scholarly journals 70 RECONSTRUCTED BOVINE BLASTOCYSTS COMPRISING NUCLEAR TRANSFER-DERIVED INNER CELL MASS AND TROPHECTODERM FROM IVF EMBRYOS DO NOT IMPROVE IN VIVO DEVELOPMENT OF CLONES

2005 ◽  
Vol 17 (2) ◽  
pp. 185
Author(s):  
H.E. Troskie ◽  
F.C. Tucker ◽  
M.C. Berg ◽  
B. Oback ◽  
D.N. Wells ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Cell Line ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Pregnancy Rates ◽  
Reconstruction Procedure ◽  
Exact Test ◽  
Significant Difference

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome) and fetal overgrowth (Lee RSF et al. 2004 Biol. Reprod. 70, 1–11). Early embryonic loss in bovine NT pregnancies may also be due to immunological rejection (Hill JR et al. 2002 Biol. Reprod. 67, 55–63). As a means of overcoming placental abnormalities and improving pregnancy outcome in bovine NT, reconstructed blastocysts were produced by combining immunosurgically isolated inner cell masses (ICM) from Day 7 NT embryos with the trophectoderm (TE) of Day 7 IVF embryos. Oocytes for the production of NT and IVF embryos were obtained from abattoir-collected ovaries of dairy cows. The semen used for IVF was from the bull from which the cell line for NT was derived. The NT blastocysts were produced as described previously (Oback B et al. 2003 Cloning Stem Cells 5, 3–12) except that two one-cell embryos were aggregated together after NT (2NT). Blastocyst reconstruction was achieved using a modified procedure (Rorie RW et al. 1994 Vet. Record 135, 186–187). Embryos from four experimental groups were transferred individually to synchronized recipient heifers on Day 8 of culture: (1) ICM from 2NT embryos reconstructed with IVF TE (R-2NT, n = 15); (2) ICM from IVF embryos reconstructed with IVF TE (R-IVF, n = 15); (3) control 2NT (n = 10); and (4) control IVF (n = 10). Pregnancy rates were recorded and treatments compared using Fisher's exact test. After slaughter between Days 149 and 161 of gestation, morphometric measurements were determined for the fetuses, fetal organ weights, fluid volumes, and placentomes. Data were rank transformed; treatments were compared using Student's t-test with standard errors calculated from the pooled variation. Pregnancy rates on Day 35 were R-2NT (60%), R-IVF (47%), 2NT (90%), and IVF (10%). Pregnancy rates on Day 150 were R-2NT (40%), R-IVF (40%), 2NT (70%), and IVF (10%). The reason for the low IVF pregnancy rate was unknown. Previously, pregnancy rates using the same sire and cell line (but using Day 7 embryo transfer) on Day 35 were 63% (n = 40) and 69% (n = 42) for IVF and single, non-aggregated NT, respectively, and 50% and 33% for IVF and NT on Day 150. The single NT pregnancy rate was not significantly different from that for the 2NT embryos. There was no significant difference in pregnancy rates on Day 35 and Day 150 between R-2NT v. 2NT, R-2NT v. R-IVF, or 2NT v. R-IVF. The blastocyst reconstruction procedure did not have any impact on fetal development or influence pregnancy rates. All fetuses recovered were male. No significant differences were found between R-2NT and 2NT fetuses in terms of fetal weight, fluid volume, total placentome weight, and placentome numbers or in the relative and absolute weights of the brain, heart, liver, and kidneys. Thus, replacement of the TE in NT embryos with TE from IVF embryos did not overcome placental abnormalities or decrease fetal overgrowth prevalence.

scholarly journals Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Craig Smith ◽  
Debbie Berg ◽  
Sue Beaumont ◽  
Neil T Standley ◽  
David N Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Cell Number ◽  
Transcript Levels ◽  
Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

scholarly journals 71 DEVELOPMENTAL DELAY OF PRE-IMPLANTATION OVINE IN VITRO CULTURED AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 185
Author(s):  
P. Tveden-Nyborg ◽  
T. Peura ◽  
K. Hartwich ◽  
P. Maddox-Hyttel
Keyword(s):  
Somatic Cell ◽  
Developmental Delay ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Germ Layers ◽  
Visceral Mesoderm

