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

OBJECTIVETo provide preimplantation genetic diagnosis(PGD) for two couples carrying thalassemia mutations and chromosomal abnormalities.

METHODSCouple 1 were both carriers of β 41/42 thalassemia mutations, while the husband has carried a reciprocal translocation with a karyotype of 46,XY,inv(9)(p11;q13),t(11;22)(q25;q13). Couple 2 were both carriers of α (-SEA) thalassemia mutation. Their chromosome karyotypes were both normal, but had two spontaneous abortions. The couples had received 1 and 3 blastocysts respectively through in vitro fertilization(IVF) cycles. Following the biopsy, the cells underwent whole genome amplification, and the amplified DNA from each embryo was subjected to genetic testing and a 23-chromosome single nucleotide polymorphism(SNP) microarray assay.

RESULTSThe embryo of couple 1 was diagnosed as carrier of β 41/42 thalassemia with euploid chromosomes. The embryo was transferred and resulted in intrauterine pregnancy. Similarly, an embryo of couple 2 was verified as carrier of α (-SEA) thalassemia with euploid chromosomes.

CONCLUSIONPGD for aneuploidy coupled with testing for single gene disorders via trophectoderm biopsy and whole genome amplification is feasible. The approach can attain diagnosis with minimal damage with sound clinical outcome.

Adult ; Aneuploidy ; Blastocyst ; cytology ; Chromosome Aberrations ; Embryo Transfer ; Female ; Fertilization in Vitro ; Genetic Testing ; Heterozygote ; Humans ; Male ; Mutation ; Pregnancy ; Preimplantation Diagnosis ; beta-Thalassemia ; diagnosis ; embryology ; genetics

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human's, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.
Animals ; Blastocyst ; Chimera ; Culture Media ; Embryo Culture Techniques ; veterinary ; Embryo, Mammalian ; Embryonic Development ; Induced Pluripotent Stem Cells ; cytology ; Mice ; Swine

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

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

OBJECTIVETo establish a novel HLA genotyping method for preimplantation genetic diagnonis (PGD) using multiple displacement amplification-polymerase chain reaction-sequencing based technique (MDA-PCR-SBT).

METHODSPeripheral blood samples and 76 1PN, 2PN, 3PN discarded embryos from 9 couples were collected. The alleles of HLA-A, B, DR loci were detected from the MDA product with the PCR-SBT method. The HLA genotypes of the parental peripheral blood samples were analyzed with the same protocol. The genotypes of specific HLA region were evaluated for distinguishing the segregation of haplotypes among the family members, and primary HLA matching was performed between the embryos.

RESULTSThe 76 embryos were subjected to MDA and 74 (97.4%) were successfully amplified. For the 34 embryos from the single blastomere group, the amplification rate was 94.1%, and for the 40 embryos in the two blastomeres group, the rate was 100%. The dropout rates for DQ allele and DR allele were 1.3% and 0, respectively. The positive rate for MDA in the single blastomere group was 100%, with the dropout rates for DQ allele and DR allele being 1.5% and 0, respectively. The positive rate of MDA for the two blastomere group was 100%, with the dropout rates for both DQ and DR alleles being 0. The recombination rate of fetal HLA was 20.2% (30/148). Due to the improper classification and abnormal fertilized embryos, the proportion of matched embryos HLA was 20.3% (15/74),which was lower than the theoretical value of 25%.

CONCLUSIONPGD with HLA matching can facilitate creation of a HLA-identical donor (saviour child) for umbilical cord blood or bone marrow stem cells for its affected sibling with a genetic disease. Therefore, preimplantation HLA matching may provide a tool for couples desiring to conceive a potential donor progeny for transplantation for its sibling with a life-threatening disorder.

Blastocyst ; cytology ; metabolism ; Female ; Genotype ; Genotyping Techniques ; methods ; HLA Antigens ; genetics ; HLA-DQ beta-Chains ; genetics ; HLA-DRB1 Chains ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; Pregnancy ; Preimplantation Diagnosis ; methods ; Reproducibility of Results ; Sequence Analysis, DNA ; methods

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.
Animals ; Blastocyst/cytology ; Cloning, Organism/*veterinary ; Culture Media/metabolism ; Dogs/*embryology ; Embryo Culture Techniques/*veterinary ; *Embryonic Development ; Nuclear Transfer Techniques/*veterinary

OBJECTIVETo estimate the value of blastocyst culture for preimplantation genetic diagnosis (PGD).

METHODSDay 3 embryos were biopsied and analyzed with fluorescence in situ hybridization (FISH) technique. Embryos with normal FISH results were cultured into blastocysts, and the ones with better morphology scores were transferred. Fourteen embryos with abnormal FISH results were cultured into blastocysts. Part of the cells taken from the blastocysts were amplified by whole genomic amplification (WGA) and assessed by array-based comparative genomic hybridization (array-CGH) analysis.

