Blastocyst Collapse Research Articles

Blastocyst collapse as an embryo marker of low implantation potential: a time-lapse multicentre study.

Spontaneous blastocyst collapse during in vitro embryo development has been suggested as a novel marker of embryo quality. Therefore, the aim of this multicentre study was to carry out a retrospective multicentre analysis to investigate the correlation between blastocyst collapse and pregnancy outcome. Here, 1297 intracytoplasmic sperm injection (ICSI)/in vitro fertilization (IVF) fresh cycles, with an elective single blastocyst transfer (eSET) were included in this study. Embryos were cultured individually in 6.0% CO2, 5.0% O2, 89.0% N2, using single step medium (GTLTM VitroLife, Sweden) or sequential medium (CookTM, Cook Medical, Australia) and selected for transfer using standard morphological criteria. With the use of time-lapse monitoring (TLM), blastocysts were analyzed by measuring the maximum volume reduction and defined as having collapsed, if there was ≥ 50% volume reduction from the expanded blastocyst and the collapse event. Following embryo replacement, each blastocyst was retrospectively allocated to one of two groups (collapsed or not collapsed). Here, 259 blastocysts collapsed once or more during development (19.9%) and the remaining 1038 either contracted minimally or not collapsed (80.1%). A significantly higher ongoing pregnancy rate (OPR) of 51.9% (95% CI 48.9-59.9%) was observed when blastocysts that had not collapsed were replaced compared with cycles in which collapsed blastocysts were transferred 37.5% (95% CI 31.6-43.4%). This study suggests that human blastocysts that collapse spontaneously during development are less likely to implant and generate a pregnancy compared with embryos that do not. Although this is a retrospective study, the results demonstrated the utility of collapse episodes as new marker of embryo selection following eSET at blastocyst stage.

Hydraulic control of mammalian embryo size and cell fate.

Size control is fundamental in tissue development and homeostasis1,2. Although the role of cell proliferation in these processes has been widely studied, the mechanisms that control embryo size-and how these mechanisms affect cell fate-remain unknown. Here we use the mouse blastocyst as a model to unravel a key role of fluid-filled lumen in the control of embryo size and specification of cell fate. We find that there is a twofold increase in lumenal pressure during blastocyst development, which translates into a concomitant increase in cell cortical tension and tissue stiffness of the trophectoderm that lines the lumen. Increased cortical tension leads to vinculin mechanosensing and maturation of functional tight junctions, which establishes a positive feedback loop to accommodate lumen growth. When the cortical tension reaches a critical threshold, cell-cell adhesion cannot be sustained during mitotic entry, which leads to trophectoderm rupture and blastocyst collapse. A simple theory of hydraulically gated oscillations recapitulates the observed dynamics of size oscillations, and predicts the scaling of embryo size with tissue volume. This theory further predicts that disrupted tight junctions or increased tissue stiffness lead to a smaller embryo size, which we verified by biophysical, embryological, pharmacological and genetic perturbations. Changes in lumenal pressure and size can influence the cell division pattern of the trophectoderm, and thereby affect cell allocation and fate. Our study reveals how lumenal pressure and tissue mechanics control embryo size at the tissue scale, which is coupled to cell position and fate at the cellular scale.

Spontaneous blastocyst collapse as an embryo marker of low pregnancy outcome: A Time-Lapse study.

Objective:In this study we investigate the correlation between spontaneous blastocyst collapse and pregnancy outcome.Methods:This is a retrospective study performed at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh, UK. Embryos were cultured individually in 6.0% CO2, 5.0% O2, 89.0% N2, using single step medium (GTL™ Vitrolife, Göteborg, Sweden) and selected for transfer using standard morphological criteria. Using the EmbryoScope™ time-lapse monitoring (TLM), blastocysts collapse was analyzed by measuring the maximum volume reduction and defined as having collapsed if there was >50% volume reduction. Couples undergoing IVF/ICSI treatment and having an elective single embryo transfer (eSET) at blastocyst stage were included in this study. After the embryo transfer, retrospectively, each blastocyst was allocated to one of two groups (collapsed or not collapsed). 62 blastocysts collapsed once or more during development (17.4%), the remaining 294 showed no collapse (82.6%).Results:A significantly higher implantation rate (IR) of 61.2% and ongoing pregnancy rate (OPR) of 53.7% was observed when blastocysts which had not collapsed were replaced compared to cycles in which collapsed blastocysts were replaced (IR rate 22.6% and OPR 17.7%).Conclusion:This study demonstrated that human blastocysts which collapse spontaneously during in vitro development are less likely to implant and generate a pregnancy compared with embryos which do not. Although this is a retrospective study, the results establish the utility of collapse episodes as new marker of embryo selection following eSET at blastocyst stage.

