Acbp is essential for decidualization during early pregnancy in mice.

Decidualization of uterine stromal cells plays an important role in the establishment of normal pregnancy. Previous studies have demonstrated that Acyl-CoA binding protein (Acbp) is critical to cellular proliferation, differentiation, mitochondrial functions, and autophagy. The characterization and physiological function of Acbp during decidualization remain largely unknown. In the present study, we conducted the expression profile of Acbp in the endometrium of early pregnant mice. With the occurrence of decidualization, the expression of Acbp gradually increased. Similarly, Acbp expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. We applied the mice pseudopregnancy model to reveal that the expression of Acbp in the endometrium of early pregnant mice was not induced by embryonic signaling. Moreover, P4 significantly upregulated the expression of Acbp, whereas E2 appeared to have no regulating effect on Acbp expression in uterine stromal cells. Concurrently, we found that interfering with Acbp attenuated decidualization, and that might due to mitochondrial dysfunctions and the inhibition of fatty acid oxidation. The level of autophagy was increased after knocking down Acbp. During induced decidualization, the expression of ACBP was decreased with the treatment of rapamycin (an autophagy inducer), while increased with the addition of Chloroquine (an autophagy inhibitor). Our work suggests that Acbp plays an essential role in the proliferation and differentiation of stromal cells during decidualization through regulating mitochondrial functions, fatty acid oxidation, and autophagy.

[Comparison of the efficiency between in-vitro maturation and in-vitro fertilization after early follicular phase GnRH agonist down-regulation in infertile women with polycystic ovary syndrome].

OBJECTIVE: To compare the outcomes of in-vitro maturation (IVM) and in-vitro fertilization (IVF) after early follicular phase gonadotropin-releasing hormone agonist (GnRH-a) down-regulation in infertile patients with polycystic ovary syndrome (PCOS). METHODS: From July 2010 to December 2012, 72 infertile patients with PCOS undergoing assisted reproductive technology treatment in the Affiliated First Hospital of Wenzhou Medical University were enrolled in this study. The patients were divided into 2 groups, which were patients with early follicular phase down-regulation IVM (36 cases) at IVM group and early follicular phase down-regulation long protocol IVF (36 cases) at IVF group. The laboratory parameters and clinical outcomes were compared between two groups. RESULTS: (1) Lab parameters: a total of 442 oocytes were retrieved in group IVM, and 560 were in group IVF. The rate of mature oocytes of 83.8% (469/560) and high-quality embryos of 70.9% (212/299) at group IVF were significantly higher than that of group IVM[54.1% (239/442) and 50.7% (73/144), retrospectively, P < 0.01]. In group IVM, the average duration of gonadotropin (Gn) was (2.8 ± 1.5) days and the average dosage of Gn was (285 ± 169) U, which were significantly lower than (11.0 ± 1.0) days and (1499 ± 165) U in group IVF (P < 0.01). The mean number of oocytes retrieved 12.8 ± 2.5, fertilization rate of 64.8% (155/239), and implantation rate of 31% (23/74) in group IVM and 15.6 ± 3.1, 65.5% (307/469), 31% (23/74) in group IVF, which did not reach statistical difference (P > 0.05) . (2) Clinical outcomes: the clinical pregnancy rate (17/31, 55%) of IVF group was not significantly higher than that 44% (14/32) at IVM group (P > 0.05). The abortion rate was 1/17 at Group IVF and 1/14 in group IVM, which did not show statistical difference. Women at IVM group has no ovarian hyper-stimulation syndrome (OHSS) cycle, group IVF has 31% (11/36) cycles presented moderate and severe OHSS. CONCLUSIONS: Infertile patients with PCOS undergoing IVM and IVF treatment after early follicular phase GnRH-a down-regulation can get satisfactory laboratory and clinical outcome. In addition to short treatment cycle, IVM can also avoid the occurrence of OHSS completely, but it has a rising trend in the abortion rate. IVF has a high incidence of OHSS, meanwhile, it increases the dosage of gonadotropins.

[Study on clinical effect on infertility women with polycystic ovary syndrome treated by in vitro maturation and in vitro fertilization-embryo transfer].

