A workflow for simultaneous DNA copy number and methylome analysis of inner cell mass and trophectoderm cells from human blastocysts.

OBJECTIVE: To establish a workflow for isolating single trophectoderm (TE) and inner cell mass (ICM) cells and to simultaneously evaluate these cells for copy number variation (CNV) as well as methylome development. DESIGN: Experimental. SETTING: Academic medical center. PATIENT(S): Donated genetically abnormal blastocysts. INTERVENTION(S): Single cells were isolated, followed by bisulfite conversion and sequencing to identify CNV and methylome profiles. MAIN OUTCOME MEASURE(S): CNV and methylation profiling. RESULT(S): Two embryos were dissociated, isolating 46 single cells, with 17 ICM and 12 TE cells selected for further downstream analysis. Chromosome ploidies and embryo sex were concordant with the results from conventional aneuploidy testing. In 3 of the 29 cells, additional aneuploidies were discovered, indicating possible mosaicism undetected by routine preimplantation genetic testing for aneuploidy. CpG methylation frequency was higher in ICM cells compared with TE cells (44.3% vs. 32.4%), respectively, while non-CpG methylation frequency was similar among both cell types. CpG methylation levels accurately distinguished ICM from TE cells epigenetically. CONCLUSION(S): We describe an effective workflow for isolating and sequencing single ICM and TE cells from human blastocysts. The use of methylation profiling can help distinguish these two cell populations better then morphologic identification alone. TE cells had significantly lower levels of DNA methylation, which may be explained in part by the fact that these cells have begun the process of differentiation and are transcriptionally more active than ICM. This approach may be used to explore the genetic complexities within human embryos, specifically among the two primary cell types seen at this stage of development.

Association between the quality of inner cell mass and first trimester miscarriage after single blastocyst transfer.

BACKGROUND: The blastocyst morphology provided valuable roles for predicting pregnancy and live birth, but was still not fully understood for evaluating miscarriage. The aim of this study was to explore the association between blastocyst morphologic evaluation and first trimester miscarriage combined with karyotype of miscarried conceptus. METHODS: This retrospective cohort study included a total of 2873 clinical pregnancy cycles with single blastocyst transfer performed from January 2013 to April 2019. Chromosome karyotype of miscarried conceptus was analyzed via single nucleotide polymorphism array analysis. Miscarriage and karyotype of miscarried conceptus associated with blastocyst morphology were analyzed by chi-square and logistic regression analysis. RESULTS: A total of 354 (12.3%) cycles resulted in first trimester miscarriage. Miscarriage rates increased with trophectoderm (TE) grade from A to C (P = 0.012), while three morphologic parameters (blastocoele expansion degree, inner cell mass (ICM) and TE) showed no statistical significance with miscarriage after multivariable analysis. The rate of aneuploidy was 47.7% (83 of 174) in total miscarried conceptuses. For euploid miscarriages, the grade B of ICM occupied a higher proportion compared with aneuploidy, with OR of 2.474, (95% CI, 1.311-4.699), P = 0.005. CONCLUSIONS: Chromosomal aberration of embryo is an important genetic factor for first trimester miscarriage, and the quality of ICM is a potential indicator for euploid miscarriage. Blastocysts with grade A of ICM should be given priority during single blastocyst transfer to reduce potential miscarriage.

The reproducibility of trophectoderm biopsies in euploid, aneuploid, and mosaic embryos using independently verified next-generation sequencing (NGS): a pilot study.

