Oocyte activation: The impact of calcium signals on fertilization 2 2 Fertilization is triggered by changes in intracellular calcium concentration. In mammals, these transients in ooplasmic calcium concentration take the form of repetitive spikes, so called calcium oscillations (Ca2+-oscillations). These oscillations are important for relieve of meiotic arrest and to induce all the other events of oocyte activation. Although a surface mediated way of oocyte activation has been proposed, there is now substantial evidence to suggest that the sperm cell induces these Ca2+-oscillations by introducing a sperm specific phospholipase C, PLCζ, in the ooplasm. Ca2+-oscillations are also observed after intracytoplasmic sperm injection (ICSI), a successful technique in human assisted reproduction. In the rare cases that no fertilization is observed following ICSI, this may be due to a deficiency in PLCζ. However, artificial activating the oocytes after ICSI by increasing the calcium concentration can restore fertilization rates in these cases and support further development, as evidenced by successful pregnancies. Further evaluation of the current protocols for assisted oocyte activation is appropriate and investigation of the future application of PLCζ is warranted. 171 174 2017/10/1 1396/7/9 2017/10/5 1396/7/13 Elke Heytens Elke Heytens Department of Reproductive Medicine, University Hospital Ghent, Ghent, Belgium elke.heytens@ugent.be Reza Soleimani Reza Soleimani Department of Reproductive Medicine, University Hospital Ghent, Ghent, Belgium Petra De Sutter Petra De Sutter Department of Reproductive Medicine, University Hospital Ghent, Ghent, Belgium Calcium Oocyte activation PLCζ ICSI Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000; 1: 11-21.##Miyazaki S, Shirakawa H, Nakada K, Honda Y. 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Injection of a soluble sperm fraction into sea-urchin eggs triggers the cortical reaction. Experientia 1985; 41: 1068-1070.##Swann K. A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs. Development 1990; 110: 1295-1302.##Dozortsev D, Rybouchkin A, De Sutter P, Qian C ,Dhont M. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Hum Reprod 1995; 10: 403-407.##Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, et al. PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development 2002; 129: 3533-3544.##Rogers NT, Hobson E, Pickering S, Lai FA, Braude P , Swann K. Phospholipase Czeta causes Ca2+ oscillations and parthenogenetic activation of human oocytes. Reproduction 2004;128: 697-702.##Kouchi Z, Fukami K, Shikano T, Oda S, Nakamura Y, Takenawa T, et al. Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs. J Biol Chem 2004; 279: 10408-10412.##Young C, Grasa P, Coward K, Davis LC , Parrington J. Phospholipase C zeta undergoes dynamic changes in its pattern of localization in sperm during capacitation and the acrosome reaction. Fertil Steril 2008; E-pub ahead of print.##Grasa P, Coward K, Young C , Parrington J. The pattern of localization of the putative egg activation factor, PLCz, in uncapacitated, capacitated, and acrosome-reacted human spermatozoa. Hum Reprod 2008; 23: 2513-2522.##Knott JG, Kurokawa M, Fissore RA, Schultz RM ,Williams CJ. Transgenic RNA interference reveals role for mouse sperm phospholipase Czeta in triggering Ca2+ oscillations during fertilization. Biol Reprod 2005; 72: 992-996.##Dupont G , Goldbeter A. CaM kinase II as frequency decoder of Ca2+ oscillations. Bioessays 1998; 20: 607-610.##https://doi.org/10.1002/(SICI)1521-1878(199808)20:8<607::AID-BIES2>3.0.CO;2-F##Madgwick S, Levasseur M , Jones KT. Calmodulin-dependent protein kinase II, and not protein kinase C, is sufficient for triggering cell-cycle resumption in mammalian eggs. J Cell Sci 2005; 118: 3849-3859.##Tesarik J, Sousa M , Testart J. Human oocyte activation after intracytoplasmic sperm injection. Hum Reprod 1994; 9: 511-518.##Sousa M , Tesarik J. Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum Reprod 1994; 9: 2374-2380.##Schirren CG, Holstein A , Schirren C. Uber die Morphogenese rundkopfiger Spermatozoen des Menschen. Andrologie 1971;3: 117-125.##Holstein AF, Schirren CG, Schirren C , Mauss J. Round headed spermatozoa: a cause of male infertility. Dtsch Med Wochenschr 1973; 98: 61-62.##Liu J, Nagy Z, Joris H, Tournaye H, Devroey P, Van Steirteghem A. Successful fertilization and establishment of pregnancies after intracytoplasmic sperm injection in patients with globozoospermia. Hum Reprod 1995; 10: 626-629.##Battaglia DE, Koehler JK, Klein NA, Tucker MJ. Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm. Fertil Steril 1997; 68: 118-122.##Rybouchkin A, Dozortsev D, Pelinck MJ, De Sutter P, Dhont M. Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes. Hum Reprod 1996; 11: 2170-2175.##Heytens E, Parrington J, Lambrecht S, Young C, Coward K, Cuvelier C, et al. First evidence of disturbed expression of the oocyte-activating factor PLCzeta in globozoospermic men. Hum Reprod 2008; 23: i103.##Ware CB, Barnes FL, Maiki-Laurila M , First NL. Age dependence of bovine oocyte activation. Gamete Res 1989; 22: 265-275.##Kline D , Kline JT. 