Despite advances in the production of somatic cell nuclear transfer (SCNT) embryos, significant embryo losses are persistent, particularly around implantation. Malformations of the placenta and in a variety of organs are seen, and have been linked to deviant epigenetic reprogramming. The aim of the present study was to examine the formation of germ layers, which are prerequisites for formation of the embryo proper and placenta, in invivo-derived (in vivo), partly in vitro-cultured (IVC), and SCNT ovine embryos. Embryos were derived as follows: In vivo embryos (n = 27) were flushed from the uterus on Days 7, 9, 11, and 13. For IVC embryos (n = 22) in vivo zygotes were flushed, followed by culture in the presence of 20% human serum, transfer to the uterus on Day 6, and flushing as in vivo embryos. SCNT embryos (n = 41) were produced by fusion of serum starved granulosa cells with enucleated oocytes, followed by activation, culture in SOF, transfer to the uterus on Day 6, and flushing as described for in vivo embryos. Recovered embryos were processed for light microscopy (LM) and transmission electron microscopy (TEM), and paraffin sections were immunohistochemically labelled for the germ layers: alpha-1-fetoprotein for potential endoderm, cytokeratin-8 for potential ectoderm, and vimentin for potential mesoderm. A consistent delay of the IVC and particularly the SCNT embryos was noted throughout all time points: On Days 7 and 9, differentiation of the inner cell mass into hypoblast and epiblast was evident in 7 out of 12 in vivo embryos, whereas this phenomenon was less prominent or absent in 9 out of 13 IVC and 13 out of 15 SCNT embryos. Furthermore, 6 of the IVC and 12 of the SCNT embryos lacked an identifiable embryonic disc. On Day 11, half of the in vivo embryos had initiated gastrulation, evidenced by localization of endoderm and mesoderm precursor cells between the hypoblast and the epiblast. This feature was noted in only a single IVC and in none of the SCNT embryos. On Day 13, all in vivo embryos had completed gastrulation including the formation of somatic and visceral mesoderm. This feature was noted in only 1 out of 3 IVC and in none of the SCNT embryos. Likewise, amniotic folds were seen in one third of the in vivo embryos at this stage, but not observed in any IVC or SCNT embryos. The immunohistochemical markers displayed the same cell lineage localization in all three groups of embryos, but a developmental delay in the IVC and in particular the SCNT embryos was evident. In conclusion, ovine IVC and SCNT embryos develop at a slower rate than in vivo embryos at least up until Day 13 of gestation.

40 EFFECT OF CHEMICAL ACTIVATION ON DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION PORCINE EMBRYOS DERIVED FROM NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 129
Author(s):  
G.-S. Im ◽  
J.-S. Seo ◽  
I.-S. Hwang ◽  
S.-W. Kim ◽  
H.-S. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Electric Pulse ◽  
Chemical Activation ◽  
Cell Mass ◽  
Cell Number ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate

Activation is one of key factors for improving developmental ability of pre-implantation nuclear transfer (NT) embryos. This study investigated the effect of chemical activation following fusion/activation on the development and apoptosis of pre-implantation porcine embryos derived from NT. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 42 to 44 h. Donor cells were prepared from a 35-day-old porcine fetus. Matured oocytes were enucleated and donor cells were introduced into the perivitelline space. Fusion/activation was conducted with two electric pulse of 1.2 kV/cm for 30 �s. Fused embryos were divided into four groups. The first one was the control without chemical activation; the other three groups were treated with thimerosal (0.2 mM for 10 min; T) and then with dithiothreitol (8 mM for 30 min; DTT), 6-dimethylaminopurine (2 mM for 3 h; 6-DMAP), or cycloheximide (10 �g/mL for 6 h; CH). Treated embryos were cultured in porcine zygote medium-3 (PZM-3) at 38.5�C under 5% CO2 in air for 6 days. Cleavage and blastocyst rate were determined on Days 3 and 6, respectively. Apoptosis was analyzed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay from day 1 to 7. Embryos treated with chemicals following fusion/activation showed significantly higher blastocyst rates compared to control embryos fused/activated by electric pulse alone (12.6% for control vs. 21.1% for DTT, 20.8% for 6-DMAP, 20.6% for CH; P < 0.05). Although total cell number of blastocysts showed no significant difference, the ratio of inner cell mass to trophectoderm was significantly higher (P < 0.05) in embryos with chemical activation than in those without it (11.9 vs. 19.4, 18.1, and 24.1%; P < 0.05). Occurrence of apoptosis was first observed on Day 3, but there was no significant difference among treatments until Day 6. It was significantly increased in embryos with chemical activation on Day 7 compared to control embryos (5.1 vs. 7.1, 7.8, and 7.8%; P < 0.05). These results indicate that chemical activation following fusion/activation could support significantly a higher blastocyst rate for pre-implantation porcine embryos derived from nuclear transfer; however, it can increase occurrence of apoptotic cells at blastocyst stage.

