{"paper_id":"29843a4f-3a72-4537-823e-d21764e2f29e","body_text":"Infertility is a fairly common problem and defined\nby World Health Organization (WHO) failure\nto conceive after 12 months of unprotected\nintercourse ( 1 ,  2 ). Infertility has different aspects\nincluding psychological and interpersonal stress\nwhich severely affects infertile couples. Globally,\nranging between 8 and 12% among women\nof childbearing age, infertility affects 50-80 million\npeople. Some of its aspects important roles\nin Eastern countries and, become a psychosocial\nburden ( 3 ,  4 ). A substantial share of the total infertility\nfalls on males ( 5 ,  6 ). Oxymetholone (OXM)\nis considered as a synthetic derivative of testosterone,\nmade by methylation of 17-alpha carbon and\nsaturation of 5-alpha carbon, and synthesized for\nthe first time by Ringold in 1959 ( 7 ). Anabolicandrogenic\nsteroids (AAS), particularly OXM, are\nused at small doses (1-5 mg/Kg) for treating anemia,\nfailure to thrive (FTT) in children, and heart\nfailure ( 8 ). This type of drug increases erythropoietin\nproduction that is followed by affecting bone\nmarrow directly to increase hemoglobin level and\nred blood cell count ( 9 ). Additionally, OXM can\npromote synthesis of protein, nitrogen retention\nand deposition of calcium in bones ( 10 ). In order\nto assume anabolic properties, these steroids must\nbe used at 10-100 times their normal (therapeutic)\ndoses in which their adverse reactions occur,\n( 11 ) having profound effects on male endocrine\nand reproductive systems. It has been found that\nAASs-induced low male infertility can be reversible. Also, researches have indicated that\npersistent hypogonadotrophic hypogonadism\ncaused by steroid abuse could be resolved after\nthe AAS withdrawal ( 12 ). Royal jelly (RJ) is a\nmilky white highly viscous substance secreted\nfrom the salivary gland of the honeybee worker,\nApismellifera (Apidae), which is essential in\nthe development of queen bees ( 13 ). This substance\npossesses many physical and chemical\nproperties, including anti-inflammatory, antioxidant,\nanti-tumor, and immunomodulatory functions\nin experimental animals ( 14 ), which are\nbeneficial to human health, leading to its wide\nuse in commercial and medical products, health\nfood, and cosmetics ( 15 ).  In vitro  fertilization\n(IVF), first successfully applied in 1978, is the\nlast option for infertile couples who failed to\nconceive. In this international method, different\nstandard therapies like surgery, fertility drugs\nand artificial insemination are applied to treat\ninfertility cases like male and immunological\ninfertility, infertility caused by endometriosis,\nand other unexplained conditions ( 16 ,  17 ). This\nstudy aims to investigate the effects of RJ on\ncatalase, total antioxidant capacity and embryo\ndevelopment in adult mice treated with OXM.\n\nIn this exprimental study, two groups of 7-9-\nweek old adult Naval Medical Research Institute\n(NMRI) mice, 32 male and 96 female, weighing\n30 ± 2 g and 27 ± 2 g, respectively, were purchased\nfrom the Animal House of Science Faculty,\nUrmia University, Urmia, Iran. All animals\nwere allowed free access to water and food ad\nlibitum in controlled conditions of temperature\n(22 ± 2˚C), relative humidity (55 ± 5%) and normal\nphotoperiod.\nOXM was used at a dose of 5 mg/kg (pilot). Drug\nwas dissolved in saline before oral administration.\nRJ was used at the dose of 100 mg/kg ( 18 ).\nIn this investigate, 32 male and 96 female with a\nratio of 1:3 for fertilization purposes were divided\ninto following 4 groups: i. control group (n=8) receiving\n0.1 ml/mice saline daily by gavage for 30\ndays, ii. OXM group (n=8) treated with 5 mg/kg\nOXM daily by gavage for 30 days, iii. RJ group\n(n=8) treated with 100 mg/kg RJ daily by gavage\nfor 30 days and iv. RJ+OXM (n=8) group treated\nwith 5 mg/kg OXM by gavage along with 100 mg/\nkg RJ daily for 30 days. All experimental procedures\nwere approved by the Ethics Committee\nfor Animal Experimentation of Urmia University,\nUrima, Iran.