Characterizing meiotic and mitotic errors in the inner cell mass and trophectoderm of poor quality preimplantation embryos

Most cell types are relatively impermeant to H+ and are able to regulate their intracellular pH by means of plasma membrane proteins, which transport H+ or bicarbonate across the membrane in response to perturbations of intracellular pH. Mouse preimplantation embryos at the 2-cell stage, however, do not appear to possess specific pH-regulatory mechanisms for relieving acidosis. They are, instead, highly permeable to H+, so that the intracellular pH in the acid and neutral range is determined by the electrochemical equilibrium of H+ across the plasma membrane. When intracellular pH is perturbed, the rate of the ensuing H+ flux across the plasma membrane is determined by the H+ electrochemical gradient: its dependence on external K+ concentration indicates probable dependence on membrane potential and the rate depends on the H+ concentration gradient across the membrane. The large permeability at the 2-cell stage is absent or greatly diminished in the trophectoderm of blastocysts, but still present in the inner cell mass. Thus, the permeability to H+ appears to be developmentally regulated.

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Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.639249 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yasumitsu Masuda ◽

Ryo Hasebe ◽

Yasushi Kuromi ◽

Masayoshi Kobayashi ◽

Kanako Urataki ◽

...

Keyword(s):

Embryo Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Three Dimensional ◽

Preimplantation Embryos ◽

Infrared Light ◽

Bovine Embryo ◽

Bovine Embryos ◽

Cell Stage ◽

Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

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Does Blastocyst Morphology Influence Live Birth Rate after Frozen-thawed Single Euploid Embryo Transfer?

10.21203/rs.3.rs-149923/v1 ◽

2021 ◽

Author(s):

Na Li ◽

Yichun Guan ◽

Bingnan Ren ◽

Yuchao Zhang ◽

Yulin Du ◽

...

Keyword(s):

Embryo Transfer ◽

Live Birth ◽

Birth Rate ◽

Inner Cell Mass ◽

Medical Center ◽

Cohort Analysis ◽

Cell Mass ◽

Live Birth Rate ◽

Poor Quality ◽

Blastocyst Quality

Abstract Objective To investigate whether the morphologic parameters of euploid blastocyst influence the live birth rate (LBR) following single frozen-thawed embryo transfer (FET) cycles? Methods A retrospective cohort analysis involving autologous single FET cycles after preimplantation genetic testing for aneuploidy (PGT-A) through next generation sequencing (NGS) by a university-based reproductive medical center that was performed from June 2017 to September 2019.Women were divided into three age groups (< 30, 30–34 and ≥ 35 years). The primary outcome measure was LBR. Outcomes were compared to determine the association between different blastocyst quality (Good, Average and Poor), inner cell mass (ICM) grade (A and B), and trophectoderm (TE) grade (A, B and C) and LBR. Results We included 232 single FET cycles for analysis, the total LBR was 48.48%. In the youngest group (< 30 years, n = 86), LBR were compared between cycles with various blastocyst quality (72.22% for good quality, 54.55% for average quality and 34.78% for poor quality; P = 0.019), ICM grade (70.59% for grade A and 42.03% for grade B; P = 0.035) and TE grade (85.71% for grade A,57.58% for grade B and 34.78 for grade C; P = 0.015). Similarly, in the 30–34 years group, LBR ranged from 50.00% for good quality to 45.45% for poor quality (P = 0.870), from 35.29% for ICM grade A to 51.22% for ICM grade B (P = 0.291), from 60.00% for TE grade A to 45.45% for TE grade C (P = 0.634). Likewise, in the oldest group (≥ 35years, n = 47), LBR were also comparable between these subgroups, but no significant differences were seen in blastocyst morphologic parameters and LBR (P > 0.05). Conclusion After single FET cycles, the LBR was associated with morphologic parameters of euploid blastocysts, especially in women < 30 years old. However, these differences were not found in women older than 30 years. We suggested that for older women whose embryos undergoing PGT-A with NGS to be euploid have the same development potential regardless of their blastocyst morphology.