PURPOSE: To assess the accuracy and reliability of comprehensive chromosome screening by next-generation sequencing (NGS) of human trophectoderm (TE) biopsy specimens. METHODS: The reliability and accuracy of diagnoses made by preimplantation genetic testing for aneuploidy (PGT-A) from TE biopsy were tested. Repeat biopsies of TE and inner cell mass (ICM) samples were obtained from thawed blastocysts previously tested by NGS. To test for the reliability of the NGS assay, biopsy samples were compared with the original PGT-A results. Prior NGS testing classified the TE samples as euploid, aneuploid, or aneuploid-mosaic. The resulting re-biopsied samples underwent SurePlex whole genome amplification followed by NGS via the MiSeq platform, with copy number value (CNV) determined using BlueFuse Multi Software. The primary outcome measure was reliability, defined as concordance between initial TE result and the repeat biopsies. Accuracy was determined by concordance between the TE and ICM samples, and compared between three chromosome types (disomic, aneuploid, and mosaic). RESULTS: Re-biopsies were performed on 32 embryos with prior PGT-A showing euploidy (10 embryos), aneuploidy of one or two chromosomes (4 embryos), or aneuploid-mosaic with one aneuploid chromosome and one mosaic chromosome (18 embryos). One hundred twenty-nine biopsy samples completed NGS (90 TE and 39 ICM biopsies) and 105 biopsy results were included in the analysis. TE biopsies provide a highly accurate test of the future fetus, with the ICM disomic concordance rate of 97.6%. Clinical concordance rates indicate that TE biopsies provide a reliable test when the result is euploid (99.5%) or aneuploid (97.3%), but less reliable when the result is mosaic (35.2%). CONCLUSION: TE biopsies predict euploidy or aneuploidy in the ICM with a high degree of accuracy. PGT-A with NGS of TE biopsies is shown to be highly reliable, with clinically relevant concordance rates for aneuploidy and euploidy over 95%. TE biopsies indicating mosaicism were less reliable (35.2%), presumably because mitotic non-disjunction events are not uniformly distributed throughout the blastocyst. However, classification of TE biopsy of PGT-A with NGS results as either aneuploid or euploid provides a highly reliable test.

Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy.

Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.

Evaluation of in vitro embryotoxicity tests for Chinese herbal medicines.

Chinese herbal medicines (CHMs) have been widely used during pregnancy, but feto-embryo safety tests are lacking. Here we evaluated in vitro embryotoxicity tests (IVTs) as alternative methods in assessing developmental toxicity of CHMs. Ten CHMs were selected and classified as strongly, weakly and non-embryotoxic. Three well validated IVTs and prediction models (PMs), including embryonic stem cell test (EST), micromass (MM) and whole embryo culture (WEC), were compared. All strongly embryotoxic CHMs were predicted by MM and WEC PM2. While all weakly embryotoxic CHMs were predicted by MM and WEC PM1. All non-embryotoxic CHMs were classified by EST, MM, but over-classified as weakly embryotoxic by WEC PM1. Overall predictivity, precision and accuracy of WEC determined by PM2 were better than EST and MM tests. Compared with validated chemicals, performance of IVTs for CHMs was comparable. So IVTs are adequate to identify and exclude embryotoxic potential of CHMs in this training set.

The clinicians´ dilemma with mosaicism-an insight from inner cell mass biopsies.

