control group (group C) received 1 ml of normal saline solution by the gavage method at 6 p.m. The experimental groups, namely low-dose (LD) and high-dose (HD), were gavaged with 2 and 10 mg/kg of MPH, respectively, at 7 p.m. for the same period. Gavaging was implemented using particular syringes in all groups (13).

3.3. Research Evaluations

On the 61st day, rats were taken out of their cages under required conditions, and then weighted and completely anesthetized in an ether container. After obtaining blood samples from the carotid artery in dry tubes, they were instantly sent to a hormone laboratory. In the next stage, the samples were centrifuged, sera were removed, and hormonal levels were measured by special kits. The levels of testosterone, Luteinizing Hormone (LH), and Follicle-Stimulating Hormone (FSH) were measured, as well.

Midline abdominal incision was performed, and the testes and epididymis were excised completely. Subsequently, the two ends of the epididymis were closed, kept in 2 mL of normal saline solution at 37ºC, and rapidly transferred to the laboratory for spermogram assessments, including sperm count and motility. Other reproductive organs, including prostate and seminal vesicles, were also excised. The samples were sent to a pathologist in separate particular containers of formalin 10% solution. The recorded data included the testes weight and histopathologic properties, including edema, congestion, necrosis, maturation arrest, Leydig cell hyperplasia, and decreased spermatogenesis. The study protocol was approved by the Medical Ethics Committee of MUMS. Besides, MPH caused no toxicity to rats.

3.4. Statistical Analysis

The biochemical and histopathologic data were analyzed by SPSS software. Group features were also analyzed using descriptive statistical methods, including central indicators of dispersion and frequency distribution. The normality of data was tested using the Shapiro-Wilk statistical test. Then, appropriate tests were used according to the normal distribution of data. The ANOVA and chi-square tests were used for the ordinal qualitative and morphologic data, respectively. A P value of less than 0.05 was considered statistically significant.

4. Results

Three groups of 18 rats with an average age of 60 days and a weight of 250 g were used in this study. The results indicated a significant difference between the control and experimental groups in LH and testosterone levels (P = 0.007 and P < 0.001, respectively), but not in FSH. Table 1 presents the differences between the groups. Concerning spermographic variables, the sperm count and motility were significantly higher in both of the experimental groups than in the control group (P < 0.001 for both). Nevertheless, there were insignificant differences between the two experimental groups in this regard. Table 1 tabulates the mean values of the variables. The body weight (P = 0.022) and testis weight (P = 0.021) were lower in the experimental groups than in the control group. The LD group (2 mL/kg) had no significant difference with the control group in terms of the mentioned variables. However, the HD group (10 mL/kg) was significantly different from the control group in this respect (Table 1). In addition, there was no significant difference between the LD and HD groups regarding the body weight and testis weight. Histopathologic studies revealed significant changes in Leydig cell hyperplasia (P = 0.001). These variations included a decrease in spermatogenesis, testis congestion, and necrosis in both of the experimental groups and the control group (P = 0.013, P < 0.001, and P < 0.001, respectively). Nonetheless, no significant changes were observed in maturation arrest and edema (Figures 1 and 2). There were four pathologic stages (stage 0 - 3) for each variable. Table 2 presents the distribution of variables in stages.

Normal seminiferous tubules in a control rat (H & E, x 100), scale bar = 250 μm.
Normal seminiferous tubules in a control rat (H & E, x 100), scale bar = 250 μm.
Figure 1.

Normal seminiferous tubules in a control rat (H & E, x 100), scale bar = 250 μm.

Edema and congestion in testis tissue of a rat from the LD group (H & E, x 100), scale bar = 250 μm.
Edema and congestion in testis tissue of a rat from the LD group (H & E, x 100), scale bar = 250 μm.
Figure 2.

Edema and congestion in testis tissue of a rat from the LD group (H & E, x 100), scale bar = 250 μm.

Table 1.