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Regulation of desmocollin transcription in mouse preimplantation embryos

Development ◽

10.1242/dev.121.3.743 ◽

1995 ◽

Vol 121 (3) ◽

pp. 743-753 ◽

Cited By ~ 9

Author(s):

J.E. Collins ◽

J.E. Lorimer ◽

D.R. Garrod ◽

S.C. Pidsley ◽

R.S. Buxton ◽

...

Keyword(s):

Protein Expression ◽

Molecular Mechanisms ◽

Inner Cell Mass ◽

Splice Variants ◽

Cell Mass ◽

Cumulus Cells ◽

Early Embryo ◽

Preimplantation Embryos ◽

Cell Stage ◽

Cleavage Stages

The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527–539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo.

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Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes

Development ◽

10.1242/dev.113.3.815 ◽

1991 ◽

Vol 113 (3) ◽

pp. 815-824 ◽

Cited By ~ 6

Author(s):

M.B. Rogers ◽

B.A. Hosler ◽

L.J. Gudas

Keyword(s):

Retinoic Acid ◽

Germ Cell ◽

Cell Fate ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Preimplantation Embryos ◽

Specific Expression

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

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P–516 Evidence for self-correction in preimplantation embryos by comparative molecular karyotyping of blastocoele fluid, trophectoderm and inner cell mass

Human Reproduction ◽

10.1093/humrep/deab130.515 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

D Zhigalina ◽

N Skryabin ◽

O Kanbekova ◽

V Artyukhova ◽

A Svetlakov ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Morphological Characteristics ◽

Preimplantation Embryos ◽

Molecular Karyotyping ◽

Embryonic Cells ◽

Developmental Potential ◽

Positive Correlation ◽

Registration Number ◽

Molecular Karyotype

Abstract Study question Does the molecular karyotype of the cell-free DNA (cfDNA) from the blastocyst fluid (BF) can predict the efficiency of self-correction of karyotype of preimplantation embryo? Summary answer Detection of aneuploidies in the BF potentially can point out on effective self-correction of blastocyst karyotype and consequently on high developmental potential of mosaic embryos. What is known already Correction of aneuploidies in the preimplantation embryos can be provided by several mechanisms, including apoptosis. The predominant death of aneuploid cells was demonstrated in mouse embryos (Bolton, 2016). A positive correlation was also shown between the concentration of cfDNA from the BF of human blastocyst and the morphology of the embryo, as well as between the activity of caspase–3 and the concentration of cfDNA (Rule, 2018). The incidence of failed amplification after WGA being significantly higher among euploid blastocysts (Magli, 2019). The capacity of abnormal cells extruding into the BF would be related to the embryo development potential (Gianaroli, 2019). Study design, size, duration This is a prospective observational study of thirty-one Day 5 human blastocysts. Cryopreserved blastocysts were received after treatment cycles at the IVF Center with informed consent obtained from couples. The average age of 15 women was 32.25±5 years. The morphological characteristics of blastocysts were estimated in accordance with the Gardner classification (Gardner, Schoolcraft, 1999). The procedure of BF aspiration and trophectoderm (TE) and ICM cells separation of the blastocysts was previously described (Tsuiko, 2018). Participants/materials, setting, methods WGA was performed by PicoPLEX kit (Rubicon Genomics, USA) or REPLI-g Mini kit (Qiagen) according to manufacturer’s protocols. The DNA of the BF, ICM and TE were analyzed separately using cCGH, aCGH and NGS. SurePrint G3 Human CGH Microarrays (8x60K, Agilent Technologies) were used according to the manufacturer’s recommendations. Image analysis was done using ISIS (v.5.5) (Metasystems) and Agilent CytoGenomics Software (v.3). VeriSeq™ PGS Kit - MiSeq® System (Illumina) was used for NGS. Main results and the role of chance Molecular karyotypes of all three samples - BF, ICM and TE, were obtained for 23 (74.2%) blastocysts. A correlation between the woman’s age and the number of aneuploidies in cfDNA (p = 0.0009) was found. A positive correlation may indicate that the number of aneuploidies in the embryonic cells increases with the age of a woman, however, the embryonic karyotype undergoes self-correcting through the elimination of aneuploid cells. It was noted that well-developing blastocysts (groups 4–5, according to Gardner’s classification) had fewer aneuploidies in ICM (p = 0.0141) and TE (p = 0.0436). In contrast, there was a tendency to an increase in the number of aneuploidies in the BF during blastocysts transition from stage 3 to 5 (p = 0.3542). We assessed the relationship between the number of aneuploidies in groups of blastocysts with different characteristics of ICM (groups “A” and “B” according to Gardner’s classification). These groups significantly differ in the number of aneuploidies in cfDNA (p = 0.0352), although the statistically significant differences between the number of aneuploidies in ICM (p = 0.5992) and in TE (p = 0.5934) was not detected. Thus, higher-quality embryos in terms of ICM morphology contain more abnormalities in the BF, since in this group the elimination of aneuploid cells is more efficient. Limitations, reasons for caution The number of embryos is limited in this study. More comprehensive studies are required to confirm the observed tendency. Wider implications of the findings: Aneuploid cells elimination can be a cause of increasing cfDNA concentration in the BF, which may be a marker of the viability of mosaic embryos when it is necessary to decide on mosaic embryo transfer. This study was supported by the RFBR (15–04–08265) and by the RSF (20–74–00064). Trial registration number Not applicable

