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The present study was undertaken to assess the effect of immunostimulation and HBV antigen treatment on testes of male swiss albino mice. This study includes the protein level in the male reproductive organ. Mice treated with immunostimulant and HBV antigen showed an increase of protein compared with controls and decrease of protein compared with immunostimulated mice. Total proteins increased in the testicular tissue of immuniostimulated mice in comparission with controls. It is shown that antigen induced reproductive toxicity in male mice may be protected by treating with immuniostimulant. Key Words: : Immunex DS, Protein , Hbs Ag, Mice REFERENCES Akbarsha, M.A., Manivannan, B., Shahul Hamid, K. and Vijayan, B. (1990). Antifertility effect of Andrographis paniculata (Nees) in male albino rat. Ind. J. Exp. Biol. 28: 421. Alter, M.J., Hadler, S.C., Margolis, H.S., Alexander, W.J., Hu, P.Y., Judson, F.N., Mares, A., Miller, J.K. and Mover, L.A. (1990). The changing epidemiology of hepatitis B in the United States: Need for alternative vaccination strategies. J. Amer. Med. Ass. 263: 1218-1222. Bhatnagar, D., Soran, H. and Durington, P.N. (2008). Hypercholesterolaemia and its maangement. BMJ. 337: a993. Citarasu, T., Sivaram, V., Immanuel, G., Rout, N. and Nurugan, V. (2006). Influence of seleced indian immunostimulant herbs against white spot syndrome virus (WSSV) infection in black tiger shrimp Penaeus monodon with reference to haematological, biochemial and immunological changes. Fish. Shellfish Immunol. 21(4): 372-384. Clark, N.L., Aagaard, J.E. and Swanson, W.J. (2006). Evolution of reproductive proteins from animals and plants. Reproduction, 131: 11-22. Dashputre, N.L. and Naikwade, N.S. (2010). Immunomodulatory activity of Abutilan indicum Linn. on albino mice. Int. J. Pharma Sci. and Res. 1(3): 178-184. Dhar, P., Singla, N. and Babbar, B.K. (2014). Effect of triptolide on vital organs, blood biochemical parameters and histomorphology of testis in male Bandicota Bengalensis. Ind. J. App. Res. 4(2): 27-30. Esin, K.F. (2008). Biochemical evidence of free radical-induced lipid peroxidation for chronic toxicityof endosulfan and malathion in liver, kidney and gonadal tissues of wistar albino rats. Fresenius Env. Bull. 17(9A): 1340-1343. Gelina, J., Yin, G., Ardo, L. and Jeney, Z. (2009). The use of immunostimulating herbs in fish. An overview of research. Fish Physiol. Biochem. 35(4): 669-676. Halder, S., Bharal, N., Parmod, K., Mediratta, Inderjeet Kaur and Krishna K. Sharma (2009). Anti-inflammatory, immunomodulatory and antinociceptive activity of Terminalia arjuna. Roxb bark powder in mice and rats. Ind. J. Exp. Biol. 47(7): 577-583. Hannesdottir, S.G., Han, X., Lund, T., Singh, M., Zee, R.V.D., Roitt, I.M. and Delves, P.J. (2004). Changes in the reproductive system of male mice immunized with a GnRH analogue conjugated to Mycobacterial hsp 70. Reproduction, 128(3): 365-371. Hatipoglu, F.S., Ozmen, O., Ata, A., Ileri-Buyukoglu, T., Sahinduran, S., Mor, F., Yildiz Gulay, O.and Gulay, M.S. (2009). Ameliorating effect of ascorbic acid on sub acute endosulfan toxicty in male Newzealand white rabbits. J. Anim. Sci. 87: E-suppl.2. Jahromi, V.H., Parivar, K. and