Aspirin (Acetylsalicylic acid) is used widely in human and veterinary as cardiovascular prophylaxis and as anti-inflammatory, anti-clotting agent and to decrease cancer risk. But there is a deficiency of information about its effects on androgenic studies histology of the testes, kidneys and livers and reproductions. In this paper we tried to collect the information about it deleterious toxicity effect on male rat reproduction and the reason of its side effects. Our finding shows that Aspirin has deadly effect the production of sperm, decrease the testis weight, decrease the production of testosterone and also affect the blood profile. Effect of aspirin on the male reproductive system may be due to being a Prostaglandins inhibitor, and According to all our findings probably it indicates that aspirin have deleterious effect on reproduction and the blood chemistry of male rats

Keywords: aspirin, toxicity, male rat, spermatogenesis, haematology

Aspirin (Acetylsalicylic acid) was introduced to treating human more than 100years ago¹ due to its anti-inflammatory and anti-pyretic benefits it is widely used in clinical settings. First of all it was reported by Vane JR² that aspirin and non-steroidal anti-inflammatory drugs inhibit the synthesis of pro-inflammatory prostaglandin E2.lattor on it was reported that aspirin has inhibitory effect on cyclooxygenase-1 (COX-1) activity by acetylating serine 530³ and COX-2.⁴

Aspirin has been reported effected response to decrease the risk of cancer,⁵ Heart problems⁶ due to its anti thombotic action.⁷ The anti-platelet effect of aspirin has been tested in various forms of coronary artery disease, pregnancy-induced hypertension and preeclampsia in angiotensin-sensitive primigravida at low dosage and showed positive results in most of the reports.⁸ Today aspirin is most widely used medicine all over world, according to estimation 4000tonnes of it is used every year.⁹

On other hand it is also reported that it has many adverse effects like long term use of it causes gastro intestinal,¹⁰ stomach problem, and cerebral problems.¹¹ The increase in incidences of infertility in male due to frequent use of a number of therapeutic drugs has made efforts to study their untoward side effects on the male reproduction. Various drugs used for treating diseases are reported to cause male infertility approximately 50% of infertility is related to problems in male.¹² One of very worse effect of aspirin is on reproduction of male. It was reported that aspirin caused a significant decrease in the weight of testis of immature rats.¹³ Decrease in the activities of sorbitol dehydrogenase and hyaluronidase.¹⁴ Decrease in the number of spermatids and increase in size of spermatocytes nuclei were observed.¹⁵ The process of spermatogenesis and the accessory reproductive organs function are dependent on androgen activity. Aspirin administration to normal rats resulted in hypercholesterolemia that’s might be due to decreased androgen production, which resulted in accumulation of cholesterol in testes.¹³ The impaired sperm dynamics, including spermatogenesis, could be an outcome of aspirin-induced alteration in cholesterol metabolism in testis.¹⁶

It was reported that during development around the 33^rd day there is an abrupt increase in testicular weight in male rats.¹⁷ Other secondary sexual characteristics mature between 41 and 54days and males reach adult body weight after 54days of age.¹⁸

The Seminiferous tubules and contents are the primary contributor to testicular weight. The process of spermatogenesis occurs in the Seminiferous tubules of the testis and within the Seminiferous tubules testosterone is the major androgen present.¹⁹ Either a decreased length of Seminiferous tubules or a decreased density of elements within a given length including decreased density due to degeneration of spermatogenic elements would affect testes weight and decreased length of Seminiferous tubules implies either fewer spermatogonial stem cells or a lower mitotic activity of these stem cells.²⁰ Testis weight are correlated with fertility because larger testicular weight have been associated with an increase in daily sperm production, daily sperm output, Sertoli cell numbers and smaller testis is associated with poorer fertility due to less no of sperm production.²¹

Aspirin decrease the testicular weight,¹³ decrease in the weight and nuclear volume of the Leydig cells indicates that Aspirin alters the steroidogenic function of the Leydig cells.

