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Optimal timing of blastocyst vitrification after trophectoderm biopsy for preimplantation genetic screening.

ABSTRACT: Is the timing of vitrification after trophectoderm (TE) biopsy associated with successful implantation and pregnancy after the embryo transfer of blastocysts subjected to preimplantation genetic screening (PGS)? In this retrospective cohort study, 1329 blastocysts from 223 patients were subjected to TE biopsy for performing array comparative genomic hybridization (CGH) tests. The PGS and frozen blastocyst transfer (FET) cycles were performed from December 2012 to May 2015. Only the good quality and expanded blastocysts on day 5 or 6 were selected for biopsy. After TE biopsy, the re-expansion grades relative to the original blastocoel were (1) collapsed blastocysts (CB), (2) 3/4 re-expansion but not full expansion (RE), and (3) full re-expansion or hatching (FE). All biopsied blastocysts were subjected to vitrification within 0.5-6 h after biopsy; the time intervals between TE biopsy and vitrification and the expansion grades at the time of vitrification were recorded. By combining two factors, namely the expansion grades and culture intervals between biopsy and vitrification, the patients were further divided into four groups, namely CB with a < 3 h culture interval (n = 34 cycles, Group I), RE and FE blastocysts with a < 3 h culture interval (n = 10 cycles, Group II); CB blastocysts with a ? 3 h culture interval (n = 6 cycles, Group III); and RE or FE blastocysts with a ? 3 h culture interval (n = 173 cycles, Group IV). The implantation (63.7%, 179/281) and clinical pregnancy (74.0%, 128/173) rates in Group IV were significantly higher than those in Group I (45.3%, 24/53; 50.0%, 17/34; P = 0.012 and 0.005, respectively). According to our findings, optimal vitrification timing > 3 hours to enable blastocysts to reach RE or FE provides improved implantation and pregnancy rates after FET.ClinicalTrials.gov NCT03065114.

SUBMITTER: Chen HH

PROVIDER: S-EPMC5628850 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Optimal timing of blastocyst vitrification after trophectoderm biopsy for preimplantation genetic screening.

Chen Hsiu-Hui HH Huang Chun-Chia CC Cheng En-Hui EH Lee Tsung-Hsien TH Chien Lee-Feng LF Lee Maw-Sheng MS

PloS one 20171005 10

Is the timing of vitrification after trophectoderm (TE) biopsy associated with successful implantation and pregnancy after the embryo transfer of blastocysts subjected to preimplantation genetic screening (PGS)? In this retrospective cohort study, 1329 blastocysts from 223 patients were subjected to TE biopsy for performing array comparative genomic hybridization (CGH) tests. The PGS and frozen blastocyst transfer (FET) cycles were performed from December 2012 to May 2015. Only the good quality ...[more]

