Present and Prospective Diagnostic and Therapeutic Options for Repeated IVF Failures 2 1 573 2 Recent evidence shows that through best standard operating procedures, chances of successful pregnancy and live birth delivery rates are less than 50%. Repeated Implantation Failure (RIF) following transfer of one or more numbers of cleavage or blastocyst stage embryos is the main cause of low pregnancy rate in IVF cycles; therefore, finding the causes and modifying treatment protocols to increase IVF success rates are cur-rently hot topics for research. RIF is defined as a failed pregnancy following transfer of ten or more high quality embryos through at least three cycles of fresh embryo transfer (1). A successful pregnancy following embryo transfer depends on the three sides of implantation triangle: a re-ceptive endometrium, a good quality embryo at the blastocyst stage and synchronization between endo-metrium and embryo through appropriate molecular dialogues. Uterine and ovarian disorders such as endometriosis, endometrial polyps, adhesions, uterine septa, submucosal and intramural fibroids, endo-metritis, adenomyosis, hydrosalpinx, leiomyoma and polycystic ovarian syndrome (PCOS) are associated with repeated implantation failure. Different studies have shown that resection of submucosal fibroids and intrauterine septum, hysterescopic polypectomy, salpingectomy, endometriosis surgery and myomectomy improve implantation and clinical pregnancy rates through molecular changes in endometrial steroid recep-tors, and changes in gene expression such as integrins, LIF, EMX2, HOXA-10 and IGFBP-1. (2).Therefore, gynecological surgery to provide a receptive endometrium is suggested as the first line of intervention in RIF due to the aforesaid uterine anomalies. Regarding the physiological role of endometrium in reproduction, due attention needs to be given to discovery of novel biomarkers for establishing the implantation window and monitoring endometrial thickness and receptivity. The aforesaid measures will change the outcome of ART cycles, especially RIF cycles. Another side of the implantation triangle and the most determinant factor in the failure of IVF cycles is embryo quality. According to cytogenetic studies, more than 60% of in vitro derived embryos have at least one aneuploid blastomere at cleavage stage, even though chromosomal anomalies can be found in embryos with normal morphology. Mosaicism and aneuploidy of embryo interfere with its subsequent development, implantation and ongoing pregnancy. Therefore, selection of embryos with higher rates of implantation and development is an effective option to reduce RIF. In most IVF clinics, selection of embryo is based on a prolonged culture and blastocyst transfer, but blastocyst transfer is not always possible due to poor culture or the low number of cleavage embryos (3). In addition, recent development in "omics" technology, such as proteomics and metabolomics, analysis of conditioned culture medium or genomics and transcriptional analysis of embryo biopsies have provided more accurate selection of the best embryos and they will be available as routine tests in IVF clinics in the near future. The third side of this triangle is the correct timing of embryo transfer for appropriate molecular dialogue between embryo and endometrium. Despite excellent status of one side of implantation triangle, the defect of one side will lead to three-side insufficiency followed by repeated implantation failure. A variable that can affect all the three sides of implantation triangle is ovarian stimulation protocols. Recent data have shown that long protocol stimulation with high doses of gonadotropins produce large numbers of oocytes and embryos with frequent chromosomal defects, poor quality of endometrium and low implantation and preg-nancy rates of the derived embryos. However, although mild stimulation regimens provide limited numbers of oocytes and embryos with lower rates of aneuploidy but higher implantation and pregnancy rates are ex-pected (4). In conclusion, decision on treatment plan for couples with repeated implantation failure should be based on the simultaneous optimization and correction of all the three sides of implantation triangle. Neglect to opti-mize and correct one side of the triangle will lead to repeated IVF cycle failures. 01 3 Mohammad Reza MR Sadeghi محمدرضا صادقی Editor-in-chief Editor-in-chief Iran No Keyword 573.pdf Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treatment of repeated implantation failure following IVF-ET. Hum Reprod. 2006;21(12):3036-43.##Cakmak H, Taylor HS. Implantation failure: molecular mechanisms and clinical treatment. Hum Reprod Update. 2011;17(2):242-53.##Assou S, Boumela I, Haouzi D, Anahory T, Dechaud H, De Vos J, et al. Dynamic changes in gene expression during human early embryo development: from fundamental aspects to clinical applications. Hum Reprod Update. 2011; 17(2):272-90.##Santos MA, Kuijk EW, Macklon NS. The impact of ovarian stimulation for IVF on the developing embryo. Repro-duction. 2010;139(1):23-34.##

Effects of Pharmaceutical Medications on Male Fertility 2 1 2 The number of couples seeking consultation for infertility problems has steadily increased over the past decade, affecting 10%-15% of the sexually active population. Abnormal semen production, a male factor infertility (MFI), is thought to be the cause of up to 50% of all infertilities in developed countries. There are potentially many different causes of male infertility, including hormonal, anatomical, and secondary to exposure to exogenous substances. In many cases of MFI, a definitive cause for abnormalities is never identified. Recently, the research community has given greater attention to identifying causes of MFI ranging from genetic Y chromosome microdeletions to mechanisms of environmental damage on sperm production. Still evolving, is a clear understanding of how many pharmaceutical medications may cause MFI, which is often treatable and reversible. In this review we will out-line the data regarding various pharmaceutical medications that have been investigated as possible causes of MFI. 03 12 Paul P R. Brezina Paul R. Brezina Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine USA Fahd F N. Yunus Fahd N. Yunus Zanvyl Kreiger School of Arts and Sciences, Johns Hopkins University Zanvyl Kreiger School of Arts and Sciences, Johns Hopkins University USA Yulian Y Zhao Yulian Zhao Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine USA Male infertilityMedicationsPregnancyReviewSperm 482.pdf Rabin DS, Qadeer U, Steir VE. A cost and outcome model of fertility treatment in a managed care environment. Fertil Steril. 1996;66(6):896-903.##Singh K, Jaiswal D. Human male infertility: a complex multifactorial phenotype. Reprod Sci. 2011;18 (5):418-25.##Thonneau P, Marchand S, Tallec A, Ferial ML, Ducot B, Lansac J, et al. Incidence and main causes of infertility in a resident population (1,850,000) of three French regions (1988-1989). Hum Reprod. 1991;6(6):811-6.##Wong EW, Cheng CY. Impacts of environmental toxicants on male reproductive dysfunction. Trends Pharmacol Sci. 2011;32(5):290-9.##Natali A, Turek PJ. An assessment of new sperm tests for male infertility. Urology. 2011;77(5):1027-34.##Baker HW. Male infertility. Endocrinol Metab Clin North Am. 1994;23(4):783-93.##World Health Organization. WHO Laboratory Manual for Examination and Processing of Human Semen. 5th ed. Geneva: World Health Organization. 2010. 287 p.##Aitken RJ. Sperm function tests and fertility. Int J Androl. 2006;29(1):69-75.##Lee Hee Y. Expenence with 3,520 infertile male patients and a clinical perspective. Seoul J Med. 1986; 27(1):1-18.##Thompson ST. Prevention of male infertility: an update. Urol Clin North Am. 1994;21(3):365-76.##Pasqualotto FF, Lucon AM, Sobreiro BP, Pasqualotto EB, Arap S. Effects of medical therapy, alcohol, smoking, and endocrine disruptors on male infertility. Rev Hosp Clin Fac Med Sao Paulo. 2004;59(6):375-82.##Pollack MH, Reiter S, Hammerness P. Genitourinary and sexual adverse effects of psychotropic medication. Int J Psychiatry Med. 1992;22(4):305-27.##Soleimani L, Lapidus KA, Iosifescu DV. Diagnosis and treatment of major depressive disorder. Neurol Clin. 2011;29(1):177-93.##IMS: Institute of Healthcare Informatics [Internet]. USA: IMS Health Incorporated; 2011. The Use of Medicines in the United States: Review of 2010; 2011 Apr [cited 2011 Jul 5]; [36 pages]. Available from: http://www.imshealth.com/deployedfiles/ims health/Global/Content/IMS Institute/Static File/IHII_UseOfMed_report.pdf##Murrough JW, Iacoviello B, Neumeister A, Charney DS, Iosifescu DV. Cognitive dysfunction in depression: Neurocircuitry and new therapeutic strategies. Neurobiol Learn Mem. 2011;96(4):553-63.##Ali MK, Lam RW. Comparative efficacy of escitalopram in the treatment of major depressive disorder. Neuropsychiatr Dis Treat. 2011;7:39-49.##Hendrick V, Gitlin M, Altshuler L, Korenman S. Antidepressant medications, mood and male fertility. Psychoneuroendocrinology. 2000;25(1):37-51.##Steiger A, von Bardeleben U, Wiedemann K, Holsboer F. Sleep EEG and nocturnal secretion of testosterone and cortisol in patients with major endogenous depression during acute phase and after remission. J Psychiatr Res. 1991;25(4):169-77.##Rubin RT, Poland RE, Lesser IM. Neuroendocrine aspects of primary endogenous depression VIII. Pituitary-gonadal axis activity in male patients and matched control subjects. Psychoneuroendocrinology. 1989;14(3):217-29.##Davies RH, Harris B, Thomas DR, Cook N, Read G, Riad-Fahmy D. Salivary testosterone levels and major depressive illness in men. Br J Psychiatry. 1992;161:629-32.