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Selection of Immature Bovine Oocytes Using Brilliant Cresyl Blue Enhances Nuclear Maturity after Vitrification
Hajarian Hadi,H. Wahid,Y. Rosnina,M. Daliri,M. Dashtizad,H. Karamishabankareh,A. Faizah,M.I. Iswadi,O. Abas Mazni
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2010.2710.2713
Abstract: Beside cooling/warming rates and composition of vitrification solution, developmental stage of immature oocytes may also affect their vitrification outcome. The aim of the present study was to evaluate the selection effect of developmentally competent immature bovine oocytes by Brilliant Cresyl Blue (BCB) on maturity of oocytes after vitrification. Oocytes were obtained from slaughterhouse ovaries. Only oocytes with 4-5 layers of cumulus cells and homogenous cytoplasm were used. After exposure to BCB stain, immature oocytes were divided into colored (BCB+) and colorless (BCB-) cytoplasm groups. Immature oocytes were equilibrated in VS1 (7.5 Ethylene Glycol (EG)+7.5% DMSO) for 10-12 min and then exposed to VS2 (15% EG+ 15% DMSO+0.5M sucrose) for 1 min. Thereafter, oocytes were loaded on Cryotop and directly plunged into liquid nitrogen. After warming, oocytes were examined for presence of polar body and nuclear maturity. Higher number of oocytes in BCB+group extruded first polar body in comparison with other vitrified groups but not significantly (p>0.05). Compared to the BCB- oocytes, there was significantly lower percentage of degeneration for BCB+oocytes (p<0.05). Within vitrified groups, reaching to the MII stage was significantly higher in BCB+group (51.5%) compared with BCB and vitrified-control groups (27.9 and 40.3%, respectively). These results indicated that selection of potent immature bovine oocytes using brilliant cresyl blue improved the nuclear maturity of immature oocytes after vitrification. In addition, this selection can be a valuable tool to improve the vitrification outcome.
ULTRASTRUCTURAL MODIFICATIONS OF THE HUMAN OOCYTES DURING IN VITRO FERTILIZATION PROCEDURE  [PDF]
I. Checiu,Maria Checiu,Delia Hutanu Checiu,Elena Vl?descu
Annals of West University of Timi?oara : Series of Biology , 2004,
Abstract: In the assisted human reproduction the quality of the gametes is very important for a successful fertilization. One of the parameters used to assess the quality of the oocytes is the degree of their maturation. After follicular puncture the oocytes can be find in three different stages of maturation: premature, mature, and postmature. The immature oocytes maturated in vitro for 24 hours in optimal conditions will be fertilized. The ultrastructural aspects of this oocytes are similar with the aspect of the typically mature oocytes The immature oocytes maturated in vitro for 24 hours in the presence of sperms will not be fertilized. The ultrastructure of this oocytes (unfertilized) presents some differences comparative with mature oocytes such as: the internal face of the zona pellucida is irregular; a large number of secretory vesicles (empty or with an amorphous electrono-aspect content ), and a high number of mitochondrias; the absence of the cortical granule in the peripheral cytoplasm; on the external face of the zona pellucida sperms partially embedded in the zona are present; new investigation at the ultrastructural level are necessary to elucidate this aspects
Maturation capacity, morphology and morphometric assessment of human immature oocytes after vitrification and in-vitro maturation
Saeedeh Nazari,Mohammad Ali Khalili,Forouzan Esmaielzadeh,Mehdi Mohsenzadeh
Iranian Journal of Reproductive Medicine , 2011,
Abstract: Background: In general, 15% of oocytes collected in ART cycles are immature. These oocytes may be cryopreserved further for use in in-vitro maturation (IVM) program.Objective: The aim of this study was to determine maturation capacity, morphometric parameters and morphology of human immature oocytes in both fresh IVM (fIVM) and vitrified-IVM (vIVM) oocytes.Materials and Methods: 93 women who underwent controlled ovarian stimulation for ART were included. The immature oocytes (n=203) were divided into two groups: the first group (n=101) directly matured in vitro; and the second group (n=102) first vitrified, then matured in vitro. All oocytes underwent IVM in Ham’s F10 supplemented with LH+FSH and human follicular fluid. After 48h of incubation, the oocyte maturation rates, as well as morphometric and morphologic characteristics were assessed using cornus imaging and were compared. Results: Oocyte maturation rates were reduced in vIVM, (40.4%), in comparison with fIVM (59.4%, p<0.001). Following morphometric assessment, there was no difference in the mean oocyte diameters (μm) between fIVM and vIVM, 156.3±6.8 and 154.07±9.9, respectively. Other parameters of perimeters, egg areas, as well as oocyte and ooplasm volumes were similar in two groups. In addition, more morphologic abnormalities, such as, vacuole, and dark oocyte were observed in vIVM oocytes.Conclusion: fIVM was more successful than vIVM groups. No statistical differences were noticed in morphometry assessment in two groups. This suggests that morphometric parameters can not be applied as prognosis factor in oocyte maturation outcome in IVM program