OBJECTIVE: To compare clinical and laboratory outcomes of in vitro maturation (IVM) with in vitro fertilization-embryo transfer (IVF-ET) in treatment of infertility associated with polycystic ovary syndrome (PCOS). METHODS: From Jan.2007 to Dec.2010, infertile patients with PCOS underwent 701 cycles in First Affiliated Hospital of Wenzhou Medical College were studied retrospectively. Those were divided into 293 cycles of IVM group and 408 cycles of IVF/intra-cytoplasmic sperm injection (ICSI) group. The average transplantation rate, mean number of retrieval oocytes, maturation rate, fertilization rate, cleavage rate, high quality embryo rate, embryo implantation rate, pregnancy rate per transfer, pregnancy outcomes and incidence of ovarian hyperstimulation syndrome (OHSS) of the two methods of treatment were compared between two groups. RESULTS: There were 275 cycles in IVM group and 342 cycles in IVF/ICSI group established embryo transfer. The transplantation rate was 93.9% (275/293) in IVM group and 83.8% (342/408) in IVF/ICSI, which reached statistical difference (P < 0.01). The maturation rate of 56.64%, cleavage rate of 88.08%, high quality embryo rate of 38.72% and embryo implantation rate of 17.8% in IVM group were significantly lower than 91.09%, 94.91%, 51.50% and 25.4% in IVF/ICSI group (all P < 0.01). The clinical pregnancy rate per transfer were 37.8% (104/275) in IVM group and 44.2% (151/342) in IVF/ICSI group, which did not show statistical difference (P > 0.05). The mean number of oocytes (12.9 ± 6.5 vs. 12.9 ± 7.9) and fertilization rate (76.52% vs. 70.75%) didn't show significant difference between IVM group and IVF/ICSI group (P > 0.05). The 21.3% (87/408) cycles presented mild to moderate OHSS and 2.0% cycles (8/408) presented severe OHSS in IVF/ICSI group. While, no OHSS cycles were observed in IVM group. CONCLUSION: IVM could get similar clinical pregnancy rates compared with IVF/ICSI in patient with PCOS, however, it can avoid occurrence of OHSS.

THBS1 regulates trophoblast fusion through a CD36-dependent inhibition of cAMP, and its upregulation participates in preeclampsia.

Preeclampsia is a pregnancy complication which threatens the survival of mothers and fetuses. It originates from abnormal placentation, especially insufficient fusion of the cytotrophoblast cells to form the syncytiotrophoblast. In this study, we found that THBS1, a matricellular protein that mediates cell-to-cell and cell-to-matrix interactions, is downregulated during the fusion of primary cytotrophoblast and BeWo cells, but upregulated in the placenta of pregnancies complicated by preeclampsia. Also, THBS1 was observed to interact with CD36, a membrane signal receptor and activator of the cAMP signaling pathway, to regulate the fusion of cytotrophoblast cells. Overexpression of THBS1 inhibited the cAMP signaling pathway and reduced the BeWo cells fusion ratio, while the effects of THBS1 were abolished by a CD36-blocking antibody. Our results suggest that THBS1 signals through a CD36-mediated cAMP pathway to regulate syncytialization of the cytotrophoblast cells, and that its upregulation impairs placental formation to cause preeclampsia. Thus, THBS1 can serve as a therapeutic target regarding the mitigation of abnormal syncytialization and preeclampsia.

The NADPH oxidase 4 protects vascular endothelial cells from copper oxide nanoparticles-induced oxidative stress and cell death.

AIMS: Nanoparticles (NPs) exposure is associated with increased risk of cardiovascular diseases, but the underlying mechanism is still obscure. In this study, we investigated the role of NADPH oxidase 4 (NOX4) in copper oxide nanoparticles (CuONPs)-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: Morphology changes were examined under the microscope. Cell viability was determined by MTS assay and Calcein AM assay. Apoptosis and the levels of superoxide anion (O2-) and hydrogen peroxide (H2O2) were measured by fluorescence activated cell sorting (FACS). Oxidative stress was detected by assaying the levels of glutathione/glutathione disulfide (GSH/GSSG) and malondialdehyde (MDA). Protein expression levels were determined by western blotting. KEY FINDINGS: We revealed that O2- rather than H2O2 was the major component of reactive oxygen species (ROS) in CuONPs-treated HUVECs. Meanwhile, CuONPs downregulated expression of O2--eliminating enzyme NOX4 both at mRNA and protein levels, but did not affect the expression of SOD2 and catalase. NOX4 knockdown caused more accumulation of O2-, and a further decrease of H2O2 in CuONPs-treated HUVECs, suggesting that NOX4 regulates the conversion of O2- to H2O2 in CuONPs-treated HUVECs. Furthermore, we revealed that NOX4 knockdown aggravated CuONPs-induced oxidative stress, characterized by a decrease of GSH/GSSG ratio, an increase of MDA level, and upregulation of HSPA5 and γH2AX. Finally, we showed that NOX4 knockdown exacerbated CuONPs-induced apoptotic cell death in HUVECs, indicating that NOX4 could protect ECs from CuONPs-induced cell death. SIGNIFICANCE: Our study provides the evidence that NOX4 protects vascular endothelial cells from CuONPs-induced oxidative stress and cell death.

