|Year : 2017 | Volume
| Issue : 2 | Page : 87-92
Antioxidants: A therapy for cryptorchidism, true or false?
Ademola A Oremosu1, Olajumoke G Osumah2, Edidiong N Akang3
1 Department of Anatomy, College of Medicine, University of Lagos, Lagos, Nigeria
2 Medical Art Center, Maryland, Lagos, Nigeria
3 Department of Anatomy, College of Medicine, University of Lagos, Lagos, Nigeria; Discipline of Clinical Anatomy, School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal, Durban, South Africa
|Date of Web Publication||4-Jun-2018|
Dr. Edidiong N Akang
Department of Anatomy, College of Medicine, University of Lagos
Source of Support: None, Conflict of Interest: None
BACKGROUND: Cryptorchidism remains a common congenital anomaly of the male genitalia, affecting 2.4%–5% of male infants.
AIM: This study investigates the effects of L-carnitine (LC), Biotin, and Vitamin D3 (Vit D) on the cryptorchid testis in Sprague-Dawley (SD) rats.
MATERIALS AND METHODS: Twenty-five adult male SD rats were divided into 5 groups of 5 each. The right testis of all rats was made cryptorchid. Group A received distilled water, Group B received LC, Group C received biotin, and Group D received Vit D while Group E received a cocktail of LC, Biotin, and Vit D. At the end 8 weeks, the animals were euthanized, and vital organs obtained, processed, and analyzed.
RESULTS: The testis of the untreated animals had marked depletion in the cells of the seminiferous tubules compared to those treated with antioxidants. They also had upregulated levels of malondialdehyde (MDA) which was inversely proportional to the levels of antioxidant enzymes. Epididymal sperm quality and testosterone level were also reduced in the untreated cryptorchid animals. These effects were, however, mitigated by the use of the antioxidants.
CONCLUSION: In this study, antioxidant therapy acted as a panacea for reversal of reactive oxygen species-induced male infertility in cryptorchid testis in SD rats. Further studies on antioxidants in comparison with hormonal supplements and surgical treatments of cryptorchid testis will be of great significance.
Keywords: Antioxidants, biotin, cryptorchidism, free radicals, L-carnitine, spermatozoa, testis, Vitamin D
|How to cite this article:|
Oremosu AA, Osumah OG, Akang EN. Antioxidants: A therapy for cryptorchidism, true or false?. J Exp Clin Anat 2017;16:87-92
| Introduction|| |
Cryptorchidism is a very common congenital anomaly of the male genitalia, affecting 2.4%–5% of male infants and is more common in premature infants (Hutson et al., 2010; Toppari et al., 2010; and Fawzy et al., 2015). Despite treatment by orchidopexy, the long-term effect remains problematic with the male fertility constantly threatened (Hadziselimovic and Herzog, 2001). Impaired fertility (33% in unilateral cases and 66% in bilateral undescended testes) and a cancer risk 5–10 times greater than normal are observed over time (Hutson et al., 2010; Thorup et al., 2010). About 10% of male infertility cases are linked with cryptorchidism and orchidopexy (Fawzy et al., 2015). Likewise, the incidence of azoospermia is 13% in unilateral cryptorchidism (Chung and Brock, 2011).
The exact cause of cryptorchidism is elusive; however, it has been linked with androgen insensitivity as a result of environmental toxins (Cobellis et al., 2014). Pregnant mothers exposed to compounds such as diethylstilboestrol are at high risk of cryptorchidism in the male child (Brucker-Davis et al., 2003). In addition, the lower temperature of the scrotum against normal body temperature makes it favorable for optimal testicular activities due to the thermoregulatory ability of the pampiniform plexus of veins and the testicular artery in the spermatic cord (Harrison and Weiner, 1949; Setchell, 1991). Hence, the high intra-abdominal temperature which is experienced by the cryptorchid testis adversely affects spermatogenesis and induces hyperproduction of reactive oxygen species (ROS), thereby increasing oxidative stress (Babaei et al., 2012, Ko et al., 2014). Furthermore, ROS hyperactivity over the testicular antioxidants will further lead to increased lipid peroxidation of the plasma membrane of the spermatozoa thereby, impairing capacitation and zeroing the spermatozoan ability to fertilize an oocyte (Akang et al., 2015; Guasti et al., 2017). Increased oxidative stress eventually leads to apoptosis (programmed cell death) of the spermatogenic cells, which explains the mechanism by which azoospermia is linked with cryptorchid testis (Kobayashi et al., 2013). Wang et al. (2007) observed a decline in sperm parameters, distorted testicular histology, and germ cell apoptosis after scrotal heating. They reported that apoptosis occurred mainly in the round spermatids and pachytene spermatocytes.