Vitrification of in vitro matured oocytes collected from antral follicles at the time of ovarian tissue cryopreservation
Giovanna Fasano, Federica Moffa, Julie Dechène, Yvon Englert, Isabelle Demeestere
Reproductive Biology and Endocrinology , 2011, DOI: 10.1186/1477-7827-9-150
Abstract: Oocyte-cumulus complexes were retrieved from freshly collected ovarian cortex by aspirating antral follicular fluid, and were matured in vitro for 24-48 h prior to vitrification. Oocytes were matured in an IVM commercial medium (Copper Surgical, USA) supplemented with 75 mIU/ml FSH and 75 mIU/ml LH and vitrified using a commercial vitrification kit (Irvine Scientific, California) in high security vitrification straws (CryoBioSystem, France). Oocyte collection and IVM rates were evaluated according to the age, the cycle period and the amount of tissue collected.Immature oocyte retrieval from ovarian tissue was carried out in 57 patients between 8 and 35 years of age, undergoing ovarian tissue cryopreservation. A total of 266 oocytes were isolated, 28 of them were degenerated, 200 were at germinal vesicle stage (GV), 35 were in metaphase I (MI) and 3 displayed a visible polar body (MII). The number of oocytes collected was positively correlated with the amount of tissue cryopreserved (p < 0.001) and negatively correlated with the age of the patients (p = 0.005). Oocytes were obtained regardless of menstrual cycle period or contraception. A total maturation rate of 31% was achieved, leading to the vitrification of at least one mature oocyte for half of the cohort.The study showed that a significant number of immature oocytes can be collected from excised ovarian tissue whatever the menstrual cycle phases and the age of the patients, even for prepubertal girls.Advances in cancer therapy have improved the long-term survival of patients suffering from malignancies. Thus, the number of young adults wishing to become parents following cancer treatment has significantly increased. However, cancer treatment often involves adverse side effects, including loss of gonadal function and sterility [1,2]. Chemotherapy using high doses of alkylating agents and radiotherapy by ionizing radiation reduces the primordial follicle reserve, which may trigger premature ovarian failure (POF)
Structural Changes in Cattle Immature Oocytes Subjected to Slow Freezing and Vitrification
H. Wahid*, M. Thein1, E.A. El-Hafez2, M.O. Abas3, K. Mohd Azam4, O. Fauziah5, Y. Rosnina and H. Hajarian
Pakistan Veterinary Journal , 2012,
Abstract: This study was conducted to evaluate the effect of different cryopreservation methods (slow-freezing and vitrification) on structural changes of bovine immature oocytes. Bovine ovaries were collected from local abattoirs. Cumulus-oocyte-complexes (COCs) were retrieved using aspiration method from 2-6 mm follicles. In Experiment 1, selected oocytes were randomly divided into 4 treatment groups namely freezing solution-exposed, frozen-thawed, vitrification solution-exposed and vitrified-thawed and then oocytes abnormalities were examined under a stereomicroscope. In Experiment 2, oocytes were randomly allocated to the same grouping as experiment 1 plus control group. Following freezing or vitrification, all oocytes were fixed in glutaraldehyde and processed for transmission electron microscopy. In experiment 1, there was a higher incidence of abnormalities in the frozen-thawed and vitrified-warmed oocytes compared to those in freezing solution and vitrification solution-exposed groups (P<0.05). In experiment 2, there were marked alterations in the perivitelline space, microvilli and vesicles of frozen-thawed and vitrified-warmed oocytes characterized by loss of elasticity and integrity of cytoplasmic processes and microvilli following cooling and warming. In conclusion, ethylene glycol-based freezing and vitrification solutions are suitable choices for cryopreservation of immature oocytes and most organelles are able to retain their normal morphology following cryopreservation and thawing processes.
Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes
Bongkoch Turathum, Kulnasan Saikhun, Parisatcha Sangsuwan, Yindee Kitiyanant
Reproductive Biology and Endocrinology , 2010, DOI: 10.1186/1477-7827-8-70
Abstract: Immature oocytes (germinal vesicles) isolated from ovaries of normal bitches (> 6 months of age) were either vitrified in open pulled straw (OPS) using 20% ethylene glycol (EG) and 20% dimethyl sulfoxide (DMSO) as vitrification solution or exposed to vitrification solution without subjected to liquid nitrogen. After warming, oocytes were investigated for nuclear maturation following in vitro maturation (IVM), ultrastructural changes using transmission electron microscopy (TEM) and gene expression using RT-PCR. Fresh immature oocytes were used as the control group.The rate of resumption of meiosis in vitrified-warmed oocytes (53.4%) was significantly (P < 0.05) lower than those of control (93.8%) and exposure (91.4%) groups. However, there were no statistically significant differences among groups in the rates of GV oocytes reaching the maturation stage (metaphase II, MII). The ultrastructural alterations revealed by TEM showed that cortical granules, mitochondria, lipid droplets and smooth endoplasmic reticulum (SER) were affected by vitrification procedures. RT-PCR analysis for gene expression revealed no differences in HSP70, Dnmt1, SOD1 and BAX genes among groups, whereas Bcl2 was strongly expressed in vitrified-warmed group when compared to the control.Immature canine oocytes were successfully cryopreserved, resumed meiosis and developed to the MII stage. The information obtained in this study is crucial for the development of an effective method to cryopreserve canine oocytes for establishment of genetic banks of endangered canid species.A major obstacle for the development of assisted reproductive technologies in canines is the low percentage of oocytes reaching the maturation stage (i.e., metaphase II, MII) following IVM. In contrast to most of other mammals that oocytes are at the MII stage when ovulated, canine oocytes released from ovaries are at the prophase I stage of the first meiotic division and they subsequently completed nuclear maturation within 60
Cryotop Device Enhances Vitrification Outcome of Immature Bovine Oocytes
H. Hajarian,H. Wahid,Y. Rosnina,M. Daliri,M. Dashtizad,T. Mirzapour,N. Yimer,M.M. Bukar,M.I. Iswadi,O. Abas Mazni
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2011.2541.2545
Abstract: The aim of this study was to evaluate the effectiveness of different cryodevices (Open Pulled Straw (OPS), Electron Microscopy Grid (EMG) and cryotop for vitrification of immature bovine oocytes. Polar body, MII stage, survivability and subsequent developmental rates were compared. Only oocytes with 4-5 layers of cumulus cells were used. Oocytes were equilibrated in the first vitrification solution (VS1; HS+10% DMSO+10% Ethylene Glycol (EG)) for 30-45 sec and then in the second vitrification solution (VS2; 20% DMSO+20% EG+0.5 M Sucrose) for 25 sec. Within 30 sec they were mounted on one of the cryodevices and directly plunged into Liquid Nitrogen (LN2) for 10 days. Immature oocytes vitrified using cryotop represented higher rate of polar body extrusion and nuclear maturity (p<0.05). The highest survivability resulted from cryotop and EMG groups and no significant difference found between them. Vitrified oocytes in cryotop group had highest cleavage and blastocyst rates. All of the mean measured rates for vitrified/warmed immature oocytes were significantly lower than that of control group (p<0.05). In conclusion, results of this study showed the superiority of cryotop device for vitrification of immature bovine oocytes which resulted in higher viability and subsequent embryo development.