STUDY QUESTION: How reliable are cleavage stage and trophectoderm (TE) biopsies compared to inner cell mass (ICM) biopsies? SUMMARY ANSWER: The reliability of TE biopsy compared to ICM biopsy is almost perfect, but only substantial between cleavage stage biopsy and ICM biopsy. WHAT IS KNOWN ALREADY: One of the prevailing reasons for implantation failure is presumed to be chromosomal aneuploidy in human preimplantation embryos. Preimplantation genetic testing for aneuploidies (PGT-A) has been introduced into assisted reproduction in an effort to increase pregnancy rates. Increasing evidence indicates that genetic results obtained following blastomere or TEbiopsy may not accurately reflect the true genetic status of the embryo due to the presence of embryonic mosaicism, and therefore the reliability of PGT is highly controversial. STUDY DESIGN, SIZE, DURATION: This was an observational descriptive study, performed in a private infertility centre from August 2016 to January 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: The mean female age was 33.9 years, ranging from 24 to 46 years, and the mean number of biopsied embryos per couple was 2.2 (range 1-7 embryos). Blastomere biopsies had been performed at cleavage stage on Day 3 (D3) due to the turnover time of genetic testing and the inability to cryopreserve embryos in accordance with the local law governing ART. To confirm the genetic results in embryos not chosen for transfer, additional biopsies of the TE at blastocyst stage (BLASTO-TE) as well as of the ICM (BLASTO-ICM) were performed on D5. Only surplus blastocysts, which had not been selected for transfer and were not cryopreserved in accordance with the law governing ART, had been included. MAIN RESULTS AND THE ROLE OF CHANCE: Comparison of all biopsies (D3/BLASTO-ICM/BLASTO-TE) per embryo demonstrated that 50 (59.5%) out of 84 embryos showed concordance in all three results (= full concordance). Thirty-four (40.4%) embryos had at least two discordant results between the three biopsies, regardless of whether the embryo diagnosis (aneuploid/euploid) was discordant or not, or in aneuploid embryos, whether the chromosomal patterns were inconsistent. Nine (= 10.7%) embryos had complete discordance between all three biopsies. False positive results between D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM were 26.4%/30.2% and 7.5%, respectively, while the Kappa agreement between the different approaches was 0.647, 0.553 and 0.857, respectively. Therefore the reliability of D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM can be interpreted as substantial, as moderate and as almost perfect. LIMITATIONS, REASONS FOR CAUTION: The limitation of this study is the possible bias in the concordance/discordance rate because embryos that had been selected for transfer did not undergo biopsy on D5. WIDER IMPLICATIONS OF THE FINDINGS: The obvious discordance between the three different approaches for PGT-A underlines the limitations of genetic testing and highlights the importance of ongoing research in order to improve the accuracy of PGT-A results. Until then reproductive specialists will continue to make challenging decisions on whether to transfer or discard an embryo in light of current evidence questioning the reliability of genetic results. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Igenomix. The funder provided support in the form of salary for R.C. The co-author R.C. is an employee of Igenomix. She participated in the blinded analysis of the samples; however the final data collection and statistical analysis of the results, as well as the decision to publish, was taken by B.L, I.E. and H.F. The authors B.L., I.E., A.L., A.B., A.A., N.D. and H.F. have no competing interests. The funder did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The commercial affiliation of R.C. did not play any role in the study. TRIAL REGISTRATION NUMBER: This study was approved by the Ethics Committee of IVIRMA Middle East Fertility Clinic, Abu Dhabi, UAE (Research Ethics Committee IVI-MEREFA009a/2017).

Assessment of aneuploidy concordance between clinical trophectoderm biopsy and blastocyst.

STUDY QUESTION: Is a clinical trophectoderm (TE) biopsy a suitable predictor of chromosomal aneuploidy in blastocysts? SUMMARY ANSWER: In the analyzed group of blastocysts, a clinical TE biopsy was an excellent representative of blastocyst karyotype in cases of whole chromosome aneuploidy, but in cases of only segmental (sub-chromosomal) aneuploidy, a TE biopsy was a poor representative of blastocyst karyotype. WHAT IS KNOWN ALREADY: Due to the phenomenon of chromosomal mosaicism, concern has been expressed about the possibility of discarding blastocysts classified as aneuploid by preimplantation genetic testing for aneuploidy (PGT-A) that in fact contain a euploid inner cell mass (ICM). Previously published studies investigating karyotype concordance between TE and ICM have examined small sample sizes and/or have utilized chromosomal analysis technologies superseded by Next Generation Sequencing (NGS). It is also known that blastocysts classified as mosaic by PGT-A can result in healthy births. TE re-biopsy of embryos classified as aneuploid can potentially uncover new instances of mosaicism, but the frequency of such blastocysts is currently unknown. STUDY DESIGN, SIZE, DURATION: For this study, 45 patients donated 100 blastocysts classified as uniform aneuploids (non-mosaic) using PGT-A by NGS (n = 93 whole chromosome aneuploids, n = 7 segmental aneuploids). In addition to the original clinical TE biopsy used for PGT-A, each blastocyst was subjected to an ICM biopsy as well as a second TE biopsy. All biopsies were processed for chromosomal analysis by NGS, and karyotypes were compared to the original TE biopsy. PARTICIPANTS/MATERIALS, SETTING, METHODS: The setting for this study was a single IVF center with an in-house PGT-A program and associated research laboratory. MAIN RESULTS AND THE ROLE OF CHANCE: When one or more whole chromosomes were aneuploid in the clinical TE biopsy, the corresponding ICM was aneuploid in 90 out of 93 blastocysts (96.8%). When the clinical TE biopsy contained only segmental (sub-chromosomal) aneuploidies, the ICM was aneuploid in three out of seven cases (42.9%). Blastocysts showing aneuploidy concordance between clinical TE biopsy and ICM were also aneuploid in a second TE biopsy in 86 out of 88 cases (97.7%). In blastocysts displaying clinical TE-ICM discordance, a second TE biopsy was aneuploid in only two out of six cases (33.3%). LIMITATIONS, REASONS FOR CAUTION: All embryos in this study had an initial classification of 'aneuploid' and not 'euploid' or 'mosaic'. Therefore, the findings of this study refer specifically to a TE biopsy predicting aneuploidy in the remaining blastocyst, and cannot be extrapolated to deduce the ability of a TE biopsy to predict euploidy in the blastocyst. No conclusions should be drawn from this study about the ability of a mosaic TE biopsy to predict the karyotype of the corresponding blastocyst. Caution should be exercised in generalizing the findings of the sample group of this study to the general IVF blastocyst population. The segmental aneuploidy group only contained seven samples. WIDER IMPLICATIONS OF THE FINDINGS: The high rate of intra-blastocyst concordance observed in this study concerning whole chromosome aneuploidy contributes experimental evidence to the validation of PGT-A at the blastocyst stage. Concomitantly, the results suggest potential clinical value in reassessing blastocysts deemed aneuploid by TE re-biopsy in select cases, particularly in instances of segmental aneuploidies. This could impact infertility treatment for patients who only have blastocysts classified as aneuploid by PGT-A available. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Zouves Foundation for Reproductive Medicine and Zouves Fertility Center. The authors have no competing interest to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass.