Mean ± Standard Deviation (SD) and P values of Comparison Between the Study Groups for Luteinizing Hormone, Follicle-Stimulating Hormone, and Testosterone Levels, Sperm Count and Motility, Rat Weight, and Testis Weight

VariableGroupsMean ± SDP Valuea
Between Each Case Group and Control GroupBetween LD and HDOverall
LHLD0.1951 ± 0.0480.1050.5490.007
HD0.2081 ± 0.0370.008
C0.1689 ± 0.014--
FSHLD0.1322 ± 0.065--0.08
HD0.1356 ± 0.067
C0.08586 ± 0.084
TestosteroneLD139.16 ± 580.040.2< 0.001
HD107.83 ± 58< 0.001
C183.66 ± 54--
Sperm count, million/mLLD15.77 ± 10.0020.566< 0.001
HD11.54 ± 0.7< 0.001
C30.55 ± 1.6--
Sperm motility. /mLLD1.44 ± 2.06< 0.0010.23< 0.001
HD0.11 ± 0.32< 0.001
C12.27 ± 3.4--
Rat weight, gLD315 ± 350.7170.150.022
HD299 ± 290.027
C328 ± 27--
Testis weight, gLD1.42 ± 0.350.7560.1230.021
HD1.33 ± 0.290.027
C1.45 ± 0.27--

aAll P values in this table resulted from the ANOVA test.

Table 2.

Distribution of Rats in Four Grades of Leydig Cell hyperplasia, Decreased Spermatogenesis, Maturation Arrest, Edema, Testis Congestion, and Necrosis in Each Group

VariableGroupsStagesP Valuea
0123
Leydig cell hyperplasiaLD180000.001
HD12600
C18000
Decreased spermatogenesisLD48600.013
HD6642
C13500
Maturation arrestLD180000.191
HD16002
C17100
EdemaLD412200.136
HD41220
C10800
Testis congestionLD01800< 0.001
HD01260
C71100
NecrosisLD16200< 0.001
HD71100
C18000

aAll P values in this table resulted from the chi-square test

5. Discussion

The results of the present study indicated significant differences between the MPH and experimental groups in terms of hormonal, spermographic, and histopathologic characteristics, as well as weight. In this regard, no significant difference was observed between experimental groups regarding LH and testosterone levels, sperm count and motility, Leydig cell hyperplasia, spermatogenesis, congestion and necrosis levels, total body weight, and testis weight. However, there was no significant difference between the control and the experimental groups considering FSH, maturation arrest, and edema levels. The HD and control groups were significantly different in terms of LH levels, body weight, and testis weight (Figures 3 and 4). However, no such difference was observed between the LD and control groups. In addition, there was no significant difference between the two experimental groups in terms of any of the evaluated variables.

Severe atrophy and disappeared germinal cells in a rat from the HD group. Very few numbers of spermatogonia (white arrow), mostly Sertoli cells (black arrow) are seen inside the tubules. Leydig cells are between tubules (quad arrow), (H & E, x 400), scale bar = 55μm.
Severe atrophy and disappeared germinal cells in a rat from the HD group. Very few numbers of spermatogonia (white arrow), mostly Sertoli cells (black arrow) are seen inside the tubules. Leydig cells are between tubules (quad arrow), (H & E, x 400), scale bar = 55μm.
Figure 3.

Severe atrophy and disappeared germinal cells in a rat from the HD group. Very few numbers of spermatogonia (white arrow), mostly Sertoli cells (black arrow) are seen inside the tubules. Leydig cells are between tubules (quad arrow), (H & E, x 400), scale bar = 55μm.

Notable height decrease in the germinative epithelium in a rat from the HD group (H & E, x 400). Black arrow: Sertoli cells, white arrow: spermatogonia, black chevron: primary spermatocytes, white chevron: spermatids, scale bar = 5
Notable height decrease in the germinative epithelium in a rat from the HD group (H & E, x 400). Black arrow: Sertoli cells, white arrow: spermatogonia, black chevron: primary spermatocytes, white chevron: spermatids, scale bar = 5
Figure 4.