Forozanfar, M. (2011). The effect of cinnamon extract on spermatogenesis. Hormonal axis of pituitary gonad in mice. Iranian. J. Applied Ani. Sci. 1(2): 99-103. Jasmin Gold, V. and Viveka Vardhani, V. (2013). Serum cholesterol in immunostimulated mice during induced Hepatitis B. The Bioscan, 8(4): 1129-1134. Jasmin Gold, V. and Viveka Vardhani, V. (2014). Effect of Hepatitis and immunostimulant on serum testosterone level in experimental mice. Biolife 2(3):808-812. Jasmin Gold, V. and Viveka Vardhani, V. (2016a). Protein level in the heart of mice during immunostimulation and hepatitis infection. Biolife 4(2):271-274. Jasmin Gold, V. and Viveka Vardhani, V. (2016b). Protective effect of immunex DS against hepatitis B in a mouse experimental model. Biolife 4(2): 337-341. Kaur, R. and Parshad, V.R. (1994). Effects of dietary selenium on differentiatio, morphology and functions of spermatozoa of the house rat, Rattus rattus L. Mutation Res. 309: 29-35. Kaur, R., Dhanju, C.K. and Kaur, K. (1999). Effects of dietary selenium on biochemical composition of rat testis. Ind. J. Exp. Biol. 37: 509-511. Kaur, S. and Dhanju, C.K. (2004). Enzymatic changes induced by some organophosphorous pesticides in female rats. Ind. J. Exp. Biol. 42: 1017-1019. Kumada, T., Toyoda, H., Kiriyama, S., Sone, Y., Tanikowa, M., Hisanaga, Y., Kanamori, A., Atsumi, H.,Takagi, M., Arakawa, T. and Fujimon, M. (2010). Incidence of hepatocellular carcinomain patients with chronic hepatitis B virus infection who have normal alanine amino transferase values. J. Med. Virol. 17, 82(4): 539-545. Porika Raju and Estari Mamidala (2015). Anti-diabetic activity of compound isolated from Physalis angulata fruit extracts in alloxan induced diabetic rats. The Ame J Sci & Med Res, 2015,1(1); Pages 1 -6. doi:10.17812/ajsmr2015.11.1. Kumar, S., Koul, M. and Rai, A. (2010). Role of immunostimulatory molecules in poultry vaccines. Recent Pat. Biotechnol. 4(3): 235-241. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with folin phenol reagent. J. Biol. Chem. 177: 751-766. Mastoi, A.A., Devrajani, B.R., Shah, S.a., Rohopoto, Q., Memon, S.A. Baloch, M., Qureshi, G.A. and Sami, N. (2010). Metabolic investigations in patients with hepatisis B and C. World J. Gastroenterol. 7: (16)5: 603-607. Michel, M.L. and Tiollais, P. (2010). Hepatitis B vaccines: protective efficacy and therapeutic potential. Pathol. Biol. 58: 288-295. Muthuviveganandave, V., Muthuraman, P., Hwang, I. And Srikumar, K. (2011). Biochemical and histologial changes induced by low dose of carbendazim on testes of male albino rat. Int. J. Pharmacy Biol. Sci. 1(4): 572-576. Petrunov, B., Nenkov, P and Shekerdjiisky, R. (2007). The role of immunostimulants in immunotherapy and immunoprophylaxis. Biotechnol. Biotechnol. 21(4): 454-462. Ruiz-Morena, M., Garcia, R. and Rua, M.J. (1993). Levamisole and interferon in children with chronic hepatitis B. Hepatol. 18(2): 264-269. Sakunthala, G., Nathanel, P.J.R. and Vardhani, V.V. (2014). Impact of immunostimulant on stomach protein and DNA activity during hepatitis B infection in mice. Ind. J. Sci. Res. Tech. 2(2): 13-17. Shapiro, C.N. and Margolis, H.S. (1990). Hepatitis B epidemiology and prevention. Epidem. Rev. 12: 221-227. Sinha, N., Narayana, R. and Saxena, D.K. (1995). Endosulfan induced biochemical changes in the testes of rat. Vet. Hum. Toxicol. 