Effect of laser-assisted hatching and necrotic blastomere removal on the development of vitrified-warmed four-cell mouse embryos.
Ontology highlight
ABSTRACT:
SUBMITTER: Fathi R
PROVIDER: S-EPMC2596673 | biostudies-other | 2008 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Blastomere biopsy is an essential technique in preimplantation genetic diagnosis (PGD), a screening test that can detect genetic abnormalities of embryos before their transfer into uterus. Our results showed that the weights of fetuses derived from biopsied embryos were lower than that of non-biopsied counterparts at E12.5, E15.5, and E18.5. The ratio of fetal/placental (F/P) weights in the biopsied group was significantly lower than that in the non-biopsied group at E18.5. At E18.5, the mRNAs for selected glucose transporters, system A amino acid transporters, system L amino acid transporters, and imprinted genes were downregulated in the placentae of biopsied group, and the GLUT1 and CAT3 protein levels were decreased too. More apoptotic cells were detected by TUNEL in the placentae of biopsied group. Placentae from biopsied embryos exhibited lower levels of SOD and GSH. Furthermore, the concentration of MDA increased in the placentae from biopsied group. The levels of IL1B, IL6, and TNFA also significantly increased in the placentae of biopsied group. This study suggested that placental function may be sensitive to blastomere biopsy procedures, and placental oxidative stress and inflammation associated with blastomere biopsy may be critical factors of abnormal placental function and further influence the fetal development.
| S-EPMC4842963 | biostudies-literature
Project description:This study shows for the first time the ability to rewarm cryopreserved zebrafish embryos that grow into adult fish capable of breeding normally. The protocol employs a single injection of cryoprotective agents (CPAs) and gold nanorods (GNRs) into the yolk and immersion in a precooling bath to dehydrate the perivitelline space. Then embryos are encapsulated within CPA and GNR droplets, plunged into liquid nitrogen, cryogenically stabilized, and rewarmed by a laser pulse. Postlaser nanowarming, embryos (n = 282) exhibit intact structure by 1 h (40%), continued development after 3 h (22%), movement after 24 h (11%), hatching after 48 h (9%), and swimming after Day 5 (3%). Finally, from fish that survives till Day 5, two larvae are grown to adulthood and spawned, yielding survival comparable to an unfrozen control. Future efforts will focus on improving the survival to adulthood and developing methods to cryopreserve large numbers of embryos for research, aquaculture, and biodiversity preservation.
| S-EPMC8627598 | biostudies-literature
Project description:BackgroundBlastomere loss is a common phenomenon that occurs following cryopreservation. To date, studies have drawn conflicting conclusions regarding the impact of blastomere loss on pregnancy outcomes. Besides, limited information is available concerning the neonatal safety of embryos with blastomere loss. In the present study, we aimed to investigate the impact of blastomere loss on pregnancy and neonatal outcomes of vitrified/warmed Day3 cleavage-stage embryos in single embryo transfer cycles.MethodsThis retrospective cohort study included all vitrified/warmed D3 cleavage-stage single frozen-thawed embryo transfer (FET) cycles between April 2015 and February 2021. We compared pregnancy and subsequent neonatal outcomes between the intact embryos group and the blastomere loss group in single FET cycles.ResultsA total of 6287 single FET cycles were included in the study, in which 5873 cycles were classified into the intact embryo group and 414 cycles were classified into the blastomere loss group. The outcomes of the blastomere loss group were significantly inferior to those of the intact embryo group, in terms of implantation/biochemical pregnancy/clinical pregnancy/ongoing pregnancy rate and live birth rate per embryo transfer cycle/per clinical pregnancy. Further binary logistic regression confirmed that blastomere loss was negatively associated with live birth. Moreover, the blastomere loss group presented with an elevated early miscarriage rate. The neonatal conditions were broadly similar between the two groups. Additionally, multiple binary logistic regression analysis demonstrated that primary infertility and intracytoplasmic sperm injection (ICSI) were common influencing factors of blastomere loss (aOR 1.447, 95% CI 1.038-2.019, P = 0.029; aOR: 1.388, 95% CI: 1.044-51.846, P = 0.024).ConclusionsThe transfer of vitrified/warmed D3 embryos with blastomere loss is related to impaired embryo developmental potentials and reduced probabilities of conception. Moreover, even if the embryos with blastomere loss have implanted and reached clinical pregnancies, they present with a lower possibility of developing to live birth owing to a higher early miscarriage rate. However, once the embryos with blastomere loss result in a live birth, no adverse neonatal outcomes are observed. Primary infertility and ICSI were found to be risk factors for blastomere loss.