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Expression of c-Jun in human granulosa cells from patients participating in in vitro fertilization programs 2 2 Background: The mitogen-activated protein kinase (MAPK) pathway is one of the major signaling pathways that transmit intracellular signals initiated by extracellular stimuli to the nucleus. The stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase is a subfamily of MAP kinases implicated in cytokine and stress responses. Objective: In this study, we have examined total and phosphorylated c-Jun in the mural and cumulus granulosa cells, and investigated also whether c-Jun can be responsible for the difference in the expression of apoptosis between mural and cumulus regions. Materials and Methods: A total of 14 consecutive couples participating in IVF program were investigated. Aspirated follicular fluid was transferred into tissue culture dishes and oocyte-cumulus cells complexes were isolated. The cells were centrifuge and fixed with Bouin’s solution and then were put on a glass slide. After fixation, the slides were stained by immunocytochemistry method. The incidence of apoptotic granulosa cells was examined by a fluorescence microscope. Results: The incidence of apoptotic granulosa cells was 1.27 ± 0.12 in the mural region and 0.38 ± 0.07 in the cumulus regions. All mural and cumulus cells expressed total c-Jun in 7 patients while phosphorylated c-Jun was also expressed in all cells of the other 7 patients. There was no difference between apoptotic and nonapoptotic cells in the expression of total and phosphorylated c-Jun. Conclusion: C-Jun may not be responsible for apoptotic effect on mural and cumulus cells. 175 180 2017/10/12017/10/1 1396/7/9 2017/10/52017/10/5 1396/7/13 Hassan Hassani Bafrani Hassan Hassani Bafrani Department of Anatomy and Embryology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran hasanhasani@yahoo.com Hidekazu Saito Hidekazu Saito Department of Obstetrics and Gynecology, School of Medicine, Yamagata University of Medical Sciences, Yamagata, Japan Apoptosis C-Jun Granulosa cells In vitro fertilization Human Kidd VJ. Proteolytic activities that mediate apoptosis. Annu Rev Physiol 1998; 60: 533-573.##Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 1999; 68: 383-424.##Kerr JFR, Gobe, GC, Winterford CM, Harmon BV. Anatomical methods in cell death. In: Schwartz LM, Osborne B (eds). Methods in cell biology, New York: Academic Press; 1998; 1-27.##Ben-Sasson SA, Sherman Y, Gavrieli Y. Identification of dying cells-in situ staining. Anatomical methods in cell death. In: Schwartz LM, Osborne B (eds). Methods in cell Biology. New York: Academic press; 1995; 29-39.##Eastman A. Assays for DNA fragmantation, endonucleases and intracellular PH and Ca2+ associated with apoptosis. Anatomical methods in cell death. In: Schwartz LM, Osborne B (eds). Methods in Cell Biology. New York: Academic Press; 1995; 41-55.##Casas C, Ribera J, Esquedra JE. Antibodies against c-Jun N-terminal peptide cross-react with neo-epitopes emerging after caspase-mediated proteolysis during apoptosis. J Neurochem 2001; 77: 904-915.##Minden A, Karin M. Regulation and function of the JNK subgroup of MAP kinases. Biochim Biophys Acta 1997; 1333: 85-104.##IP YT, Davis RJ. Signal transduction by the c-jun N-terminal kinase (JNK)-from inflammation to development. Curr Opin Cell Biol 1998; 10: 205-219.##Schaeffer HJ, Weber MJ. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 1999; 19: 2435-2444.##Boulton TG, Nye SH, Robbins DJ, IP NY, Radziejewska E, Morgenbesser SD,et al. ERKs: a family of protein-serine/threnine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 1991; 65: 663-675.##Han J, Lee JD, Bibbs L, Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 1994; 265: 808-811.##Cobb MH, Goldsmith EJ. How MAP kinases are regulated. L Biol Chem 1995; 270: 14843-14846.##Kyriakis JM, Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 1996; 18: 567-577.##Su B, Karin M. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol 1996; 8: 402-411.##Fanger GR, Gerwins P, Widmann C, Jarpe MB, Johnson GL. MEKKS, GCKs, MLKs, PAKs, TAKs, and tpls: upstream regulators of the c-jun amino-terminal kinases? Curr Opin Genet Dev 1997; 7: 67-74.##Kim JW, Chang TS, Lee JE, Huh SH, Yeon SW, Yang WS, et al. Negative regulation of the SAPK/JNK signaling pathway by presenilin 1. The Journal of Cell Biology 2001; 153: 457-464.##Bagowski PC, Xiong W, Ferrel EJ. C-jun N-terminal kinase activation in xenopus laevis eggs and embryos. The J Biol Chem 2001; 276: 1459-1465.##Estus S, Zaks WJ, Freeman RS, Gruda M, Bravo R, Johnson EM. Altered gene expression in neurons during programmed cell death: identification of c-Jun as necessary for neuronal apoptosis. J Cell Biol 1994; 127: 1717-1727.##Ham J, Babij C, Whitfield J, Pfarr CM, Lallemand D, Yaniv, et al. A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron 1995; 14: 927-939.##Wang CY, Mayo MW, Baldwin JR. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-KappaB. Science 1996; 274: 784-787.##Le-Niculescu H, Bonfoco E, Kasuya Y, Claret FX, Green DR, Karin M. Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to fas ligand induction and cell death. Mol Cell Biol 1999; 19: 751-763.##Kolbus A, Herr I, Martin S, Debatin KM, Wagner EF, Angel P. c-Jun-dependent CD95-L Expression is a rate-limiting step in the induction of apoptosis by alkylating agents. Mol Cell Biol 2000; 20: 575-582.##Leoncini L, Del Vecchio MT, Megha T, Barbini P, Galieni P, Pileri S. Correlation between apoptotic and proliferative indices in malignant non-Hodgkin's lymphoma. Am J Pathol 1993; 142: 755-763.##Wheeler Tm, Rogers E, Aihara M, Scardino PT, Thompson TC. Apoptotic index as a biomarker in prostatic intraepithelial neoplasm (PIN) and prostatic cancer. J Cell Biol 1994; 19: 202-207.##Nakahara K, Sakai N, Saito H, Naohiro T, Saito T, Hiroi M, et al. Incidence of apoptotic bodies in membrane granulosa of patients participating in an in vitro fertilization program. Fertil Steril 1997; 67: 302-308.##Amsterdam A, Koch Y, Leiberman Me, Lindner HR. Distribution of binding sites for human chorionic gonadotropin in the preovlatory follicle of the rat. J Cell Biol 1975; 67: 894-900.##Lawrence TS, Deckel N, Beers WH. Binding of human chorionic gonadotropin by rat cumuli oophori and granulosa cells: a comparative study. Endocrinology 1980; 106: 1114-1118.##Zhou J, Refuerzo J, Bondy C. Granulosa cell DNA synthesis is strictly correlated with the presence of insulinlike growth factor I and absence of c-fos/c-jun expression. Mol Endocrinol 1995; 9: 924-931.