Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome / 医学前沿

The increased levels of intracellular reactive oxygen species (ROS) in granulosa cells (GCs) may affect the pregnancy results in women with polycystic ovary syndrome (PCOS). In this study, we compared the in vitro fertilization and embryo transfer (IVF-ET) results of 22 patients with PCOS and 25 patients with tubal factor infertility and detected the ROS levels in the GCs of these two groups. Results showed that the PCOS group had significantly larger follicles on the administration day for human chorionic gonadotropin than the tubal factor group (P 0.05). PCOS group had slightly lower fertilization, cleavage, grade I/II embryo, clinical pregnancy, and implantation rates and higher miscarriage rate than the tubal factor group (P > 0.05). We further found a significantly higher ROS level of GCs in the PCOS group than in the tubal factor group (P < 0.05). The increased ROS levels in GCs caused GC apoptosis, whereas NADPH oxidase 2 (NOX2) specific inhibitors (diphenyleneiodonium and apocynin) significantly reduced the ROS production in the PCOS group. In conclusion, the increased ROS expression levels in PCOS GCs greatly induced cell apoptosis, which further affected the oocyte quality and reduced the positive IVF-ET pregnancy results of women with PCOS. NADPH oxidase pathway may be involved in the mechanism of ROS production in GCs of women with PCOS.

MicroRNA expression and its role in the cell cycle regulation in decidualized endometrial stromal cells in vitro / 中华妇产科杂志

ObjectiveTo study microRNA (miRNA) expression and role of cell cycle regulation in decidualized endometrial stormal cells (ESC) in vitro.MethodsESC was induced decasualization in vitro and matched with non-decidualized cells as controls.The expression repertoire of miRNA was measured by microarray chip and was validated by real-time PCR.Flow cytometry was used to identify ESC cycle during decidual reaction in vitro and after miRNA222 inhibitor was transfected into it.Results (1) Between decidualized and undecidualized stromal cells,there were 49 miRNAs significantly different expression by microarray chip,including 16 miRNA up-regulation and 33 miRNA down-regulation.hsa-miR-27b,30c,143,101,181 b,29b,30d,507,23 a,222,221 exhibited significantly differential expression between decicualized and undecidualized stromal cells by real-time PCR (P ＜0.05).(2) After miRNA222 inhibitor (NC-FAM) transfection to decidual ESC,ESC were cultured by FBS medium for 24 hours,the rate of transfection was 70％.ESC were transfected with miRNA 222 inhibitor and cultured for 48 hours,the percentage of ESC at Sphase of (6.2 ± 0.7 ) ％ were significantly lower than ( 10.9 ± 0.8 ) ％ in control group ( P ＜ 0.05 ) ; the percentage of ESC at G0/G1 phase increased at transfection group [ (77.5 ± 1.3 ) ％ vs.(73.0 ± 1.6) ％ at control group ],but there was no significant difference (P ＞ 0.05 ).Decasualization ESC were transfected with miRNA 222 inhibitor and cultured for 48 h,the percentage of ESC at S-phase was ( 3.3 ± 0.6) ％ in transfection group,which were significantly lower than (7.8 ± 0.9 ) ％ in control group ( P ＜ 0.05 ).The percentage of ESC at G0/G1 phase was ( 80.7 ± 1.6 ) ％ in transfection group and ( 74.9 ± 1.1 ) ％.In control group,which did not reached statistical difference ( P ＞ 0.05).ConclusionmiRNA was involved in ESC decidual process in vitro by regulating cell cycle.