##Tanrikut C, Schlegel PN. Antidepressant-associated changes in semen parameters. Urology. 2007; 69(1):185.e5-7.##Tanrikut C, Feldman AS, Altemus M, Paduch DA, Schlegel PN. Adverse effect of paroxetine on sperm. Fertil Steril. 2010;94(3):1021-6.##Relwani R, Berger D, Santoro N, Hickmon C, Nihsen M, Zapantis A, et al. Semen parameters are unrelated to BMI but vary with SSRI use and prior urological surgery. Reprod Sci. 2011;18(4):391-7.##Kumar VS, Sharma VL, Tiwari P, Singh D, Maikhuri JP, Gupta G, et al. The spermicidal and anti-trichomonas activities of SSRI antidepressants. Bioorg Med Chem Lett. 2006;16(9):2509-12.##Lu S, Zhao Y, Hu J, Li X, Zhang H, You L, et al. Combined use of phosphodiesterase-5 inhibitors and selective serotonin reuptake inhibitors for temporary ejaculation failure in couple undergoing assisted reproductive technologies. Fertil Steril. 2009;91(5):1806-8.##Ferri C, Croce G, Desideri G. Role of combination therapy in the treatment of hypertension: focus on valsartan plus amlodipine. Adv Ther. 2008;25(4): 300-20.##Benoff S, Cooper GW, Hurley I, Mandel FS, Rosenfeld DL, Scholl GM, et al. The effect of calcium ion channel blockers on sperm fertilization potential. Fertil Steril. 1994;62(3):606-17.##Aaberg RA, Sauer MV, Sikka S, Rajfer J. Effects of extracellular ionized calcium, diltiazem and cAMP on motility of human spermatozoa. J Urol. 1989;141(5):1221-4.##Kanwar U, Anand RJ, Sanyal SN. The effect of nifedipine, a calcium channel blocker, on human spermatozoal functions. Contraception.1993;48(5): 453-70.##Hong CY, Chiang BN, Turner P. Calcium ion is the key regulator of human sperm function. Lancet. 1984;2(8417-8418):1449-51.##Turner P. Recent observations on drugs and human fertility. Postgrad Med J. 1988;64(754):578-80.##Katsoff D, Check JH. A challenge to the concept that the use of calcium channel blockers causes reversible male infertility. Hum Reprod. 1997;12(7): 1480-2.##Hellstrom WJ, Sikka SC. Effects of acute treatment with tamsulosin versus alfuzosin on ejaculatory function in normal volunteers. J Urol. 2006;176(4 Pt 1):1529-33.##Hellstrom WJ, Sikka SC. Effects of alfuzosin and tamsulosin on sperm parameters in healthy men: results of a short-term, randomized, double-blind, placebo-controlled, crossover study. J Androl. 2009;30(4):469-74.##Andersson KE, Wyllie MG. Ejaculatory dysfunction: why all alpha-blockers are not equal. BJU Int. 2003;92(9):876-7.##Webber MP, Hauser WA, Ottman R, Annegers JF. Fertility in persons with epilepsy: 1935-1974. Epilepsia. 1986;27(6):746-52.##Herzog AG, Seibel MM, Schomer DL, Vaitukaitis JL, Geschwind N. Reproductive endocrine disorders in men with partial seizures of temporal lobe origin. Arch Neurol. 1986;43(4):347-50.##Shechter-Amir D, Yavetz H, Homonnai TZ, Huberman M, Cohn DF. Semen parameters among epileptic males treated with carbamazepine. Isr J Med Sci. 1993;29(10):648-9.##Taneja N, Kucheria K, Jain S, Maheshwari MC. Effect of phenytoin on semen. Epilepsia. 1994;35 (1):136-40.##Hayashi T, Yoshida S, Yoshinaga A, Ohno R, Ishii N, Yamada T. Improvement of oligoasthenozoospermia in epileptic patients on switching anti-epilepsy medication from sodium valproate to phenytoin. Scand J Urol Nephrol. 2005;39(5):431-2.##Hayashi T, Yoshinaga A, Ohno R, Ishii N, Kamata S, Watanabe T, et al. Asthenozoospermia: possible association with long-term exposure to an anti-epileptic drug of carbamazepine. Int J Urol. 2005;12 (1):113-4.##Røste LS, Taubøll E, Haugen TB, Bjørnenak T, Saetre ER, Gjerstad L. Alterations in semen parameters in men with epilepsy treated with valproate or carbamazepine monotherapy. Eur J Neurol. 2003; 10(5):501-6.##Chu C, Selwyn PA. Complications of HIV infection: a systems-based approach. Am Fam Physician. 2011;83(4):395-406.##Leone S, Gregis G, Quinzan G, Velenti D, Cologni G, Soavi L, et al. Causes of death and risk factors among HIV-infected persons in the HAART era: analysis of a large urban cohort. Infection. 2011;39 (1):13-20.##Nicopoullos JD, Almeida P, Vourliotis M, Goulding R, Gilling-Smith C. A decade of sperm washing: clinical correlates of successful insemination outcome. Hum Reprod. 2010;25(8):1869-76.##Crittenden JA, Handelsman DJ, Stewart GJ. Semen analysis in human immunodeficiency virus infection. Fertil Steril. 1992;57(6):1294-9.##van Leeuwen E, Wit FW, Prins JM, Reiss P, van der Veen F, Repping S. Semen quality remains stable during 96 weeks of untreated human immunodeficiency virus-1 infection. Fertil Steril. 2008;90(3):636-41.##van Leeuwen E, Wit FW, Repping S, Eeftinck Schattenkerk JK, Reiss P, van der Veen F, et al. Effects of antiretroviral therapy on semen quality. AIDS. 2008;22(5):637-42.##Carr A, Samaras K, Burton S, Law M, Freund J, Chisholm DJ, et al. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS. 1998;12(7):F51-8.##Ahmad G, Moinard N, Jouanolou V, Daudin M, Gandia P, Bujan L. In vitro assessment of the adverse effects of antiretroviral drugs on the human male gamete. Toxicol In Vitro. 2011;25(2):485-91.##Kehl S, Weigel M, Müller D, Gentili M, Hornemann A, Sütterlin M. HIV-infection and modern antiretroviral therapy impair sperm quality. Arch Gynecol Obstet. 2011;284(1):229-33.##Haidl G, Allam JP, Schuppe HC. Chronic epididymitis: impact on semen parameters and therapeutic options. Andrologia. 2008;40(2):92-6.##Cai T, Wagenlehner FM, Mazzoli S, Meacci F, Mondaini N, Nesi G, et al. Semen Quality in Patients with Chlamydia Trachomatis Genital Infection Treated Concurrently with Prulifoxacin and a Phytotherapeutic Agent. J Androl. 2011 Oct 6. [Epub ahead of print].##Yániz JL, Marco-Aguado MA, Mateos JA, Santolaria P. Bacterial contamination of ram semen, antibiotic sensitivities, and effects on sperm quality during storage at 15°C. Anim Reprod Sci. 2010; 122(1-2):142-9.##Farombi EO, Ugwuezunmba MC, Ezenwadu TT, Oyeyemi MO, Ekor M. Tetracycline-induced reproductive toxicity in male rats: effects of vitamin C and N-acetylcysteine. Exp Toxicol Pathol. 2008; 60(1):77-85.##Drumond AL, Weng CC, Wang G, Chiarini-Garcia H, Eras-Garcia L, Meistrich ML. Effects of multiple doses of cyclophosphamide on mouse testes: Accessing the germ cells lost, and the functional damage of stem cells. Reprod Toxicol. 2011;32(4): 395-406.##Levine J. Fertility preservation in children and adolescents with cancer. Minerva Pediatr. 2011;63(1): 49-59.##Marquis A, Kuehni CE, Strippoli MP, Kühne T, Brazzola P; Swiss Pediatric Oncology Group. Sperm analysis of patients after successful treatment of childhood acute lymphoblastic leukemia with chemotherapy. Pediatr Blood Cancer. 2010;55 (1):208-10.##Attia SM, Badary OA, Hamada FM, Hrabé de Angelis M, Adler ID. The chemotherapeutic agents nocodazole and amsacrine cause meiotic delay and non-disjunction in spermatocytes of mice. Mutat Res. 2008;651(1-2):105-13.##Rajender S, Monica MG, Walter L, Agarwal A. Thyroid, spermatogenesis, and male infertility. Front Biosci (Elite Ed). 2011;3:843-55.##Tan RS, Scally MC. Anabolic steroid-induced hypogonadism--towards a unified hypothesis of anabolic steroid action. Med Hypotheses. 2009;72 (6):723-8.##Bonetti A, Tirelli F, Catapano A, Dazzi D, Dei Cas A, Solito F, et al. Side effects of anabolic androgenic steroids abuse. Int J Sports Med. 2008;29(8): 679-87.##Lombardo F, Sgrò P, Salacone P, Gilio B, Gandini L, Dondero F, et al. Androgens and fertility. J Endocrinol Invest. 2005;28(3 Suppl):51-5.##Pomara G, Morelli G, Canale D, Turchi P, Caglieresi C, Moschini C, et al. Alterations in sperm motility after acute oral administration of sildenafil or tadalafil in young, infertile men. Fertil Steril. 2007;88(4):860-5.##Dimitriadis F, Tsambalas S, Tsounapi P, Kawamura H, Vlachopoulou E, Haliasos N, et al. Effects of phosphodiesterase-5 inhibitors on Leydig cell secretory function in oligoasthenospermic infertile men: a randomized trial. BJU Int. 2010;106(8): 1181-5.##Safarinejad MR. Effect of pentoxifylline on semen parameters, reproductive hormones, and seminal plasma antioxidant capacity in men with idiopathic infertility: a randomized double-blind placebo-controlled study. Int Urol Nephrol. 2011;43(2):315-28.##Mietens A, Tasch S, Feuerstacke C, Eichner G, Volkmann J, Schermuly RT, et al. Phosphodiesterase 5 (PDE5) inhibition, ANP and NO rapidly reduce epididymal duct contractions, but long-term PDE5 inhibition in vivo does not. Mol Cell Endocrinol. 2012;349(2):145-53.##Dimitriadis F, Tsampalas S, Tsounapi P, Giannakis D, Chaliasos N, Baltogiannis D, et al. Effects of phosphordiesterase-5 inhibitor vardenafil on testicular androgen-binding protein secretion, the maintenance of foci of advanced spermatogenesis and the sperm fertilising capacity in azoospermic men. Andrologia. 2011 Jul 27. [Epub ahead of print]##Glenn DR, McClure N, Cosby SL, Stevenson M, Lewis SE. Sildenafil citrate (Viagra) impairs fertilization and early embryo development in mice. Fertil Steril. 2009;91(3):893-9.##Glenn DR, McVicar CM, McClure N, Lewis SE. Sildenafil citrate improves sperm motility but causes a premature acrosome reaction in vitro. Fertil Steril. 2007;87(5):1064-70.##Levitas E, Lunenfeld E, Weiss N, Friger M, Har Vardi I, Koifman A, et al. Relationship between the duration of sexual abstinence and semen quality: analysis of 9,489 semen samples. Fertil Steril. 2005;83(6):1680-6.##Levitas E, Lunenfeld E, Weisz N, Friger M, HarVardi I, Potashnik G. Relationship between sexual abstinence duration and the acrosome index in teratozoospermic semen: analysis of 1800 semen samples. Andrologia. 2006;38(3):110-2.##Chen Z, Toth T, Godfrey-Bailey L, Mercedat N, Schiff I, Hauser R. Seasonal variation and agerelated changes in human semen parameters. J Androl. 2003;24(2):226-31.##Molina RI, Martini AC, Tissera A, Olmedo J, Senestrari D, de Cuneo MF, et al. Semen quality and aging: analysis of 9.168 samples in Cordoba. Argentina. Arch Esp Urol. 2010;63(3):214-22.##Lampiao F. Variation of semen parameters in healthy medical students due to exam stress. Malawi Med J. 2009;21(4):166-7.##Amann RP. The cycle of the seminiferous epithetlium in humans: a need to revisit? J Androl. 2008; 29(5):469-87.##Elia J, Imbrogno N, Delfino M, Mazzilli F. Retrograde ejaculation and abnormal hormonal profile in a subject under treatment with valproate and phenytoin. Arch Ital Urol Androl. 2010;82(4):193-4.##

