Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies.
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SUBMITTER: Capalbo A
PROVIDER: S-EPMC4677968 | biostudies-literature | 2016 Jan
REPOSITORIES: biostudies-literature
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Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies.
Capalbo Antonio A Ubaldi Filippo Maria FM Cimadomo Danilo D Maggiulli Roberta R Patassini Cristina C Dusi Ludovica L Sanges Federica F Buffo Laura L Venturella Roberta R Rienzi Laura L
Human reproduction (Oxford, England) 20151204 1
<h4>Study question</h4>Is blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and reproducible approach across different biopsy practitioners?<h4>Summary answer</h4>The blastocyst biopsy approach provides highly consistent and reproducible laboratory and clinical outcomes across multiple practitioners from different IVF centres when all of the embryologists received identical training and use similar equipment.<h4>What is known already</h4>Recen ...[more]
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Project description:PurposeTo study the impact of advanced paternal age on embryo aneuploidy.MethodsThis is a multicenter international retrospective case series of couples undergoing assisted reproduction via in vitro fertilization using donor eggs to control for maternal factors and preimplantation genetic testing for aneuploidy via next-generation sequencing at Igenomix reproductive testing centers. The main outcome measure was the prevalence of embryo aneuploidy in egg donor cycles. Semen analysis data was retrieved for a small subset of the male patients.ResultsData from 1202 IVF/ICSI egg donor cycles using ejaculated sperm (total 6934 embryos) evaluated using PGT-A between January 2016 and April 2018 in a global population across all Igenomix centers were included. No significant association was identified between advancing paternal age and the prevalence of embryo aneuploidy overall and when analyzing for each chromosome. There was also no significant association between advancing paternal age and specific aneuploid conditions (monosomy, trisomy, partial deletion/duplication) for all chromosomes in the genome.ConclusionsThis is the largest study of its kind in an international patient population to evaluate the impact of advancing paternal age on embryo aneuploidy. We conclude there is no specific effect of paternal age on the prevalence of embryo aneuploidy in the context of embryo biopsies from egg donor cycles.
| S-EPMC6823410 | biostudies-literature
Project description:Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.
| S-EPMC4463508 | biostudies-literature
Project description:Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding.
| S-EPMC11170958 | biostudies-literature
Project description:Preimplantation Genetic Testing (PGT), which encompasses both Preimplantation Genetic Diagnosis (PGD) and Preimplantation Genetic Screening (PGS), is a form of prenatal screening done on embryos conceived through assisted reproduction techniques (ART) prior to the initiation of pregnancy to ensure that only select embryos are used for transfer. PGT is typically performed on 8-cell embryos derived from either in vitro fertilization or intracytoplasmic sperm injection (ICSI) followed by extended culture. PGT requires a highly invasive embryo biopsy procedure that involves 1) incubating embryos in divalent-cation-deficient medium to disrupt cell adhesion, 2) breaching the protective zona pellucida with acid Tyrodeâs, laser drilling, or mechanical force and 3) aspirating one or two blastomeres. In this study we developed a mouse model of the embryo biopsy procedure inherent to PGT to determine the effect of various aspects of the procedure (incubation in Ca2+/Mg2+-free medium (CMF), acid Tyrodeâs treatment, blastomere aspiration), performed individually or in combination, on global patterns of gene expression in the resulting blastocysts. Experiment Overall Design: The comprehensive embryo biopsy procedure was done as outlined below and was modified according to the specific experimental treatment being performed. Experiment Overall Design: Eight-cell embryos with rounded blastomeres and no fragmentation were initially incubated in CMF-Chatot-Ziomek-Brinster medium for 5 min at 37oC in a humidified atmosphere of 5% CO2 in air to loosen cell-cell contacts and then transferred to CMF-Dulbeccoâs Phosphate-Buffered Saline. The biopsies were done on the stage of a Nikon Eclipse TE2000 microscope equipped with Hoffman optics and motorized Eppendorf micromanipulators. No more than three embryos were manipulated outside of the incubator at any time. Embryos were held in place by gentle suction on an Eppendorf Sterile VacuTip holding pipette (15 μm diameter), and a dual-holder system was used to control the pipette containing acid Tyrodeâs (6 μm diameter) and the biopsy pipette (15 μm diameter). The zona pellucida was breached by gentle expulsion of a steady stream of acid Tyrodeâs solution (pH 1.6), and a single blastomere with a clear nucleus was removed by aspiration. Following each respective treatment, embryos were cultured individually in 10 μl drops of Potassium Simplex Optimization Medium supplemented with amino acids at 37oC in a humidified atmosphere of 5% CO2 in air until 96 h post-hCG injection. Four replicates of these five experimental groups, each containing 20 embryos, were generated for a total of 20 samples and 400 embryos. Experiment Overall Design: To determine the effect of the various aspects of the embryo biopsy procedure on global patterns of gene expression, total RNA was extracted from each set of 20 embryos in each treatment group at 96 h post-hCG injection. This total RNA was then used to prepare target cRNA by linear two-round amplification as described previously. The final yield of biotinylated cRNA/20 embryos ranged from 34 μg -78 μg, of which 20 μg was fragmented and submitted to the University of Pennsylvania Microarray Facility where the samples were serially hybridized to the MOE430v2 GeneChip. Quality control parameters for all samples were within the following ranges: scale factor: 0.27-0.47, percent of genes detected: 44-51%, actin 3â/5â signal ratio: 1.5-4.2, and GAPDH 3â/5â signal ratio: 3.1-4.3. The GC-RMA algorithm was used to quantify and normalize microarray signals using Stratagene Array Assist Lite, Version 3.4. The resulting .chp files were imported to GeneSpring (v7.31, Agilent Technologies) where probe sets were filtered to retain those that were flagged as P (present) in at least 3 of the 20 samples. The data was log2-transformed and subjected to analysis using Partek Genomics Suite (v6.3, Partek, Inc).