It has been suggested that the surgical correction of cryptorchidism (orchidopexy) between the ages of 6 months to 1 year may lower subfertility (Hadziselimovic, 2012). However, this early as possible intervention before 1 year of age is not a sure proof to the complete restoration of fertility. Howbeit, such individuals may still benefit from the assisted reproduction therapy. More worrisome is the treatment of cryptorchidism at puberty as such testis may be completely depleted of spermatozoa and may never function properly even after repair (Okuyama et al., 1989, Grasso et al., 1991).
Controversies exist about the use of endocrine agonist for the treatment of cryptorchid testis as against surgery or the combination of both (Fawzy et al., 2015). On the one hand, it is believed not to be necessary while on the other hand, it is believed to be adopted as the first line of treatment for cryptorchidism, failure of this treatment may warrant the second option-surgery (Mathers et al., 2009). It has been suggested that hormonal treatment is better to be considered when the biopsy of the undescended testis had no adult dark spermatogonic cells (Hadziselimovic and Hoecht, 2008, Thorup et al., 2012),
Antioxidant therapy has been demonstrated to be of great significance in the neutralization of ROS and in the management of oxidative stress-induced male infertility (Dosumu et al., 2012, Akang et al., 2015). A study by Azari et al. (2014) reported that Vitamin C was able to improve spermatogenesis in rat unilateral cryptorchid testis. More so, antioxidant therapy has been reported to increase testosterone levels in Sprague-Dawley (SD) rats (Akang et al., 2011; Dosumu et al., 2012). L-carnitine (LC) is a vitamin-like substance that is structurally similar to amino acids (Alzahrani, 2011). It is absorbed from foods through both active and passive transport across enterocyte (intestinal cell) membranes (Rebouche, 2004). It can also be synthesized endogenously from the essential amino acids lysine and methionine (Dokmeci et al., 2006). It is well known for its lipid lowering and antioxidant activities (Dokmeci et al., 2006, Salama et al., 2015). Biotin also known as vitamin H is also known to upregulate antioxidant enzyme levels and to effectively combat ROS (Al-Qudah and Ismail, 2012) Biotin is a water-soluble vitamin that serves as an essential coenzyme for carboxylases catalyzing the transfer of a carboxyl group to targeted substrates (Zempleni and Mock, 1999; Sedel et al., 2015). Vitamin D3 (Vit D) which is currently being studied for its antioxidant property has been reported by Sharifi et al. (2014) as an adjunctive therapy to attenuate systemic inflammation and lipid peroxidation alongside other treatments. It has also been reported to attenuate DNA oxidative damage (Fedirko et al., 2010) Therefore, this study is aimed at determining the effects of LC, Biotin, and Vitamin D3 on a unilateral cryptorchid testis in SD rats.
| Materials and Methods|| |
Experimental animals and protocols
Twenty-five male SD rats weighing between 200 and 220 g were randomly selected from the Nigerian Institute of Medical Research located in Yaba, Lagos. The animals were housed in the Laboratory Animal Center, College of Medicine, University of Lagos. The animal house was well ventilated with a temperature range of 28–32°C under day/night 12–12 h photoperiodicity. The rats were fed with standard rat chow (Pfizer Nig Ltd). They had access to water ad libitum. The experimental animals were further divided into five groups of 5 rats each. Group A: Control, received 1 ml of distilled water; Group B: 400 mg/kg body weight of LC (Zambrano et al., 2013); Group C: 5.0 mg/kg body weight of biotin (Sedel et al., 2015); Group D: 10 mg/kg body weight of Vit D (Fedirko et al., 2010); and Group E: A cocktail of the same doses of the 3 antioxidants (LC, Biotin, and Vit D). All animals in groups A-E were made unilaterally cryptorchid (right testis) for 8 weeks, and the entire administration was through the oral route. At the end of the experiment, the animals were euthanized, and vital organs were obtained for subsequent analysis. All experimental protocols followed the guidelines approved by the Ethics Committee of the College of Medicine, University of Lagos, Nigeria.
