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Assisted reproductive technology (ART) is the most effective therapy for those infertile couples. In ART therapy such as IVF/ICSI-ET, both sperm and egg should be treated and fertilized in vitro. The fertilized ovum was then cultured in vitro, and the high-quality embryo was singled out for transfer. Therefore, it is very important how to evaluate the quality of sperm, egg and pre -transfer embryos. Raman spectroscopy is a non - damage detection method with the characteristics of speedliness, high sensitivity and accuracy, because the target object is not previously treated and marked. Raman spectroscopy has been used in the rapid detection of damage of biological macromolecule such as protein, nucleic acid and lipid in recent years. There are three kinds of Raman spectroscopy technologies: surface -enhanced Raman spectroscopy, confocal Raman spectroscopy and Fourier transform Raman spectroscopy. This article briefly discussed the characteristics of Raman spectroscopy and the possible application in ART for the evaluation of quality of semen, ovum and embryo, which provided us a reference for the clinical application in future. [ABSTRACT FROM AUTHOR]

Objective：To investigate the optimum blastocyst culture time after thawing of vitrificated cleaved embryos and its effect on pregnant outcome. Methods：477 patients conducting 520 cycles of blastocyst culture after thawing of vitrificated cleaved embryos were retrospectively analyzed. According to the blastocyst culture time after thawing, 520 cycles were grouped into two groups: Group D2 (the warming time: 15-16 o′clock in D2) and Group D3 (the warming time: 8-9 o′clock in D3). In both groups, blastocysts were observed on 8-9 o′clock in D5/D6. Therefore the culture times were 65 h in Group D2 and 48 h in Group D3 in D5, and 89 h and 72 h in D6. Results：There were no significant differences between Group D2 and Group D3 in the blastocyst formation rate (48.10% vs. 46.59%), good quality blastocyst formation rate (39.70% vs. 34.22%), clinical pregnancy rate (70.08% vs. 69.48%), biochemical pregnancy rate (7.48% vs. 7.98%) and implantation rate (55.93% vs. 58.06%) (P＞0.05). The blastocyst formation rates of good and non-good embryos were (30.37% vs. 13.28%) and (62.64% vs. 40.14%) in 48 h and 65 h, respectively (P<0.05). However, the blastocyst formation rates of good and non-good embryos were (32.32% vs. 27.59%) and (2.17% vs. 2.06%) in 72 h and 89 h respectively (P＞0.05). The clinical pregnancy rates and implantation rates were showed: 48 h group＞65 h group＞72 h group＞89 h group, and the biochemical pregnancy rates were showed: 89 h group＞48 h group＞65 h group＞72 h group, although there were non -significant differences in those rates among groups (P＞0.05). Conclusions：We can warm the vitrificated cleaved embryos at 15 -16 o′ clock in D2 or at 8 -9 o′ clock in D3. Majority of formed blastocysts can be transplanted into uterus in D5, if the vitrificated cleaved embryos were warmed at 15 -16 o′ clock in D2. The blastocysts of good quality embryos can be formed in D5 or D6, however those non-quality embryos could spend more time to form blastocysts until D6. [ABSTRACT FROM AUTHOR]

Human assisted reproductive technology (ART) is one of the most effective therapies for human infertility. In vitro culture condition and the strategy of embryo transfer are important factors of ART outcomes such as the live birth rate and the singleton pregnancies rate. Those conditions of in vitro culture include temperature, pH, oxygen concentration, medium and sequential or continuous culture. The strategy of embryo transfer involves the elective single or double embryo transfer, transfer in cleavage or blastocyst stage, and the index for selecting the best blastocyst. Based on recent studies, this review discussed above controversial problems. [ABSTRACT FROM AUTHOR]

