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); however, the number of retrieved oocytes was not significantly different between the two groups (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.
Abortion, Spontaneous ; epidemiology ; Acetophenones ; therapeutic use ; Adult ; Apoptosis ; drug effects ; Embryo Transfer ; Female ; Fertilization in Vitro ; Granulosa Cells ; metabolism ; Humans ; NADPH Oxidases ; antagonists & inhibitors ; Onium Compounds ; therapeutic use ; Oocyte Retrieval ; Oxidative Stress ; Polycystic Ovary Syndrome ; drug therapy ; Pregnancy ; Pregnancy Rate ; Reactive Oxygen Species ; metabolism

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.
Adult ; Cryopreservation ; methods ; Embryo Culture Techniques ; methods ; Embryo Transfer ; methods ; statistics & numerical data ; Female ; Fertilization in Vitro ; methods ; statistics & numerical data ; Humans ; Infertility ; pathology ; therapy ; Middle Aged ; Pregnancy ; Pregnancy Outcome ; Vitrification ; Young Adult

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.

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.

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.

The debate exists whether or not gonadotropin-releasing hormone (GnRH) analogs used in controlled ovarian hyperstimulation (COH) impair endometrial receptivity.Homeobox A11 (Hoxall),Meis homeobox 1 (Meisl),cadherin 1 (Cdhl),and catenin beta 1 (Ctnnbl) are well known to be involved in successful implantation.In this study,the endometrial expression of Hoxall,Meisl,Cdhl,and Ctnnbl 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 Hoxall,Meisl,Cdhl,and Ctnnbl 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 Hoxall,Meisl,Cdhl,and Ctnnbl mRNA and protein were decreased in all of the COH groups.The expression levels of Hoxall and Ctnnbl were the lowest in the GnRH agonist group,and those of Meisl and Cdbl were lower in the GnRH analog groups than the HMG group.There were positive correlations between the expression of Hoxall and Ctnnbl,as well as the expression of Meisl and Cdhl among all the groups.In conclusion,the COH protocols,particularly with GnRH analogs,suppressed Hoxall,Meisl,Ctnnbl and Cdhl 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.

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.

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.
Blastocyst/cytology ; Embryo Implantation ; Endometrium/*metabolism ; Gene Expression ; Gene Expression Regulation, Developmental ; Leukemia Inhibitory Factor/*biosynthesis ; Leukemia Inhibitory Factor/*genetics ; Medicine, Chinese Traditional ; Models, Biological ; Plant Extracts/pharmacology ; RNA, Messenger/metabolism ; Time Factors

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.
Cell Nucleus/*metabolism ; Embryo Implantation ; Embryo Transfer/*methods ; Fertilization in Vitro ; Infertility/therapy ; Models, Biological ; Oocytes/metabolism ; Ovary/*metabolism ; Pregnancy Outcome ; Spermatozoa/metabolism

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 Kun- ruing 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 L1F 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 em- bryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.