Notable height decrease in the germinative epithelium in a rat from the HD group (H & E, x 400). Black arrow: Sertoli cells, white arrow: spermatogonia, black chevron: primary spermatocytes, white chevron: spermatids, scale bar = 5

The current study revealed a lower body weight in the high-dose MPH group, which is in line with the findings of several studies (7, 14). In this regard, Carias et al. reported a decrease in food intake and body weight in rats treated with high-dose MPH. They also observed a decrease in water intake in both high- and low-dose groups. In the mentioned study, 8 and 30 ng/ml levels were considered as the peak serum concentrations in the low- and high-dose groups, respectively (15). Furthermore, Robison et al. observed that high-dose MPH decreased total weight in male and female rats while this treatment increased food intake only in females. A possible explanation for this result is that the compensation of energy loss resulted from MPH-induced hyperactivity (16). Other trials that treated rats with low doses of MPH reported insignificant weight alterations (9, 17). Similarly, our results indicated a significant difference only in the high-dose group. Based on our findings and those of the previous studies, it seems that there are some dose-dependent relationships between treatment and weight loss (17-19). Similar results have been reported in human studies. Weight reduction is the most common adverse effect of MPH in adults (20, 21). These findings may explain the relationship between obesity and ADHD (22), the role of MPH in the treatment of ADHD-related obesity (23), and the possible mechanism of decreased appetite due to the drug (24). Several pieces of evidence have shown a transient decrease following MPH administration in both humans (19, 25-30) and animals (17, 18, 31), which makes a rebound after treatment cessation (11). In line with the present results, previous studies have demonstrated a decrease in the testis weight, in addition to the prostate and seminal vesicles weight following MPH treatment in rats (11, 32). Teo et al. reported a rebound weight gain in the prostate 30 days after the last MPH administration (18). Montagnini et al. observed no significant alteration 40 days after the experiment (9). According to the blocking effect of MPH on Noradrenaline (NA) and Dopamine (DA) transporters, the presence of DA and NA receptors in testicular tissue may directly affect MPH on the testis (33, 34). Furthermore, germ cell depletion has been mentioned as the possible role player in the literature (35). The results obtained by Fazelipour et al., investigating hormonal changes, namely elevated LH, decreased testosterone, and insignificant changes of FSH, are relatively in line with our findings. They believe that despite the role of reduced testosterone secretion by Leydig cells and the subsequent rise in LH in these changes, the increased liver metabolism of the testosterone-producing enzyme could be the point and the insignificant elevation in FSH reinforces this hypothesis (6, 7, 36). The literature is indicative of the effect of MPH on pulsatile gonadotropin-releasing hormone (GnRH) release (36) and direct impairment of Leydig cells (6). However, there are also reports on the transient negative effect of MPH on testosterone, as well as body and testis weight (6, 37). There are multiple studies indicating spermatogenesis and spermiogenesis impairments, including decreased sperm count and altered morphology (7, 9, 11, 38). In an investigation carried out by Cansu et al. (11), more negative effects were observed in the high-dose group, which is in line with the dose-dependency of some MPH adverse effects. They ascribed this alteration to the increased p53 immunoreactive cell number in the high-dose group, higher testis apoptotic cell count, and lower Transforming Growth Factor (TGF)-β1 activity in high- and low-dose groups. TGF-β1 has both stimulatory and inhibitory effects on cell proliferation and spermatogenesis in rats (39-45). Nonetheless, it is not present in human seminiferous ducts (46). MPH affects dopamine transport, D2 receptors, serotonin, and noradrenaline (31, 35, 47), and D2 and α- and β-adrenergic receptors are expressed in the testis and spermatozoa of rat (33, 34). The toxicity caused during spermatogenesis could lead to apoptosis (48, 49). The increased apoptosis and p53 expression are discussed as the causes of sperm count decrease in some other studies (7, 11, 38). Due to the effect of the GnRH level on FSH (50) and the function of FSH in the primary stages of spermatogenesis (51-53), FSH changes could disrupt the spermatogenesis process. However, no significant difference was observed in the FSH levels in the current study and the one performed by Fazelipour et al. (7). In the present study, Leydig cell hyperplasia was observed in the HD group, which is contrary to the previous findings indicating the decreased Leydig cell count (8) or the absence of significant changes (9). Accordingly, further studies are needed to explain the exact effect of MPH on Leydig cells. Necrosis was another significantly increased parameter in the MPH groups. Necrosis has been rarely evaluated and discussed in the literature. Intraperitoneal injection of 30 mg/kg of hydrochloride cocaine is reported to cause necrosis and decrease the number of testis interstitial cells (54). In a study carried out by Cansu et al., necrosis was not detected (11). The responsible mechanism may be similar to the one explained for apoptosis. This finding also highlights the importance of investigating the cytotoxic effects of MPH. Congestion is also a matter of controversy that was considered in the present study. While congestion was higher in the MPH group, some studies did not detect significant congestion (11) or any associated degrees (7). To indicate limitations and strengths, we investigated a wide range of variables, including hormonal, spermatographic, and histopathologic characteristics, as well as weight. Furthermore, the two LD and HD groups were examined to illustrate dose-dependent changes. Chronic exposure could be another positive point in this regard. Some delayed or rebound effects were not presented at the termination point of medication in the study. Therefore, it is necessary to evaluate rats after medication termination. Additionally, given the probable role of weight loss in sexual dysfunction, this variable should be further investigated. In this respect, changes in weight, not the final weight, may be more accurate to assess the consequences of MPH therapy.