Induction of cryptorchidism
Cryptorchidism was induced in accordance with the method reported by Azari et al. (2014), the rats were anesthetized with intraperitoneal injection of 10 mg/kg body weight xylazine hydrochloride (Alfasan Co., Netherlands) and 100 mg/kg body weight ketamine hydrochloride (Rotexmedica, Trittau, Germany). Then, the skin of the scrotal region was shaved and prepared with a povidone-iodine solution. The right inguinoscrotal region was incised, and the ligament of the tail of epididymis was separated. The freed testis was pushed back into the abdominal cavity through the internal inguinal ring. After which, the external inguinal ring was closed by 4/0 Nylon sutured to the muscle of the abdominal wall. The animals were left for 7 days postoperation to induce oxidative stress after which antioxidants were administered orally for 8 weeks.
Testicular histology preparation
The histology of the testes was done by the modification of method reported by Akang et al. (2015). The organs were harvested and fixed in Bouin's fluid for 24 h after which, it was transferred to 70% alcohol for dehydration. The tissues were passed through 90% and absolute alcohol and xylene for different durations before they were transferred into two changes of molten paraffin wax for 1 h each in an oven at 65°C for infiltration. They were subsequently embedded and serial sections cut using rotary microtome at 5 microns. The tissues were picked up with albumenized slides and allowed to dry on a hot plate for 2 min. The slides were dewaxed with xylene and passed through absolute alcohol (2 changes); 70% alcohol, 50% alcohol and then to water for 5 min. The slides were then stained with hematoxylin and eosin. The slides were mounted in DPX. Photomicrographs were taken at a magnification of x100.
This was done using the new improved Neubauer's counting chamber (Hematocytometer). The epididymal fluid was diluted with a normal saline solution by adding 0.9 ml–0.1 ml of the crushed epididymis. This chamber was then filled with sperm fluid and placed under a binocular light microscope using an adjustable light source. The ruled part was then focused and the number of spermatozoa counted in five 16-celled squares (Keel and Webster, 1990).
The sperm motility analysis was carried out at room temperature using one epididymis of each rat. The percentage of sperm motility was calculated using the number of live sperm cells divided by the total number of sperm cells (motile and nonmotile), from two samples per epididymis of each rat. All sperm cells that were not moving at all were considered to be on motile while the rest, which displayed some movement, were considered to be motile (Yan et al., 2007; Oremosu and Akang, 2015).
Sperm morphology was evaluated by staining the sperm smears on microscope slides with two drops of 1% eosin stain after they were air dried. The slides were examined under the microscope under oil immersion with x100 objective. The abnormal sperm cells were counted, and the percentage calculated according to the method described by (Wyrobek and Bruce, 1978).
The serum levels of rat-free testosterone were measured using commercially available enzyme-linked immunoassay kit (Elabscience) according to manufacturer's instructions.
Determination of testicular malondialdehyde and antioxidant enzymes levels
The MDA level was estimated by measuring TBARS and was determined by the method of Niehaus and Samuelsson (1968). Antioxidant enzymes such as reduced glutathione (GSH) and catalase were estimated by the methods of Ellman (1959) while superoxide dismutase (SOD) was measured by methods reported by Rukmini et al.(2004).