\nEach female mouse was injected subcutaneously\nwith 10 IU pregnant mare’s serum gonadotropin\n(PMSG, Boxmeer, Netherlands) 48\nhours prior to receiving an intraperitoneal injection\nof 10 IU human chorionic gonadotropin\n(hCG, Folligon, Netherlands). The animals\nwere euthanized by dislocation of cervical vertebrae,\n12-14 hours after hCG administration.\nTheir ampulla of oviducts were removed and\ntransferred to a petri dish that contained 1 ml\nhuman tubular fluid (HTF, Sigma, St. Louis,\nUSA) medium+4 mg/ml bovine serum albumin\n(BSA, Sigma, St. Louis, USA). Using a stereo\nmicroscope (Model TL2, Olympus Co., Tokyo,\nJapan), the ampullary portion of fallopian tube\nwas found and oocytes were dissected out.\nOne day before conception, required media were\nprepared for fertilization and incubated 5% CO 2  and 37˚C for 12 hours. For each group, separate\nconception dishes were considered containing 1\nml HTF medium combined with 4 mg/ml BSA.\nA drop of 500 ml and a few drops of 100 ml per\ndish were used for washing the IVF dishes and\nthey were then covered with mineral oil (Sigma,\nSt. Louis, USA).\nMale mice were euthanized by dislocation\nof cervical vertebrae to prepare needed sperm.\nThen, the skin of the abdomen was sterilized\nwith 70% ethanol (Merck, USA) and after making\nan incision in the abdomen and removing\nthe surrounding connective tissue, caudal epididymis was isolated from testes and placed\nin a petri dish containing 1 ml HTF medium\ncombined with 4 mg/ml BSA which had reached\nequilibrium before. After making several cuts\nin the tail of the epididymis and using pressure\nin vas deferens, sperm output was placed in an\nincubator at 5% CO 2  and 37˚C for 30 minutes.\nSpermatozoa were then spread out in the medium.\nBetween 12 and 14 hours after injection of hCG\n(in the morning after), female mice were euthanized\nby dislocation of cervical vertebrae, and the\nampule of fallopian tubes were then removed and\nput in the HTF medium at 37˚C. Using dissection\ntechniques, oocytes were removed, washed\nwith the HTF, and transferred to the fertilization\ndroplets under mineral oil containing HTF+BSA.\nFollowing capacitation step, sperms (1×10 6 /1 ml\nHTF) were added to the medium, one million per\nml of culture. Fertilization is determined about 4 to\n6 hours after releasing the sperm by observing two\npre-nucleus. After granulosa cells were denuded\nand washed, these zygotes were transferred into\nthe fresh pre-equilibrated medium and cultured for\nfive days.\nEvaluation of two-cell embryos was done 24\nhours after fertilization. The percentage of blastocyst-\nstage embryos was performed on days 4 and\n5 of fertilization.\nCatalase activity was determined based on its\nability to decompose hydrogen peroxide (H 2 O 2 )\nin homogenized testicular tissue using the\nmethod of Aebi ( 19 ). Decomposition of H 2 O 2 \nis considerable by reducing the absorption at\n240 nm in an absorption spectrum. For this purpose,\n30 mM H 2 O 2  (Merck, USA) as substrate\nand 50 mM phosphate buffer solution (PBS,\nPH=7, Merck, USA) were used as an alternative\nsubstrate in the blank solution. The testis tissue\npieces were homogenized in PBS and then\ncentrifuged (Eppendorf AG 5810R, Germany)\nat 3400 rpm for 15 minutes. Subsequently 1 ml\nH 2 O 2  was added to 2 ml supernatant and absorbance\nwas measured at 240 nm in a spectrophotometer\n(pharmaciaп novaspec, and biochrom,\nEngland). The values were expressed in terms\nof U/g tissue.\nFor automatic measurement and evaluation\nof antioxidants, the ferric reducing antioxidant\npower (FRAP) was applied. Reduction of ferric\nto ferrous ions in low PH forms a colored\ncomplex of ferrous-tripyridyltriazine (FeIITPTZ).