Amount Of Embryos Research Articles

Ovarian superstimulation with FSH in Wagyu breed bovine donors impacts folicular dynamics but does not improve the amount of embryos

The increase of the world population generates the need to raise the production of food of vegetal and animal origin. In Brazil, livestock farming has evolved, demonstrating the important role of the country in the production of food. In this way, researchers of this productive chain have been looking for technologies related to production and reproduction, above all, to the use of reproduction biotechnologies, seeking to increase the production of different bovine breeds. Thus, the objective of this research was to investigate the efficacy of ovarian FSH super stimulation in bovine Wagyu oocyte donor females on follicular dynamics and in vitro embryo production. Twelve Wagyu animals, aged 12 to 24 months, randomly distributed in a crossover design were used in two groups: Group 1= animals not stimulated with FSH and Group 2= animals stimulated with FSH. The follicular, oocyte and embryonic variables were evaluated. It was observed that ovarian overstimulation in Wagyu oocyte donor cows with FSH improved the mean and large follicles but reduced the rate of oocyte recovery and, despite the best percentage of viable oocytes, there was no improvement in the amount of embryos produced in vitro.

19. PGT-A: WHEN IT IS BETTER NOT TO KNOW

Introduction PGT-A was implemented to select euploid embryos and thus, to improve IVF-outcome in terms of higher live birth rate (LBR), lower miscarriage rate or shorter time-to-pregnancy. With the implementation of refined techniques such as Next Generation Sequencing (NGS) the resolution to detect (small) chromosomal aberrations in embryos increased. This holds true for as chromosomal mosaicism as well as for segmental aberrations, and consequently the amount of embryos tested as euploid decreases. This observation highlights the complex nature of genetic (in)stability during early embryogenesis and reveals the substantial shortcomings of PGT-A. The early genetic plasticity counteracts with the primary goal to improve LBR. The aim of the study was to determine when PGT-A can be recommended considering the drop-out rate starting from non-biopsable embryos, amplification failure and drop-out due to testing as non-euploid. Patients, material & methods This retrospective single-center study (2016-2018) includes 173 hormonal stimulation cycles (71 patients). PGT-A was performed either due to three unsuccessful embryo transfers or due to three miscarriages in the patient's history. Patients with tested structural chromosomal aberrations as well as donor cycles were excluded. Blastocyst culture was performed in single step culture medium in the Embryoscope. TE-biopsy was performed on day 5-7. WGA was performed either by PicoPlex (Rubicon Genomics) or by SurPlex (BlueGenome/ Illumina). NGS was processed using VeriSeq platform with data analysis by BlueFuse Multi (Illumina). Results A total of 1,751 MII oocytes resulted in 752 blastocysts. TE-biopsy of at least one blastocyst was performed in 157 cycles (68 patients). This resulted in 609 biopsied blastocysts of which 57 showed either failure of amplification or defective sequencing. 156 (25.6%) samples showed either whole chromosome aneuploidies or uniform segmental chromosomal aberrations. Further, 117 (19.2%) biopsies revealed either chromosomal or segmental mosaicism and, 140 (23.0%) were tested with a combination of uniform and mosaic aberrations. Finally, 139 (22.8%) samples were tested as chormosomally normal. This results in mean 0.8 blastocysts/cycle tested as known euploid compared to originally 4.5 blastocysts/cycle. Conclusions High embryo losses due to non-biopsable blastocysts, amplification failure and non-interpretable results are well known but often neglected. Additionally, while NGS allows high resolution for small structural or numerical chromosomal aberrations as mosaic, the number of transferable blastocysts decreases enormous too. Chromosomal mosaicism (euploid/aneuploid) is a major problem of PGT-A, additional problems arises from segmental chromosomal aberrations that can be present as mosaic as well. When PGT-A is recommended IVF-patients, physicians have to inform about high costs for patients, the increased number of IVF-cycles needed and the high cancellation rates. When estimating a 60% baby-take home rate after PGT-A the same number of babies would be born starting with 752 untested blastocysts considering an only 11% baby-take home rate (83.4 vs. 82.7 babies). This comparison shows that counselling for PGT-A due to RIF or RM has to be well prepared.