The sperm parameters were log transformed to improve normality of data. Differences between groups were compared using one-way ANOVA and least significant difference post hoc test. This was done using the SPSS. The data were expressed as mean ± standard deviation.
| Results|| |
Histology of the testis
The cross section of the right cryptorchid testis of animals in Group A that received distilled water showed wide derangements in the germinal epithelium, distorted lumen with little or no spermatozoa. The other Groups B–E apart from Group C that received biotin and had a slight depletion of the spermatogenic cells, the rest had an intact germinal epithelium and interstitium with a marked presence of spermatozoa in the lumen [Figure 1].
|Figure 1: Testis with H and E stains ×100. (a) Control; received 1ml of distilled water. Arrows showing wide derangements in germinal epithelium. (b) received 400 mg/kg of L-carnitine; (c) received 5 mg/kg of Biotin; (d) received 10 mg/kg Vitamin D; (e) received a cocktail of L-carnitine, biotin, and Vitamin D; Groups B, D, and E depict a proper arrangement of spermatogenic cells within the germinal epithelium of each seminiferous tubule, with spermatozoa in the lumen (l) and Interstitial cells of Leydig (i) between each tubule. The arrows in group C depict slight derangements in germinal epithelium|
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Malondialdehyde and antioxidant enzymes
MDA levels significantly increased in the control animals (Group A) that received distilled water as compared to the other Groups (B–E) that received antioxidants [Table 1]. GSH and catalase levels significantly increased in animals that received Vit D (Group D) compared to Group A. SOD levels increased significantly in animals that received LC, Vit D and the cocktail of the 3 antioxidants (Groups B, D, and E) compared to the control [Table 1].
|Table 1: Effect of antioxidants on testicular oxidative stress parameters|
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Sperm motility, sperm count, and sperm morphology analysis
The sperm motility increased in all groups; however, there was no significant difference when compared to Group A (P > 0.05). The sperm count of the animals that received biotin and those that received the cocktail of the 3 antioxidants significantly increased compared to control (P < 0.05). The sperm morphology of the abnormal spermatozoa was significantly lower in the group that received biotin compared to control [Table 2].
|Table 2: Effects of antioxidants on sperm motility, sperm count, and sperm morphology|
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There was an increase in testosterone levels across the groups compared to control, but this increase was not significant (P > 0.05) as shown in [Figure 2].
| Discussion|| |
Antioxidant therapy has been used to reduce heat-induced oxidative stress in the management of infertility (Ahmad et al., 2017). In this study, LC, biotin, and Vit D were efficacious in the reduction of lipid peroxidation induced by cryptorchidism. Lipid peroxidation which is exacerbated by the excess production of free radicals (ROS) over the total antioxidant levels (Ko et al., 2014) was increased with cryptorchidism. The animals that received antioxidant supplements had an increase in GSH, SOD, and catalase levels which was inversely proportional to MDA levels. These antioxidant enzymes neutralize the free radicals produced as a result of heat-induced testicular stress. Our result is in tandem with several reports supporting the use of antioxidants to mitigate oxidative stress-induced disorders (Ahmed et al., 2014, Salama et al., 2015, and Agarwal and Majzoub, 2017).
The negative effects of lipid peroxidation in the cryptorchid testis caused a marked reduction in sperm count and increased teratozoospermia and oligospermia. Oxidative stress is directly linked with upward regulation of apoptosis leading to the demise of spermatogenic cells (Koppers et al., 2011, Aitken et al., 2013, and Kobayashi et al., 2013). This may explain the derangements observed in the seminiferous epithelium of the cryptorchid testes in this study. More so, free radicals decrease the motility of the spermatozoon by increasing peroxidation of the plasma membrane which is rich in polyunsaturated fatty acid. Subsequently, a rapid loss of intracellular adenosine triphosphate from lipid peroxidation causes axonemal damage, decreased sperm viability, and increased mid-piece sperm morphological defects, all of which contribute to decreased sperm motility (Bansal and Bilaspuri, 2010, Agarwal et al., 2014). These effects were, however, ameliorated by the antioxidant therapy. Our findings are in concert with Al-Qudah and Ismail (2012), Nikooyeh et al. (2014) and Salama et al.(2015).