OBJECTIVE: To compare the genomic profiles of blastocoel fluid (BF), inner cell mass (ICM), and trophectoderm (TE) cells derived from the same blastocyst. DESIGN: Prospective study. SETTING: Academic and in vitro fertilization units. PATIENT(S): Sixteen donated cryopreserved embryos at blastocyst stage. INTERVENTION(S): BF, TE, and ICM cells were retrieved from each blastocyst for chromosome analysis by means of next-generation sequencing (NGS). MAIN OUTCOME MEASURE(S): Aneuploidy screening and assessment of mosaicism in BF, TE and ICM samples with subsequent comparison of genomic profiles between the three blastocyst compartments. RESULT(S): Out of 16 blastocysts, 10 BF samples and 14 TE and ICM samples provided reliable NGS data for comprehensive chromosome analysis. Only 40.0% of BF-DNA karyotypes were fully concordant with TE or ICM, compared with 85.7% concordance between TE and ICM. In addition, BF-DNA was burdened with mosaic aneuploidies and the total number of affected chromosomes in BF was significantly higher compared with the TE and ICM. CONCLUSION(S): BF-DNA can be successfully amplified and subjected to NGS, but owing to increased discordance with ICM and TE, BF does not adequately represent the status of the rest of the embryo. To overcome biologic and technical challenges associated with BF sampling and processing, blastocentesis would require improvement in both laboratory protocols and aneuploidy calling algorithms. Therefore, TE biopsy remains the most effective way to predict embryonic karyotype, and the use of BF as a single source of DNA for preimplantation genetic screening is not yet advised.

Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates.

