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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access   Full Text Article                                                                                                                                            Research Article

Effects of oxycodone hydrochloride on reproductive functions in male wistar rats

Landry Martial MIGUEL^1*, Herman AKASSA¹, Archange Emmanuel MBOUNGOU MALONGA¹, Anne-Loïs NKANGA SAMBA¹, Destin MBEMBA BAHAMBOULA¹, Choupette Ravelle DOBHAT-DOUKAKINI^1,2, Didier Gesril NJILO TCHATCHOUANG^1,2, Childérick LEKANA^1,2, Donatien MOUKASSA¹, Ange Antoine ABENA^1,2

¹ Biochemical and Pharmacology Laboratory, Faculty of Health Sciences, Marien Ngouabi University, Brazzaville, Republic of Congo 

² Dénis Sassou-N’Guesso University, Kintélé, Republic of Congo

 

 

INTRODUCTION

Opioid consumption has increased considerably over the last three decades, particularly in developing countries¹. Their main indication is the management of chronic pain and palliative treatment, which partly explains the considerable increase in their use. The rise in the abuse of opioid medications has become a public health challenge globally. However, chronic opioid use can have a number of adverse effects on organ systems, including the reproductive system, significantly affecting patients quality of life^2,3. Abs and al (2000)⁴ report that opioids impair male fertility via different mechanisms. At high doses, the concentrations of testosterone decline with associated hypogonadism^4,5. Opioids have been shown to decrease reproductive parameters in male rats⁶. They increase the production of reactive oxygen species (ROS) and can adversely affect spermatogenesis, leading to sexual dysfunction or infertility³. Opioid-related adverse effects are receptor-mediated and therefore inseparable from treatment⁷. Several studies have been conducted with rodent models to investigate the adverse effects of gestational opioid consumption on the neurological development of offspring, but there is little data regarding the effects of Exposure to opioids (including oxycodone) on reproductive functions in adult rats⁸. Opioids have a pharmacological action on the hypothalamic-pituitary or gonadal axis. Some changes have been observed, such as an increase in prolactinemia, a decrease in plasma cortisol and in testosterone. Oxycodone, a μ-opioid receptor agonist, was synthesized in 1916 and introduced into clinical use in Germany in 1917⁹. It has been studied extensively as a therapeutic analgesic for acute and chronic neuropathic pain as an alternative to morphine⁶. Oxycodone emerged as a drug with widespread abuse. The therapeutic effect is mainly analgesic, anxiolytic, antitussive and sedative^3,6. In this study, we investigate the effects of repeated administration of oxycodone hydrochloride in male rats on reproductive.

MATERIALS AND METHODS

Animals and treatment

Adult male rats of the Wistar strain weighing between 100 and 300 grams were used. They came from the animal house of the Faculty of Health Sciences in Brazzaville. They were reared in polypropylene cages under standard conditions with free access to food and drinking water. All experiments were conducted in compliance with EU Directive 2010/6106/EU on the protection of laboratory animals¹⁰. Animals were divided into 3 groups of 5 animals each and treated as follows: 

• the first group received distilled water at 5 ml/kg and served as a control batch;
• the second group was treated with oxycodone hydrochloride at a dose of 5 mg/kg ;
• the third group was treated with oxycodone hydrochloride at a dose of 10 mg/kg.

The products were administered orally for 30 days. 

The doses of oxycodone hydrochloride used in this study corresponded to the therapeutic doses used in human clinical practice^2,3.

Sacrifice of animals and collection of samples

Twenty-four hours after the last treatment, all the animals were sacrificed by inhalation of ether. After incision of the carotid artery, the blood was collected in dry tubes, centrifuged and the serum obtained was used for hormone assays (FSH, LH and testosterone). After dissection, the prostate, testicles, epididymides, spermiducts and seminal vesicles were removed for morphological analysis. After sampling, the organs were immediately weighed and the weight index, corresponding to the ratio between the body weight and the mass of the organ, was calculated. The epididymis and spermiducte were excised, dilacerated and then gently crushed in 1 ml of physiological water. These organs were then removed to obtain a fixed volume of sperm suspension, which was used to determine the sperm parameters of the Wistar rat.  