Effect of the Administration of One FSH Single Dose in Superovulation Treatment in Corriedale Booroola Ewe’s Embryos Production

Embryos production allows increasing the genetic progress as well as conserving and exchanging genetic material. Nowadays production protocols based in the use of six to eight FSH crescent doses are used. This fact determines a frequent manipulation of the animals, which not only increases the costs, but also it is more difficult to carry it out in commercial farms located away from the transference centres. The objective of this research was to reduce the FSH application to one single dose. Forty-seven female, heterozygotes (FecB Fec+) Corriedale Booroola, were tested. Seven treatments were evaluated by combining different FSH doses and administration scheduling. Estro synchronization was similar in all the treatments: fourteen days of medroxiprogesterone intravaginal pessaries and the administration of 200 IU of eCG once the pessaries were retired and the administration of GnRH thirty five hours later. The treatments were T1 (Traditional Protocol), T2 and T3 were based in the administration of FSH doses of 156, 124 y 46 mg (eight decreasing doses). The treatments based in a single FSH dose (124 mg) varied in the moment of its application: 36 h, 24 h, 12 h before, and simultaneously with the pessaries extraction (0 h). The described treatments are T4, T5, T6 and T7. The obtained ovulatory rate was 10.9; 12.5; 5.0; 0; 8.9; 6.3 and 8.3 for the seven treatments respectively. The smaller rate corresponded to T3 (P<0.05) and null (without ovulation) in T4. The amount of free able embryos by treatment was 4.0; 5.0; 3.3; 0; 2.9; 4.5 and 1.7. The results shown that lower doses than those used in the Traditional Treatment gave the same amount of embryos (T1 vs T2). Likewise, a single FSH dose applied in Corriedale Booroola ewes (T6) allows producing a similar amount of good quality embryos.

Computational and functional analysis of biopharmaceutical drugs in zebrafish: Erythropoietin as a test model

The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly due to the zebrafish advantages when compared to other animal models.Erythropoietin is a glycoprotein hormone that acts principally on erythroid progenitors, stimulating their survival, proliferation and differentiation. Recombinant human erythropoietin (rhEPO) has been widely used in medicine to treat anemia and it is one of the best-selling biotherapeutics worldwide. The recombinant molecule, industrially produced in CHO cells, has the same amino acid sequence of endogenous human erythropoietin, but differs in the glycosylation pattern. This may influence efficacy and safety, particularly immunogenicity, of the final product.We employed the zebrafish embryo as a vertebrate animal model to perform in vivo pharmacological assays. We conducted a functional analysis of rhEPO alpha Eprex® and two biosimilars, the erythropoietin alpha Binocrit® and zeta Retacrit®.By in silico analysis and 3D modeling we proved the interaction between recombinant human erythropoietin and zebrafish endogenous erythropoietin receptor. Then we treated zebrafish embryos with the 3 rhEPOs and we investigated their effect on erythrocytes production with different assays. By real time-PCR we observed the relative upregulation of gata1 (2.4±0.3 fold), embryonic α-Hb (1.9±0.2 fold) and β-Hb (1.6±0.1 fold) transcripts. A significant increase in Stat5 phosphorylation was also assessed in embryos treated with rhEPOs when compared with the negative controls. Live imaging in tg (kdrl:EGFP; gata1:ds-red) embryos, o-dianisidine positive area quantification and cyanomethemoglobin content quantification revealed a 1.8±0.3 fold increase of erythrocytes amount in embryos treated with rhEPOs when compared with the negative controls. Finally, we verified that recombinant human erythropoietins did not cause any inflammatory response in the treated embryos.Our data showed that zebrafish embryo can be a valuable tool to study in vivo effects of complex pharmacological compounds, such as recombinant human glycoproteins, allowing to perform fast and reproducible pharmacological assays with excellent results.