OBJECTIVE: To determine whether blastocyst grading can predict pregnancy outcomes in the frozen-thawed embryo transfer (FET) of euploid blastocysts. DESIGN: Retrospective cohort study. SETTING: Academic medical center. PATIENT(S): Women who underwent FET of euploid embryo(s) between January 2013 and December 2015, with blastocysts were divided into four groups based on their morphologic grading before cryopreservation: excellent (≥3AA), good (3-6AB, 3-6BA, 1-2AA), average (3-6BB, 3-6AC, 3-6CA, 1-2AB, 1-2BA), and poor (1-6BC, 1-6CB, 1-6CC, 1-2BB). INTERVENTION(S): FET. MAIN OUTCOMES MEASURE(S): Ongoing pregnancy rate (OPR). RESULT(S): A total of 417 FET cycles (477 embryos) were included. Excellent-quality embryos (n = 38) yielded a statistically significantly higher OPR than poor-quality embryos (n = 106) (84.2% vs. 35.8%; adjusted odds ratio 11.0; 95% confidence interval, 3.8-32.1) and average-quality embryos (n = 197) (84.2% vs. 55.8%; adjusted odds ratio 4.8; 95% confidence interval, 1.7-13.3). Good-quality embryos (n = 76) were associated with a statistically significantly higher OPR than poor-quality embryos (61.8% vs. 35.8%). These odds ratios were adjusted for patient's age, body mass index, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of trophectoderm biopsy (5 or 6), and total number of euploid embryos for each patient. An inner cell mass grade of A yielded a statistically significantly higher OPR than ICM grade C (76.2% vs. 13.5%) or grade B (76.2% vs. 53.6%) after controlling for all confounders. CONCLUSION(S): Contrary to prior published studies, the current data suggest that blastocyst morphologic grading and particularly inner cell mass grade is a useful predictor of OPR per euploid embryo. Morphologic grading should be used to help in the selection among euploid blastocysts.

Grade and looseness of the inner cell mass may lead to the development of monochorionic diamniotic twins.

OBJECTIVE: To examine the relationship between the inner cell mass (ICM) grade and its morphological configuration on the occurrence of monochorionic diamniotic (M-D) twinning. DESIGN: Retrospective embryo cohort study. SETTING: Private IVF clinic. PATIENT(S): Evaluation of frozen-thawed single blastocyst transfers with hormone replacement treatment in 8,435. This cohort included 71 blastocysts and their ICMs observed by time-lapse photography. INTERVENTION(S): Any changes in configuration of the ICMs observed by time-lapse photography were analyzed retrospectively. MAIN OUTCOME MEASURE(S): The amount of loosening of blastomeres within the ICM was evaluated by time-lapse observations. The number of cells that were involved in the loosening process was also assessed. Both of these parameters were correlated with the type of monozygotic twinning that eventuated. RESULT(S): The M-D twinning incidence resulting from blastocysts with a high grade ICM (grade A) were transferred was 0.38% (3/796), whereas it was significantly higher, 1.38% (34/2,463), when blastocysts with a poorer (B and C) grade ICM were transferred. Among 71 transferred frozen-thawed blastocysts that were studied with time-lapse photography, there were two dichorionic diamniotic and one M-D twins. Careful observations of the embryo that resulted in the one M-D case, revealed that the ICM acquired a looser appearance due to decompaction of at least eight cells. This type of decompaction was not observed in the ICMs of other transferred blastocysts. CONCLUSION(S): The occurrence of M-D twinning may be avoided by excluding blastocysts that contain decompacting ICMs.

H3K4 Methyltransferase Set1a Is A Key Oct4 Coactivator Essential for Generation of Oct4 Positive Inner Cell Mass.

Limited core transcription factors and transcriptional cofactors have been shown to govern embryonic stem cell (ESC) transcriptional circuitry and pluripotency, but the molecular interactions between the core transcription factors and cofactors remains ill defined. Here, we analyzed the protein-protein interactions between Oct4, Sox2, Klf4, and Myc (abbreviated as OSKM) and a large panel of cofactors. The data reveal both specific and common interactions between OSKM and cofactors. We found that among the SET1/MLL family H3K4 methyltransferases, Set1a specifically interacts with Oct4 and this interaction is independent of Wdr5. Set1a is recruited to and required for H3K4 methylation at the Oct4 target gene promoters and transcriptional activation of Oct4 target genes in ESCs, and consistently Set1a is required for ESC maintenance and induced pluripotent stem cell generation. Gene expression profiling and chromatin immunoprecipitation-seq analyses demonstrate the broad involvement of Set1a in Oct4 transcription circuitry and strong enrichment at TSS sites. Gene knockout study demonstrates that Set1a is not only required for mouse early embryonic development but also for the generation of Oct4-positive inner cell mass. Together our study provides valuable information on the molecular interactions between OSKM and cofactors and molecular mechanisms for the functional importance of Set1a in ESCs and early development.