##Oktay KH, Oktay MH. Immunohistochemical analysis of tyrosine phosphorylation and AP-1 transcription factors c-Jun, Jun D, and Fos family during early ovarian follicle development in the mouse. Appl Immunohistochem Mol Morpho 2004; 12:364-369.##Peluso JJ, Luciano AM, Pappalardo A, White BA. 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Fusion and development of 2-cell bovine embryos to tetraploid blastocyst with different voltages and durations 2 2 Background: The values of embryonic stem cell and cloning are evident. Production of clone from embryonic stem cells can be achieved by introduction of stem cell into a tetraploid blastocyst. Tetraploid blastocyst can be produced in vitro by electrofusion of 2-cell embryos. Objective: The aim of this study was to assess the effect of different voltages and durations on fusion rate of bovine 2-cell embryos and their subsequent development in vitro. Material and Methods: The in vitro produced bovine 2-cell embryos were categorized into 3 groups: (1) fused group (FG); 2-cell embryos fused by exposure to different voltages (0.5, 0.75, 1, 1.25 and 1.5 kV/cm) and durations (20, 40, 60, 80 and 100 μs), (2) exposed control group (ECG); 2-cell embryos exposed to different voltages and durations but remained unfused and (3) unexposed control group (UCG); embryos cultured without exposure to any voltage. The embryos from each group were cultured and fusion, cleavage and developmental rates were compared in each group. Results: The results show that increased voltage, increases the fusion rate up to 88% for 1.5 kV/cm; however, the rate of cleavage and blastocyst formation decreases significantly to 18% and 10% respectively (p<0.05). Increased duration does not significantly increase fusion rate, however, in high voltage, increased duration decreases cleavage rate and blastocyst formation rate. Blastocyst formation rate in UCG showed a better development (32%) compared to FG (20%) or ECG (22.5%) (p<0.05). Conclusion: It can be concluded that for optimal fusion, cleavage and development, one pulse of 0.75 kV/cm for 60μs should be applied. 181 186 2017/10/12017/10/12017/10/1 1396/7/9 2017/10/52017/10/52017/10/5 1396/7/13 Mohamad Reza Darabi Mohamad Reza Darabi Department of Anatomy, Arak University of Medical Sciences, Arak, Iran Mohamad Hosein Nasr-Esfahani Mohamad Hosein Nasr-Esfahani Department of Clinical and Experimental Embryology, Reproductive Medicine Research Center, Royan Institute, Isfahan campus, ACECR Tehran, Iran mh.nasr-esfahani@royaninstitute.org Hosein Baharvand Hosein Baharvand Department of stem cell, Royan Institute, Tehran, Iran Mohmad Mardani Mohmad Mardani Department of Anatomy, Isfahan University of Medical Sciences, Isfahan, Iran Hojatolah Karimi-Jashni Hojatolah Karimi-Jashni Department of Anatomy, Jahrom University of Medical Sciences, Jahrom, Iran Bovine Embryo Development Electrofusion Tetraploi Nguyen VT, Sayaka W, Satoshi K, Hiroshi O, Takafusa H. Injection of somatic cell cytoplasm into oocytes before intracytoplasmic sperm injection impairs full-term development and increases placental weight in mice. Biol Reprod 2006; 74: 865-873.##Shinozawa T, Sugawara A, Matsumoto A, Han Y-J, Tomioka I, Inai K, et al. Development of rat tetraploid and chimeric embryos aggregated with diploid cells. Zygot 2006; 14: 287-297##Lan LI, Wei SHEN, Lingjiang MIN, Yujian SUN, Jixian DENG, Quingjie PAN. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene. Front Biol Chaina 2008; 3: 269-274.##Jingjuan JI, Tonghang GUO, Xianhong TONG, Lihua LUO, Guixiang ZHOU, Yingyun FU, et al. Experimental cloning of embryos through human-rabbit interspecies nuclear transfer. Front Biol Chaina 2007; 2: 80-84.##Polejaeva IA, Campbell KHS. New advances in somatic cell nuclear transfer: Applications in transgenesis. Theriogenology 2000; 53: 117-126.##Skrzyszowska M, Smorag Z, Slomski R, Katska-Ksiazkiewicz L, Kalak R. Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning. Biol Reprod 2006; 74: 1114-1120.##James RM, Klerkx A, Keighren M, Flockhart JH, West JD. Restricted distribution of tetraploid cells in mouse tetraploid-diploid chimaeras. Developmental Biology 1995; 167: 213- 226.##James RM, West JD. A chimaeric animal model for confined placental mosaicism. Human Genetic 1994; 93: 603-604.##Curnow EC, Gunn LM, Trounson AQ. Electrofusion of two-cell bovine embryos for the production of tetraploid blastocysts in vitro. Molecular Reproduction Development 2000; 56: 372-377.##https://doi.org/10.1002/1098-2795(200007)56:3<372::AID-MRD7>3.0.CO;2-W##Berg H. Fusion of blastomeres and blastoctsts of mouse embryos. Bioelectrochemistry Bioenergetic 1982; 9: 223-228.##Iwasaki S, Kono T, Fukatsu H, Nakahara T. Production of bovine tetraploid embryos by electrofusion and their developmental capacity in vitro. Gamete Research 1989; 24: 261-267.##Tanghe S, Soom A, Mehrzad VJ, Maes D, Duchateau L, Kruif A. Cumulus contributions during bovine fertilization in vitro. Theriogenology 2003; 60: 135-149.##Khurana NK, Nieman H. Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos. Theriogenology 2000; 54: 741-756.##Gandolfi F, Luciano MA, Modina S, Ponzini A, Pocar P, Armstrong DT, et al. The in vitro developmental competence of bovine oocytes can be related to the morphology of the ovary. Theriogenology 1997; 48:1153-1160.##Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38: 1171-1180.##Neglia G, Gasparrini B, Brienza VC, Palo RD, Campanile G, Presicce GA, et al. Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration. 