Aneuploidy rate in embryos with accelerated cleavage (day 3) in a PGS program

This work aims to establish a difference in aneuploidy rate in blastocysts generated from embryos with accelerated cleavage at day 3 compared to normally developed embryos Comparative cohort study Fifty-seven PGS cycles (50 patients) that were performed at CEGYR from June 2013 to December 2014 were considered. The average women and men age was 37.9±3.8 and 39.9±4.1 y/o, respectively. A total of 578 MII oocytes were obtained, with a 77.9% fertilization rate. From 450 fertilized oocytes, 446 (99.1%) cleaved at 48 hours, at 72 hours the cleavage rate was 100% (450). Of total cleaved embryos (72 hs), 52% (234) reached the blastocyst stage.For the analysis-interpretation of the results, 3 groups were considered according to the number of cells at day 3: (A) ≥10 cells (accelerated-cleavage), (B) =8 cells embryos from a cohort with accelerated embryos, and (C) =8 cells embryos from a cohort without accelerated embryos. Embryos that reached the blastocyst stage, either at day 5 or 6, were classified in: good, regular and low quality (degree of expansion, inner cell mass-trophectoderm and blastocoel). Blastocysts biopsies were analyzed by aCGH. In group A, the amount of embryos that reached the blastocyst stage was significantly higher (84.1%) compared than Group B (58.8%) and C (67.6%). Between groups B and C there were no significant differences. The rate of euploid blastocysts in group A was 64.9%, significantly higher than groups B (43.3%) and C (43.0%). No difference in aneuploidy rate was evidenced between day 5 or 6 blastocysts. In group A, there were no differences in blastocyst-quality and aneuploidy rate. However, in group B and C, blastocysts with better morphology correlated with lower aneuploidy rates. In our preliminary experience, we can conclude that the number of cells on day 3 could help to understand the prognosis of blastocysts aneuploidy rate on day 5 or 6. The embryos at day 3 with accelerated behavior, which usually are not preferably chosen to transfer, have lower aneuploidy rates than embryos with normal cleavage.

Proteomic analysis of wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D) — A wider perspective of the proteome

Cereal embryos are a model system to study desiccation tolerance due to their ability to survive extreme water loss during late embryogenesis. To identify proteins accumulating in mature embryos which can be used as potential markers for dehydration tolerance, we compared the embryo proteome from two durum wheat genotypes (Triticum durum Desf.), Mahmoudi (salt and drought sensitive) and Om Rabia3 (salt and drought tolerant). Total protein extracts from wheat embryos were analyzed by using conventional 2-DE and ProteomeLab PF-2D. Analysis using different pH ranges showed that a larger number of fractions were solved by LC, than by conventional 2-DE at extreme technical pHs (pH 4.0-5.0 and pH 6.5-8.0). In contrast, at intermediate pHs (pH 5.0-6.5), resolution was better in 2-DE gels. The two techniques were used in parallel to analyze total protein extracts from embryos of the two wheat varieties. Several proteins belonging to the seed storage family, LEA-type/heat shock proteins, enzyme metabolism and radical scavengers were identified by analysis of trypsin digested peptides via mass spectrometry. These proteins accumulate in different amounts in embryos of tolerant and sensitive wheat varieties. The differences in expression pattern were further validated by enzyme activity, western blotting analysis and correlated with their corresponding mRNA expression by RT-PCR analyses for the corresponding protein. We suggest that the differential expression pattern could be used as a basis for a biochemical screen of tolerance/sensitivity to drought and salt stress in wheat embryos and germplasm.