The Effect of Macromolecule Source and Type of Media During in vitro Maturation of Sheep Oocytes on Subsequent Embryo Development 2 1 2 Background: Oocyte maturation and subsequent in vitro production (IVP) of embryos are affected by diverse groups of chemicals in maturation medium which are needed for successful mammalian oocyte maturation during which the dramatic cytoplasmic and nuclear reprogramming events take place. This study was designed to evaluate the effects of protein source (fetal bovine serum, FBS, and bovine serum albumin, BSA) as well as two different maturation media during in vitro maturation of ovine oocytes on subsequent embryo development. Methods: Cumulus oocyte complexes were recovered from ovaries obtained from slaughter house and cultured for 24 hr in either TCM-199 or SOFaa maturation medium supplemented with 10% (v/v) FBS or 0.8% (w/v) BSA. Data were analyzed by one-way ANOVA using Sigma Stat (Ver. 2). A p-value smaller than 0.05 was considered statistically significant. Results: The proportions of cleavage and total blastocyst (evaluated on days 3 and 6, respectively) were significantly higher in FBS than BSA supplemented groups, though no differences were observed between the two used different maturation media. The cryotolerance of blastocysts was negatively influenced by the presence of FBS rather than BSA during IVM. The quality of produced embryos, however, was affected neither by the source of macromolecules nor the maturation medium in terms of hatching rate, total blastocyst cells and inner cell mass/total cell ratio. Conclusion: The rate of oocyte development was improved by the presence of FBS, though the cryosurvival of resulting blastocysts was negatively influenced by the presence of the serum during in vitro production of sheep oocytes. 13 20 Abolfazl A Shirazi ابوالفضل شیرازی Department of Embryology and Andrology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Department of Embryology and Andrology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad M Ansari Ardali Mohammad Ansari Ardali Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran Ebrahim E Ahmadi ابراهیم احمدی Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran Hassan H Nazari حسن نظری Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran Morteza M Mamuee Morteza Mamuee Faculty of Agriculture and Natural Resources, Ahvaz University Faculty of Agriculture and Natural Resources, Ahvaz University Iran Banafsheh B Heidari بنفشه حیدری Department of Embryology and Andrology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Department of Embryology and Andrology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran b.heidari@avicenna.ac.ir Bovine serum albuminDifferential stainingSheepVitrification 483.pdf Moor RM, Mattioli M, Ding J, Nagai T. Maturation of pig oocytes in vivo and in vitro. J Reprod Fertil Suppl. 1990;40:197-210.##Rose TA, Bavister BD. Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos. Mol Reprod Dev. 1992;31(1): 72-7.##Ali A, Sirard MA. Effect of the absence or presence of various protein supplements on further development of bovine oocytes during in vitro maturation. Biol Reprod. 2002;66(4):901-5.##Motlík J, Fulka J. Factors affecting meiotic competence in pig oocytes. Theriogenology. 1986;25(1): 87-96.##Thibault C, Szöllösi D, Gérard M. Mammalian oocyte maturation. Reprod Nutr Dev. 1987;27(5): 865-96.##Sagirkaya H, Misirlioglu M, Kaya A, First NL, Parrish JJ, Memili E. Developmental potential of bovine oocytes cultured in different maturation and culture conditions. Anim Reprod Sci. 2007;101(3-4):225-40.##Natsuyama S, Noda Y, Narimoto K, Mori T. Role of protein supplements in the culture of mouse embryos. Theriogenology. 1993;40(1):149-57.##Lonergan P, Carolan C, Mermillod P. Development of bovine embryos in vitro following oocyte maturation under defined conditions. Reprod Nutr Dev. 1994;34(4):329-39.##Korhonen K, Kananen K, Ketoja E, Matomäki J, Halmekytö M, Peippo J. Effects of serum-free in vitro maturation of bovine oocytes on subsequent embryo development and cell allocation in two developmental stages of day 7 blastocysts. Reprod Domest Anim. 2010;45(1):42-9.##Gómez E, Rodríguez A, Muñoz M, Caamaño JN, Hidalgo CO, Morán E, et al. Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification. Theriogenology. 2008;69(8):1013-21.##Leibfried-Rutledge ML, Critser ES, First NL. Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes. Biol Reprod. 1986;35(4):850-7.##Holm P, Booth PJ, Callesen H. Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media. Reproduction. 2002;123(4):553-65.##Yoshioka K, Othman AM, Taniguchi T, Yamanaka H, Sekikawa K. Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA. Theriogen-ology. 1997;48(6):997-1006.##Herrick JR, Behboodi E, Memili E, Blash S, Echelard Y, Krisher RL. Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential. Mol Reprod Dev. 2004;69(3):338-46.##Gil L, Saura S, Echegaray A, Martinez F, de Blas I, Akourki A, et al. Effect of the in vitro maturation medium on equine oocytes: comparison of follicular fluid and oestrous mare serum. Acta Vet Hung. 2005;53(2):241-8.##Mingoti GZ, Castro VS, Méo SC, Sá Barretto LS, Garcia JM. The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development. In Vitro Cell Dev Biol Anim. 2011;47(5-6):361-7.##Marco-Jiménez F, Vicente JS, Viudes-de-Castro M P. Effect of lanosterol on the in vitro maturation in semi-defined culture system of prepubertal ewe oocytes. Zygote. 2011:1-8.##Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update. 1995;1(2): 91-148.##Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil. 1972;30(3):493-7.##Shirazi A, Soleimani M, Karimi M, Nazari H, Ahmadi E, Heidari B. Vitrification of in vitro produced ovine embryos at various developmental stages using two methods. Cryobiology. 2010;60 (2):204-10.##Willis P, Caudle AB, Fayrer-Hosken RA. Equine oocyte in vitro maturation: influences of sera, time, and hormones. Mol Reprod Dev. 1991;30(4):360-8.##Saeki K, Hoshi M, Leibfried-Rutledge ML, First NL. In vitro fertilization and development of bovine oocytes matured in serum-free medium. Biol Reprod. 1991;44(2):256-60.##Zheng YS, Sirard MA. The effect of sera, bovine serum albumin and follicular cells on in vitro maturation and fertilization of porcine oocytes. Theriogenology. 1992;37(4):779-90.##Shirazi A, Shams-Esfandabadi N, Ahmadi E, Heidari B. Effects of growth hormone on nuclear maturation of ovine oocytes and subsequent embryo development. Reprod Domest Anim. 2010;45(3): 530-6.##Calder MD, Caveney AN, Sirard MA, Watson AJ. Effect of serum and cumulus cell expansion on marker gene transcripts in bovine cumulus-oocyte complexes during maturation in vitro. Fertil Steril. 2005;83 Suppl 1:1077-85.##Ikeda S, Imai H, Yamada M. Apoptosis in cumulus cells during in vitro maturation of bovine cumulusenclosed oocytes. Reproduction. 2003;125(3):369-76.##McKiernan SH, Bavister BD. Different lots of bovine serum albumin inhibit or stimulate in vitro development of hamster embryos. In Vitro Cell Dev Biol. 1992;28A(3 Pt 1):154-6.##Pinyopummintr T, Bavister BD. In vitro-matured/ in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, proteinfree culture media. Biol Reprod. 1991;45(5):736-42.##Schroeder AC, Schultz RM, Kopf GS, Taylor FR, Becker RB, Eppig JJ. Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum. Biol Reprod. 1990; 43(5):891-7.##Wrenzycki C, Herrmann D, Keskintepe L, Martins A Jr, Sirisathien S, Brackett B, et al. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod. 2001;16(5):893-901.##Niemann H, Wrenzycki C. Alterations of expression of developmentally important genes in preim-plantation bovine embryos by in vitro culture conditions: implications for subsequent development. Theriogenology. 2000;53(1):21-34.##Abe H, Yamashita S, Satoh T, Hoshi H. Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum containing media. Mol Reprod Dev. 2002;61(1): 57-66.##