Semen analysis

Sperm quality was assessed by spermogram and spermocytogram. The variables analysed were: sperm pH, leucocyte count, sperm motility, sperm count, vitality and morphology. Sperm motility was assessed by direct examination of a drop (20 μl) under a light microscope at X40 magnification. To determine sperm concentration, the sperm suspension was diluted 1:20 with 10% formalin and counted using a Mallassez cell on optical microscope. A sperm suspension in 2% eosin was used to assess sperm vitality. Live and dead sperm were counted under the light microscope. Sperm morphology was assessed after staining a sperm smear with May Grunwald Giemsa. The smear was read under a light microscope. This assessment was made according to the Kruger classification (1986)¹¹. 

Hormones levels

We used the direct ELISA (Enzyme Link Immuno Sorbent Assay) with the Cypress® kit to measure FSH, LH and testosterone, following the manufacturer's instructions. 

Statistical analysis

Data were entered into Excel 2019 (Microsoft). Results were expressed as mean ± standard error (SEM). For each variable, the groups of treated animals were compared with the control group. Comparisons of non-evolutionary parameters were made using Student's t-test with Statistical Package for Social Science (SPSS), version 20 and Microsoft Excel 2019. The significance threshold corresponds to a risk of error of p<0.05.

RESULTS

Behavioural aspects

Animals treated with oxycodone hydrochloride at doses of 5 and 10 mg/kg showed stiffness of the tail and priaprismas during the period of exposure to the drug. These manifestations are illustrated in figures 1 and 2.

 

 

 

Figure 1: Illustration of tail stiffness (A and B) in Wistar rats during exposure to oxycodone hydrochloride.

Figure 2: illustration of priapism in Wistar rats during exposure to oxycodone hydrochloride.

 

 

Figure 3: Photography of the testes of control rats (A and B) and those treated with oxycodone hydrochloride at doses of 5 and 10 mg/kg respectively (C, D, E and F).

Effects on testicular morphology

Animals treated with oxycodone hydrochloride showed testicular atrophy, the effect of which was dose-dependent. Figure 3 illustrates the morphological differences between the testes of control rats and those treated with oxycodone hydrochloride.

Organ weight 

Prolonged administration of oxycodone hydrochloride for thirty days results in a significant reduction in the weight of the prostate, testicles and epididymides. Organ weight indices are shown in Table I.

 

 

Table I: organ weight index 

Organs	ED 5 ml/kg	Oxy 5 mg/kg	Oxy 10 mg/kg
Prostate	1,64±0,33	0,53±0,16**	0,36±0,13**
Left testicle	1,73±0,63	0,56±0,39**	0,69±0,47**
Right testicle	1,76±0,63	0,58±0,39**	0,93±0,51*
Left epididymis	0,64±0,26	0,51±0,23 NS	0,34±0,13*
Right epididymis	0,58±0,24	0,50±0,20 NS	0,33±0,13*

Results are expressed as mean ± standard error. (*): p<0.05; (**): p<0.001; NS (not significant) by student's t-test. DE: distilled water; Oxy: oxycodone hydrochloride.

 

 

Sperm parameters

The effects of oxycodone hydrochloride on sperm parameters are shown in Table II. Analysis of these results shows that administration of oxycodone hydrochloride caused a significant decrease in sperm motility (p<0.001),  number of spermatozoa (p<0.05) and pourcentage of normal sperm morphology (p<0.0001).

 

Table II Effects of oxycodone hydrochloride on sperm parameters

Parameters	DE 5 ml/kg	Oxy 5 mg/kg	Oxy 10 mg/kg
pH	6,75±0,28	6,12±0,25 NS	6,37±0,25NS
Total sperm motility (%)	85,0±4,08	18.75±37,5***	17.5±3,5***
Vitality (%)	95,25±0,5	83,75±7,5NS	73,75±7,5*
Number of spermatozoa (x106/ml)	20,75±2,5	25±5,03*	31,25±2,5*
Normal morphology (%)	72,50±5,0	48,75±4,78*	46,25±11,08*

Results are expressed as mean ± standard error. (*): p<0.05; (**): p<0.001; (***): p<0.0001; NS (not significant) by Student's t-test. DE: distilled water; Oxy: oxycodone hydrochloride.

 

 

Hormones levels 

Oxycodone hydrochloride administration to rats significantly decreased testosterone levels only at 10 mg/kg (p<0.05). The effects of oxycodone hydrochloride on hormonal parameters are shown in Table III.

