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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: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.

Project description:Vitrification is an important tool to store surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and results in decreased viability. Studies have reported that L-glutathione (GSH) supplementation improves the preimplantation development of murine embryos. Glutathione constitutes the major non-protein sulphydryl compound in mammalian cells, which confers protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has yet to be reported. This study aims to determine whether GSH supplementation in culture media improves in vitro culture and vitrification outcomes, as observed through embryo morphology and preimplantation development. Female BALB/c mice aged 6−8 weeks were superovulated through an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG), followed by 10 IU of human chorionic gonadotrophin (hCG) 48 h later. The mated mice were euthanized by cervical dislocation 48 h after hCG to harvest embryos. Two-cell embryos were randomly assigned to be cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH-supplemented medium), or Group 4 (0.01 mM GSH-supplemented medium with vitrification). Non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were observed for morphological quality and preimplantation development at 24, 48, 72, and 96 h. In the non-vitrified groups, there were significant increases in the number of Grade-1 blastocysts in GSH cultures (p < 0.05). Similarly, in the vitrified groups, GSH supplementation was also seen to significantly increase blastocyst formation. Exogenous GSH supplementation resulted in a significant increase in intracellular GSH, a release of cytochrome c from mitochondria, and a parallel decrease in intracellular reactive oxygen species (ROS) levels in vitrified eight-cell embryos (p < 0.05). GSH supplementation was shown to upregulate Bcl2 expression and downregulate Bax expression in the vitrified preimplantation embryo group. The action of exogenous GSH was concomitant with an increase in the relative abundance of Gpx1 and Sod1. In conclusion, our study demonstrated the novel use and practical applicability of GSH supplementation for improving embryonic cryotolerance via a decrease in ROS levels and the inhibition of apoptotic events by improvement in oxidative status.

Project description:BackgroundIn preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.Methods and findingsWe addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.Conclusion and significanceWe conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages.

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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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