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Lipid Stores and Lipid Metabolism Associated Gene Expression in Porcine and Bovine Parthenogenetic Embryos Revealed by Fluorescent Staining and RNA-seq

International Journal of Molecular Sciences ◽

10.3390/ijms21186488 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6488

Author(s):

Arkadiusz Kajdasz ◽

Ewelina Warzych ◽

Natalia Derebecka ◽

Zofia E. Madeja ◽

Dorota Lechniak ◽

...

Keyword(s):

Gene Expression ◽

Lipid Metabolism ◽

Inner Cell Mass ◽

Mammalian Species ◽

Cell Mass ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Rna Seq ◽

Expression Of Genes ◽

Bovine Blastocyst

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

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Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

Molecular and Cellular Biology ◽

10.1128/mcb.01188-06 ◽

2007 ◽

Vol 27 (8) ◽

pp. 3123-3130 ◽

Cited By ~ 26

Author(s):

Klaus Fortschegger ◽

Bettina Wagner ◽

Regina Voglauer ◽

Hermann Katinger ◽

Maria Sibilia ◽

...

Keyword(s):

Matrix Protein ◽

Replicative Senescence ◽

Inner Cell Mass ◽

Umbilical Vein ◽

Cell Mass ◽

Preimplantation Embryos ◽

Proliferative Potential ◽

Mouse Development

ABSTRACT SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

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Does the sex ratio of singleton births after frozen single blastocyst transfer differ according to blastocyst morphology?

10.21203/rs.3.rs-17132/v1 ◽

2020 ◽

Author(s):

Hua Lou ◽

Na Li ◽

Xiaoke Zhang ◽

Ling Sun ◽

Xingling Wang ◽

...

Keyword(s):