 

 

Table III: Effects of oxycodone hydrochloride on hormonal parameters

Parameters	Distilled water 5 ml/kg	Oxy 5 mg/kg	Oxy 10 mg/kg
FSH	0,32±0,08	0,39±0,16 NS	0,32±0,12NS
LH	0,47 ±0,34	0,82±0,41**	0,80±0,37**
Testosterone	1,48± 0,35	0,41±0,13 **	0,29± 0,10***

Results are expressed as mean ± standard error. (*): p<0.05; (**): p<0.001; (***): p<0.0001; NS (not significant) by student's t-test. DE: distilled water; OxyN: oxycodone hydrochloride.

 

 

DISCUSSION

The use of opioids is known to reduce levels of male sex hormones, and it is thought that this drop in hormone levels is responsible for the reduced fertility of men who take opiates¹². Oxycodone belongs to the same family as codeine, morphine and tramadol. The aim of the present study was to evaluate the effects of oral administration of oxycodone hydrochloride at doses of 5 and 10 mg/kg for four weeks on reproductive functions in male rats.

Administration of oxycodone hydrochloride in male rats at the doses studied produced behavioural changes manifested by tail stiffness and priapism. In the investigation of male infertility, morphological evaluation of the testicles is of great importance. Indeed, it supports the hypothesis of a testicular mechanism of infertility¹³. In this study, testicular atrophy was observed in the groups of animals treated with oxycodone hydrochloride. Animals treated with oxycodone hydrochloride showed a significant decrease in testicular weight compared with control animals. The same observation was made for the prostate and epididymides.

Sperm quality is recognised as the best predictor of natural fertility¹⁴. It is assessed through sperm density, vitality, mobility and morphology. It is accepted that these parameters are directly correlated with fertility¹⁵. In the present study, treatment with oxycodone hydrochloride caused a significant decrease in the mobility, vitality and number of normal spermatozoa. Grizard (1997)¹⁶ reported that changes in pH can indicate a defect in seminal vesicle secretion (decrease in pH) or an insufficiency of prostatic secretion (increase in pH). Morphine has also been reported to increase aromatase expression in the brain and impair testicular function^17,18,19. Other work has demonstrated that exogenous opioids cause DNA fragmentation and adversely affect sperm quality. These effects are associated with all opioids²⁰. In this study, a decrease in pH was observed in the batches of animals treated with oxycodone hydrochloride. However, this decrease was not significant. This decrease in pH could be associated with a deficit in seminal fluid secretion and could explain the apparent increase in sperm density. Thus, the observed increase in sperm count would correspond rather to a concentration of spermatozoa due to hyposecretion of the seminal vesicles.

Testicular functions are regulated by the neuroendocrine axis, which comprises hypothalamic GnRH neurons and pituitary cells that secrete FSH and LH in response to GnRH. Testosterone is produced by Leydig cells in response to LH. In this study, a significant increase in LH levels was observed in animals treated with oxycodone hydrochloride at doses of 5 and 10 mg/kg. Oxycodone hydrochloride did not affect FSH levels in rats. However, there was a significant decrease in testosterone levels at 10 mg/kg. Consumption of opiates (including oxycodone) is known to reduce levels of male sex hormones^4,21. This depletion of testosterone should be compared with the testicular atrophy observed. Indeed, it has been reported that a moderate decrease in gonadotropin secretion causes testicular atrophy, as well as a decrease in sperm production²². The suggested mechanism of this testosterone depletion would involve an increase in prolactin and an inhibition of gonado-pituitary tropin secretion²¹. Furthermore, Leydig and germ cells are the sites of endogenous opioid production. Their receptors are present throughout the testis²³. The secretion of androgen transport proteins intratesticularly is also inhibited by opioids²¹. 

Other authors have also reported a decrease in FSH, LH and testosterone levels caused by opioids²⁴. Similar observations were made in rodents treated with heroin which significantly reduced sperm viability and testosterone levels²⁵.

CONCLUSION

Administration of oxycodone hydrochloride at doses of 5 and 10 mg/kg to male Wistar rats reduces testicular, prostate and epididymal weights and induces asthenospermia associated with necroteratozoospermia. This exposure also leads to an increase in LH levels and a depletion of testosterone. Further in-depth studies would help to confirm these disturbances and elucidate the molecular mechanisms involved.