RESULTSSix blastocysts with normal FISH results were transferred in 5 cycles. Four healthy babies of 3 cycles were delivered. Another one was a singleton pregnancy but with embryo growth arrest, whose villus karyotype was normal. Fourteen embryos with abnormal FISH results were cultured into blastocysts and analyzed by array-CGH. Six blastocysts were normal by array-CGH.

CONCLUSIONFISH combined with blastocyst culture may further ensure the accuracy of PGD result. Detection at the blastocyst stage can avoid false positive results and mosaic interferences on Day 3 stage and are therefore more authentic.

Adult ; Blastocyst ; cytology ; Comparative Genomic Hybridization ; methods ; Embryo Transfer ; Female ; Genetic Diseases, Inborn ; diagnosis ; embryology ; genetics ; prevention & control ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Male ; Pregnancy ; Preimplantation Diagnosis ; methods

The influence of inner cell mass (ICM) and trophectoderm (TE) score on pregnancy outcomes in frozen-thawed blastocyst transfer cycles was analyzed. A retrospective analysis of 741 cycles of frozen-thawed blastosysts transfer was performed. All cycles were divided into four groups based on the number and morphological score of blastocysts: S-ICM B/TE B group (n=91), the single blastocyst transfer of ICM B and TE B; D-ICM B/TE B group (n=579), double blastocysts transfer of ICM B/TE B; D-ICM B/TE C group (n=35), double blastocysts transfer of ICM B/TE C; and D-ICM C/TE B group (n=36), double blastocysts transfer of TE B/ICM C. The pregnancy outcomes were compared among the four groups. As compared with D-ICM B/TE C group, the clinical pregnancy rate, implantation rate and multiple pregnancy rate were increased in D-ICM B/TE B group (74.96% vs. 57.14%, 57.43% vs. 37.14%, and 48.62% vs. 25%, respectively, P<0.05 for all). Clinical pregnancy rate and implantation rate in D-ICM B/TE B group were also higher than in D-ICM C/TE B group (74.96% vs. 50%, and 57.43% vs. 33.33%, both P<0.05). Multivariable Logistic regression analysis indicated that ICM score was a better predictive parameter for clinical pregnancy (OR=3.05, CI 1.70-5.46, P<0.001), while the trophectoderm score was a better one for early abortion (OR=0.074, CI 0.03-0.19, P<0.001). Clinical pregnancy rate and multiple pregnancy rate in S-ICM B/TE B group were significantly lower than those in D-ICM B/TE B group (46.15% vs. 74.96%, and 2.38% vs. 48.62%, both P<0.05), but there was no significant difference in the implantation rate between the two groups. It was suggested that the higher score of ICM and TE may be indicative of the better pregnancy outcomes. The ICM score is a better predictor of clinical pregnancy than TE, while TE score is a better one in predicting early abortion. Single ICM B/TE B blastocyst transfer in frozen-thawed cycles can also get satisfactory pregnancy outcomes.
Adult ; Analysis of Variance ; Blastocyst ; cytology ; Blastocyst Inner Cell Mass ; cytology ; Cryopreservation ; methods ; Embryo Implantation ; Embryo Transfer ; methods ; statistics & numerical data ; Female ; Fertilization in Vitro ; methods ; statistics & numerical data ; Humans ; Pregnancy ; Pregnancy Outcome ; Pregnancy Rate ; Retrospective Studies

OBJECTIVETo investigate chromosomal euploidies in early-stage arrested human embryos.

METHODSTo determine the euploidy status of the 24 chromosomes, 13 embryos were analyzed, which included 5 arrested at 4-cell stage, 4 arrested at 8-cell stage, and 4 embryos at blastocyst stage regardless of their morphological scores. All embryos were subjected to biopsy, whole genome amplification, and array comparative genome hybridization analysis.

RESULTSChromosome euploidies of the arrested embryos can be normal, aberrant and chaotic. Mosaicism is prevalent in early stage cleavage, whilst most of the blastocysts, even with poor morphology, are normal diploid.

CONCLUSIONArrested embryo may have normal chromosomes euploidy. Mosaicism is common in cleavage stage embryos. Early stage embryo arrest may not be solely attributable to chromosomal aneuploidies and needs further research.

Adult ; Blastocyst ; cytology ; Cell Cycle Checkpoints ; Chromosome Aberrations ; Comparative Genomic Hybridization ; Embryo Loss ; genetics ; Female ; Fertilization in Vitro ; Humans ; Infertility ; genetics ; therapy ; Male ; Middle Aged ; Pregnancy