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Ultrastructural study of isolated mouse preantral follicles co-cultured with cumulus cells with leukemia inhibitory factor 2 2 Background: The ultrastructural analysis of cultured follicles could direct us to understand subcellular changes during in vitro culture. Objective: This study was done to verify the ultrastructural characteristics of in vitro cultured mouse isolated preantral follicles in co-culture system in the presence and absence of leukemia inhibitory factor (LIF) Materials and Methods: Mechanically isolated preantral follicles were divided into four groups: control without LIF, control with LIF, co-cultured group with LIF, co-cultured group without LIF. In co-culture groups the follicles were cultured with cumulus cells. After 4 days the follicles were processed and sectioned for transmission electron microscopic examination. Results: The oocytes of cultured preantral follicles in all studied groups demonstrated a homogeneous cytoplasm and they had the round or ovoid shaped mitochondria with light matrix and cristae. Their endoplasmic reticulum cisternae were in association with mitochondria and Golgi complex. The cortical granules and the aggregation of mitochondria around the germinal vesicle were prominent in both co-cultured groups. The organelle distribution in granulosa cells was normal in all groups of study and no sign of cell death was observed. In both co-cultured systems the granulosa cells contained mitochondria with tubular cristae, a well developed smooth endoplasmic reticulum and several large lipid droplets, characteristics of steroid synthesis cells. Conclusion: The oocyte and granulosa cells in co-cultured system showed more remarkable maturation features than that of control. 187 192 2017/10/12017/10/12017/10/12017/10/1 1396/7/9 2017/10/52017/10/52017/10/52017/10/5 1396/7/13 Kamran Haidari Kamran Haidari Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Mojdeh Salehnia Mojdeh Salehnia Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran mogdeh@dr.com Mojtaba Rezazadeh Valojerdi Mojtaba Rezazadeh Valojerdi Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Shahram Pourbyranvand Shahram Pourbyranvand Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Co-culture In vitro maturation Leukemia inhibitory factor Mouse preantral follicle Ultrastructure Adam AA, Takahashi Y, Katagiri S, Nagano M. In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes. J Reprod Dev 2004; 50:579-586.##Bishonga C, Takashi Y, Katagiri S, Nagano M, Ishikawa A. In vitro growth of ovarian peantral follicles and the capacity of their oocytes to develop to the blastocyst stage. J Vet Med Sci 2001; 63:619-624.##Cortvrindt R, Smitz J. Early preantral mouse follicle in vitro maturation: oocytes growth, meiotic maturation and granulose-cell proliferation. Theriogenology 1998; 49:845-859.##Yamanaka K, Aono N, Yoshida H, Sato E. Cryopreservation and in vitro maturation of germinal vesicle stage oocytes of animals for application in assisted reproductive technology. Reprod Med Biol 2007; 6:61-68.##Hasegawa A, Koyama K. In vitro growth and maturation of mouse oocyte-granulosa cell complex from cryopreserved ovaries and achievement of pup birth. Reprod Med Biol 2007; 6:77-83.##Cecconi S, Rossi G, Palmerini MG. Mouse oocyte differentiation during antral follicle development. Microsc Res Tech 2006; 69:408-414.##Boland NI, Gosden RG. Effects of epidermal growth factor on the growth and differentiation of cultured mouse ovarian follicles. J Reprod Fertil 1994; 101:369-374.##Demeestere I, Gervy C, Centner J, Devreker F, Englert Y, Delbaere A. Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice. Biol Reprod 2004; 70:1664-1669.##Demeestere I, Centner J, Gervy C, Englert Y, Delbaere A. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction 2005; 130:147-156.##Skinner MK. Regulation of primordial follicle assembly and development. Hum Reprod Update 2005; 1:461-471.##Hilton DJ, Gough NM. Leukemia inhibitory factor: a biological perspective. J Cell Biochem 1991; 46:21-26.##Senturk LM, Arici A. Leukemia inhibitory factor in human reproduction. Am J Reprod Immunol 1998; 39:144- 151.##Tsai HD, Chang CC, Hsieh YY, Hsu LW, Chang SC, Lo HY. Effect of different concentrations of recombinant leukemia inhibitory factor (LIF) on different developmental stage of mouse embryo in vitro. J Assist Reprod Genet 2000; 17:352-355.##Ptak G, Lopes F, Matsukawa K, Tischner M, Loi P. Leukaemia inhibitory factor enhances sheep fertilization in vitro via an influence on the oocyte. Theriogenology 2006; 65:1891-1899.##Morita Y, Manganaro TF, Tao XJ, Martimbeau S, Donahoe PK, Tilly JL. Requirement for phosphatidylinositol-3'-kinase in cytokine-mediated germ cell survival during fetal oogenesis in the mouse. Endocrinol 1999; 140: 941-949.##Gearing DP, Thut CJ, Van de Bos T, Gimpel SD, Delaney PB, King J, et al. leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp 130. EMBO J 1991; 6: 2839-2848.##van Eijk MJ, Mandelbaum J, Salat-Baroux J, Belaisch-Allart J, Plachot M, Junca AM, et al. Expression of leukaemia inhibitory factor receptor subunits LIFR beta and gp130 in human oocytes and preimplantation embryos. Mol Hum Reprod 1996; 2:355-360.##Nicholas J, Davidson D, Taga T, Yoshida K, Chambers I, Smith A. Complementary tissue specific expression of LIF and LIF receptor mRNA in early mouse embryogenesis. Mech Dev 1996; 57: 123-131.##Sirisathien S, Hernandez-Fonseca HJ, Bosch P, Hollet BR, Lott JD, Brackett BG. Effect of leukemia inhibitory factor on bovine embryos produced in vitro under chemically defined conditions. Theriogenology 2003; 59: 1751-1763.##Nilsson EE, Kezele P, Skinner MK. Leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition in rat ovaries. Mol Cell Endocrinol 2002; 188: 65-73.##Haidari K, Salehnia M, Rezazadeh Valojerdi M. The effects of different concentrations of leukemia inhibitory factor on development of isolated preantral follicles from fresh and vitrified ovaries. Iranian Biomed J 2006; 10: 185-190.