In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for in-vitro embryo culture. In this paper, an online monitoring system was designed for long-term culture of embryos, based on a monitoring strategy of low-magnification search and high-magnification observation. Three optical modules of 4× phase contrast, 10× and 20× Hoffman modulation phase contrast were configured in this system to meet the requirements of different fields of view, especially when the size of the embryo increases during the culture. Using an optomechanical system matching design, an error control and alignment test, the resolution of optical imaging was guaranteed, and a relief stereoscopic imaging with high contrast of embryos was obtained. Through low-magnification field of view to identify and locate embryos and high-magnification field of view to capture the details, the system realized online tracking and monitoring of embryos. In addition, we developed and verified an embryo identifying and locating algorithm based on image contour area and definition evaluation. The online monitoring system of in-vitro embryo culture proposed in this paper can track and record the morphological features of embryos without affecting the embryo development, providing a basis for the assessment of embryo development and the optimization of in-vitro culture system.

Objective: To evaluate the efficiency of the application of array comparative genomic hybridization (array-CGH) in preimplantation genetic diagnosis or screening (PGD/PGS), and compare the clinical outcomes of different stage embryo biopsy. Methods: The outcomes of 381 PGD/PGS cycles referred in the First Affiliated Hospital of Nanjing Medical University from July 2011 to August 2015 were retrospectively analyzed. There were 320 PGD cycles with 156 cleavage-stage-biopsy cycles and 164 trophectoderm-biopsy cycles, 61 PGS cycles with 23 cleavage-stage-biopsy cycles and 38 trophectoderm-biopsy cycles. Chromosomal analysis was performed by array-CGH technology combined with whole genome amplification. Single embryo transfer was performed in all transfer cycles. Live birth rate was calculated as the main clinical outcomes. Results: The embryo diagnosis rate of PGD/PGS by array-CGH were 96.9%-99.1%. In PGD biopsy cycles, the live birth rate per embryo transfer cycle and live birth rate per embryo biopsy cycle were 50.0%(58/116) and 37.2%(58/156) in cleavage-stage-biopsy group, 67.5%(85/126) and 51.8%(85/164) in trophectoderm-biopsy group (both P< 0.01). In PGS biopsy cycles, the live birth rate per embryo transfer cycle and live birth rate per embryo biopsy cycle were the same as 34.8%(8/23) in cleavage-stage-biopsy group, the same as 42.1%(16/38) in trophectoderm-biopsy group (both P> 0.05). Conclusions: High diagnosis rate and idea live birth rate are achieved in PGD/PGS cycles based on array-CGH technology. The live birth rate of trophectoderm-biopsy group is significantly higher than that of cleavage-stage-biopsy group in PGD cycles; the efficiency of trophectoderm-biopsy is better.

Objective: To investigate the effect of blastocyst quality on the strategy of single blastocyst transfer in frozen-thawed cycles., Methods: A retrospective analysis was performed in Reproductive Medicine Center, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region on clinical data of single frozen-thawed blastocyst transfer cycles from January 2008 to December 2013. All cycles were divided into four groups (AA, AB/BA, BB, BC/CB) according to the blastocyst score, then the clinical outcomes were compared between groups. And on this basis, the clinical outcomes were further explored when the group of outcomes with single blastocyst transfer wasn't ideal, which would diverted to transfer two blastocyst., Results: In single frozen blastocyst transfer cycles, the clinical pregnancy rate of each group with the blastocyst scored AA, AB/BA, BB, BC/CB were 61.4% (470/765), 51.2% (330/645), 40.5% (407/1 005), 22.9% (60/262), live births rate in each group were 52.2% (399/765), 41.2% (266/645), 30.4% (306/1 005), 13.7% (36/262), and the abortion rate were 13.6% (64/470), 16.7% (55/330), 21.4% (87/407), 35.0%(21/60), separately. This showed that the clinical pregnancy rate and live births rate decreased significantly with the decline of blastocyst quality (P<0.01), but the abortion rate showed significant upward trend (P<0.01). When single blastocyst scored ≥BB grade transferred, an acceptable clinical pregnancy rate (>40%) and live births rate (>30%) could be obtained, however, the clinical pregnancy rate of 22.9% and live births rate of 13.7% could only be acquired when blastocyst scored BC/CB only transferred one embryo, which significant lower than those of each group scored ≥BB grade (P<0.01). So, after that, the blastocyst scored BC/CB were further divided into two groups (single blastocyst transferred versus two blastocyst transferred) to investigate, then the result showed that the clinical pregnancy rate [22.9% versus 38.5%(67/174),P<0.01] and live births rate [13.7% versus 30.5%(16/67),P<0.01] were significantly increased in the group of two blastocyst transferred compared with the group of one blastocyst transferred, and the abortion rate was also significantly decreased from 35.0% to 17.9% (12/67;P<0.05). So when two blastocyst scored BC/CB were transferred, the clinical outcomes were similar to the group of one blastocyst scored BB transferred (P>0.05)., Conclusions: Of single blastocyst transfer in frozen-thawed cycles, the clinical pregnancy rate and liver births rate showed significant upward trend, but the abortion rate showed significant downward trend, with the decline of blastocyst quality. When the blastocyst scored ≥BB grade, the single blastocyst transfer could be considered to be performed.