[Clinical study on in vitro maturation of immature oocytes transferred from conventional in vitro fertilization.].

OBJECTIVE: To investigate clinical effect of in vitro maturation (IVM) of immature oocytes transferred from conventional in vitro fertilization embryo transfer (IVF-ET) cycles. METHODS: From January 2008 to June 2009, medical documents of 155 infertile patients underwent IVF-ET in the Reproductive Medical Center of First Affiliated Hospital of Wenzhou Medical College were analyzed retrospectively. If more than 20 oocytes were monitored after 5 - 7 days of ovulation induction or follicular developmental retardation were confirmed after 8 - 13 days of ovulation induction, according to patients' wish, IVM were transferred in 60 cycles (group A). In the mean time, IVF was continued in 95 cycles (group B). The mean dosage of gonadotropin, the cancellation rate of cycles, the mean numbers of oocytes retrieved and maturation, the rate of fertilization and excellent quality embryos, pregnancy outcome and the incidence rate of ovarian hyperstimulation syndrome (OHSS) were compared and analyzed. RESULTS: The rates of embryo transfer were 92% (55/60) in group A and 63% (60/95) in group B, which showed significant differences (P < 0.05). In group A, the mean dosage of the gonadotropin, the mean number of oocytes retrieved, the cleavage rate and OHSS rate were (1030 +/- 468) U, 10 +/- 6, 82.2% (231/281) and 0, and were (1544 +/- 338) U, 14 +/- 4, 94.0% (502/534) and 35% (21/60) in group B, respectively, all data above exhibited statistical difference between two groups (P < 0.05). However, the rates of fertilization and excellent quality embryos had no significant differences between two groups (P > 0.05). In group A, the rate of clinical pregnancy per transfer was 53% (29/55) and multiple pregnancy was 14% (4/29), and were 47% (28/60) and 32% (9/28) in group B, they all had no significant differences (P > 0.05). CONCLUSION: IVM of immature oocytes used in conventional IVF cycles not only obtained a high clinical pregnancy rate, but also reduced gonadotropin using dosage and avoided OHSS completely.

[A study on bisulfite sequencing method for methylation status of imprinted genes in single human oocytes].

OBJECTIVE: To establish and improve the method of bisulfite sequencing for methylation status of imprinted genes in single human oocytes. METHODS: Single superovulated immature human oocyte was embedded into low melting point agarose, followed by bisulfite treatment and polymerase chain reaction (PCR) amplification of the H19 and MEST genes. The PCR products were then subjected to TA cloning and sequencing to determine the methylation status. RESULTS: With the modified methods of embedding and bisulfite treatment, we achieved a high PCR success rate of 82.46%, with the somatic cell contamination rate as low as 7.14%. The sequencing results showed no non-CpG cytosine and exact conformity to the theoretical sequences. CONCLUSION: The bisulfite sequencing method we used to determine the methylation status of imprinted genes at the single-cell level was highly efficient and reliable, which can serve as a foundation for the further study of the influences of human assisted reproductive technology on genomic imprinting.

Establishment and characterization of two embryonic cell lines of Bombyx mori.

Two cell lines, i.e., BmE-SWU1 and BmE-SWU2, were established from silkworm embryonic tissues of the reversion phase through primary culture in Grace's medium supplemented with 20% fetal bovine serum. The BmE-SWU1 cell line mainly included diploid spindle cells and round cells, which were large and had severe heteroploidy karyotypes. The population doubling time of the 30th passage of the cell line was 58.7 hr. BmE-SWU2 cells were oblong or round, and small. The population doubling time for the 30th passage of the cell line was 46.6 hr. Of BmE-SWU2 cells 89.9% were diploid (2n=56). Both strains were attached to epithelial-like cell lines and were susceptible to Bombyx mori nucleopolyhedroviruse (BmNPV). Inter simple sequence repeat (ISSR) fingerprinting of silkworm embryonic cell line was obtained.