Artificial blastocyst collapse prior to vitrification significantly improves Na+/K+-ATPase-dependent post-warming blastocoel re-expansion kinetics without inducing endoplasmic reticulum stress gene expression in the mouse.

Blastocoel expansion during embryo development is known to be reliant on the Na+/K+-ATPase pump, but little is known about the relative contribution of active (Na+/K+-ATPase pump) and facilitated diffusion (aquaporins) water transport during blastocoel re-expansion after vitrification. The aims of this study were to examine potential effects of artificial blastocoel collapse (ABC) on markers of embryo stress and the contribution of active and facilitated diffusion water transport mechanisms to blastocoel re-expansion. Day 5 mouse embryos were vitrified using either a standard protocol, laser pulse ABC, a hyperosmotic sucrose ABC protocol or both laser pulse and sucrose. Using real-time polymerase chain reaction, no differences were found in the gene expression of the endoplasmic reticulum (ER) stress markers activating transcription factor 4 (Atf4) or heat shock protein 90-alpha (Hsp90α) 2h after warming. Similarly, expression of the Na+/K+-ATPase pump gene, ATPase, Na+/K+ transporting, beta 1 polypeptide (Atp1b1) and protein did not differ between groups. Aquaporin 8 (Aqp8) gene expression was significantly lower in the laser+sucrose ABC group than in fresh controls, and aquaporin 3 (Aqp3) expression significantly higher in standard vitrified embryos compared with all other groups. Ouabain, a potent and specific Na+/K+-ATPase pump inhibitor, inhibited blastocoel re-expansion in both standard protocol- and laser ABC-vitrified embryos, reducing both groups to the same rate of re-expansion 3h after warming. These results demonstrate that ABC before vitrification does not alter mRNA or protein expression of Na+/K+-ATPase, or mRNA levels of ER stress genes Atf4 and Hsp90α. Activity of the pump may be increased in ABC embryos, with potential compensation by AQP3 when it is compromised.

Treatment with AICAR inhibits blastocyst development, trophectoderm differentiation and tight junction formation and function in mice.

What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function? AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur. AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK. Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 μM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 μM AICAR. Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR. Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible. Not applicable. Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin. Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles. Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.

Blastocyst collapse and downgrading of inner cell mass morphology score after vitrification-warming is predictive of reduced implantation and increased early pregnancy loss

Embryo vitrification has increased confidence in embryo banking. Whether the effect of freeze-thaw on euploid blastocyst morphology impacts implantation potential is not well characterized. This study examines whether change in pre- and post-vitrification morphology correlates with clinical outcome, controlling for endometrial environment and embryo ploidy.

Does the blastocyst collapse respond to a biological need? The analysis of 1,952 embryos by time-lapse can give an answer

Does the blastocyst collapse respond to a biological need? The analysis of 1,952 embryos by time-lapse can give an answer

Blastocyst collapse. Validation of previous data

The appearance of time lapse incubators has opened the possibility of studying dynamic embryo behaviors. Blastocyst collapse has been studied in different mammal models. Strong contractions seem to be deleterious for embryo hatching. There is a high consumption of energy in the process of contraction and re-expansion, which could affect the extrusion of the blastocyst from de Zona Pellucida (ZP) and, consequently, the possibility of implantation.

Blastocyst collapse is not an independent predictor of reduced live birth: a time-lapse study

To ascertain the rate of blastocyst collapse observed by time-lapse monitoring in a retrospective cohort of unselected infertile patients undergoing single blastocyst transfer and to determine its association with live birth. Blastocyst collapse and morphokinetic variables were scored according to previously published criteria. The association between blastocyst collapse and live birth was evaluated by a multivariate logistic regression analysis including morphokinetic variables and other confounders. Private infertility clinic. Patients who underwent 277 consecutive single blastocyst transfers (mean age, 38.4 ± 3.9years; range, 28-47years) after minimal ovarian stimulation. Minimal ovarian stimulation, prolonged embryo culture in time-lapse monitoring incubator, elective vitrification with subsequent vitrified-warmed single blastocyst transfer. Live birth rate per single blastocyst transfer in different blastocyst collapse groups (no, single, multiple collapses). No, single, or multiple blastocyst collapses occurred in 54% (150/277), 22% (61/277), and 24% (66/277) of the cohort, respectively. In the multiple collapse group on average 2.9 contractions were seen (range, 2-9 contractions). Live birth rate decreased progressively between blastocyst collapse groups (36%, 31%, 14%); significantly lower if multiple collapses occurred. In a multivariate analysis, however, blastocyst collapse was not found to be a significant predictor and was confounded by stronger predictors such as morphokinetic variables t2, texpB2, and female age. Blastocyst collapse pattern should not be evaluated alone without taking into account morphokinetic variables that are stronger predictors of reproductive outcome.