37: 547-549. Sun, J., Cecic, I., Parkins. Ch.S. and Korbelik, M. (2002). Neutrophils as inflammatory and immune effectors in photodynamic therapy treated mouse –SCC VII tumors. Photochem. Photobiol. Sci. 1(9): 690. Thacker, E.L. (2010). Immuno modulators, immunostimulants and immunotherapies in small veterinary medicine. Vet. Clin. North Am. Small Anim. Pract. 40(3): 473-483. Thomas, C.E. and Kalyanaraman, B. (eds.) (1997). In: Oxygen Radials and the Disease Process, Hardwood Academic Publishers, The Netherlands. Umar, H., Ali, E., Daniel, E., Ahmed, L., Sherif, I.A. and Malgwi, I.S. (2012). Endosulfan-induced changes in sperm count, testicular weight and some erythrocyte indices in male guinea pigs. Br. J. Pharmacol. Toxicol. 3(4): 151-155. Zhang, Y., Zhou, J.H., Yin, X.L., and Wang, F.Y. (2010). Treatment of hepatitis B virus associated glomerulonephritis: a meta analysis. World J. Gastoenterol. 14: 16(6): 770-777

Six different doses of Gen Vac B Hbs Ag vaccine were given orally to 6 varied groups (A, B, C, D, E and F) of male swiss albino mice after immunostimulation. Heart protein level in all the experimental groups of mice were studied and compared with controls (group N) and immunostimulated (group I) mice 0n day 1, 3, 4 and 5 of experimental period. Mice treated alone with immunex DS (group I) showed a very high level of protein throughout the experimental period when compared to all the experimental groups (A, B, C, D, E, F, G and H). Mice of groups A (which received the lowest dose of Gene Vac B @ 0.07 ml/mouse) and E (received Gene Vac B @ 0.08 ml/mouse) showed high protein level throughout the experimental period when compared among the experimental groups. However, mice of group A showed heightened protein response compared to group E. Experimental mice of group C (which received single dose of IDS and inoculated with 0.2 ml of Gene vac B vaccine) showed very lowest protein levels in comparison with IDS treated mice (group I) and with the experimental groups. Key words: Heart, protein, immunostimulant, hepatitis, mice. REFERENCES Borzi, R.M., Dal Mote, P., Honorati, M.C.and Facchini, A. (1992). IgG subclass distribution of anti-HBs antibodies following vaccination with cDNA Hbs Ag. J. Immunol. Methodol. 146: 17-23. Bricknell, I. and Dalmo, R.A. (2005). The use of immunostimulants in fish larval aquaculture. Fish. Shell Fish Immunol. 19(5): 457-472. Dashputre, N.L. and Naikwade, N.S. (2010). Immunomodulatory activity of Abutilan indicum Linn. on albino mice. Int. J. Pharma Sci. and Res. 1(3): 178-184. Davis, E.(2006). Imunostimulants and their role as an alternative to vaccines. Immunopharmacol. Immunotoxicol. 19(9): 1-2. Gelina, J., Yin, G., Ardo, L. and Jeney, Z. (2009). The use of immunostimulating herbs in fish. An overview of research. Fish Physiol. Biochem. 35(4): 669-676. Kumada, T., Toyoda, H., Kiriyama, S., Sone, Y., Tanikowa, M., Hisanaga, Y., Kanamori, A., Atsumi, H.,Takagi, M., Arakawa, T. and Fujimon, M. (2010). Incidence of hepatocellular carcinomain patients with chronic hepatitis B virus infection who have normal alanine amino transferase values. J. Med. Virol. 