Conflicts of interest

All the authors declare that they have no conflicts of interest in relation to this work. This study is part of an end-of-study Master's dissertation and has not benefited from any funding from organisations related to the subject. 

Acknowledgements

The authors would like to thank Rudy Oyali Oye, Merveille Nguenoni, Augustin Ngape and Aurore Ouamba for animal care. 

Authors contributions

• Participated in research design: LM Miguel, AE Mboungou Malonga, AL Nkanga Samba.
• Conducted experiments: LM Miguel, AE Mboungou Malonga, C Lekana, H Akassa.
• Contributed new reagents or analytic tools: D Mbemba Bahamboula, CR Dobhat-Doukakini.
• Performed data analysis: LM Miguel, D Moukassa, AA Abena
• Wrote or contributed to the writing of the manuscript: LM Miguel, D Moukassa, AA Abena.

REFERENCES  

1. Sahan Jayawardana, Rebecca Forman, Charlotte Johnston-Webber, Allen Campbell, Stefano Berterame, Cees de Joncheere, Murray Aitken, Elias Mossialos. Global consumption of prescription opioid analgesics between 2009-2019: a country-level observational study. EClinical Medicine 2021;13:42:101198 https://doi.org/10.1016/j.eclinm.2021.101198 PMid:34820610 PMCid:PMC8599097

2. Glare PA, Walsh TC. Dose-ranging study of oxycodone for chronic pain in advanced cancer. J Clin Oncology 1993;11(5):973-8. https://doi.org/10.1200/JCO.1993.11.5.973 PMid:8487060

3. Von Korff MR. Long-term use of opioids for complex chronic pain. Best Pract Res Clin Rheumatol. 2013;27:663-72. https://doi.org/10.1016/j.berh.2013.09.011 PMid:24315147 PMCid:PMC4036624

4. Abs R, Verhelst J, Maeyaert J, Van Buyten JP, Opsomer F, Adrianensen H, … Van Acker, K. (2000). Endocrine consequences of long-term intrathecal administration of opioids. J Clin Endocrinol Metab 2000;85(6):2215-22. https://doi.org/10.1210/jcem.85.6.6615 PMid:10852454

5. Vicente Carillo A et al. The mu and delta opioid receptors modulate boar sperm motility. Mol Reprod Dev. 2017 ; 83 : 724-34. https://doi.org/10.1002/mrd.22675 PMid:27391529

6. Geppetti P and Benemei S. Pain treatment with opioids: achieving the minimal effective and the minimal interacting dose. Clin Drug Investig. 2009, 1:3-16. https://doi.org/10.2165/0044011-200929001-00002 PMid:19445550

7. Marudhai S, Patel M, Valaiyaduppu Subas S, et al. Long-term Opioids Linked to Hypogonadism and the Role of Testosterone Supplementation Therapy. Cureus 2020; 12(10): e10813. https://doi.org/10.7759/cureus.10813

8. Byrnes, EM, & Vassoler FM. Modeling prenatal opioid exposure in animals: Current findings and future directions. Frontiers in Neuroendocrinology 2017; 51:1-13 https://doi.org/10.1016/j.yfrne.2017.09.001 PMid:28965857 PMCid:PMC5649358

9. James Sammie T, Ambrish A Pandit, Bruno Machado, Nalin Payakachat & Mohamed Kamel. Opioid usage differs significantly following ureteroscopy and shockwave lithotripsy, while development of long-term usage is positively correlated with total days' supply and total MME supplied. International Urology and Nephrology 2022; 54: 2805-11. https://doi.org/10.1007/s11255-022-03313-2 PMid:35907159

10. Hartung T Comparative analysis of the revised Directive 2010/6106 / EU for the protection of laboratory animals with its predecessor 86/609 / EEEEC - t4 report. ALTEX-Alternatives to animal experimentation, 2010;27(4): 285-303 https://doi.org/10.14573/altex.2010.4.285 PMid:21240470

11. Kruger TF, Menkveld R, Stander FS et al. Sperm morphologic featuras as a prognostic factor in in vitro fertilization. Fertil. Steril., 1986;46 : 1118-23 https://doi.org/10.1016/S0015-0282(16)49891-2 PMid:2946611