Sex Ratio ◽

Inner Cell Mass ◽

Sex Ratios ◽

Cell Mass ◽

Male Infant ◽

Poor Quality ◽

Blastocyst Transfer ◽

High Quality ◽

Chi Square ◽

Live Births

Abstract Purpose: To investigate the associations between morphological blastocyst parameters and the sex ratio (male:female) of singleton live births resulting from single-blastocyst frozen embryo transfer (FET) cycles. Methods: This retrospective analysis included 1210 couples who underwent single-blastocyst FET of warmed day 5 or 6 blastocysts and achieved a singleton live birth between January 2015 and February 2019. The sex ratios in the different blastocyst groups were compared using the chi-square test. The associations of morphological blastocyst parameters, including the blastocoel expansion, inner cell mass and trophectoderm grades, with the sex ratios of live births were analyzed using multiple regression models. Results: The included female patients had an average age and body mass index of 30.5±4.5 years and 23.6±3.2 kg/m2, respectively. blastocyst transfers occurred on day 5 in 783 cases (64.7%) and day 6 in 427 cases (35.3%). Among the day 5 FET cycles, 55.4% of resulting infants were male and 44.6% were female, while among the day 6 cycles, 54.6% of infants were male and 45.4% were female ( P =0.074). Blastocysts quality was assessed according to morphological parameters, which was divided into high-quality (AA,AB,BB,BC) and poor-quality(AC,BC). High-quality blastocyst transfer was associated with a significantly higher sex ratio (60%, 383/638) relative to poor-quality transfer (49.7%, 284/572) ( P< 0.001). The sex ratio of births resulting from blastocyst transfer differed significantly with respect to the trophectoderm grade ( P <0.001). After adjusting for confounders, a grade B trophectoderm was significantly associated with a higher sex ratio among singleton live births (odds ratio: 0.591, 95% confidence interval: 0.463–0.756, P <0.001; reference: grade C trophectoderm). In contrast, the characteristics of blastocoel expansion and the score of the inner cell mass were not significantly associated with the sex ratio among singleton live births. Conclusions: The sex ratio among singleton live births is affected by the quality of blastocysts. A grade B trophectoderm is associated with a higher probability that a single-blastocyst FET cycle will result in a male infant.

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Does Blastocyst Morphology Influence Live Birth Rate after Frozen-thawed Single Euploid Blastocyst Transfer?

10.21203/rs.3.rs-131940/v1 ◽

2020 ◽

Author(s):

Na Li ◽

Yichun Guan ◽

Bingnan Ren ◽

Yuchao Zhang ◽

Yulin Du ◽

...

Keyword(s):

Live Birth ◽

Birth Rate ◽

Inner Cell Mass ◽

Statistical Significance ◽

Cohort Analysis ◽

Cell Mass ◽

Live Birth Rate ◽

Poor Quality ◽

Significant Difference ◽

Blastocyst Quality

Abstract Objective To determine whether the morphologic parameters of euploid blastocyst influence the live birth rate (LBR) following single frozen-thawed embryo transfer (FET) cycles? Methods A retrospective cohort analysis involving autologous single FET cycles after next generation sequencing (NGS) based preimplantation genetic testing for aneuploidy (PGT-A) by a large in vitro fertilization (IVF) center that was performed from June 2017 to September 2019.Women were divided into three age groups (< 30, 30–34 and ≥ 35 years old). The primary outcome measure was LBR. Outcomes were compared between different blastocyst quality (Good, Average and Poor), inner cell mass (ICM) grade (A and B), and trophectoderm (TE) grade (A, B and C). Results A total of 232 FET cycles were included, the live birth rate was 48.28%. In the youngest group (< 30 years old, n = 86), LBR were compared between cycles with various blastocyst quality (72.22% for good quality, 54.55% for average quality and 34.78% for poor quality; P = 0.019), ICM grade (70.59% for grade A and 42.03% for grade B; P = 0.035) and TE grade (85.71% for grade A,57.58% for grade B and 34.78 for grade C; P = 0.015). Nevertheless, either in the 30–34 years group (n = 99) or in the oldest group (≥ 35years, n = 47), LBR were also comparable between these subgroups, no significant difference was showed in blastocyst morphologic parameters and LBR (P > 0.05). Furthermore, in the similarly graded euploid blastocysts, there was also no statistical significance in LBR among different age subgroups (P > 0.05). Conclusions In women ≥ 30 years old, euploid blastocyst quality was not associated with the LBR in FET cycles, highlights the development competence of poor-quality euploid blastocysts.

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