17, 82(4): 539-545. Kumar, S., Koul, M. and Rai, A. (2010). Role of immunostimulatory molecules in poultry vaccines. Recent Pat. Biotechnol. 4(3): 235-241. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with folin phenol reagent. J. Biol. Chem. 177: 751-766. Lujan, H.L., Janbaih, H., Feng, H.Z., Jin, J.P and Dicarlo, S.F. (2012). Myocardial ischemia, reperfusion and infarction in chronically instrumented, intact, conscious and unrestrained mice. Am. J. Physiol. 302(12): R1384-R1400. Madhuri, D and Viveka Vardhani, V (2015). GST level in the abdominal muscles of mice treated with Immunex DS and Gene Vac B vaccine. Biolife. 3(1)26-30. Malliaras, K., Zhang, Y., Seinfeld, J., Galang, G., Tseliou, E., Cheng, K., Sun, B., Aminzadeh, M. and Marban, E. (2013). Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart. EMBO. Mol. Med. 5(2): 191-209. Malmstrom, S., Enquist, I.B. and Lindh, M. (2010). A novel geotyping method for hepatitis B virus based on Taqman real-time. PCR. J. Clin. Microbiol. 1: 27. Mastoi, A.A., Devrajani, B.R., Shah, S.a., Rohopoto, Q., Memon, S.A. Baloch, M., Qureshi, G.A. and Sami, N. (2010). Metabolic investigations in patients with hepatisis B and C. World J. Gastroenterol. 7: (16)5: 603-607. Nathanael P. J. R and Vardhani V. V (2014). The influence of immunex ds against experimental Hepatitis B vaccine on liver protein and DNA profile of Mice. Biolife. 2(1):341-347. Randle, P.J., England, P.J. and Denton, R.M. (1970). Control of tricarboxylate cycle and its interactions with glycolysis during acetate utilisation in rat heart. Biochem. 117(4): 677-695. Recchia, F.A. and Lionetti, V. (2007). Animal models of dilated cardiomyopathy for translational research. Vet. Rose Commun. 31(1): 35-41. Roger, V.L., Go, A.S., Lloyd-Jones, D.M., Benjamin, E.J., Berry, J.D., Borden, W.B., Bravata, D.M., Dai, S., Ford, E.S., Fox, C.S., Fullerton, H.J., Gillespie, C., Hailpern, S.M., Heit, J.A., Howard, V.J., Kissela, B.M., Kittner, S.J., Lackland, D.T., Lichtman, J.H., Lisabeth, L.D., Makul, D.M., Marcus, G.M., Marelli, A., Matchar, D.B, Moy, C.S., Mozaffarian, D., Mussolino, M.E., Nichol, G., Paynter, N.P., Soliman, E.Z., Sorlie, P.D., Sotoodehnia, N., Turan, T.N., Virani, S.S., Wong, N.D. and Wood, Turner, M.B. (2012). Heart disease and stroke statistics-2012 update: a report from the American heart association. Circ. 125: e2-e220. Roger, V.L., Go. A.S., Lloyd-Jones, D.M., Adams, R.J., Berry, J.D., Brown, J.D., Brown, T.M., Carnethon, M.R., Dai, S., De, S.G., Ford, E.S., Fox, C.S., Fullerton, H.J., Gillespie, C., Greenlund, K.J., Hailpern, S.M., Heit, J.A., Ho, PM., Howard, V.J., Kissela, B.M., Kittner, S.J., Lackland, D.T., Lichtman, J.H., Lisabeth, L.D., Makuc, D.M., Marcus, G.M., Marelli, A., Matchar, D.B., McDermott, M.M., Meigs, J.B., Moy, C.S., Mozaffarian, D., Mussolino, M.E., Nichol., G., Paynter, N.P., Rosamond, W.D., Sorlie, P.D., Stafford, R.S., Turan, T.N., Turner, M.B., Wong., N.D. and Wlie-Rosett, J. (2011). Heart disease and stroke statistics 2011 update: a report from the American Heart Association. Circ. 123: e18-e209. Sun, J., Cecic, I., Parkins. Ch.S. and Korbelik, M. (2002). Neutrophils as inflammatory and immune effectors in photodynamic therapy treated mouse -–SCC VII tumors. Photochem. Photobiol. Sci. 1(9): 690.