On other hand aspirin did not have any effect on body weight which is sign that aspirin was not toxic to the animals as well as non-androgenic in nature, since androgens are known to posses anabolic activities like stimulating the development and growth of the skeleton and skeletal muscles.²²

In a germ cell's path to make a spermatozoon from a spermatogonium, a spermatogonium divides by mitosis in the basal compartment, of the seminiferous tubule, to produce either stem cells or committed spermatogonia that ultimately become primary spermatocytes. These cells pass through the blood-testis barrier of the Sertoli cell tight junctions as they move into the adluminal compartment. They continue their development in the immunologic-protected site of the adluminal compartment.²³ Hyper activation of sperm is essential prerequisite for penetration through the cumulus mass and zona pellucid.²⁴ A variety of agents in mild to moderate concentrations are known to inhibit sperm hyper activation in vitro including amino acids neurb transmitters calcium channel blockers, calmodulin antagonists and analgesics.²⁵

Sperm maturation within the epididymis of the rat has been shown by the greater ability of spermatozoa collected from the cauda epididymidis, over those from the caput, to fertilize eggs after insemination into the uterus.26 But according to founding rat treated with aspirin had decreased no of sperm.¹³

The effects of aspirin in the total number, motility, and effectiveness of the sperm are the most important factors affecting fertility, Increase the number of dead sperm to half of the total leads to complete infertility.¹⁴ The ability of aspirin to affect the motility is the sign that aspirin was able to permeate the blood-testis barrier with a resultant alteration in the microenvironment of the inner part of the wall of the Seminiferous tubules.²² Aspirin not only decreases the no of sperm and its motility but also including to it causes the decreased in increase in percentage of morphological abnormal sperm cell that may be due to interference of aspirin in spermatogenesis process or due to interaction of testosterone on hypothalamic release factor which can cause changes in spermatogenesis.²⁷ Aspirin can uncouple oxidative phosphorylation in addition to its COX inhibiting activity,²⁸ this pathway of action can also contribute for the aspirin induced impairment of sperm hyper activation.

Gholami M et al.²⁹ explained that giving aspirin to adult mice cause a decrease in the activity of certain enzymes such as Hyalurounidase, which is secreted by sperm and contributes to the process of ovarian Penetrate rubble during the process of fertilization. The effect of aspirin can be through the impact on the flow of calcium into sperm which is necessary for the formation, activation, and movement of sperm.³⁰

Aspirin-induced reduction in sperm dynamics might be an outcome of depressed levels of androgens. Interestingly, subchronic aspirin administration influenced androgen dependent parameters including that of reduced sperm count, motility, and density. The observed reduced sperm activity profile might be an outcome of androgen depletion at target level, particularly in the cauda epididymis, thereby affecting physiological maturation of the sperm.¹³ Decrease in activity of hyaluronidase, decrease in number of spermatid and increase in size of spermatocytes nuclei is the sign that aspirin cause impairment on later stage of spermatogenesis.

Testosterone is the principle male sex hormones in mammals, birds and other vertebrates; it is produced in the leydig cells within the testes.³¹ Primary action of testosterone is anabolic growth, spermatogenesis promotion and promotion of secretion from the accessory sex glands. Testosterone plays an important role in increasing the Fertilization portability of sperm in vivo and in vitro.³² Testosterone receptor (androgen receptor) are present on Leydig cells, peritubular cells and Sertoli cells but in germ cells of the mature testis have no any receptor³³ that’s why testosterone don’t target germ cell directly, instated of that testosterone target the sertoli cell and these sertoli cell nourish the germ cell to differentiate in spermatozoa.³⁴ Testosterone is the maintenance and reforming of the blood-testis barrier that blood–testis barrier provides a specialized environment for the development of the germ cells and prevents immunogenic germ cell antigens from reaching the immune system.³⁵ Aspirin treated group is possibly an indication of testosterone deficiency.³⁶ Aspirin treatment causes a decrease in Leydig cell function, change in Sertoli cell morphology,³⁷ so it can cause decrease in testosterone production.²² Testosterone levels in the adult testis of rat should be likely stable and high because germ cell development does not progress beyond the pachytene stage of meiosis if its level is unstable or low.³⁸ It was found that aspirin has anti androgenic effect; alter masculinisation and potent inhibitors of prostaglandin (PG).³⁹

When LH binds to the Leydig cells, it stimulates the cellular messenger cAMP to activate protein kinase A. Protein kinase A undergoes a series of phosphorylations that in turn activate a series of enzymes that synthesis testosterone from the cholesterol base molecule.⁴⁰ Aspirin decrease the production of luteinizing hormones which lead to decrease in decrease testosterone hormone, a significant effect in decrease the effectiveness of sperm, as that testosterone hormone increases the secretions of the epididymis and seminiferous tubules which has an important role in transmission of sperm.¹⁴ Including to it testosterone is also required to release of sperm, In the absence of testosterone, mature sperm are phagocytized by the Sertoli cells.⁴¹