The purpose of this study is to evaluate the embryotoxicity of alkaloids in Senecionis Scandentis Hebra on in vitro cultured mouse embryos. Mouse whole embryo culture (WEC) was applied in this study. Post-implantation (8.5 d) mouse embryos were isolated from their mothers, and cultured in medium of immediately centrifuged serum (ICS) with different concentrations of seneciphylline (target concentrations were 100, 50, 25 and 12.5 μg x mL(-1)) or senkirkine (target concentrations were 50, 25 and 12.5 μg x mL(-1)) for 48 h. After culturing completed, the development and organic morphodifferentiation of the cultured embryos were evaluated microscopically. Treatment with seneciphylline and senkirkine had adverse effects on the development and organic morphodifferentiation of embryos. The effect also had clear dose-response. Alkaloidals in Senecionis Scandentis Hebra had embryotoxicity on cultured embryos, which indicated that pregnant people exposed to Senecionis Scandentis Hebra may get potential risk on fetus.

Objective: To investigate the clinical use of array comparative genomic hybridization (aCGH) with fluorescence in situ hybridization (FISH) in preimplantion genetic diagnosis (PGD) for reciprocal and Robertsonian translocation carriers., Methods: From Jan. 2012 to Jun. 2013, a total of 220 PGD cycles from 151 reciprocal translocation and 62 Robertsonian translocation carrier couples, including 33 cycles for reciprocal translocation carriers and 22 cycles for Robertsonian translocation carriers performed using array CGH, and 119 cycles for reciprocal translocation carriers and 46 cycles for Robertsonian translocation carriers performed using FISH were retrospectively studied. The rate of accurate diagnosis was compared between two methods., Results: Normal and/or balance rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 38.20% (123/322) and 67.20% (127/189), significantly higher than 15.39% (195/1 267) and 30.75% (202/657) by FISH (all P < 0.05). Abnormal rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 59.32% (191/322) and 30.69% (58/189), significantly lower than 83.03% (1 052/1 267) and 67.43% (443/657) by FISH (all P < 0.05). And the rate of aneu ploidy in non-translocated chromosome from reciprocal translocation embryos was 20.19% (65/322), which was significantly lower than 38.62% (13/189) from Robertsonian translocation embryos (P < 0.01)., Conclusions: Normal and/or balance rates of the two translocated chromosomes detected by array CGH were significantly higher than FISH. And the rate of aneuploidy in non-translocated chromosomes from reciprocal translocation embryos was significantly lower than that from Robertsonian translocation embryos.