[Meiotic segregation results of male reciprocal chromosome translocations].

OBJECTIVE: To analyze the meiotic segregation results of male reciprocal chromosome translocation by fluorescence in situ hybridization (FISH). METHODS: Multi-color FISH using 3 combined probes located in any 3 chromosome segments on both sides of two breakpoints was performed on the de-condensed sperm head to analyze the sperm chromosomal contents and segregation patterns. RESULTS: Four male reciprocal translocation carriers were included in the study, with the karyotypes of 46, XY, t(2;18) (p16; q23); 46, XY, t(4;6) (q34;q21); 46, XY, t(8;13) (q23;q21) and 46, XY, t(4;5) (4q31;5q13), respectively. The results showed that 4 carriers had different proportions of various segregated spermatozoa. The spermatozoa of alternate, adjacent-1, adjacent-2, 3:1, non-disjunction in meiosis II, and 4:0 or diploidy accounted for 27.1%-49.4%, 26.9%-37.6%, 2.7%-15.7%, 8.6%-32.7%, 0.2%-1.9%, and 0.1%-0.4%, respectively. CONCLUSION: For each-reciprocal translocation carrier seems to have a particular meiotic segregation results, FISH analysis on sperm head should be done for each carrier in order to provide an accurate genetic counseling.

Outcomes of in vitro fertilization cycles among patients with polycystic ovary syndrome following ovarian puncture for in vitro maturation.

OBJECTIVE: To investigate the effects of ovarian puncture for in vitro maturation (IVM) on subsequent in vitro fertilization (IVF) embryo transfer cycles in patients with polycystic ovary syndrome (PCOS). METHODS: A retrospective study included data from patients admitted to the First Affiliated Hospital of Wenzhou Medical University, China, between January 1, 2008 and December 31, 2014. Patients with PCOS undergoing IVF cycles after having been treated with IVM unsuccessfully were included as the study group and an IVF-procedure data-matched control group of patients undergoing their first IVF cycles was included in a 1:4 ratio. Patients with reproductive anomalies were excluded. Endocrine-hormone levels and antral follicle counts were measured and fertilization-related outcomes were evaluated. RESULTS: There were 49 patients included in the study group and 196 included in the control group. Within the study group, basal luteal-hormone, testosterone, and antral follicle count levels were significantly lower following IVM treatment. The total gonadotropin dose was lower (P<0.001) and the duration of stimulation was shorter (P<0.001) in the study group compared with the control group. The clinical-pregnancy rate was higher in the study group (P=0.018) and no difference was observed between the groups in ovarian hyper-stimulation syndrome (P=0.633). CONCLUSIONS: Previous IVM resulted in improved endocrine profiles and increased clinical-pregnancy rates among patients with PCOS undergoing IVF cycles.

Prediction of clinical pregnancy in vitrified-warmed single blastocyst transfer cycles by pre-freeze morphology.

BACKGROUND: The selection of blastocyst warmed for transfer is based on pre-freeze morphology in vitrified-warmed single blastocyst transfer cycles. But, it is controversial which parameter of blastocyst morphology most closely related to the clinical outcomes. OBJECTIVE: To estimate the effect of blastocoele expansion, trophectoderm (TE) morphology grade, and inner cell mass (ICM) morphology grade on clinical pregnancy in vitrified-warmed single blastocyst transfers. MATERIALS AND METHODS: There were 172 vitrified-warmed single blastocyst transfer cycles during the year 2012 included in this analysis. Comparison of clinical results between pregnancy and no pregnancy group based on patient and blastocyst morphology characteristics was done. Then stepwise logistic regression analysis was used to select the best morphological predictor for clinical pregnancy. Last, comparison of patient characteristics and clinical outcomes separated by the best independent morphological predictor was done. RESULTS: Comparison of clinical results between pregnancy and no pregnancy group and logistic regression showed the clinical pregnancy rate was affected by ICM. Comparison of patient characteristics separated by ICM grade, ICM grade A cycles got higher clinical pregnancy rate than ICM grade B cycles (54.3% vs. 35.0% respectively, p=0.037). CONCLUSION: Blastocyst with good ICM morphology could increase clinical pregnancy rate in vitrified-warmed single blastocyst transfer cycles.

Effect of sperm DNA fragmentation on clinical outcome of frozen-thawed embryo transfer and on blastocyst formation.

During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles.