291 embryos (Blastocyst/Morula) from 20 donor sows were vitrified by two step method with OPS (open pulled straw) in solution I (TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3min, and solution II (TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4mol/L SUC) for 1min, stored in liquid nitrogen for 3 months, and transferred into 8 recipient sows after warming, one recipient sow was pregnant and 8 alive piglets were born. This is the first paper to report getting alive piglets by vitrification in China.
Animals ; Blastocyst ; physiology ; Cryopreservation ; methods ; veterinary ; Embryo Transfer ; Female ; Pregnancy ; Swine ; embryology

Embryonic stem cells (ESCs) is one of the best cell types for regenerative medicine. It is derived from inner cell mass of the blastocyst stage and characterized by self-renewal and pluripotency, which are regulated by kinds of signal molecules, such as the Wnt/β-catenin signaling pathway. β-catenin is a multifunctional protein and plays a key role in Wnt/β-catenin signaling pathway. β-catenin involves self-renewal of ESCs and promotes the differentiation of ESCs into three primary germ layers in space and time. Elucidating the mechanisms of β-catenin in regulating the self-renewal and pluripotency of ESCs will pave the way to use it in research and application.
Blastocyst ; cytology ; Cell Differentiation ; Embryonic Stem Cells ; cytology ; Humans ; Wnt Signaling Pathway ; beta Catenin ; physiology

OBJECTIVETo compare the development of abnormal pronuclear zygotes after intracytoplasmic sperm injection (ICSI) and analyze their genetic polymorphism.

METHODSFour hundred and ninety three abnormal pronuclear zygotes after ICSI were divided into three groups based on the number of pronuclei: 347 nonpronuclear oocytes, 71 monopronuclear zygotes and 75 multipronuclear zygotes. All of them were cultured in the medium of Vitrolife G5 series(TM). Sixteen short tandem repeats (STR) of seven blastocysts were then analyzed by ABI3100.

RESULTSThe cleavage rate of nonpronuclear group (25.4%) was lower than that of the others (P<0.01), the proportion of blocked embryos in nonpronuclear group (48.9%) was significantly higher than that of the others (P<0.05), but the blastocyst rate showed no significant difference in three groups (P>0.05). The genetic polymorphism of the 16 STRs showed that the blastocysts from the nonpronuclear and multipronuclear were diploid, and one of the blastocysts from nonpronuclear oocyte was Y-bearing.

CONCLUSIONThe zygotes with abnormal pronuclei after ICSI might have development potential, and the blastocysts from nonpronuclear oocytes and multipronuclear zygotes could be diploid.

Blastocyst ; physiology ; Cell Nucleus ; physiology ; Embryonic Development ; genetics ; physiology ; Female ; Fertilization in Vitro ; adverse effects ; Humans ; Male ; Oocytes ; physiology ; Sperm Injections, Intracytoplasmic ; adverse effects ; Tandem Repeat Sequences ; Zygote ; physiology

UNLABELLEDObtaining human blastocysts is a prerequisite for cell replacement therapy using embryonic stem cells. We established an interspecies somatic cell nuclear transfer (iSCNT) technique for producing blastocysts without sacrificing human oocytes. Human foetal fibroblasts were used as donor cells injected into the enucleated bovine oocytes in nuclear transfer, whereas bovine foetal fibroblasts were used to produce intraspecies embryos. We also examined the fate of human and bovine mitochondrial DNA (mtDNA) during preimplantation development after nuclear transfer by PCR. PCR analysis for the detection of human and bovine mtDNA was done at the 2,8-morula, and blastocyst stages of the embryos.

RESULT2.8% interspecies embryos developed to blastocysts after cultured in an SOF medium, while blastocyst rate of intraspecies embryos were 10.1%. Both human and bovine mtDNAs existed until the morula stage, whereas only the bovine mtDNA was found at the blastocyst stage. These results indicated that interspecies cloning without using human oocytes could generate human blastocysts. Because of the incoordination between bovine mtDNA and human nuclear gene, developmental rate of interspecies embryos was significantly lower than intraspecie. Whether the embryonic stem cell could be used for cell replacement therapy need further research.

Animals ; Blastocyst ; cytology ; physiology ; Cattle ; Cloning, Organism ; DNA, Mitochondrial ; genetics ; Embryonic Development ; physiology ; Female ; Fibroblasts ; physiology ; Humans ; Nuclear Transfer Techniques ; Oocytes ; physiology ; Species Specificity

The expression of tumor suppressor gene p16INK4a in mouse endometrium during early pregnancy and its possible role in blastocyst implantation were investigated in the present study. Real-time fluorescent quantitative PCR (FQ-PCR) and immunohistochemistry were applied to detect p16INK4a mRNA and protein expressions in endometrium of un-pregnant and pregnant mice on day 2, 3, 4, 5, 7, respectively. In addition, p16INK4a antibody was injected into the horns of uteri in pregnant mice on day 3 and its effect during blastocyst implantation was detected in vivo. The higher expressions of p16INK4a mRNA and protein were observed in pregnant mice compared with that in un-pregnant mice, with a steady increase from day 2 to day 5 and reaching the maximal level on day 5 of pregnancy and then decreasing. p16INK4a antibody decreased the number of implanted blastocysts compared with that of saline-injected group. The results suggest that p16INK4a may be associated with apoptosis of luminal epithelial cells and decidual cells, coordinating decidualization of endometrium and invasion of trophoblastic cells. Thus, we presume that p16INK4a participates in the process of blastocyst implantation in mice.
Animals ; Blastocyst ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; physiology ; Embryo Implantation ; Endometrium ; physiology ; Female ; Immunohistochemistry ; Mice ; Pregnancy ; RNA, Messenger ; Real-Time Polymerase Chain Reaction

By the method of single preimplantation embryos differential display polymerase chain reaction (SPEDDRT-PCR), 25 reprogramming cDNA fragments were obtained from single 2-cell, 8-cell embryos and blastula. After cloning and sequencing, five of them were identified by reverse-Northern and characterized with stage-specific expression during reconstructed embryo development. This results will help to isolate full length reprogramming genes and study their function during embryonic development.
Animals ; Blastocyst ; metabolism ; physiology ; Blotting, Northern ; Embryo, Mammalian ; metabolism ; Embryonic Development ; genetics ; physiology ; Female ; Gene Expression Regulation, Developmental ; genetics ; physiology ; Polymerase Chain Reaction ; Pregnancy ; Rabbits

BackgroundDespite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization (IVF) therapy, it is not yet clear which blastocyst morphological parameters best predict the outcomes of single blastocyst transfer. In addition, most of the previous studies did not exclude the effect of embryo aneuploidy on blastocysts transfer. Thus, the present study investigated the predictive value of various parameters on the pregnancy outcomes achieved via the transfer of frozen euploid blastocysts.

MethodsThe study retrospectively analyzed 914 single euploid blastocyst transfer cycles that were performed at the Peking University Third Hospital Reproductive Medical Center between June 2011 and May 2016. The expansion, trophectoderm (TE), and inner cell mass (ICM) quality of the blastocysts were assessed based on blastocyst parameters, and used to differentiate between "excellent", "good", "average", and "poor"-quality embryos. The relationship between these embryo grades and the achieved pregnancy outcomes was then analyzed via the Chi-square and logistic regression tests.

ResultsFor embryo grades of excellent, good, average and poor, the clinical pregnancy rates were 65.0%, 59.3%, 50.3% and 33.3%, respectively; and the live-birth rates were 50.0%, 49.7%, 42.3% and 25.0%, respectively. Both the clinical pregnancy rate (χ = 21.28, P = 0.001) and live-birth rate (χ = 13.50, P < 0.001) increased with the overall blastocyst grade. Both rates were significantly higher after the transfer of a blastocyst that exhibited either an A-grade or B-grade TE, and similarly, an A-grade ICM, than after the transfer of a blastocyst that exhibited a C-grade TE and/or ICM. The degree of blastocyst expansion had no apparent effect on the clinical pregnancy or live-birth rate. All odds ratio were adjusted for patient age, body mass index, length (years) of infertility history, and infertility type.

ConclusionsA higher overall euploid blastocyst quality is shown to correlate most strongly with optimal pregnancy outcomes. The study thus supports the use of the described TE and ICM morphological grades to augment current embryo selection criteria.

Blastocyst ; cytology ; physiology ; Chi-Square Distribution ; Embryo Transfer ; Female ; Humans ; Logistic Models ; Odds Ratio ; Pregnancy ; Pregnancy Outcome ; Retrospective Studies

Somatic cell nucleus transfer (SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer (NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts (P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells (85% to 91%) than those at 6 and 12 months (66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group (8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.
Animals ; Blastocyst ; physiology ; Cell Proliferation ; Cells, Cultured ; Ear ; embryology ; growth & development ; Fibroblasts ; cytology ; physiology ; transplantation ; Nuclear Transfer Techniques ; Swine ; Swine, Miniature ; anatomy & histology ; embryology ; growth & development

Coordination of cell division and cell fate is crucial for the successful development of mammalian early embryos. Aurora kinases are evolutionarily conserved serine/threonine kinases and key regulators of mitosis. Aurora kinase B (AurkB) is ubiquitously expressed while Aurora kinase C (AurkC) is specifically expressed in gametes and preimplantation embryos. We found that increasing AurkC level in one blastomere of the 2-cell embryo accelerated cell division and decreasing AurkC level slowed down mitosis. Changing AurkB level had the opposite effect. The kinase domains of AurkB and AurkC were responsible for their different ability to phosphorylate Histone H3 Serine 10 (H3S10P) and regulate metaphase timing. Using an Oct4-photoactivatable GFP fusion protein (Oct4-paGFP) and fluorescence decay after photoactivation assay, we found that AurkB overexpression reduced Oct4 retention in the nucleus. Finally, we show that blastomeres with higher AurkC level elevated pluripotency gene expression, which were inclined to enter the inner cell mass lineage and subsequently contributed to the embryo proper. Collectively, our results are the first demonstration that the activity of mitotic kinases can influence cell fate decisions in mammalian preimplantation embryos and have important implications to assisted reproduction.
Animals ; Aurora Kinase B ; metabolism ; Aurora Kinase C ; metabolism ; Blastocyst ; metabolism ; Gene Expression Regulation, Developmental ; physiology ; Histones ; metabolism ; Mice ; Phosphorylation ; physiology

Assisted hatching (AH), which is known to improve the hatching potential of mammalian embryos, has been used to increase the pregnancy rate in in vitro fertilization cycles. However, the effect of AH on a trypsin-like protease, which is known to be associated with the hatching process, has not been studied. In this study, we evaluate whether the intactness of zona pellucida affects the secretion of a trypsin-like protease from mouse blastocyst. Four- to 8-cell stage mouse embryos were collected at 66- to 68 hr after hCG injection and divided into 3 groups according to the manipulation of zona pellucida. The groups are no treatment (control), drilling of zona pellucida (ZD) and thinning of zona pellucida (ZT). The activity of a trypsin-like protease, blastocyst development and hatching rate were compared among the three groups at 110 and 135 hr after hCG injection, respectively. The protease activity and blastocyst development were not significantly different among control, ZD and ZT groups at 110 and 135 hr after hCG injection, respectively. However, the hatching rate of ZD and ZT groups was significantly higher than that of control group at each time, respectively (p>0.001). Even in the zona pellucida removed embryos, the protease activity did not differ from the control group. In conclusion, the secretion of a trypsin-like protease from mouse blastocyst does not seem to be affected by the intactness of zona pellucida.
Animal ; Blastocyst/secretion ; Blastocyst/enzymology* ; Female ; Fertilization in Vitro/methods ; Gonadotropins, Chorionic/pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Pregnancy ; Serine Endopeptidases/secretion ; Serine Endopeptidases/metabolism* ; Zona Pellucida/physiology* ; Zona Pellucida/drug effects