PMID: 28982142

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Project description:A systematic review of the literature showed that trophectoderm biopsy could assist in the selection of healthy embryos for uterine transfer without affecting implantation rates. However, previous studies attempting to establish the relationship between trophectoderm gene expression profiles and implantation competency using either microarrays or RNA sequencing strategies, were not sufficiently optimized to handle the exceptionally low RNA inputs available from biopsied material. In this pilot study, we report that differential gene expression in human trophectoderm biopsies assayed by an ultra-sensitive next generation RNA sequencing strategy could predict blastocyst implantation competence. RNA expression profiles from isolated human trophectoderm cells were analysed with established clinical pregnancy being the primary endpoint. Following RNA sequencing, a total of 47 transcripts were found to be significantly differentially expressed between the trophectoderm cells from successfully implanted (competent) versus unsuccessful (incompetent) blastocysts. Of these, 36 transcripts were significantly down-regulated in the incompetent blastocysts, including Hydroxysteroid 17-Beta Dehydrogenase 1 (HSD17B1) and Cytochrome P450 Family 11 Subfamily A Member 1 (CYP11A1), while the remaining 11 transcripts were significantly up-regulated, including BCL2 Antagonist/Killer 1 (BAK1) and KH Domain Containing 1 Pseudogene 1 (KHDC1P1) of which the latter was always detected in the incompetent and absent in all competent blastocysts. Ontological analysis of differentially expressed RNAs revealed pathways involved in steroidogenic processes with high confidence. Novel differentially expressed transcripts were also noted by reference to a de novo sequence assembly. The selection of the blastocyst with the best potential to support full-term pregnancy following single embryo transfer could reduce the need for multiple treatment cycles and embryo transfers. The main limitation was the low sample size (N = 8). Despite this shortcoming, the pilot suggests that trophectoderm biopsy could assist with the selection of healthy embryos for embryo transfer. A larger cohort of samples is needed to confirm these findings. Abbreviations: AMA: advanced maternal age; ART: assisted reproductive technology; CP: clinical pregnancy; DE: differential expression; FDR: false discovery rate; IVF: in vitro fertilization; LD PCR: long distance PCR; qRT-PCR: quantitative real-time PCR; SET: single embryo transfer; TE: trophectoderm.

Project description:ObjectiveTo validate the performance of our laboratory-developed whole-genome screening assay within clinical preimplantation genetic testing environments.DesignPerform a laboratory-developed whole-genome assay on both cell lines and trophectoderm biopsies, subsequently employing the next-generation sequencing procedure to reach a sequencing depth of 30X. Adhere to the Genome Analysis Toolkit best practices for accuracy, sensitivity, specificity, and precision calculations by comparing samples with references. Our assay was then applied to cell lines and biopsies harboring known pathogenic variants, aiming to ascertain these changes solely from the next-generation sequencing data, independent of parental genome information.SettingsClinical laboratory.PatientsCoriell cell lines and research embryos with known chromosomal or genetic variants. Research trophectoderm biopsies from a couple that are heterozygous carriers for distinct variants in the same autosomal recessive gene (HOGA1).InterventionNot applicable.Main outcome measuresAccuracy, sensitivity, specificity, and precision were assessed by comparing the samples to their references. For samples with known variants, we calculated our sensitivity to detecting established variants. For the research embryos, noncarrier, carrier, and compound heterozygous states of inherited HOGA1 variants were distinguished independently of parental samples.ResultsAmplification of DNA from cell lines and embryos yielded success rates exceeding 99.9% and 98.2%, respectively, although maintaining an accuracy of >99.9% for aneuploidy assessment. The accuracy (99.99%), specificity (99.99%), sensitivity (98.0%), and precision (98.1%) of amplified genome in the bottle (reference NA12878) and embryo biopsies were comparable to results on genomic DNA, including mitochondrial heteroplasmy. Using our assay, we achieved >99.99% sensitivity when examining samples with known chromosomal and genetic variants. This encompassed pathogenic CFTR, BRCA1, and other variants, along with uniparental isodisomies and microdeletions such as DiGeorge syndrome. Our research study identified noncarrier, carrier, and compound heterozygous states within trophectoderm biopsies while simultaneously screening for 1,300 other severe monogenic diseases.ConclusionTo our knowledge, this is the first clinical validation of whole-genome embryo screening. In this study, we demonstrated high accuracy for aneuploidy calls (>99.9%) and genetic variants (99.99%), even in the absence of parental genomes. This assay demonstrates advancements in genomic screening and an extended scope for testing capabilities in the realm of preimplantation genetic testing.

Project description:INTRODUCTION:Preimplantation 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 DESCRIPTION:An infertile couple, with family history of ?-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 EVALUATION:Preconception 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. CONCLUSIONS:This 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.

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Optimal timing of blastocyst vitrification after trophectoderm biopsy for preimplantation genetic screening.

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Optimal timing of blastocyst vitrification after trophectoderm biopsy for preimplantation genetic screening.

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