\nReactive oxygen species (ROS) that are\npotentially harmful are produced as byproducts\nof normal aerobic metabolism and usually removed\nor disabled by antioxidants groups in in\nvivo. In low PH, ferric tripyridyltriazine (FeШ-\nTPTZ) complex is reduced to form the Feп and\nto produces an intense blue color with a high\nabsorption at 593 nm. FRAP reagent (with a ratio\nof 10:1:1) including 25 ml of (300 mM PBS,\n10 mM TPTZ in 40 mM HCL, Merck, USA) and\n2.5 ml of 0.0540 g FeCl3 (Merck, USA) were\npoured into a flask and brought to volume of\n10 ml with distilled water sample was weighted\ndissolved into 1.5 M of 10% (W/V) KCL buffer\n(Merck, USA) and grounded in a mortar. The\nsolution was then poured in a micro-tube and\ncentrifuged at 1000 rpm for 5 minutes. One\nhundred ml of supernatant was poured into a\ntest tube, mixed with 3 mL of FRAP reagent and\nincubated in water bathat 37˚C for 7-10 minutes.\nThe absorbance of blue complex was then\ndetermined at 593 nm. Data are expressed in\nmmol tissue weight (FRAP value) ( 20 ,  21 ).\nAll data were analyzed using the one-way ANOVA\nby Tukey-Kramer test. The level of significance\nwas considered at P<0.05. All analysis for\neach sample were done individually.\n\nResults show that OXM caused a significant decrease\nin catalase enzyme compared to the control group (P<0.05). However RJ+OXM group partially\ncompensated this lack attributed to the OXM\ngroup ( Table 1 ).\nIn the OXM group, total antioxidant concentration\ndropped as compared with the control group,\nwhile in the RJ group, there was a significant increase\nin total antioxidant concentration in comparison\nwith the control group (P<0.05). The\nOXM+RJ showed a significant increase in this regard\nas compared with the control and OXM group\n(P<0.05,  Table 1 ).\nIn this study, OXM caused a significant decrease\nin fertilization rate and percentage of\nblastocysts (P<0.05) and a significant increase\nin the percentage of arrested embryos (P<0.05),\nbut did not affect percentage of the two-cell\nembryos. RJ caused an increase in fertilization\nrate and percentage of blastocyst and a decrease\nin percentage of the arrested embryos (P<0.05)\nand two-cell embryo ( Table 2 , Figes .1 ,  2 ).\nEffects of OXM, RJ as well as OXM and RJ on catalase activity and FRAP level in adult male mice\nValues are expressed as mean ± standard deviation (n=8).\na ; Significant differences as compared with the control group (P<0.05),  b ; Significant differences as compared with the OXM group (P<0.05),\nOXM; Oxymetholone, RJ; Royal jelly and FRAP; Ferric reducing antioxidant power.\nEffect of OXM , RJ as well as OXM and RJ on fertilization and embryonic development rates\nValues are expressed as mean ± standard deviation (n=8).\na ; Significant differences as compared with the control group (P<0.05),  b ; Significant differences as compared with the OXM group (P<0.05),\nOXM; Oxymetholone and RJ; Royal jelly.\nFertilized and infertilized oocytes in OXM group (×200).\nOXM; Oxymetholone.\nBlastocyst embryos in control group (×200).\n\nAAS alter the function of the hypothalamicpituitary-\ngonadal (HPG) axis that results in affecting\nthe target reproductive tissues. Acting on the\nhypothalamus, circulating testosterone regulates\nthe gonadotropin-releasing hormone (GnRH) release.\nIntroducing exogenous AAS to the body\nincreases androgen levels that cause a decrease\nin luteinizing hormone (LH) release from the pituitary,\nresulting in the suppression of endogenous\ntestosterone production ( 22 ,  23 ), which is found\nto be reversible ( 12 ). ROS induced damage is major\nfactors in male infertility ( 24 ,  25 ). In the last\ndecade, numerous evidences have been gathered\nabout etiology of oxidative stress (OS) in male infertility\n( 26 ,  27 ). Our findings indicated that OXM\ncauses a significant decrease in catalase activity\nand also total antioxidant concentration. The total\nantioxidant potential is a measure of the individual’s\nability to cope with or prevent a state of\nOS. It is noted that a state of disequilibrium is\nthe rate of generation of ROS that overwhelms an\nindividual’s ability to remove them ( 28 ). Our findings\nalso found that OXM reduces the fertilization\nrate and percentage of blastocysts, but increases\npercentage of arrested embryos which is probably\ndue to the effects of OS system. MacLeod ( 29 ),\nobserved that human spermatozoa produces ROS,\ni.e. highly reactive derivatives of oxygen such\nas H 2 O 2 , and have harmful effects on different\nsperm functions. ROS involvement in etiology of\nmale infertility is now well-established ( 30 ,  31 ).