Modulation of Cx43 and Gap Junctional Intercellular Communication by Androstenedione in Rat Polycystic Ovary and Granulosa Cells in vitro 2 1 2 Background: Gap-junctional intercellular communication (GJIC) is implicated in physicological processes and it is vitally important for granulosa cell (GC) differentiation and oocyte growth. We investigated the expression of connexin 43 (Cx43), a gap junctional protein, in normal and androstenedione-induced polycystic ovary (PCO), the effects of androstenedione on Cx43 expression, GJIC and progesterone production in granulosa cells in vitro. Methods: Isolated GCs from rat ovary were supplemented with FSH and dripped with EHS-matrix (EHS-drip) in culture media, were treated with physiological (10-7 M) or pathological (10-5 M) androstenedione concentrations to induce differentiation. Cx43 protein levels were assessed by Western blotting. Immunohistochemistry was also used to determine the localization of Cx43 in GCs and corpus luteum (CL) of controls and PCOs. Differentiation of GCs was determined by progesterone assay and Lucifer yellow dye transfer for GJIC status. The degree of significance of variations between the results was analyzed by ANOVA using SPSS (version 11.5; 2002). Results: Cx43 localized in the GC layer of both the control and PCOs. Its protein levels were upregulated in PCO rat ovaries. GCs in culture with or without androstenedione had a punctate membranous distribution of Cx43. However, androstenedione increased GJIC and upregulated progesterone and Cx43 protein levels. Inhibiting GJIC by 18- GA in androstenedione-treated GCs caused partial inhibition of progesterone production, suggesting a possible role of GJIC in mediating the action of androstenedione on GC differentiation. Conclusion: This study presented a suitable culture model for polycystic ovary syndrome and showed that Cx43 and GJIC might contribute to the pathogenesis of polycystic ovary syndrome. 21 33 Rabih R Talhouk Rabih Talhouk Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Lebanon rtalhouk@aub.edu.lb Charbel C Tarraf Charbel Tarraf Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Lebanon Laila L Kobrossy Laila Kobrossy Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Lebanon Abdallah A Shaito Abdallah Shaito Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Lebanon Samer S Bazzi Samer Bazzi Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Lebanon Dana D Bazzoun Dana Bazzoun Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB) Lebanon Marwan M El-Sabban Marwan El-Sabban Department of Anatomy, Cell Biology and Physiology, Faculty of Medicine, American University of Beirut (AUB) Department of Anatomy, Cell Biology and Physiology, Faculty of Medicine, American University of Beirut (AUB) Lebanon me00@aub.edu.lb AndrostenedioneConnexinsExtracellular matrixGap junction intercellular communicationGranulosa cellOvaryPolycystic ovary 485.pdf Stein IF, Leventhal ML. Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynecol. 1935;29:181-91.##Jones MR, Chua A, Chen YD, Li X, Krauss RM, Rotter JI, et al. Harnessing expression data to identify novel candidate genes in polycystic ovary syndrome. PLoS One. 2011;6(5):e20120.##Pigny P, Merlen E, Robert Y, Cortet-Rudelli C, Decanter C, Jonard S, et al. Elevated serum level of anti-mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab. 2003;88(12):5957-62.##Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature. 1996;383(6600):531-5.##Matzuk MM. Revelations of ovarian follicle biology from gene knockout mice. Mol Cell Endocrinol. 2000;163(1-2):61-6.##Gougeon A, Busso D. Morphologic and functional determinants of primordial and primary follicles in the monkey ovary. Mol Cell Endocrinol. 2000;163 (1-2):33-42.##McNatty KP, Fidler AE, Juengel JL, Quirke LD, Smith PR, Heath DA, et al. Growth and paracrine factors regulating follicular formation and cellular function. Mol Cell Endocrinol. 2000;163(1-2):11-20.##Samoto T, Maruo T, Matsuo H, Katayama K, Barnea ER, Mochizuki M. Altered expression of insulin and insulin-like growth factor-I receptors in follicular and stromal compartments of polycystic ovaries. Endocr J. 1993;40(4):413-24.##Pierro E, Andreani CL, Lazzarin N, Cento R, Lanzone A, Caruso A, et al. Further evidence of increased aromatase activity in granulosa luteal cells from polycystic ovary. Hum Reprod. 1997;12(9): 1890-6.##Doldi N, Grossi D, Destefani A, Gessi A, Ferrari A. Polycystic ovary syndrome: evidence for reduced 3 beta-hydroxysteroid dehydrogenase gene expression in human luteinizing granulosa cells. Gynecol Endocrinol. 2000;14(1):32-7.##Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol. 1991;124:43-101.##Vlodavsky I, Lui GM, Gospodarowicz D. Morphological appearance, growth behavior and migratory activity of human tumor cells maintained on extracellular matrix versus plastic. Cell. 1980;19 (3):607-16.##Ben-Rafael Z, Orvieto R. Cytokines--involvement in reproduction. Fertil Steril. 1992;58(6):1093-9.##Giudice LC, Chandrasekher YA, Cataldo NA. The potential roles of intraovarian peptides in normal and abnormal mechanisms of reproductive physiology. Curr Opin Obstet Gynecol. 1993;5(3):350-9.##Kol S, Adashi EY. Intraovarian factors regulating ovarian function. Curr Opin Obstet Gynecol. 1995;7(3):209-13.##Aten RF, Kolodecik TR, Behrman HR. A cell adhesion receptor antiserum abolishes, whereas laminin and fibronectin glycoprotein components of extracellular matrix promote, luteinization of cultured rat granulosa cells. Endocrinology. 1995;136 (4):1753-8.##Aharoni D, Meiri I, Atzmon R, Vlodavsky I, Amsterdam A. Differential effect of components of the extracellular matrix on differentiation and apoptosis. Curr Biol. 1997;7(1):43-51.##Amsterdam A, Rotmensch S, Furman A, Venter EA, Vlodavsky I. Synergistic effect of human chorionic gonadotropin and extracellular matrix on in vitro differentiation of human granulosa cells: progesterone production and gap junction formation. Endocrinology. 1989;124(4):1956-64.##Grazul-Bilska AT, Redmer DA, Reynolds LP. Cellular interactions in the corpus luteum. Semin Reprod Endocrinol. 1997;15(4):383-93.##Munari-Silem Y, Lebrethon MC, Morand I, Rousset B, Saez JM. Gap junction-mediated cell-to-cell communication in bovine and human adrenal cells. A process whereby cells increase their responsiveness to physiological corticotropin concentrations. J Clin Invest. 1995;95(4):1429-39.##Wiesen JF, Midgley AR Jr. Changes in expression of connexin 43 gap junction messenger ribonucleic acid and protein during ovarian follicular growth. Endocrinology. 1993;133(2):741-6.##Gittens JE, Mhawi AA, Lidington D, Ouellette Y, Kidder GM. Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin 43 in early stages of folliculogenesis. Am J Physiol Cell Physiol. 2003;284(4):C880-7.##Tong D, Gittens JE, Kidder GM, Bai D. Patchclamp study reveals that the importance of connexin43-mediated gap junctional communication for ovarian folliculogenesis is strain specific in the mouse. Am J Physiol Cell Physiol. 2006;290(1): C290-7.##Juneja SC, Barr KJ, Enders GC, Kidder GM. Defects in the germ line and gonads of mice lacking connexin43. Biol Reprod. 1999;60(5):1263-70.##Ackert CL, Gittens JE, O'Brien MJ, Eppig JJ, Kidder GM. Intercellular communication via connexin 43 gap junctions is required for ovarian folliculogenesis in the mouse. Dev Biol. 2001;233(2):258-70.##Gittens JE, Barr KJ, Vanderhyden BC, Kidder GM. Interplay between paracrine signaling and gap junctional communication in ovarian follicles. J Cell Sci. 2005;118(Pt 1):113-22.##Gittens JE, Kidder GM. Differential contributions of connexin37 and connexin43 to oogenesis revealed in chimeric reaggregated mouse ovaries. J Cell Sci. 2005;118(Pt 21):5071-8.##Grümmer R, Traub O, Winterhager E. Gap junction connexin genes cx26 and cx43 are differentially regulated by ovarian steroid hormones in rat endometrium. Endocrinology. 1999;140(6):2509-16.##Kilarski WM, Hongpaisan J, Semik D, Roomans GM. Effect of progesterone and oestradiol on expression of connexin43 in cultured human myometrium cells. Folia Histochem Cytobiol. 2000;38 (1):3-9.##Habermann H, Chang WY, Birch L, Mehta P, Prins GS. Developmental exposure to estrogens alters epithelial cell adhesion and gap junction proteins in the adult rat prostate. Endocrinology. 2001;142(1): 359-69.##Chang X, Patiño R, Yoshizaki G, Thomas P, Lee VH. Hormonal regulation and cellular distribution of connexin 32.2 and connexin 32.7 RNAs in the ovary of Atlantic croaker. Gen Comp Endocrinol. 2000;120(2):146-56.##Sasseville' M, Gagnon MC, Guillemette C, Sullivan R, Gilchrist RB, Richard FJ. Regulation of gap junctions in porcine cumulus-oocyte complexes: contributions of granulosa cell contact, gonadotropins, and lipid rafts. Mol Endocrinol. 2009;23(5):700-10.##Boone DL, Tsang BK. Identification and localization of deoxyribonuclease I in the rat ovary. Biol Reprod. 1997;57(4):813-21.##Anderson E, Little B, Lee GS. Androgen-induced changes in rat ovarian granulosa cells in vitro. Tissue Cell. 1987;19(2):217-34.##Henmi H, Endo T, Nagasawa K, Hayashi T, Chida M, Akutagawa N, et al. Lysyl oxidase and MMP-2 expression in dehydroepiandrosterone-induced polycystic ovary in rats. Biol Reprod. 2001;64(1): 157-62.##Stener-Victorin E, Lundeberg T, Waldenström U, Manni L, Aloe L, Gunnarsson S, et al. Effects of electro-acupuncture on nerve growth factor and ovarian morphology in rats with experimentally induced polycystic ovaries. Biol Reprod. 2000;63 (5):1497-503.##Lee MT, Anderson E, Lee GY. Changes in ovarian morphology and serum hormones in the rat after treatment with dehydroepiandrosterone. Anat Rec. 1991;231(2):185-92.##Mahesh VB, Mills TM, Bagnell CA, Conway BA. Animal models for study of polycystic ovaries and ovarian atresia. Adv Exp Med Biol. 1987;219:237-57.##Roy S, Mahesh VB, Greenblatt RB. Effect of dehydroepiandrosterone and delta4-androstenedione on the reproductive organs of female rats: production of cystic changes in the ovary. Nature. 1962;196:42-3.##Streuli CH, Edwards GM, Delcommenne M, Whitelaw CB, Burdon TG, Schindler C, et al. Stat5 as a target for regulation by extracellular matrix. J Biol Chem. 1995;270(37):21639-44.##El-Sabban ME, Sfeir AJ, Daher MH, Kalaany NY, Bassam RA, Talhouk RS. ECM-induced gap junctional communication enhances mammary epithelial cell differentiation. J Cell Sci. 2003;116(Pt 17): 3531-41.##el-Fouly MH, Trosko JE, Chang CC. Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res. 1987;168(2):422-30.##Rana B, Mischoulon D, Xie Y, Bucher NL, Farmer SR. Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors. Mol Cell Biol. 1994;14(9): 5858-69.##Rosenkranz HS, Pollack N, Cunningham AR. Exploring the relationship between the inhibition of gap junctional intercellular communication and other biological phenomena. Carcinogenesis. 2000; 21(5):1007-11.##Moorby CD. A connexin 43 mutant lacking the carboxyl cytoplasmic domain inhibits both growth and motility of mouse 3T3 fibroblasts. Mol Carcinog. 2000;28(1):23-30.##Kidder GM, Mhawi AA. Gap junctions and ovarian folliculogenesis. Reproduction. 