| S-EPMC9116052 | biostudies-literature
Project description:Endometrial Mesenchymal Stromal Cells (endMSCs) are multipotent cells with immunomodulatory and pro-regenerative activity which is mainly mediated by a paracrine effect. The exosomes released by MSCs have become a promising therapeutic tool for the treatment of immune-mediated diseases. More specifically, extracellular vesicles derived from endMSCs (EV-endMSCs) have demonstrated a cardioprotective effect through the release of anti-apoptotic and pro-angiogenic factors. Here we hypothesize that EV-endMSCs may be used as a co-adjuvant to improve in vitro fertilization outcomes and embryo quality. Firstly, endMSCs and EV-endMSCs were isolated and phenotypically characterized for in vitro assays. Then, in vitro studies were performed on murine embryos co-cultured with EV-endMSCs at different concentrations. Our results firstly demonstrated a significant increase on the total blastomere count of expanded murine blastocysts. Moreover, EV-endMSCs triggered the release of pro-angiogenic molecules from embryos demonstrating an EV-endMSCs concentration-dependent increase of VEGF and PDGF-AA. The release of VEGF and PDGF-AA by the embryos may indicate that the beneficial effect of EV-endMSCs could be mediating not only an increase in the blastocyst's total cell number, but also may promote endometrial angiogenesis, vascularization, differentiation and tissue remodeling. In summary, these results could be relevant for assisted reproduction being the first report describing the beneficial effect of human EV-endMSCs on embryo development.
| S-EPMC5912768 | biostudies-literature
Project description:At present, there is no standardised protocol for assisted hatching (AH) and the field is beset with contradictory data. We hypothesised that such contradiction may be related to inconsistencies in clinical practice. This study aimed to investigate the application, preferences, and variations of AH in current clinical practice prior to embryo transfer (AHpET) and biopsy (AHpBP). An online voluntary survey, consisted of 25 questions regarding different aspects of AH, was circulated amongst different fertility centres via newsletters between October 2019 and March 2020. One-hundred twenty-nine different fertility centres participated in the survey. AHpBP was widely used (90.6% [48/53]) amongst these centres, especially for trophectoderm biopsy (92.2% [47/51]). In contrast, only 64.6% (73/113) of centres administrated AHpET; the application of AHpET was even lower in UK-based centres (36.6% [15/41]). Although laser pulses have become the predominant technique for AH, significant variation existed in the precise strategy. Zona pellucida (ZP) drilling was the main method for AHpBP, whilst both ZP drilling and ZP thinning were applied equally for AHpET. Furthermore, the ZP manipulation varied widely with regards to the size of the ZP opening and the extension of ZP thinning. This is the first representative survey relating to the current practice of AH. Laser-assisted AH is used extensively, especially for AHpBP. However, there is significant disparity in clinical practice across different centres. Future research should aim to create a standardised protocol for AH to help reduce the evident variation in clinical practice and investigate the true value of AH.
| S-EPMC9444829 | biostudies-literature
Project description:INTRODUCTION:Recent data suggest a higher clinical pregnancy rate performing assisted hatching (AH) on previously cryopreserved embryos but fail to demonstrate significant effects on live birth rate. However, current evidence is based on studies with a small sample size and may hide a type II error. Moreover, poor attention has been given to the specific effect of AH on frozen/thawed blastocysts. To shed light on this topic, we developed the present protocol for a randomised trial to investigate the benefits of the laser-mediated partial removal of the zona pellucida in vitrified/warmed blastocysts. METHODS AND ANALYSIS:The pArtiaL zonA pelluciDa removal by assisteD hatchINg of blastocysts (ALADDIN) study is a multicentric prospective comparative study with a parallel randomised controlled design aiming to investigate whether AH performed on warmed blastocysts before embryo transfer can improve live birth rate. Women allocated to the control group will undergo embryo transfer of blastocysts not previously subjected to AH. Two infertility units will be involved in the study. Enrolment of patients will last 18 months with quarterly monitoring and the entire study is foreseen to be closed in 36 months. Secondary outcomes include: proportion of transferred blastocysts/thawed blastocyst, morphological features of blastocysts before embryo transfer, implantation, biochemical pregnancy, clinical pregnancy (ultrasound visible gestational sac), miscarriage, multiple pregnancy, preterm birth (<37 weeks of gestation), obstetrical and neonatal complications and congenital anomaly rates. ETHICS AND DISSEMINATION:This protocol received a favourable ethical opinion from the Ethical Committee of IRCCS San Raffaele Scientific Institute and the Ethical Committee Area 2 Milan. Each participant will provide written consent to participate and remain encoded during the study. The trial results will be published in peer-reviewed journals and presented at conferences. TRIAL REGISTRATION NUMBER:NCT03623659; Pre-results.