Contrary to the view that orchidopexy remains the only therapy for the treatment of cryptorchidism (Hadziselimovic, 2012), our study though did not include the administration of any exogenous hormone proved that the use of antioxidants upregulated the testosterone synthesis. This is markedly expressed in the group that received the combined therapy. More so, the photomicrographs of the cryptorchid testis treated with antioxidants, especially the combined therapy showed sufficient availability of interstitial cells of Leydig. These cells are the cells responsible for the synthesis of testosterone (Shima et al., 2012) which is necessary for the steady flow of spermatogenesis and the maintenance of a rich presence of cells of the germinal epithelium (Heller et al., 2016) as seen in this study.
| Conclusion|| |
Cryptorchidism disrupts spermatogenesis through the antioxidant-oxidant pathway. The hyper production of free radicals in the testis leads to the death of spermatozoa. Antioxidant therapy of LC, Biotin, and Vit D mitigates the deleterious effects of heat-induced ROS activity, leading to upregulation of testosterone, and subsequent enhancement of spermatogenesis. Contrary to the view that hormonal therapy may serve as first-line therapy in the treatment of cryptorchidism, we are of the opinion that antioxidant therapy may suffice for both the reduction of redox activities and testicular hormonal requirements for spermatogenesis. However, further research needs to be done to compare outcomes with hormonal supplements and surgical treatments.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Agarwal A., Majzoub A. (2017). Role of antioxidants in assisted reproductive techniques. World J Mens Health 35. [Doi: 10.5534/wjmh. 2017.35.e3].
Agarwal A., Virk G., Ong C., Du Plessis S.S. (2014). Effect of oxidative stress on male reproduction. World J Mens Health 32:1-17.
Ahmad G., Agarwal A., Esteves S., Sharma R., Almasry M., Al-Gonaim A., et al.
(2017). Ascorbic acid reduces redox potential in human spermatozoa subjected to heat-induced oxidative stress. Andrologia, e12773. Doi: 10.1111/and. 12773.
Ahmed M.M., Ibrahim Z.S., Alkafafy M., El-Shazly S.A. (2014). L-carnitine protects against testicular dysfunction caused by gamma irradiation in mice. Acta Histochem 116:1046-55.
Aitken R., Smith T., Lord T., Kuczera L., Koppers A., Naumovski N., et al
. (2013). On methods for the detection of reactive oxygen species generation by human spermatozoa: Analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid. Andrology 1:192-205.
Akang E., Oremosu A., Dosumu O., Ejiwunmi A. (2011). Telfairia occidentalis, a prophylactic medicine for alcohol's damaging effect on the testis. Maced J Med Sci 4:;380-7.
Akang E., Oremosu A., Osinubi A., Dosumu O., Kusemiju T., Adelakun S., et al
. (2015). Histomorphometric studies of the effects of Telfairia occidentalis
on alcohol-induced gonado-toxicity in male rats. Toxicol Rep 2:968-75.
Al-Qudah K.M., Ismail Z.B. (2012). The relationship between serum biotin and oxidant/antioxidant activities in bovine lameness. Res Vet Sci 92:138-41.
Alzahrani H.A. (2011). Protective effect of l-carnitine against acrylamide-induced DNA damage in somatic and germ cells of mice. Saudi J Biol Sci 18:29-36.
Azari O., Gholipour H., Kheirandish R., Babaei H., Emadi L. (2014). Study of the protective effect of Vitamin C on testicular tissue following experimental unilateral cryptorchidism in rats. Andrologia 46:495-503.
Babaei H., Azari O., Kheirandish Abshenas J., Mohammadi N. (2012). Zinc therapy improves deleterious effects of experimental unilateral cryptorchidism: Histopathological evaluation of testes. Iran J Vet Surg 5:77-88.
Bansal A.K., Bilaspuri G.S. (2010). Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int 2010. pii: 686137.
Brucker-Davis F., Pointis G., Chevallier D., Fenichel P. (2003). Update on cryptorchidism: Endocrine, environmental and therapeutic aspects. J Endocrinol Invest 26:575-87.
Chung E., Brock G.B. (2011). Cryptorchidism and its impact on male fertility: A state of art review of current literature. Can Urol Assoc J 5:210-4.