Objective: To establish a suitable protocol for activating mouse somatic cell nuclear transfer embryos with strontium chloride (SrCl2)., Methods: We constructed and identified mouse nuclear transfer (NT) embryos. After nuclear injection, we activated the NT embryos using the following chemical activation methods: exposing the NT embryos to 5 and 10 mmol/L SrCl2 strontium for 1 -8 h, activating the NT embryos with 1-20 mmol/L SrCl2 strontium at 4 and 6 h, treating the NT embryos with 10 mmol/L SrCl2 strontium in different activating media, and exposing the NT embryos to 10 mmol/L SrCl2 strontium combined with 6-dimethylaminopurine (6-DMAP) and cytochalasin B (CB). After activation, the NT embryos were cultured in vitro in the cleavage medium., Results: When the NT embryos were treated with SrCl2 at the concentration of 5 mmol/L, the cleavage rate was remarkably higher at 6 h (38.9%) than at 1 h (6.7%), 2 h (22.8%), 3 h (22.8%) and 4 h (25.6%) (P < 0.05), but with no significant differences from those at 5 h (28.9%), 7 h (34.4%) and 8 h (28.9%) (P > 0.05). When the NT embryos were treated with SrCl2 for 6 h, the rates of cleavage and blastulation were 68.9% and 7.2% at 10 mmol/L, markedly higher than at 1 mmol/L (28.3% and 0%), 2.5 mmol/L (35.6% and 0%), 5 mmol/L (37.8% and 1.1%), 7.5 mmol/L (60.6% and 2.2%), 15 mmol/L (51.7% and 1.1%), and 20 mmol/L (41.7% and 1.1%) (P < 0.05). The cleavage rate of the NT embryos cultured in the Ca2+ and Mg2+ KSOM medium was 27.8%, significantly lower than in the Ca(2+)-free KSOM (69.4%), Ca2+/Mg(2+)-free KSOM (66.1%), and Ca2+/Mg(2+)-free + EDTA KSOM (68.3%) (P < 0.05). The total cell blastocyst number was significantly larger in the NT embryos treated with SrCl2 + CB (45.40 +/- 2.23) than in those treated with SrCl2 (30.15 +/- 1.12), 6-DMAP (34.95 +/- 1.38), and 6-DMAP + CB (37.45 +/- 1.43) (P < 0.05)., Conclusion: Six-hour treatment with 10 mmol/L SrCl2 in Ca2+ alone or in combination with CB can well activate NT embryos in mice.

Objective: To investigate the role of TGF-beta1 in embryo implantation and development in vitro in mice., Methods: Mouse embryos at 2-cell stage were cultured in the media of M16 with exposure to different levels of TGF-beta1 (0, 1, 10 and 50 ng/mL). The percentage of embryos reaching fixed stages (early blastocyst, expanding blastocyst and hatched blastocyst) was monitored 68 h and 92 h after the culture. The expanding blastocys cultured for 68 h in M16 without TGF-beta1 and those with 10 ng/mL of TGF-beta1 were transferred to pseudopregnant mice. On the 6th day post transfer, the successful rates of implantation were counted. The level of IL-10/IFN-gamma in the serum and maternal-fetus interface of the mice was detected by ELISA on the 6th day post transfer., Results: TGF-beta1 improved embryo growth in vitro. TGF-beta1 at a level of 10 ng/mL had the maximum impact, with 15.6%, 68.09%, 1.42% of embryos reaching early, expanding, and hatched stage, respectively, 68 h after culture, and 6.38%, 28.37%, 53.19% of embryos reaching early, expanding, and hatched stage, respectively, 92 h after culture. The promoting effect declined when TGF-beta1 reached 50 ng/mL. The successful rate of implantation of embryos cultured in M16 with TGF-beta1 was significantly higher than those cultured in M16 without TGF-beta1 (35. 2% vs. 17.19%, P < 0.05). The embryos cultured in M16 with TGF-beta1 had significantly lower level of IFN-gamma in the maternal-fetus interface than those cultured in M16 without TGF-beta1 [(30.89 +/- 11.31) pg/mL vs. (43.23 +/- 18. 09) pg/mL, P < 0.053., Conclusion: TGF-beta1 at an appropriate dose improves embryo implantation in mice in vitro. The mechanism may involve the improvement of the quality of embryos and their development, and decrease of IFN-gamma synthesis in maternal-fetal interface, a chemical that could cause Th2 bias.