Testis is composed of seminiferous tubules and interstitial tissues. Seminiferous tubules are the site for spermatogenesis and they contain three types of cells: male germ cells, Sertoli cells, and myoid cells, while Leyding cell are locate between neighboring Seminiferous tubules.⁴² According to developmental progression at the base of the Seminiferous tubules have Spermatogonia, spermatocytes in the middle, and spermatids near the apex of the Seminiferous epithelium.²³ The aspirin treated rats showed cytological and nuclear degenerative changes in Seminiferous leading to shrinkage to Seminiferous tubules.¹³

In Seminiferous tubules Sertoli cells are the most important cell as because germ cell proliferation depend on it, it provides the nutrition to growing germ cell including to it Sertoli cells are involved in the release of spermatids into the Seminiferous tubule lumen⁴³ blood test is barrier, regulating the testicular vasculature.⁴⁴ leyding cell are involve in production of reproductive hormones.¹⁹ It was reported that aspirin toxicity caused the reduction primary spermatogonia, secondary spermatogonia, spermatocytes, and Leydig’s cell.¹³

Ablating Sertoli cells germ cells, seminiferous tubules leyding cell results in reduction in testis size. The impairment of spermatogenesis partly originates from abnormal quantitative increment or functional maturation of Sertoli cells.⁴⁵ Decreasing in number of Sertoli cell result decreased intratesticular vascular volume, decrease in number of germ cell, increased permeability of the blood-testis barrier, and structural changes in the Leydig cells and decreased testosterone secretion.⁴⁶ Decreased testicular vasculature leads to a reduction in fluid exchange between the vasculature and testicular interstitial, which reduces gonadotropins-stimulated circulating testosterone concentrations, indicative of reduced Leydig cell stimulation and/or reduced secretion of testosterone into the vasculature leading to infertility.⁴⁴

Blood is a complex two-phase fluid, made up of plasma and formed elements. The formed elements consist of RBCs or erythrocytes, white blood cells (WBCs or leukocytes) and platelets.

The primary function of RBCs is the transportation and delivery of oxygen to the peripheral tissues. However, their mechanical and flow properties are responsible for the complex fluid dynamics which occur in micro vessels.⁴⁷ As a corollary, altered biomechanical properties of RBCs can result in impaired oxygen and nutrient supply to peripheral tissues.⁴⁸ According to finding of¹⁴ low doses aspirin don’t have any significant effect on production of RBCs, but on other hand it significantly reduces the production of Leukocytes.¹³ Leukocytes in addition to their primary role in inflammation and immune system also play a part in modulating blood flow.⁴⁹ So suggests that decrease in number of WBC the compromised immune system and significant reduction in monocyte count probably indicates that the phagocytic function of the body has been compromised by aspirin.⁵⁰

Aspirin causes significant reductions in packed cell volume (PCV) and haemoglobin(Hb) values¹³ and it induction of anaemia and decrease in oxygen- carrying capacity of the blood as well as the amount of oxygen delivered to the tissues.⁵¹ Including to all this it is also reported that sub-chronic aspirin treatment in both low-dose and in low-followed-by-higher dose significantly potentiated the glucose lowering effect of.⁵²

Aspirin is worldwide extensively used medicine both in human and veterinary side. But Aspirin toxicity has deleterious effect on the blood chemistry and reproductive efficiency of Male. So, it is recommended that caution should be exercised in the use of aspirin.

None.

Author declares that there is no conflict of interest.