Cobellis G., Noviello C., Nino F., Romano M., Mariscoli F., Martino A., et al.
(2014). Spermatogenesis and cryptorchidism. Front Endocrinol (Lausanne) 5:63.
Dokmeci, D., Akpolat, M., Aydogdu, N., Uzal, C., Doganay, L. and Turan, F.N., (2006). The protective effect of L-carnitine on ionizing radiation-induced free oxygen radicals. Scandinavian Journal of Laboratory Animal Sciences, 33(2), p.75-83.
Dosumu O.O., Akinola O.B., Akang E.N. (2012). Alcohol-induced testicular oxidative stress and cholesterol homeostasis in rats – The therapeutic potential of virgin coconut oil. Middle East Fertil Soc J 17:122-8.
Ellman G.L. (1959). Tissue sulfhydryl groups. Arch Biochem Biophys 82:70-7.
Fawzy, F., Hussein, A., Eid, M.M., El Kashash, A.M. and Salem, H.K., (2015). Cryptorchidism and fertility. Clinical medicine insights. Reproductive health, 9, p.39.
Fedirko V., Bostick R.M., Long Q., Flanders W.D., McCullough M.L., Sidelnikov E., et al.
(2010). Effects of supplemental vitamin D and calcium on oxidative DNA damage marker in normal colorectal mucosa: A randomized clinical trial. Cancer Epidemiol Biomarkers Prev 19:280-91.
Grasso M., Buonaguidi A., Lania C., Bergamaschi F., Castelli M., Rigatti P. (1991). Postpubertal cryptorchidism: Review and evaluation of the fertility. Eur Urol 20:126-8.
Guasti P.N., Monteiro G.A., Maziero R.R., Carmo M.T., Dell'Aqua J.A. Jr., Crespilho A.M., et al.
(2017). Pentoxifylline effects on capacitation and fertility of stallion epididymal sperm. Anim Reprod Sci 179:27-34.
Hadziselimovic F. (2012). Cryptorchidism: Management and Implications. Berlin: Springer Science and Business Media.
Hadziselimovic F., Herzog B. (2001). The importance of both an early orchidopexy and germ cell maturation for fertility. Lancet 358:1156-7.
Hadziselimovic F., Hoecht B. (2008). Testicular histology related to fertility outcome and postpubertal hormone status in cryptorchidism. Klin Pädiatr 220:302-7.
Harrison R., Weiner J. (1949). Vascular patterns of the mammalian testis and their functional significance. J Exp Biol 26:304-16.
Heller C.G., Nelson W.O., Hill I.B., Henderson Maddock W.O., Jungck E.C., Paulsen C.A., et al
. (2016). Improvement in spermatogenesis following depression of the human testis with testosterone. Fertil Steril 1:415-22.
Hutson J.M., Balic A., Nation T., Southwell B. (2010) Cryptorchidism. Semin Pediatr Surg 19:215-24.
Keel B.A., Webster B.W. (1990). Handbook of the Laboratory Diagnosis and Treatment of Infertility. Boston: CRC Press.
Ko E.Y., Sabanegh E.S., Agarwal A. (2014). Male infertility testing: Reactive oxygen species and antioxidant capacity. Fertil Steril 102:1518-27.
Kobayashi H., Nagao K., Nakajima K. (2013). Therapeutic advances in the field of male infertility: Stem cell research. Adv Stud Med Sci 1:39-54.
Koppers A.J., Mitchell L.A., Wang P., Lin M., Aitken R.J. (2011). Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. Biochem J 436:687-98.
Mathers M.J., Sperling H., Rübben H., Roth S. (2009). The undescended testis: Diagnosis, treatment and long-term consequences. Dtsch Arztebl Int 106:527-32.
Niehaus W., Samuelsson B. (1968). Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur J Biochem 6:126-30.
Nikooyeh B., Neyestani T.R., Tayebinejad N., Alavi-Majd H., Shariatzadeh N., Kalayi A., et al.
(2014). Daily intake of Vitamin D- or calcium-vitamin D-fortified Persian yogurt drink (doogh) attenuates diabetes-induced oxidative stress: Evidence for antioxidative properties of Vitamin D. J Hum Nutr Diet 27 Suppl 2:276-83.