To study the molecular mechanism of Larix somatic embryogenesis, a differentially expressed cDNA library of Larix somatic embryo in the period of maturation was constructed using suppression subtractive hybridization (SSH). The cDNA from the cultures at the stage of somatic embryo maturation of embryogenic cell line Y35 of L. leptolepis xL. principis-rupprechtii was used as the tester and the cDNA from its subcultured callus was used as the driver. Eight hundreds randomly selected positive clones were sequenced, and 468 UniGenes were obtained finally. According to their function, these ESTs were classified into 19 categories and were involved in many biological process related to plant growth and development such as metabolism, transcription, signal transduction, transport facilitation, cell growth and division, cell structure, cell fate, protein synthesis or degradation, defense etc. Real-time PCR results of several ESTs showed that they were all differentially expressed at the different stages during cell line Y35 somatic embryo maturation.

Morulaes and blastocysts obtained from Guanzhong dairy goats 6-7 days after mating were treated with whole embryo cultivaton, enzymatic digestion and immunosurgery separately. The goat embryonic stem cells (ESC) were isolated and cultured on a feeder layer of mitomycin-inactivated mouse embryo fibroblasts (MEF). The characteristics of goat ESCs were analyzed by immunohistochemisty, RT-PCR and inducing differentiation in vitro. The results indicated that the embryos were easier to attach the culture dish and form primary colonies with whole embryo method. There were colonies that maintained undifferentiated for 18 passages. The ESCs expressed the protein of Nanog, Oct4 and SSEA-3, whereas the protein of SSEA-4 was absent and the protein of SSEA-1 was weakly expressed. In addition, the genes of Nanog, Oct4, TERT and CD117 were expressed in goat ESCs. The cells also could differentiate to myocardial cells when induced in vitro by DMSO. These results suggest that the goat ESCs have characteristics of ESCs.

Objective: To study the developmental toxicity of Caowu and its mechanism on rat embryos., Methods: Whole embryo culture was applied to test the effects of Caowu on rat embryos during the critical period of organogenesis. All embryos isolated on gestational day 9.5 were exposed to different concentrations of Caowu. The growth and differentiation of the embryos at the end of the 48 h culture period were observed., Results: The embryonic growth and development were adversely affected by Caowu at the concentration of 2.5 mg/mL, which were represented as reduced yolk sac diameters, decreased number of somites, and lower morphological scores. With the increase of the concentrations of Caowu, more severe teratogenic effects appeared, including cardiac defect (undivided cardiac tube and inflated pericardial cavity), irregular somites, and brain malformation such as narrow brain vesicles., Conclusion: Caowu has direct embryotoxic effects on rats during the organogenesis period. We suggest that pregnant women in the first trimester use Caowu with cautions before a conclusion has been made.

Objective: Human embryonic stem (hES) cells are considered to be a valuable resource for research in regenerative medicine, drug screening, and developmental studies. However, hES cells are usually established and maintained on mouse embryonic fibroblast feeder layers, and the risk of animal origin contamination from feeder layer generally excludes the clinical use of these hES cells. The main emphasis over the last several years has been in finding defined serum-and feeder layer-free system for derivation and culture of hES cells to enable the clinical use of hES cell for cell transplantation.

Objective: To compare the effects of vitrification with slow-freezing on the developmental ability of day 3 cleavage stage embryos., Methods: Patients who had no less than 4 high quality embryos were included in this study. These embryos were cryopreserved using the methods of vitrification or slow-freezing. In the cryopreserved embryo transfer cycles, the embryos which were cryopreserved using one of the methods were chosen randomly. The developmental ability of embryos was compared between these two groups., Results: A total of 80 patients were included in this study with 160 embryos. In the group of slow-freezing, 73 (91%) embryos were survived and achieved 15 (38%) clinical pregnancies. Among these, 3 were twins and the implantation rate was 25% (18/73). In the group of vitrification, 71 (89%) embryos were survived and achieved 19 (48%) clinical pregnancies. Among these, 9 were twins and the implantation rate was 39% (28/71), which was significantly higher than the slow-freezing group (P < 0.05). Otherwise, the clinical pregnant rate and multiple pregnant rate was higher in the group of vitrification than the slow-freezing group, but had no significance., Conclusion: Vitrification is more benefit for the developmental ability of the thawed embryos and more suitable for the cryopreservation of day 3 cleavage stage embryos.