Micromanipulation of equine blastocysts to allow vitrification.

Embryo cryopreservation presents an essential method for banking of valuable genetics. However, in equine species the cryopreservation of embryos is complicated by three interacting factors: (1) the late entry of the embryo into the uterus (~6 days after ovulation); (2) the rapid expansion of the blastocyst; and (3) the formation of the equine embryonic capsule, a glycoprotein membrane that forms between the embryo and zona. Efforts to freeze or vitrify equine expanded blastocysts were initially met with little success. In addition, it was thought that breaching the capsule led to loss of embryo viability. We found that micromanipulation with the Piezo drill to puncture the capsule and collapse the blastocyst before vitrification provided a means for successful cryopreservation of equine expanded blastocysts, and that this can be done successfully using a standard sperm injection pipette. Modification of cryoprotectants and methods for vitrification and warming resulted in a technique that allowed successful vitrification of expanded equine blastocysts up to 650 µm diameter, with pregnancy rates approaching those for fresh embryos. After blastocyst collapse, vitrification is performed with ethylene glycol and galactose as cryoprotectants, and the embryo is cooled in a low-volume micropipette tip. Vitrification of expanded equine blastocysts provides a valuable tool for use in exotic equids to preserve genetics.

Collapse of blastocysts is strongly related to lower implantation success: a time-lapse study.

Is there an association between blastocyst collapse patterns and implantation potential? Embryos that exhibit collapse are as likely to hatch as those that do not, but are less likely to implant and should not be replaced if alternatives are available. Studies of blastocyst collapse in different species of mammals have found that most blastocysts, that experience consecutive weak contractions, hatch successfully whereas those that exhibit strong contractions or collapse, fail to hatch. Retrospective cohort study. Seven hundred and fifteen transferred blastocysts were analyzed from July 2012 to May 2013. University-affiliated infertility center. Participant patients were recipients of oocyte donation and autologous IVF cycles (n = 460). Embryo development was analyzed with a time-lapse imaging system. Variables studied included blastocyst collapse (defined as the separation of ≥50% of the surface of the trophectoderm of the blastocyst from the zona pellucida), kinetic variables, embryo morphology, implantation and clinical pregnancy rates. A total of 139 blastocysts presented collapse (19.4%), of these 8 presented 2 collapses and 2 presented 3 collapses. The timings of embryo cleavages and the time taken to reach the morula stage and blastulation were significantly shorter in embryos with collapse than in those without. Implantation rates were compared in cycles where either all or none of the embryos replaced, implanted so that implantation data were known for every embryo. Among 408 embryos without collapse and 94 embryos with at least one collapse (94) the implantation rates were 48.5% (95% confidence intervals (CI) 46.7-53.4%) and 35% (95% CI 25.3-44.9%), respectively. The percentage of embryos that hatched was similar in both groups 28.7 and 31%, respectively. The retrospective nature of the study limits its potential value. Study the collapse pattern of the blastocyst, may assist selection of the blastocysts most likely to implant and increase IVF/ICSI success rates.

Successful cryopreservation of expanded equine blastocysts

Effective cryopreservation of expanded equine blastocysts (> 300 μm in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 μm) were vitrified in fine-diameter microloader pipette tips using dimethylsulfoxide-containing medium (DM) or ethylene glycol-containing medium (EG). A third group was vitrified with EG, but was warmed using sucrose (EG/s). Embryos in the DM and EG/s treatments grew in culture after vitrification, and established pregnancies after transfer (3 of 12 and 3 of 6, respectively). Expanded blastocysts 300–730 μm in diameter were then biopsied and vitrified; rates of normal pregnancy (detection of embryonic heartbeat) after warming and transfer were 2 of 16 (13%) and 6 of 13 (46%) for DM and EG/s treatments, respectively (P = 0.05). Within the EG/s treatment, it appeared that greater loss of blastocoele fluid after biopsy was associated with higher survival. Therefore, an altered (“Central”) biopsy technique was used to aspirate blastocoele fluid, followed by vitrification in EG/s. Pregnancy rates were 1 of 8 (13%) for embryos cultured after warming and 4 of 7 (57%) for embryos transferred immediately after warming (P = 0.1). Finally, expanded blastocysts 407 to 565 μm in diameter were biopsied from the periphery, and blastocoele fluid was removed with gentle suction. After vitrification with EG/s, this resulted in a rate of normal pregnancy of 5 of 7 (71%). These findings demonstrated that blastocoele collapse and vitrification in fine-diameter pipettes allowed successful cryopreservation of expanded equine blastocysts.