##Haidari K, Salehnia M, Rezazadeh Valojerdi M. The effect of Leukemia inhibitory factor and co-culture on the in vitro maturation and ultrastructure of vitrified and non vitrified isolated mouse preantral follicles. Fertil Steril 2008; 90: 2389-2397.##Mazoochi T, Salehnia M, Rezazadeh Valojerdi M, Mowla SJ. Morphologic, ultrastructural, and biochemical identification of apoptosis in vitrified-warmed mouse ovarian tissue. Fertil Steril 2008; 90: 1480-1486.##Quinn P, Margalit R. Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos. J Assist Reprod Genet 1996; 13: 9-14.##Kohata Y, Gupta PD, Yasuzumi F. Stereo-electron microscopy of the ovarian follicles of cat and mouse. Okajimas Folia Anat Jpn 2007; 83: 97-106.##Makabe S, Naguro T, Stallone T. Oocyte-follicle cell interactions during ovarian follicle development, as seen by high resolution scanning and transmission electron microscopy in humans. Micros Res Tech 2006; 69: 436-449.##Nishi Y, Takeshita T, Sato K, Araki T. Change of the mitochondrial distribution in mouse ooplasm during in vitro maturation. J Nippon Med Sch 2003; 70: 408-415.##Nilsson E, Parrott JA, Skinner MK. Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis. Mol Cell Endocrinol 2001; 175: 123-130.##Santos RR, Rodrigues PR, Costa SHF, Silva JRV, Matos MHT, Lucci CM, et al. Histological and ultrastructural analysis of cryopreserved sheep preantral follicles. Anim Reprod Sci 2006; 91: 246-263.##Silva JR, Bao SN, Lucci CM, Carvalho FC, Andrade ER, Ferreira MA, et al. Morphological and ultrastructural changes occurring during degeneration of goat preantral follicles preserved in vitro. 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Ultrastructural characteristics of human granulosa cells in a coculture system for in vitro fertilization. Microsc Res Tech 2006; 69: 508-516.## ##

Antispasmodic effect of Physalis alkekengi fruit extract on rat uterus 2 2 Background: Studies have shown that Physalis alkekengi reduces implantation and induces antifertility in rat. In Iranian traditional medicine it is believed that this plant has abortifacient and antifertility activities. Objective: The goal of this study was to evaluate the effect of Physalis alkekengi ripe fruit hydroalcoholic extract (PFE) on uterine contractility and its possible mechanism(s). Materials and Methods: Extraction of Physalis alkekengi fruit was carried out by maceration method (70% alcohol). Uterus was dissected out from adult non-pregnant rat (Wistar) and contracted by KCl (60mM) or oxytocin (10mU/ml) in an organ bath containing De Jalon solution and the effect of PFE on the uterine contractions was investigated. Furthermore, the role of α- and β-adrenoceptors, opioid receptors, nitric oxide and cyclic guanosine monophosphate synthesis inhibitors on the extract effects were evaluated. Results: KCl- and oxytocin-induced uterine contractions were inhibited (p<0.001) by the cumulative concentrations of the extract in a concentration dependent manner. Incubation of uterus with propranolol (1μM) and L-NAME (100μM) attenuated the PFE antispasmodic effect (p<0.05). But the PFE effect was unaffected by phentolamine (1μM), naloxone (1μM) or methylene blue (10μM). In Ca2+-free with high potassium (60mM) De Jalon solution, cumulative concentrations of CaCl2 (0.1-0.5mM) induced uterine contraction concentration-dependently (p<0.001). Uterus incubation (5min) with PFE (0.25-1.75mg/ml) attenuated the CaCl2–induced contractions (p<0.05). Conclusion: It seems that the extract induced antispasmodic effect mainly via calcium influx blockade and partially through blocking β-adrenoceptors and nitric oxide (NO) synthesis. However, neither α-adrenoceptors nor opioid receptors or cGMP synthesis were involved. 193 198 2017/10/12017/10/12017/10/12017/10/12017/10/1 1396/7/9 2017/10/52017/10/52017/10/52017/10/52017/10/5 1396/7/13 Mohammad Kazem Gharib Naseri Mohammad Kazem Gharib Naseri Physiology Research Center, Department of Physiology, School of Medicine, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran Gharibnaseri_m@yahoo.com Maryam Mohammadian Maryam Mohammadian Physiology Research Center, Department of Physiology, School of Medicine, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran Zahra Gharib Naseri Pharm Zahra Gharib Naseri Pharm Physiology Research Center, Department of Physiology, School of Medicine, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran Rat Uterus Physalis alkekengi Antispasmodic Amini A, Dictionary of therapeutic plants. Tehran University Publication, Tehran, 2004; 157-159.##Zargari A. Medical Plants, Tehran University Publications, Tehran, 2003; 3595-3596.##Saadat M, Saadat I. The anti-bacterial effect of Physalis alkekengi L. Armaghane-Danesh, Journal of Yasuj University of Medical Sciences 1997; 7-8: 9-15.##Vessal M, Rasti M. Estradiol antagonistic effects of winter cherry extract on pituitary and hypothalamic G6PD activities. Iranian Journal of Medical Sciences 1995; 20: 152- 158.##Vessal M, Akmali M, Bambaee Row N. Thin layer chromatographic detection of steroid and alkaloid glycosides in an ethanolic extract of winter cherry (Physalis alkekengi) fruit. Arch Iran Med 1999; 3: 128-130.##Schumer E, Friedman FD, Vessal M, Yazdanian M. Comparison of the effects of an aqueous extract of Physalis alkekengi fruits and/or various doses of 17-β-estradiol on rat estrous cycle and uterine glucose 6-phasphate dehydrogenase activity. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 1995; 112: 229-236.##Pintea A, Varga A, Stepnowski P, Socaciu C, Culea M, Diehl HA. Chromatographic analysis of carotenol fatty acid esters in Physalis alkekengi and Hippophae rhamnoides. Phytochem Anal 2005; 16: 188-195.##Qiu L, Zhao F, Jiang ZH, Chen LX, Zhao Q, Liu HX, et al. Steroids and flavonoids from Physalis alkekengi var. franchetii and their inhibitory effects on nitric oxide production. J Nat Prod 2008 71: 642-646.