Selection and vitrification of embryos with a poor morphological score: A proposal to avoid embryo wastage / 华中科技大学学报（医学）（英德文版）

Embryos with a poor morphological score at cleavage stage are usually discarded because they are considered unsuitable for transfer and cryopreservation. This study examined the in vitro blastocyst development after extended culture of these embryos and the clinical outcomes after transfer of these blastocysts in warming cycles. A total of 597 blastocysts (24.7%) were obtained from 2421 embryos with low morphological scores after extended culture. One hundred and sixty blastocysts (6.6%) with optimal morphology were vitrified. Embryo utilization rate was increased from 30.8% to 32.6%. After warming, 61 out of 92 blastocysts (66.3%) survived and were transferred in 44 cycles. The clinical pregnancy rate and the implantation rate were 40.9% (18/44) and 32.8% (20/61) respectively. Thirteen healthy babies were born, and 5 pregnancies aborted spontaneously. Our study suggested that some blastocysts derived from embryos with a poor morphological score can be successfully vitrified and give rise to live births. Selection and vitrification of viable embryos after extended culture of embryos with a poor morphological score may constitute a proposal to avoid embryo wastage.

Selection and vitrification of embryos with a poor morphological score: a proposal to avoid embryo wastage / 华中科技大学学报（医学）（英德文版）

Embryos with a poor morphological score at cleavage stage are usually discarded because they are considered unsuitable for transfer and cryopreservation. This study examined the in vitro blastocyst development after extended culture of these embryos and the clinical outcomes after transfer of these blastocysts in warming cycles. A total of 597 blastocysts (24.7%) were obtained from 2421 embryos with low morphological scores after extended culture. One hundred and sixty blastocysts (6.6%) with optimal morphology were vitrified. Embryo utilization rate was increased from 30.8% to 32.6%. After warming, 61 out of 92 blastocysts (66.3%) survived and were transferred in 44 cycles. The clinical pregnancy rate and the implantation rate were 40.9% (18/44) and 32.8% (20/61) respectively. Thirteen healthy babies were born, and 5 pregnancies aborted spontaneously. Our study suggested that some blastocysts derived from embryos with a poor morphological score can be successfully vitrified and give rise to live births. Selection and vitrification of viable embryos after extended culture of embryos with a poor morphological score may constitute a proposal to avoid embryo wastage.

Association of controlled ovarian hyperstimulation treatment with down-regulation of key regulators involved in embryonic implantation in mice / 华中科技大学学报（医学）（英德文版）

The debate exists whether or not gonadotropin-releasing hormone (GnRH) analogs used in controlled ovarian hyperstimulation (COH) impair endometrial receptivity. Homeobox A11 (Hoxa11), Meis homeobox 1 (Meis1), cadherin 1 (Cdh1), and catenin beta 1 (Ctnnb1) are well known to be involved in successful implantation. In this study, the endometrial expression of Hoxa11, Meis1, Cdh1, and Ctnnb1 during the peri-implantation period was investigated in an in vitro fertilization (IVF) mouse model by real-time RT-PCR and Western blot to evaluate the relationship between Hoxa11, Meis1, Cdh1, and Ctnnb1 expression and the impact of the COH on endometrial receptivity. The mimic COH protocols included GnRH agonist plus human menopausal gonadotropin (HMG) (GnRH agonist group), GnRH antagonist plus HMG (GnRH antagonist group), and HMG alone (HMG group). The expression levels of Hoxa11, Meis1, Cdh1, and Ctnnb1 mRNA and protein were decreased in all of the COH groups. The expression levels of Hoxa11 and Ctnnb1 were the lowest in the GnRH agonist group, and those of Meis1 and Cdh1 were lower in the GnRH analog groups than the HMG group. There were positive correlations between the expression of Hoxa11 and Ctnnb1, as well as the expression of Meis1 and Cdh1 among all the groups. In conclusion, the COH protocols, particularly with GnRH analogs, suppressed Hoxa11, Meis1, Ctnnb1 and Cdh1 expression, in mouse endometrium during the peri-implantation period. Our data reveal a novel molecular mechanism by which the COH protocols might impair endometrial receptivity.

