Screening of miRNAs in human follicular fluid reveals an inverse relationship between microRNA-663b expression and blastocyst formation.

RESEARCH QUESTION: Are miRNAs found in follicular fluid related to blastocyst formation from the corresponding oocytes? DESIGN: In this study, 91 individual follicular fluid samples from single follicles containing mature oocytes from 91 women were collected and classified into group 1 (n = 38) with viable blastocysts, and group 2 (n = 53) with no blastocyst. TaqMan human miRNA cards and quantitative reverse transcription polymerase chain reaction were used to identify differently expressed follicular fluid miRNAs between the two groups. RESULTS: We found MIR-663B to be significantly differentially expressed in follicular fluid of oocytes that yielded viable blastocysts versus those that did not develop into blastocysts (14.16 ± 7.00 versus 23.68 ± 17.02; P = 0.019), as well as for those which develop into blastocysts with good morphology versus those with poor morphology (11.69 ± 3.49 versus 20.16 ± 9.33; P = 0.003). CONCLUSIONS: MIR-663B expression levels in human follicular fluid samples were significantly negatively related to viable blastocyst formation and may become an objective evaluation criterion for embryo development potential after IVF.

[Effects of different doses of dexmedetomidine combined with ropivacaine for brachial plexus nerve block in children undergoing polydactyly surgery].

OBJECTIVE: To observe the anesthetic effect and safety of different doses of dexmedetomidine combined with ropivacaine for brachial plexus nerve block in children undergoing polydactyly surgery. METHODS: Eighty children undergoing polydactyly surgery were randomized into 4 groups to receive brachial plexus nerve block with dexmedetomidine at 0.25, 0.50 or 0.75 µg/kg combined with 0.25% ropivacaine (0.20 mL/kg) (D1, D2, and D3 groups, respectively) or with 0.25% ropivacaine (0.20 mL/kg) only (control group). The onset time, duration of brachial plexus nerve block, awakening time, success rate, and incidence of complications were compared among the groups. Results In D2 and D3 groups, the onset time and awakening time were shorter and anesthesia lasted longer than those in the control group. The onset time and awakening time were shorter and anesthesia maintenance time was longer in D3 group than in D1 group. The success rates of brachial plexus nerve block were significantly higher in D1-3 groups than in the control group (P<0.05). Hematoma was found in one of the patients. In each of the 4 groups, laryngeal nerve block occurred in 1 child and respiratory depression in another; 2 or 3 patients had Horner syndrome, and 1 patient in D3 group experienced an episode of lowered heart beat to below 70 min-1. All the complications were managed properly and the patients all recovered uneventfully. CONCLUSION: Brachial plexus nerve block with 0.5 µg/kg dexmedetomidine combined with 0.25% ropivacaine (0.20 mL/kg) is safe for effective anesthesia in children undergoing surgery for polydactyly.

[Application of human oocyte morphometric parameters in assessment of fertilization and embryo development].

OBJECTIVE: To investigate the correlation of human oocyte morphometric parameters with fertilization and embryo development in the intracytoplasmic sperm injection (ICSI) cycles. METHODS: The morphometric parameters of oocytes collected and submitted to evaluation using OCTAX Eye-ware software just before ICSI. Oocyte diameter (OD), perivitelline space width (PSW), zonapellucida thickness (ZPT) and the shape of first polar body (FPB) (intact or fragmented) were analyzed. A stepwise multivariate Logistic regression was used to test the association between the morphometric parameters and fertilization and embryo development. RESULTS: In the study, 436 oocytes were measured and 370 were fertilized (84.9%), 225 fertilized oocytes were developed to high-quality embryos (60.8%). ZPT and PSW were associated with fertilization. The oocytes fertilized had thicker ZPT [(18.0±2.3) µm vs. (16.9±2.7) µm] and wider PSW [(14.4±3.3) µm vs. (13.2±3.9) µm]. The OD and shape of FPB were associated with embryos development. The oocytes developed to high-quality embryos had larger OD [(116.6±3.7) µm vs. (114.7±3.6) µm] and more intact FPB (86.2% vs. 66.7%). CONCLUSION: The morphometric parameters of oocytes can indicate fertilization and embryo development.

Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF.

The aim of this study was to investigate whether the sperm chromatin structure assay (SCSA) results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitro fertilization (IVF). A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and sperm chromatin structure assay results were collected. Fertilization rate, embryo quality and IVF success rates of different DNA fragmentation index (DFI) subgroups and high DNA stainability (HDS) subgroups were compared. There were no significant differences in fertilization rate, clinical pregnancy or delivery rates between the DFI and HDS subgroups. However, the group with abnormal DFI had a lower good embryo rate. So, we concluded that the SCSA variables, either DFI or HDS after swim-up preparation, were not valuable in predicting fertilization failure or pregnancy rate, but an abnormal DFI meant a lower good embryo rate following IVF.

[A 10-year survey on birth defects after in vitro fertilization-embryo transfer in Shanghai].

OBJECTIVE: To survey birth defects of neonates conceived by using various types of in vitro fertilization and embryo transfer (IVF-ET) between 1998 and 2007 in Shanghai. METHODS: From 1998 to 2007, 8507 neonates from 6551 pregnancies conceived through assistant reproductive technology (ART) from 7 reproductive medicine center in Shanghai were enrolled in this retrospective study, including Shanghai Ji-Ai Genetics and IVF Institute, Shanghai Jiaotong University School of Medicine affiliated Renji Hospital, Ruijin Hospital, China Welfare Institute International Maternal and Infant Health Hospital, Shanghai First Maternity and Infant Health Hospital, Shanghai the Ninth People's Hospital and the Second Military Medical University affiliated Changhai Hospital. The clinical data about the type and incidence of birth defect were analyzed. Meanwhile, the factors associated with birth defect were investigated, such as various ART, gender, mother age, numbers of gestational sac, the source and quality of sperm. RESULTS: The birth defect rate was 1.08% (92/8507) and the most remarkable malformation occurred in circulation system [34% (31/92)]. The rates of major congenital malformations were 1.21% (34/2799) in fresh conventional IVF-ET, 1.07% (20/1871) in IVF-frozen-thawed embryo transfer (IVF-FET), 1.04% (23/2212) in fresh intracyto plasmic sperm injection (ICSI)-ET and 0.92% (15/1625) in ICSI-FET, which did not show statistical difference (P > 0.05). There was also no significantly different incidence of malformations between 1.12% (49/4371) in male neonates and 1.02% (42/4136) in female neonates (P > 0.05). However, the occurrence of congenital malformation is positively related with maternal age, the rates were 0.84%(41/4884) in mother's age less than 30 years and 1.77% (16/902) in mother' age more than 35 years, which reached statistical difference (P < 0.05). It also showed remarkable different incidence among 0.53% (25/4679) in singletons, 1.59% (57/3576) in twins and 3.97% (10/252) in triplets (P < 0.05). No remarkable difference of malformation rate among sperm sources used in fertilization were observed, including 1.09% (81/7419) in ejaculated sperm, 1.21% (7/579) in percutaneous epididymal aspiration (PESA) and 0.79% (4/509) in donor sperm (P > 0.05). CONCLUSIONS: The overall incidence of birth defect in neonates from ART is similar to those conceived naturally and is not associated with different types of IVF-ET, embryo cryopreservation, sperm sources. However, maternal age and multiple pregnancies confer the higher possibility of birth defect.

[Factors related to occurrence of twin pregnancy after double-embryo transfer in vitro fertilization cycles].