5.1. Conclusions

Sexual hormones, especially, methylphenidate have the potential to cause infertility. These changes may result from the effect of drugs on the central nervous system, including hypophysis or hypothalamus, the direct effect of drugs on the testis, or both of them. These findings suggest that methylphenidate must be used just in indicated patients, and the overuse of this drug can increase the infertility rate in society. Awareness about its adverse effects may decrease the infertility rates.

References

  • 1.

    Berger I. Diagnosis of attention deficit hyperactivity disorder: much ado about something. IMAJ-Israel Medical Association Journal. 2011;13(9):571.

  • 2.

    Sharma A, Couture J. A review of the pathophysiology, etiology, and treatment of attention-deficit hyperactivity disorder (ADHD). Annals of Pharmacotherapy. 2014;48(2):209-25.

  • 3.

    Polanczyk G, De Lima MS, Horta BL, Biederman J, Rohde LA. The worldwide prevalence of ADHD: a systematic review and metaregression analysis. American journal of psychiatry. 2007;164(6):942-8.

  • 4.

    Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reproductive Biology and Endocrinology. 2015;13(1):37.

  • 5.

    Delashoub M, Ziaee M, Khorrami A, Banan-Khojasteh SM. Comparison of the Effects of Clofibrate and Silafibrate on Sperm Parameters Quality and Sex Hormones in Male Rats. Urol J. 2018;15(2):38-43. [PubMed ID: 29299890]. https://doi.org/10.22037/uj.v0i0.3954.

  • 6.

    Adriani W, Leo D, Guarino M, Natoli A, Di Consiglio E, De Angelis G, et al. Short‐term effects of adolescent methylphenidate exposure on brain striatal gene expression and sexual/endocrine parameters in male rats. Annals of the New York Academy of Sciences. 2006;1074(1):52-73.

  • 7.

    Fazelipour S, Tootian Z, Saremi ZG, Shafii M, Sheibani MT, Kiaei SB, et al. Evaluation of histopathologic and histomorphometric changes of testicular tissue and gonadotropin levels following consumption of methylphenidate in male mice. Turkish journal of medical sciences. 2014;44(4):554-9.

  • 8.

    Fazelipour S, Jahromy MH, Tootian Z, Kiaei SB, Sheibani MT, Talaee N. The effect of chronic administration of methylphenidate on morphometric parameters of testes and fertility in male mice. J Reprod Infertil. 2012;13(4):232-6. [PubMed ID: 23926551]. [PubMed Central ID: PMCPmc3719348].

  • 9.

    Montagnini BG, Silva LS, dos Santos AH, Anselmo-Franci JA, Fernandes GS, Mesquita Sde F, et al. Effects of repeated administration of methylphenidate on reproductive parameters in male rats. Physiol Behav. 2014;133:122-9. [PubMed ID: 24866909]. https://doi.org/10.1016/j.physbeh.2014.05.016.