P5 Correlation between preimplantation genetic screening for chromosomal aneuploidies and amount of embryos in IVF cycles

P5 Correlation between preimplantation genetic screening for chromosomal aneuploidies and amount of embryos in IVF cycles

A Study on the Occurrence of Polyovular Follicles in Porcine Ovaries with Particular Reference to Intrafollicular Hormone Concentrations, Quality of Oocytes and their in vitro Fertilization

The aim of this study was to identify the occurrence of polyovular follicles in porcine ovaries. We investigated the presence of such follicles in relation to age, and compared the intrafollicular concentrations of steroid hormones between poly- and uniovular follicles. Then we measured the size, viability and the in vitro fertilizing ability of the oocytes from polyovular follicles. Histological examinations documented the occurrence of polyovular follicles in pigs at various stages of follicular growth. Within antral follicles, the number of polyovular follicles was higher in the ovaries of gilts than in sows (P < 0.01). We noticed differences in the viability and size of oocytes recovered from the same follicles. We noted a higher concentration of oestradiol-17beta and a lower concentration of progesterone in polyovular follicles as compared with uniovular follicles (P < 0.01). The amount of embryos after in vitro-fertilization of oocytes from polyovular follicles was significantly lower than that from uniovular ones. Nevertheless, we found that some oocytes from polyovular follicles also have the capacity to be fertilized in vitro and be developed to the blastocyst stage.

The MADS-Domain Transcriptional Regulator AGAMOUS-LIKE15 Promotes Somatic Embryo Development in Arabidopsis and Soybean

The MADS-domain transcriptional regulator AGAMOUS-LIKE15 (AGL15) has been reported to enhance somatic embryo development when constitutively expressed. Here we report that loss-of-function mutants of AGL15, alone or when combined with a loss-of-function mutant of a closely related family member, AGL18, show decreased ability to produce somatic embryos. If constitutive expression of orthologs of AGL15 is able to enhance somatic embryo development in other species, thereby facilitating recovery of transgenic plants, then AGL15 may provide a valuable tool for crop improvement. To test this idea in soybean (Glycine max), a full-length cDNA encoding a putative ortholog of AGL15 was isolated from soybean somatic embryos. Subsequently, the corresponding genomic region of the gene was obtained. This gene, designated GmAGL15, encodes a protein with highest similarity to AGL15 from Arabidopsis (Arabidopsis thaliana) and Brassica napus that accumulates to its highest amount in embryos in these species. Like Arabidopsis and Brassica AGL15, GmAGL15 was preferentially expressed in developing embryos. When ectopically overexpressed the soybean protein was able to enhance somatic embryo development in soybean.

Comparison of nuclear replication activity and protein expression patterns during tomato seed germination

Abstract Flow cytometric determination of DNA amounts in embryos of fully matured dry tomato (Lycopersicon esculentum Mill.) seeds revealed mainly 2C signals. Apparently, most embryonic cells had arrested the cell cycle activity during maturation at the presynthetic G1 phase of nuclear division. During imbibition of tomato seeds, both in water and in PEG solution, an augmentation in the 4C signal of tissues at the embryonic root tip region occurred. This increase was found to be related to cells entering the synthetic phase of nuclear division leading to a doubling of the chromosomal material. In the root tip cells, 4C 2C ratios rose 1 day after imbibition in water while radicle emergence started 2 days later. Apparently, DNA synthesis preceded germination. The induction of nuclear activities was compared with changes in protein expression patterns during seed imbibition. Using chemiluminescent immunodetection after Western blotting, we identified changes in β-tubulin expression during germination and priming of tomato seeds. Tubulin was found to be synthesized 8 h after the start of imbibition both in water and in PEG. The start of tubulin synthesis considerably preceded DNA replication, cell division and germination. The induction of cell cycle activities may be an early indicator of the initiation of cell division and the start of germination.

Expression of catenins during mouse embryonic development and in adult tissues.