1. Wu K. Aspirin and salicylate. Circulation. 2000;102(17):2022‒2023.
2. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol. 1971;231(25):232‒235.
3. Roth GJ, Majerus PW. The mechanism of the effect of aspirin on human platelets Acetylation of a particulate fraction protein. J Clin Invest. 1975;56(3):624‒632.
4. Kurumbail RG, Stevens AM, Gierse JK, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature. 1996;384(6610):644‒648.
5. Manzano A, Segura PP. Colorectal cancer chemoprevention is this future of colorectal cancer prevention. Scientific World Journal. 2012;2012:327341.
6. Nayak MK, Dash A, Singh N, et al. Aspirin delimits platelet life span by proteasomal inhibition. PLoS One. 2014;9(8):e105049.
7. Larran AA, Pereira A, Guglielmelli P, et al. Antiplatelet therapy versus observation in low-risk essential thrombocythemia with CALR mutation. Haematologica. 2016;101(8):926‒931.
8. Wurtz M. Aspirin in coronary artery disease an appraisal of functions and limitations. Dan Med J. 2015;62(4):B5011.
9. Macdonald S. Aspirin use to be banned in under 16year olds. BMJ. 2002;325(7371):988.
10. Ma N, Liu XW, Yang YJ, et al. Preventive effect of aspirin eugenol ester on thrombosis in carrageenan-induced rat tail thrombosis model. PLoS One. 2015;10(7):1‒14.
11. Pignatelli P, Di Santo S, Barilla F, et al. Multiple anti-atherosclerotic treatments impair aspirin compliance effects on aspirin resistance. J Thromb Haemost. 2008;6(10):1832‒1834.
12. Ahmadi R, Ahmadifar M, Safarpour E, et al. The Effects of Levofloxacin on Testis Tissue and Spermatogenesis in Rat. Cell J. 2016;18(1):112‒116.
13. Vyas A, Ram H, Purohit A, et al. Adverse Effects of Subchronic Dose of Aspirin on Reproductive Profile of Male Rats. J Pharm. 2016;2016:1‒9.
14. Al-Taei BSD. Effect of Aspirin on Sperm Specification and Some Hematological Parameters in Male Albino White Rat. J Biotechnol Res Cent. 2014;8(4):34‒38.
15. Didolkar AK, Patel PB, Roychowdhury D. Effect of aspirin on spermatogenesis in mature and immature rats. Int J Androl. 1980;3(5):585‒593.
16. Bedwal RS, Edwards MS, Katoch M, et al. Histological and biochemical changes in testis of zinc deficient BALB/c strain of mice. Indian J Exp Biol. 1994;32(4):243‒247.
17. Zemunik T, Peruzović M, Čapkun V, et al. Reproductive ability of pubertal male and female rats. Brazilian J Med Biol. 2003;36(7):871‒877.
18. Gabriel SM, Roncancio JR, Ruiz NS. Growth hormone plasticity and the endocrine milieu during sexual maturation in male and female rats. Neuroendocrinolog. 1992;56(5):619‒625.
19. Walker WH. Molecular mechanisms of testosterone action in spermatogenesis. Steroids. 2009;74(7):602‒607.
20. Calvert C, Bradford GE. Reduced male reproductive capacity in mice with high genetic potential for post-weaning growth. J Reprod Fertil. 1989;87(1):33‒38.
21. Thompson TL, Berndtson WE. Testicular weight, Sertoli cell number, daily sperm production, and sperm output of sexually mature rabbits after neonatal or prepubertal hemicastration. Biol Reprod. 1993;I48(5):952‒957.
22. Oyedeji KO, Bolarinwa AF, Adigun AK. Effect of Aspirin on reproductive functions in male Albino rats. IOSR J Pharm Biol Sci. 2013;4(6):49‒54.                
23. Johnson L, Thompson DL, Varner DD. Role of Sertoli cell number and function on regulation of spermatogenesis. Anim Reprod Sci. 2008;105(1‒2):23‒51.
24. Mbizvo MT, Burkman LJ, Alexander NJ. Human follicular fluid stimulates hyper activated motility in human sperm. Fertile Ster. 1990;54(4):708‒712.
25. Ratnasooriya WD, Jayakody JR. Long-term administration of large doses of paracetamol impairs the reproductive competence of male rats. Asian J Androl. 2000;2(4):247‒255.
26. Dyson AL, Orgebin-Crist MC. Effect of hypophysectomy castration and androgen replacement upon the fertilizing ability of rat epididymal spermatozoa. Endocrinol. 1973;93(2):391‒402.
27. Al-Inany HG, Youssef MA, Ayeleke RO, et al. Gonadotrophin-releasing hormone antagonists for assisted reproductive technology. Cochrane Database Syst Rev. 2011;11(5):CD001750.
28. Vane J. The evolution of non-steroidal anti-inflammatory drugs and their mechanisms of action. Drugs. 1987;33(Suppl 1):18‒27.