In order to research developmental competence of transgenic somatic cell by serial nuclear transplantation, goat cloned embryos were compared with recloned embryos in ability of in vitro development. Fetal fibroblasts including human finger-domain lacking t-PA gene was microinjected into cytoplasm of the MII oocytes. Goat embryos (G0) were cloned by this procedure. A single blastomere from 16 - 64-cell goat cloned embryos (G0) was microinjected into Intracytoplasm of the MII oocytes. Goat embryos (G) were cloned by this procedure. Goat embryos (G2, G3) were recloned by using 16 - 64-cell recloned embryos. The developmental time of donor embryo affected the developmental rate of recloned embryos (G1, G2). The results show: the cleavage rate of cloned embryos (G0) (76.45% +/- 1.17%) was no difference significantly with recloned embryos (G1 G2 G3) (72.18% +/- 1.97%, 76.05% +/- 2.38%, 75.99% +/- 2.84%); the developmental rate of morulae and blastocysts of cloned embryos (47.20% +/- 2.93%, 11.00% +/- 1.42%) were higher than these of recloned embryos(34.99% +/- 2.66%, 28.23% +/- 2.00%, 23.34% +/- 1.99%) (3.87% +/- 0.67%, 2.08% +/- 1.66%, 0); the morulae rate(29.57% +/- 1.53%, 24.43% +/- 1.87%) and blastocysts rate(1.96% +/- 1.31%, 2.01% +/- 1.34%) of recloned embryos (G1 G2) from 16-cell recloned embryos were lower than those(34.32% +/- 1.31%, 29.76% +/- 1.66% and 3.86% +/- 1.03%, 3.48% +/- 0.34% )from 32 - 64-cell recloned embryos (P > 0.05). In conclusion, nuclear transfer embryos should not were recloned mostly; and the embryos recloned by using 32 - 64-cell embryos achieved higher developmental ability compared with using 16-cell embryos.

Objective: To determine whether cleavage developmentally retarded embryos have not cleaved during a 24 hour period could develop into blastocysts and produce hESC cell lines., Methods: A total of 120 such embryos were cultured to blastocyst stage by sequential culture. Blastocysts formation rate and quality of blastocyst were detected under microscope. The relation between blastocyst formation rate and blastomere number, the fragment of blastomere and blastomere symmetry were analyzed by stepwise Logistical regression analysis. Inner cell masses (ICMs) were isolated by immunosurgery. Colonies derived from the ICMs were passed every 4 - 7 days and the derivatives were passaged and identified., Results: A total of 22 blastocysts were obtained from 120 embryos. The blastulation rate was 18.7%. Early blatocyst, blastocyst, full blastocyst, expanded blastocyst, hatching blastocyst and hatched blastocyst accounted for 5.9%, 23.5%, 35.3%, 23.5%, 5.9%, and 5.9% respectively. The grade of ICM and trophoblast was mostly scored C or B. Blastocyst formation rate was related to cell number and blastomere symmetry but not fragment. Immunosurgery resulted in the formation of 7 ICMs and 3 primary colonies, which produced 2 cell lines. The cell lines satisfied the criteria that characterize pluripotent hESC cells. Undifferentiated cells were positive for AKP, SSEA-4, TRA-1-60, and TRA-1-81. It could continue to proliferate in vitro and form embryoid bodies when cultured in suspension. It had capability to form teratoma in SCID mice. Both cell lines had normal karyotypes after 45 and 34 passages respectively., Conclusions: Our results suggest that a subset of developmentally retarded embryos can form blastocysts and give rise to hESC cell lines.