81 INDUCED BLASTOCOEL COLLAPSE IMPROVES SURVIVAL RATES OF PORCINE BLASTOCYSTS AFTER VITRIFICATION

Cryopreservation of porcine blastocysts is regarded as a demanding task because of the high chilling sensitivity and low survival rates. Ultrarapid vitrification techniques combined with the previous artificial induction of collapse of the blastocoel have been successfully applied in mouse and human. The purpose of our work was to use the Cryotop (Kitasato Supply Co., Tokyo, Japan) vitrification method combined with induced blastocyst collapse for cryopreservation of porcine blastocysts produced with parthenogenetic activation of in vitro-matured oocytes. A total of 1200 slaughterhouse-derived porcine oocytes were matured for 41–42 h. Zona-intact or zona-free oocytes (after pronase digestion) were activated with a single electric impulse of 63 or 43 V cm–1, respectively, and incubated in 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide for 4 h (Du et al. 2007 Cryobiology 54, 181–187). Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2, and 90% N2 and maximum humidity. On Day 6, collapse was induced manually in approximately half of the blastocysts with the use of a fire-polished holding pipette and injection pipette (Cook Australia, Brisbane, Queensland, Australia). Ten min after the manipulation, collapsed and control (not manipulated) blastocysts were vitrified with the Cryotop, and then warmed and diluted in 1, 0.5, and 0 m sucrose, respectively (Kuwayama et al. 2005, Reprod. BioMed. Online 11, 300–308). Reexpansion of blastocysts was evaluated under a stereomicroscope after 16–18 h of culture under conditions described above. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical calculations; values with P &lt; 0.05 were regarded as significant. Reexpansion rates of blastocysts are shown in Table 1. Our results indicate that manually induced collapse of the blastocoel increases reexpansion rates of both zona-intact and zona-free porcine blastocysts after Cryotop vitrification. The combination of these simple techniques may help to improve the efficiency of cryopreservation of pig embryos for both experimental and practical applications. Table 1. Reexpansion rates after vitrification of blastocysts with or without induced blastocoel collapse The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen, and ClausWillemoes for their help and excellent technical assistance.

Vitrification of human blastocysts using the cryoloop: laser-induced blastocyst collapse prior to vitrification

Vitrification of human blastocysts using the cryoloop: laser-induced blastocyst collapse prior to vitrification

Ouabain sensitivity and expression of Na/K-ATPase alpha- and beta-subunit isoform genes during bovine early development.

The fluid movements that arise during blastocyst formation (cavitation) are, at least in part, driven by the Na/K-ATPase. In this study, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to survey bovine pre-attachment embryos for transcripts encoding known isoforms of the Na/K-ATPase alpha- and beta-subunits, including isoforms not previously detected during the first week of mammalian development. Transcripts encoding the Na-K-ATPase alpha 1, alpha 2, alpha 3 and beta 2 isoforms were detected throughout bovine preattachment development. This is the first indication that alpha 2, alpha 3 and beta 2 mRNAs are expressed during this early developmental interval. As in the mouse, beta 1-subunit transcripts were not detected until the morula stage and were also present in blastocysts. Thus, in two mammalian species an increase in abundance of beta 1 isoform transcripts in the morula stage is coincident with the onset of cavitation. Transcripts encoding the recently characterized alpha 4 isoform were not detected. The sensitivity of bovine blastocysts to ouabain (a potent inhibitor of Na/K-ATPase) was determined by assessing the ability of bovine blastocysts to recover in ouabain supplemental culture medium following cytochalasin-induced blastocyst collapse. Re-expansion of bovine blastocysts was inhibited in all ouabain concentrations down to 10(-9) M. Mouse blastocysts, in contrast, were sensitive to ouabain at or above 10(-3)M. These results have established that transcripts encoding multiple isoforms of both the alpha and beta subunits of the Na/K-ATPase are expressed throughout early bovine development and that bovine blastocysts display a greater sensitivity to ouabain than murine blastocysts. Future analysis will determine the possible individual and collective roles of these isoforms during blastocyst formation.