2002;123 (5):613-20.##Simon AM, Goodenough DA, Li E, Paul DL. Female infertility in mice lacking connexin 37. Nature. 1997;385(6616):525-9.##Yen SS. The polycystic ovary syndrome. Clin Endocrinol (Oxf). 1980;12(2):177-207.##Anderson E, Lee MT, Lee GY. Cystogenesis of the ovarian antral follicle of the rat: ultrastructural changes and hormonal profile following the administration of dehydroepiandrosterone. Anat Rec. 1992;234(3):359-82.##Dunaif A. Adrenal disorders and polycystic ovary syndrome. Horm Res. 1992;37 Suppl 3:39-44.##Beyer EC, Kistler J, Paul DL, Goodenough DA. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol. 1989; 108(2):595-605.##Goodenough DA, Simon AM, Paul DL. Gap junctional intercellular communication in the mouse ovarian follicle. Novartis Found Symp. 1999;219: 226-35; discussion 235-40.##Huet C, Monget P, Pisselet C, Hennequet C, Locatelli A, Monniaux D. Chronology of events accompanying follicular atresia in hypophysectomized ewes. Changes in levels of steroidogenic enzymes, connexin 43, insulin-like growth factor II/mannose 6 phosphate receptor, extracellular matrix components, and matrix metalloproteinases. Biol Reprod. 1998;58(1):175-85.##Grazul-Bilska AT, Redmer DA, Johnson ML, Jablonka-Shariff A, Bilski JJ, Reynolds LP. Gap junctional protein connexin 43 in bovine corpora lutea throughout the estrous cycle. Biol Reprod. 1996;54 (6):1279-87.##Itahana K, Morikazu Y, Takeya T. Differential expression of four connexin genes, Cx-26, Cx-30.3, Cx-32, and Cx-43, in the porcine ovarian follicle. Endocrinology. 1996;137(11):5036-44.##Lenhart JA, Downey BR, Bagnell CA. Connexin 43 gap junction protein expression during follicular development in the porcine ovary. Biol Reprod. 1998;58(2):583-90.##Schreiber JR, Beckmann MW, Polacek D, Davies PF. Changes in gap junction connexin-43 messenger ribonucleic acid levels associated with rat ovarian follicular development as demonstrated by in situ hybridization. Am J Obstet Gynecol. 1993; 168(4):1094-102; discussion 1102-4.##Mayerhofer A, Garfield RE. Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary. Mol Reprod Dev. 1995;41(3):331-8.##Safieh-Garabedian B, Mouneimne GM, El-Jouni W, Khattar M, Talhouk R. The effect of endotoxin on functional parameters of mammary CID-9 cells. Reproduction. 2004;127(3):397-406.##Mahesh VB, Greenblatt RB. Isolation of dehydro-epiandrosterone and 17alpha-hydroxy-delta5-pregenolone from the polycystic ovaries of the Stein Leventhal syndrome. J Clin Endocrinol Metab. 1962;22:441-8.##Anderson E, Selig M, Lee G, Little B. Androgeninduced changes in ovarian granulosa cells from immature rats in vitro. Adv Exp Med Biol. 1987; 219:259-74.##Yildiz BO, Azziz R. The adrenal and polycystic ovary syndrome. Rev Endocr Metab Disord. 2007; 8(4):331-42.##Ehrmann DA, Barnes RB, Rosenfield RL. Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev. 1995;16(3):322-53.##Rosenfield RL, Barnes RB, Cara JF, Lucky AW. Dysregulation of cytochrome P450c 17 alpha as the cause of polycystic ovarian syndrome. Fertil Steril. 1990;53(5):785-91.##Rosenfeld CS, Wagner JS, Roberts RM, Lubahn DB. Intraovarian actions of oestrogen. Reproduction. 2001;122(2):215-26.##Yu W, Dahl G, Werner R. The connexin43 gene is responsive to oestrogen. Proc Biol Sci. 1994;255 (1343):125-32.##Hillier SG. Intrafollicular paracrine function of ovarian androgen. J Steroid Biochem. 1987;27(1-3):351-7.##Kanemitsu MY, Lau AF. Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase. Mol Biol Cell. 1993;4(8):837-48.##Wood KW, Sarnecki C, Roberts TM, Blenis J. Ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992;68(6):1041-50.##Kim DY, Kam Y, Koo SK, Joe CO. Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation. J Biol Chem. 1999;274(9):5581-7.##El-Sabban ME, Abi-Mosleh LF, Talhouk RS. Developmental regulation of gap junctions and their role in mammary epithelial cell differentiation. J Mammary Gland Biol Neoplasia. 2003;8(4):463-73.##Godwin AJ, Green LM, Walsh MP, McDonald JR, Walsh DA, Fletcher WH. In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C. Mol Cell Biochem. 1993;127-128:293-307.##Yogo K, Ogawa T, Akiyama M, Ishida-Kitagawa N, Sasada H, Sato E, et al. PKA implicated in the phosphorylation of Cx43 induced by stimulation with FSH in rat granulosa cells. J Reprod Dev. 2006;52(3):321-8.##Cooper CD, Lampe PD. Casein kinase 1 regulates connexin-43 gap junction assembly. J Biol Chem. 2002;277(47):44962-8.##Hsueh AJ, Adashi EY, Jones PB, Welsh TH Jr. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev. 1984;5 (1):76-127.##Furman A, Rotmensch S, Kohen F, Mashiach S, Amsterdam A. Regulation of rat granulosa cell differentiation by extracellular matrix produced by bovine corneal endothelial cells. Endocrinology. 1986;118(5):1878-85.##Furger C, Cronier L, Poirot C, Pouchelet M. Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions. Mol Hum Reprod. 1996;2(8):541-8.##Romanello M, Moro L, Pirulli D, Crovella S, D'Andrea P. Effects of cAMP on intercellular coupling and osteoblast differentiation. Biochem Biophys Res Commun. 2001;282(5):1138-44.##Martin W, Zempel G, Hülser D, Willecke K. Growth inhibition of oncogene-transformed rat fibroblasts by cocultured normal cells: relevance of metabolic cooperation mediated by gap junctions. Cancer Res. 1991;51(19):5348-51.##Munari-Silem Y, Rousset B. Gap junction-mediated cell-to-cell communication in endocrine glands--molecular and functional aspects: a review. Eur J Endocrinol. 1996;135(3):251-64.##D'Andrea P, Paschini V, Vittur F. Dual mechanism for cAMP-dependent modulation of Ca2 signalling in articular chondrocytes. Biochem J. 1996; 318( Pt 2):569-73.##Okuma A, Kuraoka A, Iida H, Inai T, Wasano K, Shibata Y. Colocalization of connexin 43 and connexin 45 but absence of connexin 40 in granulosa cell gap junctions of rat ovary. J Reprod Fertil. 1996;107(2):255-64.##

Acute Effects of Ruta graveolens L. on Sperm Parameters and DNA Integrity in Rats 2 1 2 Background: Increase in world population is one of the serious and threatening issues in this century. Therefore, it is vitally important to find safe and effective contraceptive methods, especially for men which already have few choices in this regard. Medicinal plants that were used for contraception in ancient times could be good sources of investigation in this filed. Ruta graveolens L. is one the plants introduced in the Iranian traditional medicine as an oral male contraception to be used before intercourse. In this study we tried to investigate the probable effects of the plant on the spermatozoa of male rats. Methods: Ruta graveolens L. aqueous extract (5 g/kg) was administered orally to five groups of male rats and sperm motility was checked after half, one, two, four and six hours later. Moreover, one group of rats served as the control group. Subsequently, viability of cells (Eosin-Nigrosin staining), morphological changes (Diff-Quick staining), DNA status (acridine orange dye) and serum testosterone levels were assessed in the treated groups which had significant immotile spermatozoa. For statistical analysis, Student’s t-test and one-way ANOVA with Tukey's post-hoc test were employed for comparison between groups. Results: A significant reduction in sperm motility was seen one hour after administration of the extract in the case groups compared to the controls (36% vs. 68.15%, respectively, p <0.01). The motility gradually increased afterwards, and by 6 hours, it was the same as the control group (65.43% and 68.15%, respectively). No significant changes were seen in viability, morphology or DNA structure of spermatozoa in each group. Testosterone levels did not show any significant changes in the treated groups when compared with the controls. Conclusion: Since a significant temporary immobility of spermatozoa without any adverse effects on other sperm characteristics occurred upon the administration of Ruta graveolens L. aqueous extract, it seems that this plant might have the potential to be used for the suggested male contraception. 33 39 Iman I Halvaei Iman Halvaei Department of Physiology, Faculty of Medicine, Medical Sciences, Tehran University Department of Physiology, Faculty of Medicine, Medical Sciences, Tehran University Iran Hamid Reza HR Sadeghipour Roodsari حمیدرضا صادقی‌پور رودسری Department of Physiology, Faculty of Medicine, Medical Sciences, Tehran University Department of Physiology, Faculty of Medicine, Medical Sciences, Tehran University Iran sadeghipour@sina.tums.ac.ir Zhila Zh Naghibi Harat Zhila Naghibi Harat IVF laboratory of Arash Women's Hospital, School of Medicine, Tehran University of Medical Sciences IVF laboratory of Arash Women's Hospital, School of Medicine, Tehran University of Medical Sciences Iran Male contraceptionRatRuta graveolens L.Sperm function assaySpermatozoaIranian traditional medicine 493.pdf United Nations, Department of Economic and Social Affairs, Population Division. World Population Prospects: The 2010 Revision, Volume I: Comprehensive Tables. New York: United Nations; 2011. p. xvii.##United Nations Population Division. World population prospects: the 2002 revision: Volume III: Analytical report. New York: United Nations; 2002. p. xvi.##Henshaw SK. Unintended pregnancy in the United States. Fam Plann Perspect. 1998;30(1):24-9, 46.##Page ST, Amory JK, Bremner WJ. Advances in male contraception. Endocr Rev. 2008;29(4):465-93.##Hoffer AP, Agarwal A, Meltzer P, Herlihy P, Naqvi RH, Lindberg MC, et al. Ultrastructural, fertility, and spermicidal studies with isomers and derivatives of gossypol in male hamsters. Biol Reprod. 1987;37 (4):909-24.##Garg S, Doncel G, Chabra S, Upadhyay SN, Talwar GP. Synergistic spermicidal activity of neem seed extract, reetha saponins and quinine hydrochloride. Contraception. 