12. Abd HH, Ahmed HA, & Mutar TF. Moringa oleifera leaves extract modulates toxicity, sperms alterations, oxidative stress, and testicular damage induced by tramadol in male rats. Toxicology Research. 2020; 9(2): 101-6. https://doi.org/10.1093/toxres/tfaa009 PMid:32440341 PMCid:PMC7233315

13. Braik Rebiha. Exploration biologique de l'infertilité masculine.Thèse Université MOULOUD MAAMERI TIZI-OUZOU UMMTO, 2021; 174 p

14. Saïdi Hichem, Gabrielle M Pagé, Aline Boulanger, Mark A Ware, and Manon Choinière. Effectiveness of long-term opioid therapy among chronic non-cancer painpatients attending multidisciplinary pain treatment clinics: A Quebec Pain Registry study. Canadian Journal Of Pain/Revue Canadienne De La Douleur 2018; 2(1): 113-24 https://doi.org/10.1080/24740527.2018.1451252 PMid:35005371 PMCid:PMC8730575

15. Oshio S, Kaneko R. Iizuka & H. Mohri. Effects of Gradient Centrifugation on Human Sperm. Archives of Andrology Journal of Reproductive Systems1987; 19 (1): 85-93. https://doi.org/10.3109/01485018708986804 PMid:3426342

16. Grizard G, Jimenez C. Les examens du sperme dans l'exploration de la fertilité masculine. Progrès en Urologie. 1997; 7 : 496-504.

17. Aloisi AM, Aurilio C, Bachiocco V, Biasi G, Fiorenzani P, Pace MC, … Ceccarelli I. Endocrine consequences of opioid therapy. Psychoneuroendocrinology 2009;34 (Suppl 1) 2009:S162-S168. https://doi.org/10.1016/j.psyneuen.2009.05.013 PMid:19540049

18. Aloisi AM, Ceccarelli I, Fiorenzani P, Maddalena M, Rossi A, Tomei V, … Giordano A. Aromatase and 5-alpha reductase gene expression: Modulation by pain and morphine treatment in male rats. Molecular Pain 2010; 6: 69. http://www.molecularpain.com/content/6/1/69  https://doi.org/10.1186/1744-8069-6-69 PMid:20977699 PMCid:PMC2978140

19. Aloisi AM, Ceccarelli I, Carlucci M, Suman A, Sindaco G, Mameli S., … Pari G. Hormone replacement therapy in morphine-induced hypogonadic male chronic pain patients. Reproductive Biology and Endocrinology 2011;9:26. https://doi.org/10.1186/1477-7827-9-26 PMid:21332999 PMCid:PMC3049183

20. Subira'n, N., Casis, L., & Irazusta, J. Regulation of male fertility by the opioid system. Molecular medicine (Cambridge, Mass.) 2011;17(7-8), 846-853. https://doi.org/10.2119/molmed.2010.00268 PMid:21431247 PMCid:PMC3146603

21. Ajayi AF & Akhigbe RE. The physiology of male reproduction: Impact of drugs and their abuse on male fertility. Andrologia. 2020:1-25. https://doi.org/10.1111/and.13672 PMid:32542870

22. Tousson E., El-Moghazy M., Massoud A. et al. Histopathological and immunohistochemical changes in the testes of rabbits after injection with the growth promoter boldenone. Reprod. Sci 2012;19:253-9. https://doi.org/10.1177/1933719111418126 PMid:22383777

23. Albert O, Desdoits-Lethimonier C, Lesné L, Legrand A, Guillé F, Bensalah K., … Jégou, B. (2013). Paracetamol, aspirin and indomethacin display endocrine disrupting properties in the adult human testis in vitro. Human Reproduction 2013;28: 1890-8. https://doi.org/10.1093/humrep/det112 PMid:23670170

24. Abdellatief R. B., Elgamal D. A. and Mohamed E. E. M., Effects of chronic tramadol administration on testicular tissue in rats: an experimental study. Andrologia 2015;47:674-9. https://doi.org/10.1111/and.12316 PMid:25228095

25. Fazelipour S, Kiaei SB, & Tootian Z. Adverse effect of heroin hydrochloride on selected male reproductive parameters in mice. Comparative Clinical Pathology 2010;19: 565-9. https://doi.org/10.1007/s00580-009-0925-5