\nSince sperm functions are extremely sensitive to\nROS -because of high content of polyunsaturated\nfatty acids (PUFA) and limited ability of DNA\nrepairthe role of ROS and OS in human sperm\nfunction and pathophysiology of male infertility\nhave been intensively investigated ( 32 ,  33 ). Very\nrecently, scientists revealed that ROS not only affects\nthe fertilization process negatively, but also\nraises serious questions about the health and wellbeing\nof the progeny since the male germ cell is\nexposed to high oxidants concentrations ( 34 ,  35 ).\nFurthermore the concept of OS causing damage\nto spermatozoa by impairing various functions\nof sperm such as motility, acrosome reaction or\nDNA integrity is widely accepted nowadays ( 26 ,\n 36 ,  37 ). Several studies reported positive effects of\nRJ which is a homogeneous substance secreted by\nworker honey bees for feeding young larvae and\nthe adult queen bee on animal reproduction ( 38 ).\nThe physical and chemical properties of RJ have\nbeen described in detail elsewhere ( 39 ,  40 ). Intramuscular\nor oral administration of RJ has proven\nto be effective in improving estrus responses and pregnancy rate ( 41 ). The results of this study indicate\nthat RJ increases catalase activity and total\nantioxidant capacity. Antioxidants including the\nenzymes superoxide dismutase, catalase and glutathione\nperoxidase that are constantly produced\nduring normal metabolic and physiological processes\n( 42 ) counteract with the deleterious effects\nof ROS mammalian spermatozoa with rich\npolyunsaturated fatty acids are very susceptible\nto ROS attack which results in decreased sperm\nmotility, axonemal damage, decreased sperm\nviability, and increased midpiece morphology\ndefects with deleterious effects on sperm capacitation\nand acrosome reaction that is presumably\ndue to rapid loss of intracellular ATP ( 43 ).\nThe key mechanism of this ROS-induced sperm\ndamage is considered to be lipid peroxidation of\nsperm membrane that leads to infertility ( 44 ).\nSeveral studies using various antioxidants alone\nor in combination with others have shown a significant\nreduction in seminal ROS levels ( 45 ,  46 )\nand improvement in sperm count and motility\n( 47 ,  48 ), while other studies have found contrary\nresults ( 49 ,  50 ). We also found that RJ increases\nfertility-related parameters such as fertilization\nrate and blastocysts percentage and also reduces\npercentage of arrested embryos which is probably\ndue to its effect on the total antioxidant capacity.\nAntioxidants levels in seminal plasma of\ninfertile men are significantly low ( 51 ), Renard et\nal. ( 52 ) reported that intravaginal administration\nof Egyptian bee honey and RJ might be a reasonable\nand effective method to treat infertility\ncaused by asthenozoospermia. Amino acid content\nof both honey and RJ may play a main role\nin fertility either by enhancing acrosome reaction\nand sperm motility or by improving fertilization.\nAlso sperm motility may be further enhanced by\nthe short chain fatty acids specific to RJ, especially\n10-hydroxy2-decenoic acid ( 45 ).\n\nIt can be concluded that with the decline in total\nantioxidant capacity, OXM reduces fertility. Due\nto beneficial biological properties of its components,\nRJ showed to increase antioxidant capacity;\ntherefore, it prevents embryo toxicity manifested\nby OXM when given after OXM administration.\nAs the result, RJ with its antioxidant properties increases\nfertility.","source_license":"CC-BY-4.0","license_restricted":false}