##Mirheydar H. Herbal information: usage of plants in prevention and treatment of diseases. Islamic Culture Publishing Center, Tehran, 2003; 5: 39-42.##Vessal M, Mehrani HA, Omrani GH. Effects of an aqueous extract of Physalis alkekengi fruit on estrus cycle, reproduction and uretine creatine kinase BB-isozyme in rats. J Ethnopharmacol 1991; 34: 69-78.##Montaserti A, Pourheydar M, Khazaei M, Ghorbani R. Anti-fertility effects of Physalis alkekengi alcoholic extract in female rat. IJRM 2007; 5: 13-16.##Gharib Naseri MK, Handali S, Hoseini H. Antispasmodic activity of Physalis alkekengi leaf hydroalcoholic extract on rat ileum. Iranian Journal of Medicinal and Aromatic Plants 2007; 23: 340-349.##Kitazawa T, Kajiwara T, Kiuchi A, Hatakeyama H, Taneike T. Muscle layer- and region-dependent distributions of oxytocin receptors in the porcine myometrium. Peptides 2001; 22: 963-974.##Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano MS, Harada KI, et al. Calcium movements, distribution, and functions in smooth muscle. Pharmacol Rev 1997; 49: 157-230.##Cantabrana B, Fernandez A, Baamonde A, Andres-Trelles F, Hidalgo A. Effects of some inhibitors of arachidonic acid metabolism in rat uterus in vitro. Methods Find Exp Clin Pharmacol 1991; 13: 187-192.##Tezuka N, Ali M, Chwalisz K, Garfield RE. Changes in transcripts encoding calcium channel subunits of rat myometrium during pregnancy. Am J Physiol 1995; 269: 1008-1017.##Mershon JL, Mikala G, Schwartz A. Changes in the expression of the L-type voltage- dependent calcium channel during pregnancy and parturition in the rat. Biol Reprod 1994; 51: 993-999.##Gilani AH, Aziz N, Khurram IM, Chaudhary KS, Iqbal A. Bronchodilator, spasmolytic and calcium antagonist activities of Nigella sativa seeds (Kalonji): a traditional herbal product with multiple medicinal uses. J Pak Med Assoc 2001; 51: 1120.##Sanborn BM. Hormones and calcium: mechanisms controlling uterine smooth muscle contractile activity. Exp Physiol 2001; 86: 223-237.##Wassdal I, Nicolaysen G, Iversen JG. Bradykinin causes contraction in rat uterus through the same signal pathway as oxytocin. Acta Physiol Scand 1998; 164: 47-52.##Mhaouty-Kodja S, Houdeau E, Legrand C. Regulation of myometrial phospholipase C system and uterine contraction by β-adrenergic receptors in midpregnant rat. Biol Reprod 2004; 70: 570-576.##Matthew A, Kupittayanant S, Burdyga T, Wray S. Characterization of contractile activity and intracellular Ca2+ signaling in mouse myometrium. J Soc Gynecol Investig 2004; 11: 207-212.##Arthur P, Taggart MJ, Mitchell BF. Oxytocin and parturition: a role for increased myometrial calcium and calcium sensitization? Front Biosci 2007; 12: 619-633.##Perez-Hernandez N, Ponce-Monter H, Medina JA, Joseph-Nathan P. Spasmolytic effect of constituents from Lepechinia caulescens on rat uterus. J Ethnopharmacol 2008; 115: 30-35.##Ducza E, Gaspar R, Marki A, Gyula P, Bottka S, Falkay G. Use of antisense oligonucleotides to verify the role of the α1A-adrenergic receptor in the contractility of the rat uterus post partum. Mol Pharmacol 2001; 59: 1235-1242.##Ohia SE, Laniyonu AA. Naloxone-insensitive inhibitory and excitatory effects of opioid agonists in the rat isolated uterus. J Pharm Pharmacol 1989; 41: 168-172.##Okawa T, Vedernikov YP, Saade GR, Garfield RE. Effect of nitric oxide on contractions of uterine and cervical tissues from pregnant rats. Gynecol Endocrinol 2004; 18:186-193.##Diamond J. Lack of correlation between cyclic GMP elevation and relaxation of nonvascular smooth muscle by nitroglycerin, nitroprusside, hydroxylamine and sodium azide. J Pharmacol Exp Ther1983; 225: 422-426.##Bradley KK, Buxton ILO, Barber JE, McGaw T, Bradley ME. Nitric oxide relaxes human myometrium by a cGMP-independent mechanism. Am J Physiol Cell Physiol 1998; 275: C1668-C1673.##Mesiano S, Welsh TN. Steroid hormone control of myometrial contractility and Parturition. Semin Cell Dev Biol 2007; 18: 321-331.##Pinto RM, Lerner U, Pontelli H. The effect of progesterone on oxytocin-induced contraction of the three separate layers of human gestational myometrium in the uterine body and lower segment. Am J Obstet Gynecol 1967; 98: 547-554.##Mueller A, Siemer J, Schreiner S, Koesztner H, Hoffmann I, Binder H, et al. Role of estrogen and progesterone in the regulation of uterine peristalsis: results from perfused non-pregnant swine uteri. 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Determination of effective dosage of FSH and hCG in the maturation of preantral follicles and enclosed oocytes in mice 2 2 Background: In vitro maturation (IVM) of oocytes reduces the costs and averts the side-effects of gonadotropin stimulation for in vitro fertilization (IVF). Reliable IVM is an intellectual, scientific and clinical challenge with a number of potential applications. Objective: The effect of hCG was evaluated on the timing and regulation of in vitro ovulation for the Syrian mice oocytes in the presence and absence of FSH. Materials and Methods: Preantral follicles, isolated from the ovaries of 6 weeks-old mice, were cultured in TCM-199 medium. The effect of 10-200 mIU/ml FSH and 1.5 IU/ml hCG was seen on the follicle maturation, as well as the changes in ovulation capacity of enclosed oocytes, after the incubation period of 6 days at 37 °C, 92% humidity and 5% CO2 in air. Results: 100 mIU/ml FSH showed increased follicle diameter, survival, germinal vesicle breakdown (GVBD) and oocyte maturation rates (p<0.0001). Significantly higher number of follicles showed cumulus attachment when ovulation started within 16-24 hours post hCG (97% and 80% respectively; p<0.0001) as compared to the cultures without hCG or when the ovulation time increased from 24 hours post hCG. Combination of FSH and hCG showed 97% (p<0.0001) ovulation as compared to that seen for FSH-containing medium only (81%) or control (10%). Conclusion: The combined administration of 1.5 IU/ml hCG and 100 mIU/ml FSH induces the in vitro follicle maturation, ovulation capacity and proper timing of mice oocytes. 