  • 10.

    [No authors listed]. Reproductive toxicology. Methylphenidate hydrochloride. Environ Health Perspect. 1997;105(Suppl 1):319-20. [PubMed ID: 9114343]. [PubMed Central ID: PMCPmc1470256].

  • 11.

    Cansu A, Ekinci Ö, Ekinci Ö, Serdaroglu A, Erdoğan D, Coşkun ZK, et al. Methylphenidate has dose-dependent negative effects on rat spermatogenesis: decreased round spermatids and testicular weight and increased p53 expression and apoptosis. Human & experimental toxicology. 2011;30(10):1592-600.

  • 12.

    Grant JE, Redden SA, Lust K, Chamberlain SR. Nonmedical Use of Stimulants Is Associated With Riskier Sexual Practices and Other Forms of Impulsivity. J Addict Med. 2018. [PubMed ID: 30095567]. https://doi.org/10.1097/adm.0000000000000448.

  • 13.

    Ahmadnia H, Akhavan Rezayat A, Hoseyni M, Sharifi N, Khajedalooee M, Akhavan Rezayat A. Short-Period Influence of Chronic Morphine Exposure on Serum Levels of Sexual Hormones and Spermatogenesis in Rats. Nephrourol Mon. 2016;8(4). e38052. [PubMed ID: 27713869]. [PubMed Central ID: PMC5045526]. https://doi.org/10.5812/numonthly.38052.

  • 14.

    Gulati D, Hope E, Mounce R, Russell S. Methylphenidate hydrochloride. Environmental Health Perspective. 1997;105(Supplement 1).

  • 15.

    Carias E, Fricke D, Vijayashanthar A, Smith L, Somanesan R, Martin C, et al. Weekday-only chronic oral methylphenidate self-administration in male rats: Reversibility of the behavioral and physiological effects. Behavioural brain research. 2019;356:189-96.

  • 16.

    Robison LS, Michaelos M, Gandhi J, Fricke D, Miao E, Lam C, et al. Sex differences in the physiological and behavioral effects of chronic oral Methylphenidate treatment in rats. Frontiers in behavioral neuroscience. 2017;11:53.

  • 17.

    Beckman DA, Schneider M, Youreneff M, Tse FL. Juvenile toxicity assessment of d, l‐methylphenidate in rats. Birth Defects Research Part B: Developmental and Reproductive Toxicology. 2008;83(1):48-67.

  • 18.

    Teo S, Stirling D, Thomas S, Hoberman A, Kiorpes A, Khetani V. A 90-day oral gavage toxicity study of d-methylphenidate and d, l-methylphenidate in Sprague–Dawley rats. Toxicology. 2002;179(3):183-96.

  • 19.

    Golub M, Costa L, Crofton K, Frank D, Fried P, Gladen B, et al. NTP‐CERHR Expert Panel Report on the reproductive and developmental toxicity of methylphenidate. Birth Defects Research Part B: Developmental and Reproductive Toxicology. 2005;74(4):300-81.

  • 20.

    Poulton ACCT, Cowell CT. Slowing of growth in height and weight on stimulants: a characteristic pattern. Journal of paediatrics and child health. 2003;39(3):180-5.

  • 21.

    Ptacek R, Kuzelova H, Paclt I. Effect of stimulants on growth of ADHD children: a critical review. Activitas Nervosa Superior. 2009;51(4):140-6.

  • 22.

    Cortese S, Angriman M, Maffeis C, Isnard P, Konofal E, Lecendreux M, et al. Attention-deficit/hyperactivity disorder (ADHD) and obesity: a systematic review of the literature. Critical reviews in food science and nutrition. 2008;48(6):524-37.

  • 23.

    Levy LD, Fleming JP, Klar D. Treatment of refractory obesity in severely obese adults following management of newly diagnosed attention deficit hyperactivity disorder. International Journal of Obesity. 2009;33(3):326.

  • 24.