1994;50(2):185-90.##Khillare B, Shrivastav TG. Spermicidal activity of Azadirachta indica (neem) leaf extract. Contraception. 2003;68(3):225-9.##Bai JP, Shi YL. Inhibition of Ca(2 ) channels in mouse spermatogenic cells by male antifertility compounds from Tripterygium wilfordii Hook. f. Contraception. 2002;65(6):441-5.##Delgado NM, Taboada Ramírez J, Ortega Hernández A, Merchant-Larios H, Sánchez-Vázqueź ML, Ramírez G, et al. Effects of a purified fraction from Echeveria gibbiflora aqueous crude extract on guinea-pig spermatozoa. Phytother Res. 1999;13 (1):46-9.##Reyes R, Merchant-Larios H, Ortega-Hernández A, Delgado NM. Male contraception, IV: hypotoniclike effect from Echeveria gibbiflora on human sperm. Arch Androl. 2002;48(6):443-9.##Chakrabarti K, Pal S, Bhattacharyya AK. Sperm immobilization activity of Allium sativum L. and other plant extracts. Asian J Androl. 2003;5(2): 131-5.##Lohiya NK, Kothari LK, Manivannan B, Mishra PK, Pathak N. Human sperm immobilization effect of Carica papaya seed extracts: an in vitro study. Asian Journal of Andrology. 2000;2(2):103-10.##Chattopadhyay D, Dungdung SR, Mandal AB, Majumder GC. A potent sperm motility-inhibiting activity of bioflavonoids from an ethnomedicine of Onge, Alstonia macrophylla Wall ex A. DC, leaf extract. Contraception. 2005;71(5):372-8.##Sandhyakumary K, Bobby RG, Indira M. Antifertility effects of Ricinus communis (Linn) on rats. Phytother Res. 2003;17(5):508-11.##Paul D, Bera S, Jana D, Maiti R, Ghosh D. In vitro determination of the contraceptive spermicidal activity of a composite extract of Achyranthes aspera and Stephania hernandifolia on human semen. Contraception. 2006;73(3):284-8.##Naghibi Harat Z, Kamalinejad M, Sadeghi MR, Sadeghipour HR, Eshraghian MR. [A review on Ruta graveolens L.; its usage in traditional medicine and modern research data]. J Med Plants. 2009;8(30):1-19. Persian.##Avicenna. [The Canon of Medicine]. Sharafkandi A, editor. 2nd ed. Tehran: Soroush Press; 1997. p. 1825 . Persian.##Azam Khan NJM. [Eksir-e-azam]. Dehli, Publisher: Nami Monshi Nolkshur; 1905. p. 761. Persian.##Khouri NA, El-Akawi Z. Antiandrogenic activity of Ruta graveolens L in male Albino rats with emphasis on sexual and aggressive behavior. Neuro Endocrinol Lett. 2005;26(6):823-9.##Sailani MR, Moeini H. Effect of Ruta graveolens and Cannabis sativa alcoholic extract on spermatogenesis in the adult wistar male rats. Indian J Urol. 2007;23(3):257-60.##Harat ZN, Sadeghi MR, Sadeghipour HR, Kamalinejad M, Eshraghian MR. Immobilization effect of Ruta graveolens L. on human sperm: a new hope for male contraception. J Ethnopharmacol. 2008; 115(1):36-41.##Clarke GN, McCoombe SG, Short RV. Sperm im mobilizing properties of lemon juice. Fertil Steril. 2006;85(5):1529-30.##Gijon E, Cartas L, Lorenzana-Jimenez M, Garcia X. Immobilization of frog sperm by exposure to Ruta chalepensis extract. Proc West Pharmacol Soc. 1995;38:53-4.##Opdyke DL. Monographs on fragrance raw materials. Food Cosmet Toxicol. 1975;13(5):545-54.##WHO. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. United Kingdom: Cambridge University Press; 1999. p. 9-10.##Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril. 1984;42(1):87-91.##Mirghazanfari SM, Naghibi Harat Z, Kamalinejad M. Effect of ruta graveolens L leaf extract on motility and viability of spermatozoa in male rats. Proceeding of 4th young medics international conference; 2007 Sept 20-23; Yerevan. Armenia: Armenian Medical Association; 2007. p. 50.##Rahim F, Saki G, Bazrafkan M. Effect of alcohol extracts of the Ruta graveolens L. on the count, motility and in vitro fertilization capacity of rat's sperm. Asian J Plant Sci. 2010; 9(1):63-6.##

Correlates and Determinants of Reproductive Behavior among Female University Students in Tehran 2 1 2 Background: This paper aims to examine the reproductive health and behaviors which might expose young people at risks of STIs/HIV and potential correlates of such behaviors among female college students in Tehran. Methods: This paper focuses on the study conducted on a sample of 1743 female undergraduate students in four multidisciplinary universities in Tehran during 2005-2006 using a two-stage stratified cluster sampling. The main focus was to determine the predictors of premarital heterosexual reproductive behavior among female students. Results: The mean age of the unmarried students was 21.4 years. Low self-efficacy (OR=7.87, p <0.001), perceived peers' liberal attitude on virginity (OR= 4.33), perception of parents' liberal attitude towards relationship with the opposite sex and poor family atmosphere (OR=3.04 and 2.20, p <0.001, respectively) were predictors of ever having any type of sexual experience after controlling for other factors. The only predictors of penetrative sex remained in the logistic model were older age (OR=5.95), low self-efficacy (OR=10.86), poor family atmosphere (OR= 2.96), liberal parental attitude (OR=4.29) and liberal peer norms on virginity (OR= 4.90). Conclusion: Interventional programs need to be designed at various levels such as enhancing self-efficacy, informing families of the protective role of a balanced control and monitoring over adolescents' behavior and choices of peer network against premarital sexual activity. 39 52 Farideh F Khalajabadi Farahani Population Studies and Research Center in Asia and the Pacific Population Studies and Research Center in Asia and the Pacific Iran John J Cleland John Cleland Department of Epidemiology and Population Health, Centre for Population Studies, London School of Hygiene and Tropical Medicine Department of Epidemiology and Population Health, Centre for Population Studies, London School of Hygiene and Tropical Medicine England Amir Hooshang AH Mehryar Amir Hooshang Mehryar Department of Behavioural Science, Institute for Research on Managment and Planning Department of Behavioural Science, Institute for Research on Managment and Planning Iran DeterminantsHIV/STIReproductive behaviorRisk taking behavioursYoung people 484.pdf Mohammadi MR, Mohammad K, Khalajabadi Farahani F, Alikhani S, Zare M, Tehrani FR, et al. Reproductive knowledge, attitudes and behavior among adolescent males in Tehran, Iran. Int Fam Plan Perspect. 2006;32(1):35-44.##SCI: Statistical Ceter of Iran [Internet]. Tehran: Statistical Center of Iran; 2012. [Population and Housing Census]; 2006 May [cited 2006 May]; [about 2 screens]. Available from: http://www.amar.org.ir/ Default.aspx?tabid=549. Persian.##Abbasi-Shavazi MJ. Recent changes and the future of fertility in Iran. In: United Nations. Expert Group meeting on completing the fertility transition. New York: Population Division, D.E.S.A., United Nations Secretariat; 2002. p. 389.##Mohammad K. [Attitude regarding relationship between boys and girls among college students]. 1996. 80 p. Located at: Tehran University of Medical Science; Persian.##Mohammad K, Farahani FK, Mohammadi MR, Alikhani S, Zare M, Tehrani FR, et al. Sexual risktaking behaviors among boys aged 15-18 years in Tehran. J Adolesc Health. 2007;41(4):407-14.##Khalaj Abadi Farahani F, Cleland J. Norms, attitude and sexual conduct of female college students in Tehran; Implications for sexual and reproductive health policy and research in Iran [PhD Thesis]. [London]: London School of Hygiene and Tropical Medicine; 2008. 325 p.##Khalaj Abadi Farahani F, Cleland J, Mehryar AH. Associations between family factors and premarital heterosexual relationships among female college students in Tehran. Int Perspect Sex Reprod Health. 2011;37(1):30-9.##De Vaus D. Surveys in social research. 5th ed. London: Routledge, Taylor & Francis Group; 2002. p. 180.##Streiner DL, Norman GR. Health measurement scales: a practical guide to their development and use. 3rd ed. New York: Oxford University Press; 2003. Chapter 6, Biases in Responses, p. 72.##Barahini MN, [Assessment of reliability and validity of the Eysenck Personality Questionnaire]. Paper presented at: The 1st Seminar on Psychology and Psychiatry; 1993 Dec 20-23; Tehran, Iran. Persian.##Jafari F, Poorzarrabi P, Tarokh A. [The study of sexual problems among male college students and associated factors]. Paper presented at: the 2nd Congress on Family and Sexual Disorders of Shahed University; 2005 Nov 23-24; Tehran, Iran. Persian.##Saboor-parsa M, Tabatabaee A. [Religion, family and sexual behaviour in a university population]. Paper presented at: the 2nd Congress on Family and Sexual Disorders of Shahed University; 2005 Nov 23-24; Tehran, Iran. Persian.##Rahnama A. [The comparative study of sexual attitudes among female and male students of secondary school in Tehran]. Paper presented at: the 2nd Congress on Family and Sexual Disorders of Shahed University; 2005 Nov 23-24; Tehran, Iran. Persian.##Mirmolaee ST. [The study of sexual behaviours and related factors among female students of governmental schools in Tehran]. Paper presented at: the 2nd Congress on Family and Sexual Disorders of Shahed University; 2005 Nov 23-24; Tehran, Iran. Persian.##Abraham L, Kumar KA. Sexual experiences and their correlates among college students in Mumbai City, India. Int Fam Plan Perspect. 1999;25(3):139-52.##Odimegwu CO, Solanke LB, Adedokun A. Parental characteristics and adolescent sexual behaviour in Bida Local Government Area of Niger State, Nigeria. Afr J Reprod Health. 2002;6(1):95-106.##Magnani RJ, Karim AM, Weiss LA, Bond KC, Lemba M, Morgan GT. Reproductive health risk and protective factors among youth in Lusaka, Zambia. J Adolesc Health. 2002;30(1):76-86.##Kiragu K, Zabin LS. The correlates of premarital sexual activity among school-age adolescents in Kenya. Int Fam Plan Perspect. 1993;19(3):92-7.