199 204 2017/10/12017/10/12017/10/12017/10/12017/10/12017/10/1 1396/7/9 2017/10/52017/10/52017/10/52017/10/52017/10/52017/10/5 1396/7/13 Aisha Javed Aisha Javed Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Saeed Rezaei-Zarchi Saeed Rezaei-Zarchi Department of Biology, Payam-e-Noor University, Yazd, Iran Morteza Anvari Morteza Anvari Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Madiha Javeed Ghani Madiha Javeed Ghani Department of Bioinformatics, Government College University, Faisalabad, Pakistan Fatemeh Barzegari Firouzabadi Fatemeh Barzegari Firouzabadi Department of Biology, Payam-e-Noor University, Yazd, Iran Amer Jamil Amer Jamil Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan Seyed Mehdi Kalantar Seyed Mehdi Kalantar Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran smkalantar@yahoo.com Habibollah Nazem Habibollah Nazem Department of Biology, Payam-e-Noor University, Yazd, Iran Follicle stimulating hormone HCG Preantral follicles Oocyte maturation GVBD. Weon-Young S, Jin-Tae C, Ri-Cheng C, Belen H, Ezgi D, Shai E, et al. 38 h interval between hCG priming and oocyte retrieval increases in vivo and in vitro oocyte maturation rate in programmed IVM cycles. Human Reprod 2008; 23: 2010-2016.##Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000; 15: 165-170.##Lin YH, Hwang JL, Huang LW, Mu SC, Seow KM, Chung J, et al. Combination of FSH priming and hCG priming for in-vitro maturation of human oocytes. Hum Reprod 2003; 18: 1632-1636.##Son WY, Lee SY, Yoon SH, Lim JH. Pregnancies and deliveries after transfer of human blastocysts derived from in vitro matured oocytes in in vitro maturation cycles. Fertil Steril 2007; 87: 1491-1493.##Smitz JEJ, Cortvrindt GR. The earliest stages of folliculogenesis in vitro. Reprod 2002; 123: 185-202.##O'Brien MJ, Pendola JK, Eppig JJ. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod 2003; 68: 1682-1686.##Liu X, Andoh K, Mizunuma H, Kamijo T, Kikuchi N, Yamada K, et al. Effects of recombinant human FSH (rhFSH), urinary purified FSH (uFSH) and hMG on small preantral follicles and tertiary follicles from normal adult and androgen-sterilized female mice. Fertil Steril 2000; 73: 372-380.##Abel MH, Wootton AN, Wilkins V, Huhtaniemi I, Knight PG, Charlton HM. The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction. Endocrinol 2000; 141: 1795-1803.##Wang Y, Rippstein PU, Tsang BK. Role and gonadotrophic regulation of X-linked inhibitor of apoptosis protein expression during rat ovarian follicular development in vitro. Biol Reprod 2003; 68: 610-619.##Tan SL, Child TJ, Gulekli B. In vitro maturation and fertilization of oocytes from unstimulated ovaries: predicting the number of immature oocytes retrieved by early follicular phase ultrasonography. Am J Obstet Gynecol 2002; 186: 684-689.##Son WY, Yoon SH, Lee SW, Ko Y, Yoon HG, Lim JH. Blastocyst development and pregnancies after IVF of mature oocytes retrieved from un-stimulated patients with PCOS after in-vivo HCG priming: case report. Hum Reprod 2002; 17: 134-136.##Mikkelsen AL, Lindenberg S. Influence of the dominant follicle on in-vitro maturation of human oocytes: a prospective non-randomized study. RBM Online 2001; 3: 199-204.##Conti M. Specificity of the cyclic adenosine 3', 5'-monophosphate signal in granulosa cell function. Biol Reprod 2002; 67: 1653-1661.##Mahmoudi R, Subhani A, Pasbakhsh, Abolhasani F, Amiri I, Salehnia M, et al. The Effects of cumulus cells on in vitro maturation of mouse germinal vesicle stage oocytes. Ir J Reprod Med 2005; 3: 74-78.##Javed A, Jamil A, Rezaei-Zarchi S, Anvari M, Nazem H, Kalantar SM. An in vitro comparative study of follicle stimulating hormone (FSH) and activin A effects on the maturation of preantral follicle-enclosed oocytes from immature Syrian mice. Ir Biomed J 2008; 12: 85-92.##Javed A, Rezaei-Zarchi S, Jamil A, Kalantar SM. An in vitro comparative study of the effects of ascorbic acid and FSH on the maturation of Syrian mice follicles. Mid East Fretil Soc J 2008; 13: 135-142.##Mitchell LM, Kennedy CR, Hartshorne GM. Effects of varying gonadotrophin dose and timing on antrum formation and ovulation efficiency of mouse follicles in vitro. Hum Reprod 2002; 17: 1181-1188.##Bishonga C, Takashi Y, Katagiri S, Nagano M, Ishikawa A. In vitro growth of ovarian peantral follicles and the capacity of their oocytes to develop to the blastocyst stage. J Vet Med Sci 2001; 63: 619-624.##Albertini DF, Combelles CM, Benecchi E, Carabatsos MJ. Cellular basis for paracrine regulation of ovarian follicle development. Reprod 2001; 121: 647-653.##Mao J, Wu G, Smith MF, McCauley MC, Cantley TC, Prather RS, et al. Effects of culture medium, serum type, and various concentrations of follicle-stimulating hormone on porcine preantral follicular development and antrum formation in vitro. Biol Reprod 2002; 67: 1197-1203.##Demeestere I, Centner J, Gervy C, Englert Y, Delbaere A. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reprod 2005; 130: 147-156.##Cecconi S, Rossi G, Coticchio G, Macchiarelli G, Borini A, Canipari R. Influence of thyroid hormone on mouse preantral follicle development in vitro. Fertil Steril 2004; 81: 919-924.##Demeestere I, Gervy C, Centner J, Devreker F, Englert Y, Delbaere A. Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation and embryo development in mice. 