OBJECTIVE: To analysis high risk factors of twin pregnancy after double-embryo transfer in fresh in vitro fertilization-embryo transfer (IVF-ET) cycles. METHODS: From Jan. 2003 to Dec. 2007, 275 infertile cases underwent IVF-ET or intracytoplasmatic sperm injection (ICSI) and obtained clinical pregnancy in Reproductive Medical Center, Ruijin Hospital affiliated to Shanghai Jiaotong University. A total of 280 cycles were performed, which were classified into single pregnancy group (198 cycles) and twin pregnancy group (82 cycles). The general information, patient and embryo characteristics were compared between those two groups, then univariate and multivariate regression were analyzed. RESULTS: (1) There was no statistical difference in the following clinical features between single and twin pregnancy groups, such as patients ages, the ratio of secondary infertility, period and possible causes of infertility (P > 0.05). (2) When comparing basal level of follicle stimulating hormone (FSH), mean numbers of follicles, mean obtained ovum, ovarian responsibility (ratio of follicle stimulation hormone dose/number of oocyte retrieved), endometrial thickness given by human chorionic gonadotropin (hCG), no significant difference were observed between two groups (P > 0.05). Twin pregnant group had fewer cycles of in vitro fertilization treatment when compared with single pregnancy group (0.18 +/- 0.16 vs. 0.22 +/- 0.21, P = 0.03). (3) No significant difference was observed in the following clinical index, including fertilization approaches, mean numbers of embryo, mean score of transferred embryo, developmental stage of top quality embryo, morphological score of embryo, morphological score of the second best embryo transferred (P > 0.05). The number of top-quality embryos and the development stage score of the second best embryo transferred were higher than those of single pregnant group (3.8 +/- 3.3 vs. 2.9 +/- 2.5, P < 0.05 and 3.7 +/- 0.2 vs. 3.4 +/- 0.2, P < 0.05). (4) Multivariate regression analysis showed that four variables was correlated independently with twin pregnancy including first treatment cycle of IVF-ET (OR = 1.82, P = 0.02), number of good quality embryos (OR = 1.35, P = 0.01), development stage score of the second best embryo (OR = 1.55, P = 0.009) and ovarian responsibility (OR = 0.96, P = 0.04). CONCLUSIONS: It is advisable to perform single embryo transfer. If patients are at high risk factors of twin pregnancy including initial IVF-ET treatment, good ovarian responsibility, more number of top-quality embryos and development stage score of the second best embryo transferred.

Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines.

Parthenogenetic embryonic stem (pES) cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2af1-rs1, Peg3, Impact, Zfp127, Dlk1 and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activation of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.

Temporal and parental-specific expression of imprinted genes in a newly derived Chinese human embryonic stem cell line and embryoid bodies.

Although the study of imprinted genes in human development is very important, little is known about their expression and regulation in the early differentiation of human tissues due to lack of an appropriate model. In this study, a Chinese human embryonic stem (hES) cell line, SHhES1, was derived and fully characterized. Expression profiles of human imprinted genes were determined by Affymetrix Oligo micro-array in undifferentiated SHhES1 cells and SHhES1-derived embryoid bodies (EBs) at day 3, 8, 13 and 18. Thirty-two known human imprinted genes were detected in undifferentiated ES cells. Significantly, differential expression was found in nine genes at different stages of EB formation. Expression profile changes were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction in SHhES1 cells as well as in another independently derived hES cell line, HUES-7. In addition, the monoallelic expressions of four imprinted genes were examined in three different passages of undifferentiated ES cells and EBs of both hES cell lines. The monoallelic expressions of imprinted genes, H19, PEG10, NDNL1 and KCNQ1 were maintained in both undifferentiated hES cells and derived EBs. More importantly, with the availability of maternal peripheral blood lymphocyte sample, we demonstrated that the maternal expression of KCNQ1 and the paternal expression of NDNL1 and PEG10 were maintained in SHhES1 cells. These data provide the first demonstration that the parental-specific expression of imprinted genes is stable in EBs after extensive differentiation, also indicating that in vitro fertilization protocol does not disrupt the parental monoallelic expression of the imprinted genes examined.