Temporal and spatial expression of Wnt5a in prenatal female mouse reproductive tract / 解剖学报

ObjectiveTo develop a method of location of prenatal female mouse reproductive tract based on paraffin block serial sections of female fetuses, and get high quality paraffin sections of fetuses containing female reproductive tract (FRT).And to investigate the role of Wnt5a during the development of FRT of prenatal mice. Methods The sex of fetuses at gravidity 15.5 days( G15.5d), G17.5d, G19.5d) and G21.5d was identified by polymerase chain reaction(PCR),and the female fetuses were embedded in paraffin block, the specimen was sectioned serially . One of every four sections was stained by hematoxylin-eosin(HE), and the next one being used later.The images of specimen were taken with digital camera and the serial sections were obtained. The paramesonephric duct /uterus were located and recognized. Immunohistochemistry was used to determine the location and intensity of Wnt5a staining in the middle of the paramesonephric duct / uterus. Results The morphology of the paramesonephric duct /uterus was recognized first and the sections of fetuses with paramesonephric ducts were obtained. The intensity of Wnt5a immunohistochemical staining was increased from G15.5d to G21.5d in mesenchymal cells(P<0.01).Conclusion Wnt5a plays a marked role in early period of female mouse reproductive tract, and is possible to be a key factor to induce uterus differentiation and development.

Polo-like kinase-1 regulates first cleavage of one-cell embryos in culture during assisted reproduction

To investigate the role of polo-like kinase -1 [PLKl] in the first cleavage of a zygote in culture. This experiment took place in the Reproductive Medicine Center of Tongji Hospital, Wuhan, China, from 1st June 2009 to 20th November 2009. First, we detected the expression of PLKl during the first zygotic division by using Western blotting, and then we reduced the expression of PLKl during the first zygotic division by ribonucleic acid [RNA] interference [including 4 groups: PLKl small interfering RNA [siRNA], siRNA control, mock transfection, and only zona pellucida [ZP] removal], finally we evaluated and compared the first cleavage rates of the 4 groups. The expression of PLKl peaked in the first M phase of zygotic cleavage [3 samples/group, 100 zygotes/sample]. The relative amount of PLKl of the mouse zygotes was reduced significantly after siRNA transfection. The first cleavage rate of the PLKl siRNA group was significantly less than that of other groups [siRNA control, mock transfection, and only ZP removal, p=0.000]. The PLKl plays a crucial role during the first cleavage of one-cell embryos, and the zygotes are unable to divide successfully without functional PLKl

Relationship between pronuclear scoring and embryo quality and implantation potential in IVF-ET / 华中科技大学学报（医学）（英德文版）

To assess the relationship between pronuclear scoring and day-3 embryo quality and pregnancy outcome and to determine the clinical value of pronuclear stage scoring system in human in vitro fertilization-embryo transfer (IVF-ET) program, a pronuclear scoring system was used to score zygotes 16-20 h after insemination during conventional IVF or intracytoplasmic sperm injection (ICSI). The embryos were classified into groups Z1, Z2, Z3 and Z4. Comparisons were made of the rates of arrested embryos and excellent embryos on day 3. Comparisons of pregnancy outcome were made only in those patients in whom cohorts of similarly Z-scored embryos were transferred. The results showed that there were less arrested embryos and more excellent embryos on day 3 in groups Z1 and Z2 than those in group Z3 and Z4. More embryos arrested and less excellent embryos developed in group Z4 than group Z3. The clinical pregnancy rates resulting from the transfer of single pronuclear score homologous embryo types were similar among groups Z1, Z2 and Z3. Implantation rates of group Z1 were higher (P<0.05) than that of group Z3. These findings suggests that pronuclear scoring can predict developmental ability on day 3 and implantation potential. A evaluation that combines the Z-score and day 3 embryo morphology is useful in the determination of the most viable embryos and the number of embryos for transfer.

Effect of Bushenantai recipe on the expression of endometrial LIF in mice with embryonic implantation dysfunction / 华中科技大学学报（医学）（英德文版）

In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor (LIF) in mice with embryonic implantation dysfunction (EID), 120 Kunming mice post coition were randomized into three groups: normal control group, model group and traditional Chinese medicine group (TCM group) (n=40 in each group). Uterus was collected on the pregnancy day (Pd) 4, 5, 6 after an intravenous injection of Evan's blue. The endometrium was dyed by Evan's blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of LIF mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve embryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.