Objective: To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos., Methods: ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced., Results: Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development., Conclusion: Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

The effects of reproductive hormone on mouse embryo sexes were studied with in vitro fertilization (IVF), in vitro culture (IVC) and sex identification. Results showed that it had no adverse effects on embryos cleavage and development after the embryos were incubated with the fertilization media with 17beta-E2, FSH and progesterone respectively at the concentration of this study. Mice embryos sex ratios significantly deviated from 1:1(P < 0.01) and skewed in favor of males at the concentration of 17beta-E2 attained 70 pg/mL and progesterone attained 99 pg/mL in IVF culture media respectively. 0.01-0.04 IU/mL FSH had no effects on the mouse embryo sexes.

Objective: To assess the feasibility and clinical value of cryopreservation of human oocytes., Methods: Human oocytes were collected during stimulated cycle and then vitrificated with cryoloop. Surviving oocytes were thawed and then inseminated by intracytoplasmic sperm injection (ICSI). The fertilized oocytes were further cultured in vitro for 48 - 72 hours. High-quality embryos were transferred to the patients of infertility with informed consent., Results: A total of 235 oocytes were vitrificated and thawed. The survival and fertilization rates of the oocytes were 71.9% and 72.5% respectively. Forty-one embryos were transferred to 11 patients. Two of the 11 patients achieved pregnancy, one of which gave birth of 2 healthy female twin babies and the other was at the 16 th week of pregnancy. The mother and babies were all healthy., Conclusion: Human oocyte vitrification is safe and feasible.

This study was undertaken to systematically examine effects of different donor cells and passages on the development of nuclear transfer porcine embryos, so as to establish a preliminary procedure for porcine cloning. Porcine oocytes obtained from ovaries at slaughter were matured in vitro for 40-44h and then enucleated in manipulation medium containing 5 microg/mL cytochalasin B. Fibroblast cells (FC), oviduct epithelial cells (OEC), granulosa cells (GC) and cumulus cells(CC) after 3-9 passages in TCM + 10% FBS were treated by serum starvation (0.5% FBS for 2-9 days), 0.1 microg/mL aphidiconlin (APD) for 1 day and 0.5% FBS for 2-9 days or continue culturing in 10% FBS for 2-9 days, then, were transferred into enucleated oocytes by microinjection or electronic fusion (100 V/mm, 30 micros and 1 pulses). Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP, and cultured for 6 days, to evaluate their cleavage and embryonic development. The cleavage rate of embryos reconstructed with FC and GC pretreated with 0.1 microg/mL APD + 0.5% FBS were significantly higher than that of serum starvation group and control group (P<0.01). There was significantly difference in the cleavage rate and embryonic development among embryos derived from GC, CC and FC, OEC pretreated with 0.1 microg/mL APD + 0.5% FBS. The cleavage rate of embryos reconstructed with GC by electrofusion was significantly higher than that by microinjection (P<0.05), but no difference was found in their proportion developing to blastocysts. 75% to 85% of GC at 3 and 6 passages, and FC at 6 and 10 passages had normal karyotype, which did not show significant difference among them (P>0.05). When GC at G3, G4, G5 and G6 of passages were used as donor cells, the cleavage rate and blastocyst rate was similar, moreover, FC at G6, G7, G8 and G9 of passages also resulted in a similar cleavage rate and blastocyst development. These results indicate that: (1) FC and GC can be cultured up to 9 passages and keep relatively stable karyotepe; (2) Using 0.1 microg/mL APD to treat donor cells prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both of FC and GC cells can be used as the donor karyoplasts for nuclear transfer, and their efficiency are not influenced by the culture passages; (4) The development of reconstructed embryos by electrofusion is higher than that by microinjection, but there is no difference in the total efficiency between the two methods.