##Murray NJ, Zabin LS, Toledo-Dreves V, Luengo Charath X. Gender differences in factors influencing first intercourse among urban students in Chile. Int Fam Plan Perspect. 1998;24(3):139-44, 152.##Meekers D. Sexual initiation and premarital childbearing in Sub-Saharan Africa. Population Studies. 1994;48(1):47-64.##Park IU, Sneed CD, Morisky DE, Alvear S, Hearst N. Correlates of HIV risk among Ecuadorian adolescents. AIDS Educ Prev. 2002;14(1):73-83.##Slap GB, Lot L, Huang B, Daniyam CA, Zink TM, Succop PA. Sexual behaviour of adolescents in Nigeria: cross sectional survey of secondary school students. BMJ. 2003;326(7379):15.##Gupta N. Sexual initiation and contraceptive use among adolescent women in northeast Brazil. Stud Fam Plann. 2000;31(3):228-38.##Han S, Choe MK, Lee MS, Lee SH. Risk-taking behavior among high school students in South Korea. J Adolesc. 2001;24(4):571-4.##Viner RM, Haines MM, Head JA, Bhui K, Taylor S, Stansfeld SA, et al. Variations in associations of health risk behaviors among ethnic minority early adolescents. J Adolesc Health. 2006;38(1):55.##Nonnemaker JM, McNeely CA, Blum RW; National Longitudinal Study of Adolescent Health. Public and private domains of religiosity and adolescent health risk behaviors: evidence from the National Longitudinal Study of Adolescent Health. Soc Sci Med. 2003;57(11):2049-54.##Rew L, Wong YJ. A systematic review of associations among religiosity/spirituality and adolescent health attitudes and behaviors. J Adolesc Health. 2006;38(4):433-42.##Lacson RS, Theocharis TR, Strack R, Sy FS, Vincent ML, Osteria TS, et al. Correlates of sexual abstinence among urban students in the Philippines. International Family Planning Perspectives. 1997; 23:168-72.##Fatusi AO, Blum RW. Predictors of early sexual initiation among a nationally representative sample of Nigerian adolescents. BMC Public Health. 2008; 8:136.##Mott FL, Haurin RJ. Linkages between sexual activity and alcohol and drug use among American adolescents. Fam Plann Perspect. 1988;20(3):128-36.##Ohene SA, Ireland M, Blum RW. The clustering of risk behaviors among Caribbean youth. Matern Child Health J. 2005;9(1):91-100.##Weden MM, Zabin LS. Gender and ethnic differences in the co-occurrence of adolescent risk behaviors. Ethn Health. 2005;10(3):213-34.##Takakura M, Wake N, Kobayashi M. Relationship of condom use with other sexual risk behaviors among selected Japanese adolescents. J Adolesc Health. 2007;40(1):85-8.##Jessor R. Risk behavior in adolescence: a psychosocial framework for understanding and action. J Adolesc Health. 1991;12(8):597-605.##Doljanac RF, Zimmerman MA. Psychosocial factors and high-risk sexual behavior: race differences among urban adolescents. J Behav Med. 1998;21 (5):451-67.##Millstein SG, Moscicki AB. Sexually-transmitted disease in female adolescents: effects of psychosocial factors and high risk behaviors. J Adolesc Health. 1995;17(2):83-90.##Shafer MA, Boyer CB. Psychosocial and behavioral factors associated with risk of sexually transmitted diseases, including human immunedeficiency virus infection, among urban high school students. J Pediatr. 1991;119(5):826-33.##Robins LN, Wish E. Childhood Deviance as a Developmental Process: A Study of 223 Urban Black Men From Birth to 18. Soc Forces. 1977;56(2): 448-73.##Sack AR, Keller JF, Hinkle DE. Premarital sexual intercourse: A test of the effects of peer group, religiosity, and sexual guilt. J Sex Res. 1984;20(2): 168-85.##Ingham R, Zessen GV. From individual properties to interactional processes. In: Campenhoudt LV, Cohen M, Guizzardi G, Hausser D, editors. Sexual interactions and HIV risk: new conceptual perspectives in European research. London: Taylor & Francis; 1997. p. 83-99.##Stanton BF, Fitzgerald AM, Li X, Shipena H, Ricardo IB, Galbraith JS, et al. HIV risk behaviors, intentions, and perceptions among Namibian youth as assessed by a theory-based questionnaire. AIDS Educ Prev. 1999;11(2):132-49.##DiIorio C, Dudley WN, Kelly M, Soet JE, Mbwara J, Sharpe Potter J. Social cognitive correlates of sexual experience and condom use among 13- through 15-year-old adolescents. J Adolesc Health. 2001;29(3):208-16.##Blum RW, Mmari KN. Risk and protective factors for adolescent sexual reproductive health in developing countries. Switzerland: World Health Organization and John Hopkins Bloomberg School of Public Health; 2004. 13 p. Report No.: 9241593652.##

Effects of Vitrification on Immature and in vitro Matured, Denuded and Cumulus Compact Goat Oocytes and their Subsequent Fertilization 2 1 2 Background: Vitrification has proven to be more effective than slow freezing methods to cryopreserve mammalian oocytes. The objectives of this study were to evaluate the effects of vitrification on immature and in vitro matured, denuded and cumulus compact goat oocytes and their subsequent fertilization. Methods: Oocytes were either cryopreserved as immature cumulus compact (IMCC) (n=98 Exp 1; 102 Exp 2) and immature denuded (IMDN) (n=127 Exp 1; 109 Exp 2) or were first matured in vitro for 28 h and then cryopreserved as mature cumulus compact (MCC) (n=109 Exp 1; 89 Exp 2) or mature denuded (MDN) (n=112 Exp 1; 110 Exp 2) oocytes in four groups. The vitrification solution comprised of Dulbecco’s phosphate buffered saline supplemented with 0.5% sucrose, 0.4% bovine serum albumin and 8 M propylene glycol. After 7 days of cryopreservation in liquid nitrogen, oocytes in all groups were evaluated for normal morphologic survival and in vitro maturation (Experiment 1) and fertilization in vitro using epididymal buck spermatozoa (Experiment 2). Results: The number of oocytes retaining normal morphology was significantly higher (p <0.05) for cumulus compact oocytes (IMCC: 94.12% vs. IMDN: 89.22%, experiment 1 and MCC: 87.80% vs. MDN: 82.17%, experiment 2) compared to the denuded oocytes. The in vitro maturation of oocytes was highest for non-vitrified control oocytes. The maturation of vitrified IMCC oocytes was significantly higher than IMDN and their fertilizability was higher than MCC and MDN oocytes. Conclusion: The results suggest that immature cumulus compact goat oocytes better tolerate cryopreservation stress by vitrification in terms of fertilization rate. 53 60 Govind Narayan GN Purohit Govind Narayan Purohit Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences India gnpobs@gmail.com Harikesh H Meena Harikesh Meena Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences India Kanika K Solanki Kanika Solanki Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences India GoatIn Vitro fertilizationIn vitro maturationOocytesVitrification 492.pdf Nakagata N. High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil. 1989;87(2):479-83.##Otoi T, Yamamoto K, Koyama N, Tachikawa S, Suzuki T. Cryopreservation of mature bovine oocytes by vitrification in straws. Cryobiology. 1998;37(1): 77-85.##Chen SU, Lien YR, Chen HF, Chao KH, Ho HN, Yang YS. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod. 2000;15(12):2598-603.##Liebermann J, Tucker MJ. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reproduction. 2002;124(4):483-9.##Maclellan LJ, Carnevale EM, Coutinho da Silva MA, Scoggin CF, Bruemmer JE, Squires EL. Pregnancies from vitrified equine oocytes collected from super-stimulated and non-stimulated mares. Theriogenology. 2002;58(5):911-9.##Begin I, Bhatia B, Baldassarre H, Dinnyes A, Keefer CL. Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods. Theriogenology. 2003;59(8):1839-50.##Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature. 1985;313(6003):573-5.##Vajta G. Vitrification of the oocytes and embryos of domestic animals. Anim Reprod Sci. 2000;60-61: 357-64.##Massip A, Van Der Zwalmen P, Scheffen B, Ectors F. Pregnancies following transfer of cattle embryos preserved by vitrification. Cryo-letters. 1986;7:270-3.##Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev. 1998; 51(1):53-8.##Dattena M, Ptak G, Loi P, Cappai P. Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts. Theriogenology. 2000;53(8): 1511-9.##Isachenko V, Alabart JL, Dattena M, Nawroth F, Cappai P, Isachenko E, et al. New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos. Theriogenology. 2003;59(5-6):1209-18.##Garg N, Purohit GN. Effect of different cryoprotectant concentrations for ultrarapid freezing of immature goat follicular oocytes on their subsequent maturation and fertilization in vitro. Anim Reprod. 2007;4:113-8.##Yadav RC, Sharma A, Garg N, Purohit GN. Survival of vitrified water buffalo cumulus-oocytes complexes and their subsequent development in vitro. Bulg J Vet Med. 2008;11(1):55-64.##Johnson MH, Pickering SJ. The effect of dimethylsulphoxide on the microtubular system of the mouse oocyte. Development. 1987;100(2):313-24.##Széll A, Zhang J, Hudson R. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at -180 degrees C. Reprod Fertil Dev. 1990;2(6):613-8.##Le Gal F. In vitro maturation and fertilization of goat oocytes frozen at the germinal vesicle stage. Theriogenology. 1996;45(6):1177-85.##Traldi AS. Vitrification of goat in vivo and in vitro produced embryos. In: Gruner L, Chabert Y, editors. Proceedings of the 7th International Conference on Goats; 2000 May 15-18; Tours, France: Institut de l'elevage; 2000. p. 1031.##Branca A, Galllus M, Dattena M, Cappai P. Preliminary study of vitrification of goat embryos at different stages of development. In: Gruner L, Chabert Y, editors. Proceedings of the 7th International Conference on Goats; 2000 May 15-18; Tours; France: Institut de l'elevage; 2000. p. 1032.##Traldi AS, Leboeuf B, Cognié Y, Poulin N, Mermillod P. Comparative results of in vitro and in vivo survival of vitrified in vitro produced goat and sheep embryos. Theriogenology. 1999;51:175.