    Wax PM. Analeptic use in clinical toxicology: a historical appraisal. Journal of Toxicology: Clinical Toxicology. 1997;35(2):203-9.

  • 25.

    Charach A, Figueroa M, Chen S, Ickowicz A, Schachar R. Stimulant treatment over 5 years: effects on growth. Journal of the American Academy of Child & Adolescent Psychiatry. 2006;45(4):415-21.

  • 26.

    Spencer TJ. OROS methylphenidate treatment for ADHD: long term effect on growth. 50th Annual Meeting of the American Academy of Child and Adolescent Psychiatry, Miami, October. 2003. p. 14-9.

  • 27.

    Klein RG, Mannuzza S. Hyperactive boys almost grown up: III. Methylphenidate effects on ultimate height. Archives of General Psychiatry. 1988;45(12):1131-4.

  • 28.

    Satterfield JH, Cantwell DP, Schell A, Blaschke T. Growth of hyperactive children treated with methylphenidate. Archives of general psychiatry. 1979;36(2):212-7.

  • 29.

    Faraone SV, Biederman J, Morley CP, Spencer TJ. Effect of stimulants on height and weight: a review of the literature. Journal of the American Academy of Child & Adolescent Psychiatry. 2008;47(9):994-1009.

  • 30.

    Hechtman L. Long-term treatment of children and adolescents with attention-deficit/hyperactivity disorder (ADHD). Current psychiatry reports. 2006;8(5):398-408.

  • 31.

    Gray JD, Punsoni M, Tabori NE, Melton JT, Fanslow V, Ward MJ, et al. Methylphenidate administration to juvenile rats alters brain areas involved in cognition, motivated behaviors, appetite, and stress. Journal of Neuroscience. 2007;27(27):7196-207.

  • 32.

    Manjanatha MG, Shelton SD, Dobrovolsky VN, Shaddock JG, McGarrity LG, Doerge DR, et al. Pharmacokinetics, dose‐range, and mutagenicity studies of methylphenidate hydrochloride in B6C3F1 mice. Environmental and molecular mutagenesis. 2008;49(8):585-93.

  • 33.

    Otth C, Torres M, Ramírez A, Fernandez JC, Castro M, Rauch MC, et al. Novel identification of peripheral dopaminergic D2 receptor in male germ cells. Journal of cellular biochemistry. 2007;100(1):141-50.

  • 34.

    Adeoya-Osiguwa SA, Gibbons R, Fraser LR. Identification of functional α2-and β-adrenergic receptors in mammalian spermatozoa. Human Reproduction. 2006;21(6):1555-63.

  • 35.

    Lanning LL, Creasy DM, Chapin RE, Mann PC, Barlow NJ, Regan KS, et al. Recommended approaches for the evaluation of testicular and epididymal toxicity. Toxicologic pathology. 2002;30(4):507-20.

  • 36.

    Chatterjee-Chakrabarty S, Miller BT, Collins TJ, Nagamani M. Adverse effects of methylphenidate on the reproductive axis of adolescent female rats. Fertility and sterility. 2005;84:1131-8.

  • 37.

    Kianifard D, Hasanzadeh S, Kianifard L. The study of time dependent administration of methylphenidate on the microscopic indices of spermatogenesis and sperm analysis in adult rats. Journal of Experimental & Integrative Medicine. 2013;3(2).

  • 38.

    Yang GS, Wang W, Wang YM, Chen ZD, Wang S, Fang JJ. Effect of cocaine on germ cell apoptosis in rats at different ages. Asian J Androl. 2006;8(5):569-75. [PubMed ID: 16752006]. https://doi.org/10.1111/j.1745-7262.2006.00191.x.

  • 39.

    Konrad L, Keilani MM, Laible L, Nottelmann U, Hofmann R. Effects of TGF-betas and a specific antagonist on apoptosis of immature rat male germ cells in vitro. Apoptosis. 2006;11(5):739-48. [PubMed ID: 16532270]. https://doi.org/10.1007/s10495-006-5542-z.

  • 40.