The present study aimed to illustrate the effect of the immunostimulant, immunex DS and IDS + hepatitis B infection on serum testosterone level in male swiss albino mice. Testosterone level was increased significantly in IDS treated mice and IDS + vaccine treated mice from day 1 to 5 of infection when compared with controls. Viral toxicity led to the abnormal increase and release of testosterone in the blood of all the experimental groups of mice due to improper spermatogenesis. Key words: Immunex DS, Hepatitis B infection, Serum testosterone, Mice

Additional file 1. Description of the accurate analyzed region in each tested gene

STUDY QUESTION Has PGD-HLA been successful relative to diagnostic and clinical efficacy? SUMMARY ANSWER The diagnostic efficacy of PGD-HLA protocols was found lower in this study in comparison to published PGD-HLA protocols and to that reported for general PGD by ESHRE (78.5 vs 94.1% and vs 92.6%, respectively), while the clinical efficacy has proven very difficult to assess due to inadequate follow-up of both the ART/PGD and HSCT procedure outcomes. WHAT IS KNOWN ALREADY The first clinical cases for PGD-HLA were reported in 2001. It is now a well-established procedure, with an increasing number of cycles performed every year. However, PGD-HLA is still offered by relatively few PGD centres, the currently available data is fragmented and most reports on PGD-HLA applications are limited in number and scope. Published systematic details on methodology, diagnostic results, overall ART success and haematopoietic stem cell transplantation (HSCT) outcomes are limited, precluding an evaluation of the true clinical utility of PGD-HLA cycles. STUDY DESIGN, SIZE, DURATION This retrospective multi-centre cohort study aimed to investigate the diagnostic and clinical efficacy of the PGD-HLA procedure and the aspects of PGD-HLA cycles influencing positive outcomes: birth of genetically suitable donor-baby (or babies) and HSCT. In April 2014, 32 PGD centres (Consortium members and non-members) with published/known PGD-HLA activity were invited to participate. Between February and September 2015, 14 centres submitted their data, through a custom-designed secure database, with unique login access for each centre. Data parameters covered all aspects of PGD-HLA cycles (ART, embryology and genetic diagnosis), donor-babies born and HSCT. PARTICIPANTS/MATERIALS, SETTING, METHODS From 716 cycles submitted by 14 centres (performed between August 2001 and September 2015), the quality evaluation excluded 12 cycles, leaving 704, from 364 couples. The online database, based on REDCap, a free, secure, web-based data-capture application, was customized by Centre for Clinical Epidemiology and Outcomes Research (CLEO), Athens. Continuous variables are presented using mean, standard deviation, median and interquartile range, and categorical variables are presented as absolute and relative frequencies. MAIN RESULTS AND THE ROLE OF CHANCE The data included 704 HLA-PGD cycles. Mean maternal age was 33.5 years. Most couples (81.3%) requested HLA-typing with concurrent exclusion of a single monogenic disease (58.6% for beta-thalassaemia). In 92.5% couples, both partners were fertile, with an average 1.93 HLA-PGD cycles/couple. Overall, 9751 oocytes were retrieved (13.9/cycle) and 5532 embryos were analysed (7.9/cycle). Most cycles involved fresh oocytes (94.9%) and Day 3 embryo biopsy (85.3%). In 97.5% of cycles, the genotyping method involved PCR only. Of 4343 embryos diagnosed (78.5% of analysed embryos), 677 were genetically suitable (15.4% of those analysed for HLA alone, 11.6% of those analysed for HLA with exclusion of monogenic disease). Of the 364 couples, 56.6% achieved an embryo transfer (ET) and 598 embryos were transferred in 382 cycles, leading to 164 HCG-positive pregnancies (pregnancy rate/ET 41.3%, pregnancy rate/initiated cycle 23.3%) and 136 babies born (live birth rate/ET 34.3%, live birth rate/initiated cycle 19.3%) to 113 couples. Data analysis identified the following limitations to the overall success of the HLA-PGD procedure: the age of the mother undergoing the treatment cycle, the number of oocytes collected per cycle and genetic chance. HSCT was reported for 57 cases, of which 64.9% involved combined umbilical cord-blood and bone marrow transplantation from the HLA-identical sibling donor; 77.3% of transplants reported no complications. LIMITATIONS REASONS FOR CAUTION The findings of the study may be limited as not all PGD centres with PGD-HLA experience participated. Reporting bias on completion of the online database may be another potential limitation. Furthermore, the study is based on retrospective data collection from centres with variable practices and strategies for ART, embryology and genetic diagnosis. WIDER IMPLICATIONS OF THE FINDINGS This is the first multi-centre study evaluating the clinical utility of PGD-HLA, indicating variations in practice and outcomes throughout 15 years and between centres. The study highlights parameters important for positive outcomes and provides important information for both scientists and couples interested in initiating a cycle. Above all, the study underlines the need for better collaboration between all specialists involved in the ART-PGD/HLA procedure, as well as the need for comprehensive and prospective long-term data collection, and encourages all specialists to aim to properly evaluate and follow-up all procedures, with the ultimate aim to promote best practice and encourage patient informed decision making. STUDY FUNDING/COMPETING INTEREST(S) The study wishes to acknowledge ESHRE for funding the customization of the REDCap database. There are no competing interests. TRIAL REGISTRATION NUMBER N/A. © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.