Analysis of the Phospholipid Profile of Metaphase II Mouse Oocytes Undergoing Vitrification  [PDF]
Jaehun Jung, Hyejin Shin, Soyoung Bang, Hyuck Jun Mok, Chang Suk Suh, Kwang Pyo Kim, Hyunjung Jade Lim
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0102620
Abstract: Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18:2/16:0} [M?H]? and phosphatidylglycerol (PG) {14:0/18:2} [M?H]? were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38:3} [M+H]+ and SM {d34:0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.
The Beneficial Effects of Antifreeze Proteins in the Vitrification of Immature Mouse Oocytes  [PDF]
Jun Woo Jo, Byung Chul Jee, Chang Suk Suh, Seok Hyun Kim
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0037043
Abstract: Antifreeze proteins (AFPs) are a class of polypeptides that permit organismal survival in sub-freezing environments. The purpose of this study was to investigate the effect of AFP supplementation on immature mouse oocyte vitrification. Germinal vesicle-stage oocytes were vitrified using a two-step exposure to equilibrium and vitrification solution in the presence or absence of 500 ng/mL of AFP III. After warming, oocyte survival, in vitro maturation, fertilization, and embryonic development up to the blastocyst stage were assessed. Spindle and chromosome morphology, membrane integrity, and the expression levels of several genes were assessed in in vitro matured oocytes. The rate of blastocyst formation was significantly higher and the number of caspase-positive blastomeres was significantly lower in the AFP-treated group compared with the untreated group. The proportion of oocytes with intact spindles/chromosomes and stable membranes was also significantly higher in the AFP group. The AFP group showed increased Mad2, Hook-1, Zar1, Zp1, and Bcl2 expression and lower Eg5, Zp2, Caspase6, and Rbm3 expression compared with the untreated group. Supplementation of the vitrification medium with AFP has a protective effect on immature mouse oocytes, promoting their resistance to chilling injury. AFPs may preserve spindle forming ability and membrane integrity at GV stage. The fertilization and subsequent developmental competence of oocytes may be associated with the modulation of Zar1, Zp1/Zp2, Bcl2, Caspase6, and Rbm3.
Comparison of Different Vitrification Procedures on Developmental Competence of Mouse Germinal Vesicle Oocytes in the Presence or Absence of Cumulus Cells  [PDF]
Saeed Zavareh,Mojdeh Salehnia,Adel Saberivand
International Journal of Fertility & Sterility , 2009,
Abstract: Background: An evaluation of the developmental competence of vitrified mouse germinal vesicle(GV) oocytes with various equilibration and vitrification times; in the presence or absence ofcumulus cells and by comparison between the cryotop method and straws was performed.Materials and Methods: Mouse GV oocytes were considered in cumulus-denuded oocytes(CDOs) and cumulus-oocyte complexes (COCs) groups. Their survival and developmental rateswere studied in the following experiments: (I) exposure to different equilibration times (0, 3 and5 minutes) and vitrification (1, 3 and 5 minutes) without plunging in LN2 as toxicity tests, (II)oocytes were vitrified using straws followed by exposure to equilibration solution for 0, 3 and 5minutes and vitrification solution for 1 and 3 minutes, and (III) oocytes were vitrified by cryotopfollowing exposure to equilibration for 5 minutes and vitrification for 1 minute, respectively.Results: Maturation and developmental rates of the COCs were higher than CDOs in the nonvitrifiedgroup (p<0.05). The survival and maturation rates were low in all oocytes exposed tovitrification solution for 5 minutes (p <0.05). In vitrified CDOs and COCs using straws, the survivalrates ranged from 56.9% to 85.4% and 44.0% to 84.5%, and the maturation rates from 35.3% to56.8% and 25.8% to 56.2%, respectively; which were lower than non-vitrified samples (p <0.05).Cryotop vitrified oocytes showed higher survival, maturation and fertilization rates when comparedto straw in both CDOs and COCs (p <0.05).Conclusion: The presence of cumulus cells improves developmental competence of GV oocytesin control groups but it did not affect the vitrified group. Vitrification of mouse GV oocytes usingcryotop was more effective than straws, however both vitrification techniques did not improve thecleavage rate.
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