Okuyama A., Nonomura N., Nakamura M., Namiki M., Fujioka H., Kiyohara H., et al.
(1989). Surgical management of undescended testis: Retrospective study of potential fertility in 274 cases. J Urol 142:749-51.
Oremosu A.A., Akang E.N. (2015). Impact of alcohol on male reproductive hormones, oxidative stress and semen parameters in Sprague-Dawley rats. Middle East Fertil Soc J 20:114-8.
Rebouche C.J. (2004). Kinetics, pharmacokinetics, and regulation of l-carnitine and acetyl-l-carnitine metabolism. Ann N
Y Acad Sci 1033:30-41.
Rukmini M.S., D'Souza B., D'Souza V. (2004). Superoxide dismutase and catalase activities and their correlation with malondialdehyde in schizophrenic patients. Indian J Clin Biochem 19:114-8.
Salama A.F., Kasem S.M., Tousson E., Elsisy M.K. (2015). Protective role of L-carnitine and Vitamin E on the testis of atherosclerotic rats. Toxicol Ind Health 31:467-74.
Sedel F., Papeix C., Bellanger A., Touitou V., Lebrun-Frenay C., Galanaud D., et al.
(2015). High doses of biotin in chronic progressive multiple sclerosis: A pilot study. Mult Scler Relat Disord 4:159-69.
Cupps PT. (1991). Reproduction in Domestic Animals, San Diego: Academic Press, 221-49.
Sharifi N., Amani R., Hajiani E., Cheraghian B. (2014). Does Vitamin D improve liver enzymes, oxidative stress, and inflammatory biomarkers in adults with non-alcoholic fatty liver disease? A randomized clinical trial. Endocrine 47:70-80.
Shima Y., Miyabayashi K., Haraguchi S., Arakawa T., Otake H., Baba T., et al.
(2012). Contribution of leydig and sertoli cells to testosterone production in mouse fetal testes. Mol Endocrinol 27:63-73.
Thorup J., McLachlan R., Cortes D., Nation T.R., Balic A., Southwell B.R., et al.
(2010). What is new in cryptorchidism and hypospadias – A critical review on the testicular dysgenesis hypothesis. J Pediatr Surg 45:2074-86.
Thorup J., Petersen B.L., Kvist K., Cortes D. (2012). Bilateral undescended testes classified according to preoperative and postoperative status of gonadotropins and inhibin B in relation to testicular histopathology at bilateral orchiopexy in infant boys. J Urol 188 4 Suppl: 1436-42.
Toppari J., Virtanen H.E., Main K.M., Skakkebaek N.E. (2010). Cryptorchidism and hypospadias as a sign of Testicular Dysgenesis Syndrome (TDS): Environmental connection. Birth Defects Res A Clin Mol Teratol 88:910-9.
Wang C., Cui Y.G., Wang X.H., Jia Y., Sinha Hikim A., Lue Y.H., et al.
(2007). Transient scrotal hyperthermia and levonorgestrel enhance testosterone-induced spermatogenesis suppression in men through increased germ cell apoptosis. J Clin Endocrinol Metab 92:3292-304.
Wyrobek A.J., Bruce W.R. (1978). The induction of sperm-shape abnormalities in mice and humans. Chem Mutagens 5:257-85.
Yan J.G., Agresti M., Bruce T., Yan Y.H., Granlund A., Matloub H.S. (2007). Effects of cellular phone emissions on sperm motility in rats. Fertil Steril 88:957-64.
Zambrano S., Blanca A.J., Ruiz-Armenta M.V., Miguel-Carrasco J.L., Revilla E., Santa-María C., et al.
(2013). The renoprotective effect of L-carnitine in hypertensive rats is mediated by modulation of oxidative stress-related gene expression. Eur J Nutr 52:1649-59.
Zempleni J., Mock D.M. (1999). Bioavailability of biotin given orally to humans in pharmacologic doses. Am J Clin Nutr 69:504-8.
[Figure 1], [Figure 2]
[Table 1], [Table 2]