##Baril G, Traldi AL, Cognié Y, Leboeuf B, Beckers JF, Mermillod P. Successful direct transfer of vitrified sheep embryos. Theriogenology. 2001;56(2): 299-305.##Kharche SD, Taru Sharma G, Majumdar AC. In vitro maturation and fertilization of goat oocytes vitrified at the germinal vesicle stage. Small Rumin Res. 2005;57(1):81-4.##Taru Sharma G, Kharche SD, Majumdar AC. Vitrification of in vitro matured goat oocytes and the effect on in vitro fertilization. Small Rumin Res. 2006;64(1-2):82-6.##Nagar D, Purohit GN. Effect of epidermal growth factor on maturation and fertilization in vitro of goat follicular oocytes in a serum free or serum supplemented medium. Vet Arhiv. 2005;75(6):459-67.##Purohit GN, Brady MS, Sharma SS. Influence of epidermal growth factor and insulin-like growth factor 1 on nuclear maturation and fertilization of buffalo cumulus oocyte complexes in serum free media and their subsequent development in vitro. Anim Reprod Sci. 2005;87(3-4):229-39.##Kumar D, Purohit GN. Effect of epidermal and insulin-like growth factor-1 on cumulus expansion, nuclear maturation and fertilization of buffalo cumulus oocyte complexes in simple serum free media DMEM and Ham’s F-10. Vet Arhiv. 2004; 74(1):13-25.##Garcia-Alvarez O, Marroto-Morales A, Martinez Pastor F, Grade J, Romon M, Fernandez-Santos M, et al. Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection. Theriogenology. 2009;72(2):160-8.##Palomo MJ, Izquierdo D, Mogas T, Paramio MT. Effect of semen preparation on IVF of prepubertal goat oocytes. Theriogenology. 1999;51(5):927-40.##Chauhan MS, Singla SK, Palta P, Manik RS, Madan ML. In vitro maturation and fertilization, and subsequent development of buffalo (Bubalus bubalis) embryos: effects of oocyte quality and type of serum. Reprod Fertil Dev. 1998;10(2):173-7.##Modina S, Beretta M, Lodde V, Lauria A, Luciano AM. Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells. Eur J Histochem. 2004;48(4): 337-46.##Agarwal KP. Cryo-preservation of caprine oocytes by vitrification. Indian J Anim Reprod. 1999;20:6-8.##Tharasanit T, Colleoni S, Galli C, Colenbrander B, Stout TA. Protective effects of the cumulus-corona radiata complex during vitrification of horse oocytes. Reproduction. 2009;137(3):391-401.##Suo L, Zhou GB, Meng QG, Yan CL, Fan ZQ, Zhao XM, et al. OPS vitrification of mouse imamture oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development. Zygote. 2009;17(1):71-7.##Zhang J, Nedambale TL, Yang M, Li J. Improved development of ovine matured oocyte following solid surface vitrification (SSV): effect of cumulus cells and cytoskeleton stabilizer. Anim Reprod Sci. 2009;110(1-2):46-55.##Ge L, Sui HS, Lan GC, Liu N, Wang JZ, Tan JH. Coculture with cumulus cells improves maturation of mouse oocytes denuded of the cumulus oophorus: observations of nuclear and cytoplasmic events. Fertil Steril. 2008;90(6):2376-88.##Shaw JM, Oranratnachai A, Trounson AO. Cryopreservation of oocytes and embryos. In: Trounson AO, Gardner D, editors. Handbook of IVF. Boca Raton: CRC press; 1999. p. 373.##Parks JE, Ruffing NA. Factors affecting low temperature survival of mammalian oocytes. Theriogenology. 1992;73(1):59-73.##Otoi T, Yamamoto K, Koyama N, Suzuki T. Invitro fertilization and development of immature and mature bovine oocytes cryopreserved by ethylene glycol with sucrose. Cryobiology. 1995;32(5): 455-60.##Park SE, Son WY, Lee SH, Lee KA, Ko JJ, Cha KY. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril. 1997;68(5):920-6.##Kasai M, Iritani A, Chang MC. Fertilization in vitro of rat ovarian oocytes after freezing and thawing. Biol Reprod. 1979;21(4):839-44.##Schroeder AC, Champlin AK, Mobraaten LE, Eppig JJ. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J Reprod Fertil. 1990;89(1):43-50.##Men H, Monson RL, Rutledge JJ. Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation. Theriogenology. 2002;57(3):1095-103.##Rojas C, Palomo MJ, Albarracín JL, Mogas T. Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments. Cryobiology. 2004;49(3):211-20.##Rodríguez-González E, López-Bejar M, Izquierdo D, Paramio MT. Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation. Reprod Nutr Dev. 2003;43(2):179-87.##Urdaneta A, Jiménez-Macedo AR, Izquierdo D, Paramio MT. Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test. Zygote. 2003;11 (4):347-54.##Martino A, Mogas T, Palomo MJ, Paramio MT. In vitro maturation and fertilization of prepubertal goat oocytes. Theriogenology. 1995;43(2):473-85.##Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril. 1990;54 (1):102-8.##Le Gal F, De Roover R, Verhaeghe B, Etienne D, Massip A. Development of vitrified matured cattle oocytes after thawing and culture in vitro. Vet Rec. 2000;146(16):469-71.##

Placenta Accreta Causing Uterine Rupture in Second Trimester of Pregnancy after in vitro Fertilization: A Case Report 2 1 2 Background: Placenta accreta is a rare obstetrical condition that mainly occurs in the third trimester leading to life-threatening complications. Hereby, a case of uterine rupture due to placenta accreta occuring in the second trimester is presented. Case Presentation: A forty-year old patient who conceived after in vitro fertilization treatment (oocyte donation and embryo transfer) presented in emergency department in the nineteen weeks of gestation with acute abdominal pain, heamoperitoneum and fetal death. Emergency laprotomy showed uterine rupture along with placenta accreta for which the patient underwent subtotal hysterectomy. Conclusion: Although, an uncommon occurrence, physicians in assisted reproductive techniques (ART) clinics should consider placenta accreta in gravid patients who present with acute abdominal pain and shock, considering the fact that they usually have associated high risk factors for abnormal placentation. 61 64 Priya P Dahiya Priya Dahiya Akanksha IVF Center, Mata Chanan Devi Hospital, Janak Puri Akanksha IVF Center, Mata Chanan Devi Hospital, Janak Puri India Kanad K D. Nayar Kanad D. Nayar Akanksha IVF Center, Mata Chanan Devi Hospital, Janak Puri Akanksha IVF Center, Mata Chanan Devi Hospital, Janak Puri India Amar A J.S.Gulati Amar J.S.Gulati Department of General Surgery, Mata Chanan Devi Hospital, Janak Puri Department of General Surgery, Mata Chanan Devi Hospital, Janak Puri India Kiran K Dahiya Kiran Dahiya Department of Biochemistry, Pt. B. D. Sharma Post Graduale Institute of Medical Sciences (PGIMS) Department of Biochemistry, Pt. B. D. Sharma Post Graduale Institute of Medical Sciences (PGIMS) India kirandahiya_2002@yahoo.com IVFPlacenta accretaSecond trimester pregnancyUterine rupture 488.pdf Khong TY. The pathology of placenta accreta, a worldwide epidemic. J Clin Pathol. 2008;61 (12): 1243-6.##LeMaire WJ, Louisy C, Dalessandri K, Muschenheim F. Placenta percreta with spontaneous rupture of an unscarred uterus in the second trimester. Obstet Gynecol. 2001;98(5 Pt 2):927-9.##Levrant SG, Wingate M. Midtrimester uterine rupture. A case report. J Reprod Med. 1996;41(3):186-90.##Kupferminc MJ, Tamura RK, Wigton TR, Glassenberg R, Socol ML. Placenta accreta is associated with elevated maternal serum alpha-fetoprotein. Obstet Gynecol. 1993;82(2):266-9.##Höpker M, Fleckenstein G, Heyl W, Sattler B, Emons G. Placenta percreta in week 10 of pregnancy with consecutive hysterectomy: case report. Hum Reprod. 2002;17(3):817-20.##Endres LK, Barnhart K. Spontaneous second trimester uterine rupture after classical cesarean. Obstet Gynecol. 2000;96(5 Pt 2):806-8.##Morken NH, Henriksen H. Placenta percreta--two cases and review of the literature. Eur J Obstet Gynecol Reprod Biol. 2001;100(1):112-5.##

Detection of Congenital Mullerian Anomalies by Real-time 3D Sonography 2 1 2 Mullerian duct anomalies (MDAs) are relatively common disorders that are particularly responsible for obstetrical complications and have a prevalence of about 3%-4% in the general population (1, 2). Uterine malformations may result from arrested development of the mullerian ducts, failure of fusion of the mullerian ducts or failure of resorption of the median septum (1, 2). 65 67 Firoozeh F Ahmadi Firoozeh Ahmadi Department of Reproductive Imaging, Reproductive Biomedicine Research Center, Royan Institute, ACECR Department of Reproductive Imaging, Reproductive Biomedicine Research Center, Royan Institute, ACECR Iran f_ahmadi@royaninstitute.org Hadieh H Haghighi Hadieh Haghighi Department of Reproductive Imaging, Reproductive Biomedicine Research Center, Royan Institute, ACECR Department of Reproductive Imaging, Reproductive Biomedicine Research Center, Royan Institute, ACECR Iran No Keyword 491.pdf Salem S, Wilson SR. Gynecologic Ultrasound. In: Rumack CM, Wilson SR, Charboneau JW, editors. Diagnostic ultrasound, Vol. 1. Calgary: Elsevier; 2005. p. 527-87.##Raga F, Bauset C, Remohi J, Bonilla-Musoles F, Simón C, Pellicer A. Reproductive impact of congenital Müllerian anomalies. Hum Reprod. 1997;12(10): 2277-81.##Troiano RN, McCarthy SM. Mullerian duct anomalies: imaging and clinical issues. Radiology. 2004; 233(1):19-34.##Deutch TD, Abuhamad AZ. The role of 3-dimensional ultrasonography and magnetic resonance imaging in the diagnosis of müllerian duct anomalies: a review of the literature. J Ultrasound Med. 2008;27(3):413-23.##Rai R, Clifford K, Regan L. The modern preventative treatment of recurrent miscarriage. Br J Obstet Gynaecol. 1996;103(2):106-10.##Caliskan E, Ozkan S, Cakiroglu Y, Sarisoy HT, Corakci A, Ozeren S. Diagnostic accuracy of real-time 3D sonography in the diagnosis of congenital Mullerian anomalies in high-risk patients with respect to the phase of the menstrual cycle. J Clin Ultrasound. 2010;38(3):123-7.##Dahiya N. Three-dimensional ultrasound: technique and applications revisited. Ultrasound Clin. 2009;4 (3):291-306.##