2008-06-11 | E-GEOD-11206 | biostudies-arrayexpress
Project description:Potential use of preimplantation genetic testing for aneuploidy (PGT-A) is increasing. Patients who have excess embryos cryopreserved at the blastocyst stage may desire PGT-A but there is little data available on options for these patients. We compared the efficacy and safety of the timing on the cryopreservation and trophectoderm(TE) biopsy for preimplantation genetic testing for aneuploidy (PGT-A) program associated with the better outcomes after frozen blastocyst transfer. Retrospective analysis of patients who underwent PGT-A cycles from January 2016 to December 2019 was carried out. 2684 blastocysts from cycles were subjected to TE biopsy for performing array comparative genomic hybridization test and Next-generation sequencing. All cycles were divided into two according to the timing of biopsy: biopsy-first (n = 211 cases/ 232 transfers) versus freeze all-first (n = 327 cases/ 415 transfers). In the biopsy-first group, embryos were cultured to expanded blastocyst and proceed to TE biopsy on day 5 or day 6 followed by cryopreservation. In the freeze all-first, blastocysts were vitrified and warmed before biopsy. Rates of clinical pregnancy (52.3% vs. 38.7%, P = 0.09) and ongoing pregnancy (44.3% vs. 34.5%, P = 0.07) in biopsy-first were significantly higher than those in freeze all-first. Biopsy-first showed comparable miscarriage rate with freeze all-first (15.2% (33/217) vs.11.1% (10/90), respectively). Rate ratio (RR) for clinical pregnancy was lower in freeze all-first group (adjusted RR = 0.78, 95% confidence interval: 0.65, 0.93). The RRs for miscarriage and live birth was also lower but it did not reach statistical significance. Our result supported performing TE biopsy of blastocyst for PGT-A before vitrification and warming. This finding would contribute to more evidence-based decision in PGT-A cycles.
| S-EPMC9512210 | biostudies-literature
Project description:IntroductionPreimplantation genetic diagnosis and/or screening (PGD/PGS) allow the assessment of the genetic health of an embryo before transferring it into the uterus. These techniques require the removal of cellular material (polar bodies, blastomere(s) or trophectoderm cells) in order to perform the proper genetic analysis. We report the implantation and live birth outcome of a vitrified-warmed blastocyst developed after triple biopsy and double vitrification procedures at oocyte, cleavage embryo and blastocyst stage.Case descriptionAn infertile couple, with family history of β-thalassemia, searched for IVF procedure and PGD. First polar bodies biopsy with subsequent vitrification was uninformative due to meiotic crossing-over, so oocytes were inseminated after warming. Two embryos were obtained and blastomere biopsy was performed on day 3 with inconclusive results on their genetic status. Their culture resulted in one expanded blastocyst stage on day 7 that underwent trophectoderm biopsy and vitrification. This embryo showed to be normal. It was then warmed and transferred in an artificial cycle.Discussion and evaluationPreconception genetic analysis by removal and analysis of the first polar body is technically possible, but the genetic information that we can obtain at this stage may be limited and the oocytes to be inseminated is not predictable. Compared to blastomere biopsy, trophectoderm biopsy has more diagnostic efficiency with respect to both chromosomal mosaicism and PCR accuracy, reducing the problems of amplification failure and allele drop out. Moreover, embryos biopsied at the cleavage stage seem to have lower implantation rate than biopsied blastocyst.ConclusionsThis is the first case report of a live birth obtained from a three step biopsy and double vitrification procedures of a blastocyst. This case report seems also to suggest the harmlessness of all these procedures if carefully performed by a skilled biologist in an IVF lab with quality management system. Finally, our study highlight that blastocyst cryopreserved on day 7 have clinically important potential and embryos that not reach blastocyst stage on day 6 should not to be discharged because they may result in an ongoing pregnancy.
| S-EPMC4300308 | biostudies-literature
Project description:PurposeA subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy.MethodsFour cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms.ResultsWith the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy).ConclusionsBy demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.