Stress exposure during the preimplantation period affects blastocyst lineages and offspring development.

We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life.

DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic.

Trophectoderm (TE) biopsy and DNA microarray have become the new technologies for preimplantation genetic diagnosis in humans. In this study, we comprehensively examined aneuploid formation in human blastocysts produced in vitro with microarray and investigated the clinical outcome after transfer of euploid embryos. Biopsied cells from either TE or inner cell mass (ICM) were processed for microarray to examine the errors in 23 pairs of chromosomes and the consistency between TE and ICM. It was found that 56.6% of blastocysts were aneuploid. Further analysis indicated that 62.3% of aneuploid blastocysts had single and 37.7% had multiple chromosomal abnormalities. Chromosome errors could occur in any chromosome, but errors in chromosome 21 accounted for the most (11.3%) among the 23 pairs of chromosomes. Transfer of array-screened blastocysts produced high pregnancy (70.2%) and implantation (63.5%) rates. Microarray of TE and ICM cells in the same blastocysts revealed that high proportions of aneuploid blastocysts (69.2%) were mosaic, including aneuploid TE and euploid ICM, inconsistent anomalies between ICM and TE, or euploid TE cells and aneuploid ICM in the same blastocyst. These results indicate that high proportions of human blastocysts produced in vitro from women of advanced maternal age are aneuploid and mosaic. Errors can occur in any of the 23 pairs of chromosomes in human blastocysts. Biopsy from TE in blastocysts does not exactly predict the chromosomal information in ICM if the embryos are aneuploid. Some mosaic blastocysts have euploid ICM, which may indicate important differentiate mechanism(s) of human preimplantation embryos.

Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

BACKGROUND: A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. CONCLUSIONS: Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

Embryonic toxicity of sanguinarine through apoptotic processes in mouse blastocysts.

In this study, we examined the cytotoxic effects of sanguinarine, a phytoalexin with antimicrobial, anti-oxidant, anti-inflammatory and pro-apoptotic effects, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro and in vivo implantation via embryo transfer. Blastocysts treated with 0.5-2 µM sanguinarine exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with sanguinarine were lower than that of their control counterparts. Moreover, in vitro treatment with 0.5-2 µM sanguinarine was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that sanguinarine induces apoptosis and retards early post-implantation development in vitro and in vivo. In addition, sanguinarine induces apoptotic injury effects on mouse blastocysts through intrinsic and extrinsic apoptotic signaling processes to impair sequent embryonic development. However, the extent to which sanguinarine exerts teratogenic effects on early human development is not known at present, and further studies are required to establish effective protection strategies against its cytotoxic effects.

Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor.

The hypothesis that high concentrations of IGF1 can impair embryo development was investigated in a bovine in vitro model to reflect conditions in polycystic ovary syndrome (PCOS) patients. Embryos were either cultured in the absence or presence of a physiological (100  ng/ml) or supraphysiological (1000  ng/ml) IGF1 concentration. Cell allocation, apoptosis, transcript and protein expression of selected genes involved in apoptosis, glucose metabolism and the IGF system were analysed. Supraphysiological IGF1 concentration did not improve blastocyst formation over controls, but induced higher levels of apoptosis, decreased TP53 protein expression in the trophectoderm and increased the number of cells in the inner cell mass (ICM). The increase in ICM cells corresponded with an increase in IGF1 receptor (IGF1R) protein in the ICM. A small, but significant, percentage of blastocysts displayed a hypertrophic ICM, not observed in controls and virtually absent in embryos treated with physiological concentrations of IGF1. Physiological IGF1 concentrations increased total IGF1R protein expression and upregulated IGFBP3 transcripts leading to an increase in blastocyst formation with no effects on cell number or apoptosis. In conclusion, the results support the hypothesis of detrimental effects of supraphysiological IGF1 concentrations on early pregnancy. However, our results do not support the premise that increased apoptosis associated with high levels of IGF1 is mediated via downregulation of the IGF1R as previously found in preimplantation mouse embryos. This in vitro system with the bovine preimplantation embryo reflects critical features of fertility in PCOS patients and could thus serve as a useful model for in-depth mechanistic studies.

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.