The effects of murine cytomegalovirus on the maturation, fertilization, cleavage and blastula formation of mouse oocytes in vitro / 华中科技大学学报（医学）（英德文版）

To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID(50), 10 TCID(50) and 1 TCID(50)). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID(50) of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.

Regulatory effect of estrogen, progestin and HB-EGF on the expression of HOXA10 gene in Ishikawa cells / 华中科技大学学报（医学）（英德文版）

HOXA10 gene plays an essential role in differentiation of the endometrium and in human reproduction. The aim of this study was to investigate the regulatory effect of sex steroids and HB-EGF on HOXA10 gene in Ishikawa cells. Ishikawa cells were incubated with 17-beta estradiol (10(-8) mol/L), medroxyprogesterone acetate (MPA) (10(-6) mol/L), RU486 (10(-5) mol/L) or HB-EGF (10 ng/mL) for 48 h respectively. The expression of HOXA10 gene was detected by immunofluorescence, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Our results showed that either estrogen alone, progestin alone or progestin combined with estrogen could significantly increase the expression of HOXA10 gene 48 h after the treatment (P<0.05). But estrogen combined with progestin and RU486 could inhibit the up-regulation by estrogen and progestin. HB-EGF could elevate the expression of HOXA10 gene 48 h after the treatment (P<0.05). It is concluded that both estrogen and progestin can up-regulate the expression of HOXA10 gene in Ishikawa cells, but RU486 can inhibit the effect and HB-EGF can elevate the expression level of HOXA10 gene.

Expression of angiopoietin-1/-2 in the process of mouse embryo implantation / 华中科技大学学报（医学）（英德文版）

This study examined the expression and distribution of angiopoietin-1/-2 (Ang-1/-2) in the endometrium of early pregnant mice. The expression of Ang-1/-2 was detected by immunohisto-chemical staining and in situ hybridization respectively. Computerized image analysis system was used to measure the average optical intensity of Ang-1/-2 in endometria at different time points after gestation. Mice were randomly divided into 5 groups: control group, D2 group (2 days after pregnancy), D4 group (4 days after pregnancy), D6 group (6 days after pregnancy) and D8 group (8 days after pregnancy), each containing 15 mice. The results showed that the expression of Ang-1 and Ang-2 was very different among 4 groups (P<0.01). Immunohistochemical staining revealed that Ang-1 was localized in the cytoplasma of stromal cells 2 days after pregnancy (day 2), and in luminal epithelial cells on day 4. The protein of Ang-2 was mainly expressed in the cytoplasma of glandular epithelia and stromal cells. With gestation time, the positive reactions of Ang-1/-2 were stronger in the endometria of the pregnant mice (P<0.01). In situ hybridization showed Ang-1 mRNA in stromal cells on day 2. Hybridization signal was localized in both stromal cells and vessel epithelial cells on day 4; Ang-2 mRNA was expressed in stromal cells and glandular epithelia on day 2; high mRNA levels appeared in stromal cells, glandular epithelia and vascular endothelia on day 4; an increasing in mRNA expression of Ang-1/-2 was observed on day 6 and day 8 (P<0.01). It is suggested that Ang-1/-2 may play an important role in the cross-talk between blastocyst and maternal endometrium during the process of embryo implantation.

Establishment of a simple and useful way for preimplantation genetic diagnosis of chromosomal diseases / 华中科技大学学报（医学）（英德文版）

In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further development capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P0.05). The hybridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chromosomal diseases.

Apoptosis and expression of protein TRAIL in granulosa cells of rats with polycystic ovarian syndrome / 华中科技大学学报（医学）（英德文版）

The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the control rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regulating the apoptosis of granulosa cells in PCOS rats.

Expression of Angiopoietin-1/-2 in the Process of Mouse Embryo Implantation / 华中科技大学学报（医学）（英德文版）

This study examined the expression and distribution of angiopoietin-1/-2 (Ang-1/-2) in the endometrium of early pregnant mice. The expression of Ang-1/-2 was detected by immunohistochemical staining and in situ hybridization respectively. Computerized image analysis system was used to measure the average optical intensity of Ang-1/-2 in endometria at different time points after gestation. Mice were randomly divided into 5 groups: control group, D2 group (2 days after pregnancy), D4 group (4 days after pregnancy), D6 group (6 days after pregnancy) and D8 group (8 days after pregnancy), each containing 15 mice. The results showed that the expression of Ang-1 and Ang-2 was very different among 4 groups (P＜0.01). Immunohistochemical staining revealed that Ang-1 was localized in the cytoplasma of stromal cells 2 days after pregnancy (day 2), and in luminal epithelial cells on day 4. The protein of Ang-2 was mainly expressed in the cytoplasma of glandular epithelia and stromal cells. With gestation time, the positive reactions of Ang-1/-2 were stronger in the endometria of the pregnant mice (P＜0.01). In situ hybridization showed Ang-1 mRNA in stromal cells on day 2. Hybridization signal was localized in both stromal cells and vessel epithelial cells on day 4; Ang-2 mRNA was expressed in stromal cells and glandular epithelia on day 2; high mRNA levels appeared in stromal cells, glandular epithelia and vascular endothelia on day 4; an increasing in mRNA expression of Ang-1/-2 was observed on day 6 and day 8 (P＜0.01). It is suggested that Ang-1/-2 may play an important role in the cross-talk between blastocyst and maternal endometrium during the process of embryo implantation.

Apoptosis and Expression of Protein TRAIL in Granulosa Cells of Rats with Polycystic Ovarian Syndrome / 华中科技大学学报（医学）（英德文版）

The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P＜0.01, P＜0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P＞0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the control rats (P＜0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regulating the apoptosis of granulosa cells in PCOS rats.

Establishment of a Simple and Useful Way for Preimplantation Genetic Diagnosis of Chromosomal Diseases / 华中科技大学学报（医学）（英德文版）

In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD)of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further development capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%)(P＞0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P＜0.05). There was no significant difference in cytoplasm remains between methanol/acetic acid method and Tween/HCL method (P＞0.05). The hybridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P＞0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chromosomal diseases.

Regulatory Effect of Estrogen, Progestin and HB-EGF on the Expression of HOXA10 Gene in Ishikawa Cells / 华中科技大学学报（医学）（英德文版）

HOXA10 gene plays an essential role in differentiation of the endometrium and in human reproduction. The aim of this study was to investigate the regulatory effect of sex steroids and HB-EGF on HOXA10 gene in Ishikawa cells. Ishikawa cells were incubated with 17-beta estradiol (10-8 mol/L), medroxyprogesterone acetate (MPA) (10-6 mol/L), RU486 (10-5 mol/L) or HB-EGF (10 ng/mL) for 48 h respectively. The expression of HOXA10 gene was detected by immunofluorescence,reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Our results showed that either estrogen alone, progestin alone or progestin combined with estrogen could significantly increase the expression of HOXA10 gene 48 h after the treatment (P＜0.05). But estrogen combined with progestin and RU486 could inhibit the up-regulation by estrogen and progestin. HB-EGF could elevate the expression of HOXA10 gene 48 h after the treatment (P＜0.05). It is concluded that both estrogen and progestin can up-regulate the expression of HOXA10 gene in Ishikawa cells, but RU486 can inhibit the effect and HB-EGF can elevate the expression level of HOXA10 gene.

The Effects of Murine Cytomegalovirus on the Maturation,Fertilization, Cleavage and Blastula Formation of Mouse Oocytes In Vitro / 华中科技大学学报（医学）（英德文版）

To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID50, 10 TCID50 and 1 TCID50). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID50 of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.

Experimental study on meiotic spindles and chromosomes of mouse mature (M II) stage oocytes under laser scanning confocal microscopy / 华中科技大学学报（医学）（英德文版）

Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope (LSCM) was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional (3D) image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM. The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of M II oocytes.