To investigate the expression patterns of histone acetyltransferase (GCN5) and histone deacetylase 1 (HDAC1) in preimplantation mouse embryos and the effects of in-vitro cultures on their expressions, immunocytochemistry was used to detect the expressions of GCN5 and HDAC1 in mouse embryos at the stages of two-cell, four-cell, eight-cell, morula and blastocyst in both in-vivo and invitro groups. In in-vivo group, the obvious expressions of GCN5 were observed in the cytoplasm of all kinds of mouse embryos but blastocysts. Meanwhile, HDAC1 was highly expressed in the nuclei of four and eight-cell embryos and morula but mainly seen in the cytoplasm of two-cell embryos. In blastocysts, the HDAC1 fluorescence was limited to the nuclei of trophoblast cells. In in-vitro group, there were no obvious GCN5 expressions in all kinds of embryos and the expression of HDAC1 was significantly reduced although its expression pattern was similar to that of in-vivo group. Those results showed that in -vitro culture environments could inhibit GCN5 expression and decrease the expression of HDAC1 in mouse preimplantation embryos, which might affect the correct embryonic gene expression.

Objective: To assess the outcome of thawing human cleaved embryos from cryopreservation by vitrification., Methods: A total of 957 day 2 or day 3 embryos from 219 patients were thawed after vitrification with ethylene glycol 5.5 solution and 0.25 ml straw between Jan 2003 and Jun 2005, 514 embryos were recovered and transferred in 178 patients., Results: The survival rate of thawing embryos and the clinical pregnancy rate after transfer was 72.2% and 19.7% respectively. Twenty-two healthy babies were born from 16 deliveries, including 12 girls and 10 boys, 6 pregnancies ended in miscarriage and another 13 are ongoing., Conclusion: Vitrification method is an alternative for cryopreservation of human cleaved embryos because of high effectiveness, convenience and good cost efficiency.

Objective: To evaluate the developmental toxicity of ginsenoside Rb1 and the potential mechanism of it in vitro., Methods: Whole embryo culture system was used in this research. Embryos were exposed to various concentrations of ginsenoside Rb1, and scored for growth and development at the end of the 48h culture period., Results: With the concentration of Rb1 rising, the total morphological scores significantly decreased; Developments of forebrain, midbrain and optic system were relatively sensitive to Rb1 and were affected at the concentration of 30 microg/ml; hindbrain, Branchial arch and limb buds were affected at 50 microg/ml; Yolk sac was affected at the low concentration of 15 microg/ml and the indexes of embryonic growth and development decreased at the high concentration of 50 microg/ml. Moreover, the rate of embryo teratogenicity were obviously increased., Conclusion: Ginsenoside Rb1 displayed teratogenic action in mice. The damage on structure and function of yolk sac might be part of teratogenic mechanisms of it.

Objective: To investigate the effects of leptin on development of mouse preimplantation embryos in vitro., Methods: (1) Groups of 2-cell stage embryos randomly selected were placed in drops of CZB medium with or without recombinant leptin (10, 50, 100 and 500 ng/ml) and were cultured to the hatched blastocyst stage, and then embryo-transfer was carried out and the implantation rate was observed and determined. (2) Groups of 2-cell stage embryos randomly selected were placed in drops of leptin free CZB medium and cultured to morula stage. Then we changed the medium, randomized the embryos into CZB medium with or without recombinant leptin (10, 50, 100 and 500 ng/ml), and cultured them to the hatched blastocyst stage., Results: Addition of leptin to embryo culture media promoted the development from 2-cell stage embryo to morula, blastocyst and hatched blastocyst and improved the implantation rate. Leptin at 0, 10, 50 ng/ml concentrations caused a dose-dependent promotion, nevertheless Leptin at even higher concentrations (100 and 500 ng/ml) only brought on the same promotion as what the Leptin at concentration of 50 ng/ml did (P>0.05). Addition of leptin to embryo culture media seemed to have no effects on the in vitro development from morula to blastocyst and hatched blastocyst., Conclusion: Leptin plays an important role in the development of preimplantation embryo. It can promote embryo development and embryo implantation.