29. Gholami M, Saki G, Hemadi M, et al. Melatonin improves spermatogonial stem cells transplantation efficiency in azoospermic mice. Iran J Basic Med. 2014;17(2):93‒99.
30. Garcia MB, Gonzalez-Fernandez L, Loux SC, et al. Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm. Reproduction. 2015;149(1):87‒99.
31. McLachlan RI, O’Donnell L, Meachem SJ, et al. Identification of specific sites of hormonal regulation in spermatogenesis in rats monkeys and man. Recent Prog Horm Res. 2002;57:149‒179.
32. Defalco T, Saraswathula A, Briot A, et al. Testosterone levels influence mouse fetal Leydig cell progenitors through notch signaling. Biol Reprod. 2013;88(4):91.
33. Lyon MF, Glenister PH, Lamoreux ML. Normal spermatozoa from androgen-resistant germ cells of chimaeric mice and the role of androgen in spermatogenesis. Nature. 1975;258(5536):620‒622.
34. O’Shaughnessy PJ, Morris ID, Huhtaniemi I, et al. Role of androgen and gonadotrophins in the development and function of the Sertoli cells and Leydig cells data from mutant and genetically modified mice. Mol Cell Endocrinal. 2009;306(1‒2):2‒8.
35. Meng J, Holdcraft RW, Shima JE, et al. Androgens regulates the permeability of the blood-testis barrier. Proc Natl Acad Sci. 2005;102(46):16696‒1700.
36. Biswas NM, Sanyal S, Patra PB. Antispermatogenic effect of aspirin and its prevention by prostaglandin E2. Andrologia. 1978;10(2):137‒141.
37. Guittot MS, Nicolaz CN, Lethimonier C, et al. Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent. J Clin Endocrinal Metab. 2013;98(11):1757‒1767.
38. De Gendt K, Swinnen JV, Saunders PTK, et al. A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad. 2004;101(5):1327‒1332.
39. Kristensen DM, Lesne L, Le Fol V, et al. Paracetamol (acetaminophen), aspirin (acetylsalicylic acid) and indomethacin are anti-androgenic in the rat foetal testis. Int J Androl. 2012;35(3):377‒384.
40. Cheng J, Watkins SC, Walker WH. Testosterone activates mitogen-activated protein kinase via Src kinase and the epidermal growth factor receptor in sertoli cells. Endocrinology. 2007;148(5):2066‒2074.
41. Davey RA, Grossmann M. Androgen Receptor Structure, Function and Biology from Bench to Bedside. Clin Biochem Rev. 2016;37(1):3‒15.
42. Elftman H. Sertoli cells and testis structure. Am J Anat. 1963;113(1):25‒33.
43. Hay Y, Hou J, Liu Y, et al. The roles and regulation of Sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis. Semin Cell Dev Biol. 2014;29:66‒75.
44. Rebourcet D, Wu J, Cruickshanks L, et al. Sertoli cells modulate testicular vascular network development structure and function to influence circulating testosterone concentrations in adult male mice. Endocrinology. 2016;157(6):2479‒2488.
45. Li N, Mruk DD, Lee WM, et al. Is toxicant-induced Sertoli cell injury in vitro a useful model to study molecular mechanisms in spermatogenesis. Semin Cell Dev Biol. 2016;59:141‒156.
46. Marettova E, Maretta M, Legath J. Toxic effects of cadmium on testis of birds and mammals a review. Anim Reprod Sci. 2015;155:1‒10.
47. Salvagno GL, Gomar SF, Picanza A, et al. Red blood cell distribution width: A simple parameter with multiple clinical applications. Crit Rev Clin Lab Sci. 2015;52(2):86‒105.
48. Da L, Suner L, Galimand J, et al. Diagnostic tool for red blood cell membrane disorders  Assessment of a new generation ektacytometer. Blood Cells Molecules and Diseases. 2016;56(1):9‒22.
49. Agrawal R, Sherwood J, Chhablani J, et al. Red blood cells in retinal vascular disorders. Blood Cells. 2016;56(1):53‒61.
50. Adewusi EA, Afolayan AJ. Safety evaluation of the extract from the roots of Pelargonium reniforme Curtis in male wistar rats. Afr J Pharm Pharmacol. 2009;3(8):368‒373.
51. Kim A, Fung E, Parikh SG, et al. Isocitrate treatment of acute anemia of inflammation in a mouse model. Blood Cells Mol Dis. 2016;56(1):31‒36.
52. Bag S, Das S, Bagchi C, et al. Aspirin potentiates blood glucose lowering effect of glimepiride-pioglitazone combination in streptozotocin induced diabetic rats. Indian J Pharmacol. 2014;46(5):562‒564.