| S-EPMC7371140 | biostudies-literature
Project description:Blastomere cytofragmentation in mammalian embryos poses a significant problem in applied and clinical embryology. Mouse two-cell-stage embryos display strain-dependent differences in the rate of cytofragmentation, with a high rate observed in C3H/HeJ embryos and a lower rate observed in C57BL/6 embryos. The maternally inherited genome exerts the strongest effect on the process, with lesser effects mediated by the paternally inherited genome and the ooplasm. The effect of the maternal genome is transcription dependent and independent of the mitochondrial strain of origin. To identify molecular mechanisms that underlie cytofragmentation, we evaluated transcriptional activities of embryos possessing maternal pronuclei (mPN) of different origins. The mPN from C57BL/6 and C3H/HeJ strains directed specific transcription at the two-cell stage of mRNAs corresponding to 935 and 864 Affymetrix probe set IDs, respectively. Comparing transcriptomes of two-cell-stage embryos with different mPN revealed 64 transcribed genes with differential expression (1.4-fold or greater). Some of these genes occupy molecular pathways that may regulate cytofragmentation via a combination of effects related to apoptosis and effects on the cytoskeleton. These results implicate specific molecular mechanisms that may regulate cytofragmentation in early mammalian embryos. The most striking effect of mPN strain of origin on gene expression was on adenylate cyclase 2 (Adcy2). Treatment with dibutyryl cAMP (dbcAMP) elicits a high rate and severe form of cytofragmentation, and the effective dbcAMP concentration varies with maternal genotype. An activator of exchange proteins directly activated by cAMP (EPACs, or RAPGEF 3 and 4) 8-pCPT-2'-O-methyl-cAMP, elicits a high level of fragmentation while the PKA-specific activator N6-benzoyl-cAMP does not. Inhibition of A kinase anchor protein activities with st-Ht31 induces fragmentation. Inhibition of phosphatidylinositol 3-kinase signaling also induces fragmentation. These results reveal novel mechanisms by which maternal genotype affects cytofragmentation, including a system of opposing signaling pathways that most likely operate by controlling cytoskeletal function.
| S-EPMC2867389 | biostudies-literature
Project description:BackgroundEmbryo cryopreservation is the process that water is removed from the cell by cryoprotectant materials, and embryos are stored at temperature below zero. This process may affect the viability and developmental potential of embryos.ObjectiveIn this study, the effect of the vitrification cryotop method on the expression level of Oct4 and Mest developmental genes in mouse blastocysts was examined.Materials and methodsThe collected 2-cell embryos of superovulated mouse by oviduct flushing were divided into non-vitrified and vitrified groups. These embryos were cultured to the blastocyst stage directly in the non-vitrified group and in the vitrified group, these embryos were cultured to 4-8 cell embryos, vitrified with cryotop in these stages and after 2-6 months, warmed and cultured to blastocyst embryos. Quantitative expression of two developmental genes, namely Oct4 and Mest, were performed in these groups, using RNA purification and Real-time RT-PCR.ResultsQuantitative PCR analysis showed that the expression level of both genes, Oct4 and Mest, was reduced significantly in the vitrified-warmed group relative to the control group (p=0.046 and p=0.001).ConclusionThis study revealed that morphologically normal embryos show a reduced amount of Oct4 and Mest transcripts which indicate that the vitrification method negatively effects the expression level of these two developmental genes.
| S-EPMC3941331 | biostudies-literature
Project description:PurposeThis study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring.MethodsThe prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR.ResultsThe drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta.ConclusionThese observations suggested that drilling used for hatching of in vitro cultured mouse blastocysts may improve the production of offspring.
| S-EPMC7714794 | biostudies-literature
Project description:ObjectiveThe aim of this study is to compare the amplification efficiency and the genomic profiles of blastocoel fluid (BF) derived by laser-assisted hatching and trophectoderm (TE) cells derived from the same blastocyst.MethodsFifty-four fresh blastocysts underwent shrinkage by laser-assisted hatching, and each BF sample was collected individually. BF and TE cells were retrieved from each blastocyst for chromosome analysis through multiple annealing and looping-based amplification cycles (MALBAC) and next-generation sequencing (NGS).ResultsFifty-four BF samples and 32 TE samples were retrieved for this study. Out of the 54 BF samples, only 35 provided reliable NGS data for comprehensive chromosome analysis (64.8%), while all 32 TE samples did (100%). Finally, there were 23 pairs of BF and TE samples from the same blastocyst. Only 17.4% of the BF-DNA karyotypes were completely agreeable with the TE samples (4/23).ConclusionBlastocoel fluid derived by laser-assisted hatching is easy to operate, and BF-DNA can be successfully amplified and subjected to NGS. Due to the low amplification efficiency and increased discordance with TE, BF does not adequately represent the status of the rest of the blastocyst. The use of BF as a single source of DNA for preimplantation genetic screening (PGS) is not yet advised.
| S-EPMC9216213 | biostudies-literature