| S-EPMC5125146 | biostudies-literature
Project description:ObjectiveThe aim of this study was to investigate whether blastocyst morphology and developmental rate are associated with euploidy and live birth rates (LBRs) in single euploid frozen-thawed embryo transfer (FET) cycles.DesignRetrospective cohort study.MethodsThis study included 431 preimplantation genetic testing for aneuploidy (PGT-A) cycles followed by 393 FET cycles performed at our center from June 2017 to March 2021. All cycles were analyzed for euploidy based on blastocyst morphology (good, average and poor), developmental stage (day 5 and 6) and maternal age (< 35 and ≥ 35 years old). Multivariate logistic analysis models were used to identify the independent effects of conventional blastocyst morphology, developmental rate and morphological parameters (degree of blastocoele expansion, and grade of inner cell mass and trophectoderm (TE)) on LBRs.ResultsIn the group of women aged < 35 years, compared with poor-quality blastocysts, good-quality blastocysts (62.90% vs. 32.46%; odds ratio (OR) 3.163, 95% confidence interval (CI) 2.247-4.451; P < 0.001) and average-quality blastocysts (46.70% vs. 32.46%; OR 1.665, 95% CI 1.287-2.154; P < 0.001) had significantly higher euploidy rates. Additionally, day 5 blastocysts were associated with higher euploidy rates than day 6 blastocysts (49.28% vs. 35.02%; OR 1.506, 95% CI 1.191-1.903; P= 0.001). In the group of women aged ≥ 35 years, euploidy rates were also associated with blastocyst morphology, with 41.86%, 45.65% and 24.39% of good, average and poor-quality embryos, respectively, exhibiting euploidy. However, no relationship was seen between euploidy and blastocyst developmental rate. Multiple logistic regression analysis show that overall blastocyst morphology of euploid embryos was not associated with LBR, only embryos with A-grade TE had significantly higher LBRs than those with C-grade TE (62.71% vs. 45.40%; OR 2.189, 95% CI 1.166-4.109; P=0.015). Similarly, LBRs were significantly higher when day 5 blastocysts were transferred than when day 6 blastocysts were transferred (57.75% vs. 41.67%; OR 2.132, 95% CI 1.370-3.318; P = 0.001).ConclusionPoor-quality embryos have reduced rates of euploidy. However, blastocyst developmental rate only significantly associates with euploidy rates in women aged younger than 35. Furthermore, only TE grade and blastocyst developmental rate are significantly associated with LBRs following FET cycles.
| S-EPMC9044033 | biostudies-literature
Project description:BackgroundTo investigate whether the noninvasive preimplantation genetic testing (niPGT) complement conventional preimplantation genetic testing (PGT) in the embryos for aneuploidy.Results40 spent culture medium (SCM) samples from routine embryo culture were collected, and half of each SCM (10 µL) sample was used for whole genome amplification, while the other half was stored at -80 °C for 3-6 months. Thirty-six out of 40 fresh SCM samples were successfully amplified and sequenced. Thirty-six paired frozen-thawed SCM samples showed 100% concordance with the freshly amplified SCM samples. Then, SCM and trophectoderm (TE) samples from 149 blastocysts from 51 couples were collected. A 98.0% successful SCM sample amplification rate (146/149) was achieved. For the 146 paired TE biopsy and SCM samples, the overall concordance rate was 82.9% (121/146). Ten embryos with aneuploid TE results but euploid niPGT results were donated. A 70.0% (7/10) true negative rate was achieved by niPGT with respect to the inner cell mass (ICM) results (TE-positive embryos).ConclusionsThese results suggested that SCM stored at -80 °C for 6 months without affecting niPGT results based on NICSInst amplification.
| S-EPMC11846158 | biostudies-literature
Project description:PurposeThe study aims to contrast the efficacy of trophectoderm biopsy preimplantation genetic screening (PGS)/vitrification (VTF)-all cycles to past treatment protocols. Specifically, do these applied technologies increase live birth rates on a per cycle/first transfer basis?Materials and methodsAn observational, retrospective cohort study of first transfer outcomes was performed in two groups. Group 1 (PGS) included PGS/VTF-all cycles, and group 2 (no PGS) included the first transfer from non-PGS fresh cycles or VTF-ALL cycles. In group 1, all blastocysts were biopsied on days 5/6, vitrified and array CGH performed. Group 2 patients had embryo transfers on day 3 or day 5. All blastocysts were vitrified and warmed according to μS-VTF protocols. Clinical pregnancies and implantation were confirmed by ultrasound and live birth information attained. Results were stratified by age with donor cycles excluded, and to eliminate bias, the same groups were then validated on a per cycle basis. Chi-squared used to determine significance.ResultsAnalyzing 287 embryo transfers and 1,000+ PGS-tested blastocysts, an overall 97 % increase in live births favored group 1 (PGS). When utilizing PGS/VTF-ALL cycles, patients under 43 years old exhibited higher implantation, clinical pregnancy, and ongoing/live birth rates. Re-analyzing the data to include all cycles initiated revealed higher live birth rates in group 1 age groups ≤34 and 38-40 years old.ConclusionValidating PGS on a per cycle basis eliminated data bias by including patients without blastocysts to biopsy or euploid embryos. Clearly, PGS uses blastocysts more efficiently to achieve success, while many women over 40 may benefit most by understanding why some failures occur.SupportNone.
| S-EPMC5125149 | biostudies-literature