Classical cadherins are cell-surface glycoproteins that mediate calcium-dependent cell adhesion. The cytoplasmic domain of these glycoproteins is linked to the cytoskeleton through the catenins (alpha, beta and gamma). The catenins are intracellular polypeptides that are part of a complex sub-membranous network modulating the adhesive ability of the cells. One approach to elucidate the role of these molecules in the cell is to investigate their distribution during mouse development and in adult tissues. This study reports that catenins are widely expressed but in varying amounts in embryos and adult tissues. The expression of all three catenins is most prominent in the adult heart muscle and in epithelia of all developmental stages. In other embryonic and adult tissues, lower expression of catenins was detected, e.g., in smooth muscle or connective tissue. Catenins are coexpressed with various cadherins in different tissues. Gastrulation is the first time during embryogenesis when a discrepancy occurs between the expression of catenins and E-cadherin. E-cadherin expression is suppressed in mesodermal cells but not the expression of catenins. This discrepancy suggests that another cadherin may interact with catenins. Similarly, E-cadherin is generally expressed in adult liver but not in the regions surrounding the central veins. In contrast, catenins are uniformly expressed in the liver, suggesting that they are associated with other cadherins in E-cadherin negative cells. Finally, the three catenins are not always concurrently expressed. For example, in peripheral nerves, only beta-catenin is observable, and in smooth muscle plakoglobin is not detectable.

Yolk Phosphoprotein Metabolism during Early Development of the Fish, Oryzias latipes: (Fish egg/Yolk/Phosphoprotein/Oryzias latipes/Fish development).

Changes in yolk phosphoproteins of Oryzias latipes embryos during early development were examined by electrophoresis. Four phosphoproteins were identified in the yolk of blastulae by polyacrylamide gel electrophoresis. Two of them were high molecular weight phosphoproteins containing 0.7% (w/w) phosphorus and with similar amino acid compositions to that of vitellogenin. The other two were low molecular weight phosphoproteins, characterized by high contents of phosphorus [12.2% (w/w)] and serine (44.8 mole%) and low contents of aromatic amino acid residues. From these characteristics, together with their behaviors on DEAE-cellulose chromatography and electrophoresis and low stainability with dyes, the latter two were concluded to be phosvitins. These phosvitins were isolated and partially characterized. The yolk phosphoproteins, especially the phosvitins, were degraded, their amounts in embryos decreased throughout early development. Studies on the mechanism of endogenous phosphoprotein degradation strongly suggested the participation of some protease(s) that was precipitated on centrifugation of the egg homogenate at 14,000 × g for 10 min.

Harvesting large amounts of Drosophila embryos at precisely defined stages

A simple method is described which makes it possible to eliminate the retained eggs ofDrosophila melanogaster and to harvest a large amount of embryos at a defined stage of development. It is based upon carbon dioxide anesthesia, which brings about the expulsion of eggs remaining in the uterus.

(14C)Ethylenethiourea: distribution, excretion, and metabolism in pregnant rats.

Following administration of single oral doses of [14C]ethylenethiourea (ETU) to pregnant rats maternal blood maintained peak radioactivity for 2 h, and the radioactivity was dispersed uniformly between the red blood cells and plasma. The level of radioactivity was distributed equally among several maternal tissues but was present in lower amounts in embryos. Twenty-four hours after treatment all tissues examined, except blood, were relatively clear of radioactivity and 72.8% of the total radioactivity given had been excreted in the urine. Elution patterns of metabolites from Sephadex separation suggested that ethylenethiourea was degraded very little. The teratological mechanism is discussed.

The Presence of the Virus of Visceral Lymphomatosis in Embryonated Eggs of Normal Appearing Hens

INTRODUCTIONNUMEROUS investigators have reported evidence for and against the transmission of lymphomatosis from parent to offspring through the egg. Cottral et al. (1954) recently reviewed this subject and presented evidence that the causative agent of visceral lymphomatosis can be transmitted from dam to progeny through the egg and that normal appearing chickens are carriers of this agent. Investigations on this phase of the transmission of lymphomatosis have continued at the Regional Poultry Research Laboratory.The purpose of this manuscript is to report data which give additional evidence that the virus of visceral lymphomatosis is present in significant amounts in embryos of eggs laid by normal appearing hens and that such carrier hens are much more numerous in susceptible lines of an infected population than in resistant lines of the same population.MATERIALS AND METHODSGeneral.—The method used in testing for the presence of the virus in embryonic tissue …