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Neonatal mortality of low birth weight infants in Yazd, Iran 2 2 Background: Low birth weight (LBW) is one of the major determinants of neonatal survival as well as postnatal morbidity. Objective: The main objective of the present study was to determine neonatal mortality rate (NMR) in LBW infants in Yazd, Iran. Materials and Methods: In a prospective-cohort study, all births in the maternity hospitals of Yazd, Iran in 2004 were evaluated and mortality rate in LBW population over the course of the first month of extra uterine life was determined. Results: In total, 8.4% (507 of 6016 births) of all newborns were LBW and 18.7% (95/507) of all LBW neonates died. Neonatal mortality rate in Yazd was 24/1000 live births .Two- third (95 /143) of all neonatal deaths occurred in LBW. Neonatal mortality rate (NMR) in LBW, Moderately low birth weight (MLBW), Very low birth weight (VLBW) and Extremely low birth weight (ELBW) were 23, 11.5 , 62.5 and 117 times more than that of normal weight newborns, respectively. Nearly 65% of all LBW neonatal deaths occurred in first 24 hours after birth. Overall NMR, Early Neonatal mortality rate (ENMR) and Late Neonatal mortality rate (LNMR) in LBW were 187, 118 and 9.8 in 1000 live births, respectively. The main causes of mortality among LBW in order of prevalence were respiratory distress syndrome (RDS) (59%), asphyxia (20%), septicemia (12%) and congenital malformation (9%). Conclusion: Neonatal mortality rate in Yazd is high and LBW accounted for two-third of neonatal deaths. Therefore, effort should be intensified to implement effective strategies for the reduction of LBW births and improving the care of these vulnerable neonates. 205 208 2017/10/12017/10/12017/10/12017/10/12017/10/12017/10/12017/10/1 1396/7/9 2017/10/52017/10/52017/10/52017/10/52017/10/52017/10/52017/10/5 1396/7/13 Motahhareh Golestan Motahhareh Golestan Department of Pediatrics, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Iran mogolestan@yahoo.com Razieh Fallah Razieh Fallah Department of Pediatrics, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Sedighah Akhavan Karbasi Sedighah Akhavan Karbasi Department of Pediatrics, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Low Birth Weight Neonatal Mortality Rate Neonat American Academy of Pediatrics and the American College of Obstetricians and Gynecologists. Guidelines for Perinatal Care. American College of Obstetricians and Gynecologists. Washington DC; 2002; 138- 145.##Hamilton BE, Minin AM, Martin JA, Kochanek KD, Strobino DM, Guyer B. Annual summary of vital statistics: 2005. Pediatrics 2007; 119: 345-360.##Darmstadt GL, Lawn JE, Costello A. Advancing the state of the world's newborns. Bull World Health Organ .2003; 81: 224-225.##Kruger C. The missing link: neonatal care in rural communities. Bull World Health Organ 2002; 80:759-760.##Mathews TJ, Mac Dorman MF. Infant mortality statistics from the 2003 period linked birth/infant death data set. Natl Vital Stat Rep 2006; 54: 1 -32.##Centers for Disease Control and Prevention (CDC). Unregistered deaths among extremely low birth weight infants--Ohio, 2006. MMWR Morb Mortal Wkly Rep 2007; 56: 1101-1103.##Mufti p, Setna F, Nazir K. Early neonatal mortality: effects of intervention on survival of low birth babies weighing 1000-2000g. J Pak Med Assoc 2006; 56:174-176.##Corchia C, Orzalesi M . 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The effects of vitrification on spindle apparatus of in vitro matured germinal vesicle in mice 2 2 Background: Routine oocytes cryopreservation remained an elusive technique in the wide ranges of available assisted reproductive technologies. The microtubules of oocytes are vulnerable to cryoprotectants and thermal change during cryopreservation. Objective: The effects of a vitrification protocol were investigated on the spindle and chromosome configurations of mice oocytes cryopreserved at the germinal vesicle stage. Materials and Methods: Germinal vesicle with cumulus cells were transferred to vitrification solution which was composed of 30% (v/v) ethylene glycol, 18% Ficoll-70 and 0.3 M Sucrose either by single step or in step-wise way. Following vitrification and in vitro maturation (MII), the matured oocytes were immonostained for meiotic spindles and chromosomes, before visualization using fluorescent microscopy. Results: A statistically significant increase was observed in the survival and maturation rate in step-wise vitrification (88.96% and 71.23% respectively) compared to single step vitrification (70.6% and 62.42% respectively) (p<0.05). Normal spindle morphology after vitrification-thawing in step-wise vitrification group (77.26%) was higher than single step vitrification group (64.24%) but lower than control group (94.75%) (p<0.05). Conclusion: The results suggest that vitrification with step-wise procedure on mice germinal vesicle oocytes has positive effects on survival and maturation rate and normal spindle configuration compare with single step vitrification procedure. 209 215 2017/10/12017/10/12017/10/12017/10/12017/10/12017/10/12017/10/12017/10/1 1396/7/9 2017/10/52017/10/52017/10/52017/10/52017/10/52017/10/52017/10/52017/10/5 1396/7/13 Reza Mahmoudi Reza Mahmoudi Department of Anatomy, Faculty of Medicine, Yasouj University of Medical Sciences, Yasuj, Iran Iraj Amiri Iraj Amiri Department of Anatomy, Faculty of Medicine, Hamedan University of Medical Sciences, Hamedan, Iran Parichehr Pasbakhsh Parichehr Pasbakhsh Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Iraj Ragardi Kashani Iraj Ragardi Kashani Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Mehdi Abbasi Mehdi Abbasi Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Farid Aboulhasani Farid Aboulhasani Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Tooba Mehrannia Tooba Mehrannia Department of Anatomy, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran Aligholi Sobhani Aligholi Sobhani Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Sobhania@tums.ac.ir Vitrification Germinal vesicle oocyte Mice Microtubule Wennerholm WB. 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