    Olaso R, Pairault C, Boulogne B, Durand P, Habert R. Transforming Growth Factor β1 and β2 Reduce the Number of Gonocytes by Increasing Apoptosis*. Endocrinology. 1998;139(2):733-40. https://doi.org/10.1210/endo.139.2.5765.

  • 41.

    Lui WY, Lee WM, Cheng CY. TGF-betas: their role in testicular function and Sertoli cell tight junction dynamics. Int J Androl. 2003;26(3):147-60. [PubMed ID: 12755993].

  • 42.

    Godin I, Wylie CC. TGF beta 1 inhibits proliferation and has a chemotropic effect on mouse primordial germ cells in culture. Development. 1991;113(4):1451-7. [PubMed ID: 1811953].

  • 43.

    Hakovirta H, Kaipia A, Söder O, Parvinen M. Effects of activin-A, inhibin-A, and transforming growth factor-beta 1 on stage-specific deoxyribonucleic acid synthesis during rat seminiferous epithelial cycle. Endocrinology. 1993;133(4):1664-8. https://doi.org/10.1210/endo.133.4.8404607.

  • 44.

    Sanchez-Capelo A. Dual role for TGF-beta1 in apoptosis. Cytokine Growth Factor Rev. 2005;16(1):15-34. [PubMed ID: 15733830]. https://doi.org/10.1016/j.cytogfr.2004.11.002.

  • 45.

    Roberts AB, Frolik CA, Anzano MA, Sporn MB. Transforming growth factors from neoplastic and nonneoplastic tissues. Fed Proc. 1983;42(9):2621-6. [PubMed ID: 6303865].

  • 46.

    Zhang Y, He X, Zhang J, Wang R, Zhou J, Xu R. Stage-specific localization of transforming growth factor beta1 and beta3 and their receptors during spermatogenesis in men. Asian Journal of Andrology. 2004;6(2):105-9. [PubMed ID: 15154083].

  • 47.

    Kuczenski R, Segal DS. Effects of Methylphenidate on Extracellular Dopamine, Serotonin, and Norepinephrine: Comparison with Amphetamine. Journal of Neurochemistry. 1997;68(5):2032-7. https://doi.org/10.1046/j.1471-4159.1997.68052032.x.

  • 48.

    Creasy DM, Flynn JC, Gray TJ, Butler WH. A quantitative study of stage-specific spermatocyte damage following administration of ethylene glycol monomethyl ether in the rat. Experimental and Molecular Pathology. 1985;43(3):321-36. https://doi.org/10.1016/0014-4800(85)90069-3.

  • 49.

    Creasy DM. Pathogenesis of male reproductive toxicity. Toxicol Pathol. 2001;29(1):64-76. [PubMed ID: 11215686]. https://doi.org/10.1080/019262301301418865.

  • 50.

    Ciechanowska M, Łapot M, Mateusiak K, Przekop F. Neuroendocrine regulation of GnRH release and expression of GnRH and GnRH receptor genes in the hypothalamus-pituitary unit in different physiological states. Reproductive Biology. 2010;10(2):85-124. https://doi.org/10.1016/S1642-431X(12)60054-0.

  • 51.

    Meachem SJ, Wreford NG, Stanton PG, Robertson DM, Mclachlan RI. Follicle‐stimulating hormone is required for the initial phase of spermatogenic restoration in adult rats following gonadotropin suppression. Journal of Andrology. 1998;19(6):725-35.

  • 52.

    Boitani C, Politi MG, Menna T. Spermatogonial cell proliferation in organ culture of immature rat testis. Biology of reproduction. 1993;48(4):761-7.

  • 53.

    Ding L, Yan G, Ge Q, Yu F, Zhao X, Diao Z, et al. FSH acts on the proliferation of type A spermatogonia via Nur77 that increases GDNF expression in the Sertoli cells. FEBS letters. 2011;585(15):2437-44.

  • 54.

    Barroso-Moguel R, Mendez-Armenta M, Villeda-Hernandez J. Testicular lesions by chronic administration of cocaine in rats. J Appl Toxicol. 1994;14(1):37-41. [PubMed ID: 8157868].