136 INVESTIGATION OF LEPTIN ON DEVELOPMENT OF MOUSE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 160
Author(s):  
A. C. Taskin ◽  
A. Kocabay ◽  
M. Yucel
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Development Rate ◽  
Mouse Embryos ◽  
In Vitro Development ◽  
Development Rates ◽  
Significant Difference ◽  
Vitro Development

Leptin is a hormone-like protein of 167 amino acids. As an adipocyte-related hormone it has an important role in weight regulation and physical fitness but also has effects on reproductive and other physiological mechanisms. The aim of the present study was to investigate the effects of different concentrations of leptin added to the culture media, the quality, in vitro development rate, and in vivo rate of mouse embryos. Superovulated CB6F1 (C57BL/6XBalb/c) hybrid female mice (5–6 weeks of age) were killed ~18 to 20 h after hCG administration. Single-cell embryos were flushed from the oviducts of the dead mice with human tubal fluid medium supplemented with HEPES and 3 mg mL–1 of BSA. They were cultured in Quinn's cleavage medium supplemented with 4 mg mL–1 of BSA in 5% CO2, 37°C until reaching 2-cell stage. The 2-cell embryos were randomly divided into 4 groups and cultured in Quinn's blastocyst medium supplemented with 4 mg mL–1 BSA + 0, 10, 50, and 100 ng mL–1 leptin (L0, L10, L50, and L100) in 5% CO2, 37°C until the blastocyst stage. Some of the developing blastocysts were used for differential staining for the inner cell mass and trophectoderm (TE) cells [Mallol et al. 2013 Syst. Biol. Reprod. Med. 59,117–122]. Some of them were transferred into pseudopregnant females (CD1) on the 2.5 to 3.5th days and kept until the 13.5th day of pregnancy for the in vivo development rate. The results were evaluated using one-way ANOVA with Bonferroni post-hoc test in SPSS 22.0. The P-values <0.05 were considered statistically significant. Each experiment was repeated at least 4 times. The blastocyst development rates of L0, L10, L50, and L100 were 92.57% (162/175), 97.16% (205/211), 97.80% (178/182), and 97.85% (182/186), respectively. The in vitro development rates were significantly higher in the L10, L50, and L100 compared with L0 (P < 0.05). The inner cell mass cells of L0, L10, L50, and L100 were 13.17, 14, 16, and 15.43. There was no significant difference between the groups in terms of inner cell mass cells (P > 0.05). The TE cells of L0, L10, L50, and L100 were 47, 56.4, 53.7, and 58.57, respectively. The TE numbers were significantly increased in the presence of L10 and L100 compared with L0 (P < 0.05). The in vivo development rates of L0, L10, L50, and L100 were 13.51% (5/37), 48.72% (19/39), 15.38% (6/39), and 41.03% (16/39), respectively. The in vivo development rates of L10 and L100 were significantly higher than for L0 and L50 (P < 0.05). The resorption rates of L0, L10, L50, and L100 were 10.8% (4/37), 30.8% (12/39), 12.8% (5/39), and 20.5% (8/39), respectively. There was no significant difference between the groups in terms of the resorption rates (P > 0.05). This study found that L10, L50, and L100 were supporting the development of the embryos in the in vitro culture. The L10, L50, and L100 significantly increased the total cell numbers. The L10 and L100 were particularly effective on the number of the TE cells. In conclusion, 10 and 100 ng mL–1 leptin have a positive effect on the in vitro, quality and in vivo development of the mouse embryo. Therefore, leptin seems to play an important role on the embryo development and in vivo development. Research supported by TUBITAK-113O223.

scholarly journals 175 ESTABLISHMENT OF PORCINE EMBRYONIC STEM CELL LINE DERIVED FROM IN VIVO BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 238 ◽  
Author(s):  
S.-A. Ock ◽  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
L.-Y. Shi ◽  
S.-L. Lee ◽  
...  
Keyword(s):  
Cell Line ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Feeder Layer ◽  
Es Cell ◽  
Stem Cell Line

A porcine embryonic stem (ES) cell line was established from an in vivo-flushed blastocyst. The present study evaluated the effectiveness of IVP, parthenotes and in vivo-produced embryos on establishment of an ES cell line. IVP blastocysts were produced from slaughterhouse ovaries based on the previously reported protocols (2000 Theriogenology 54, 787–797) with minor modifications. Parthenote blastocysts were produced by activation of oocytes matured in vitro with electric stimulation of 2 DC pulses at 2.0 kV/cm for 30 μsec in 0.3 M mannitol solution containing 100 μM CaCl2 and 100 μM MgCl2 in vivo blastocysts were recovered on Day 7 after AI (Day = 0) by flushing the uterus with D-PBS containing 10% FBS from three females. After removal of zona pellucida with 0.2% pronase, the blastocysts were subjected to immunosurgical treatment with 10% rabbit anti-pig serum to isolate the inner cell mass (ICM) as previously reported (1975 PNAS 72, 5099–5102). The ICM was seeded onto the feeder layer of STO which was inactivated by treatment with 10 μg/mL mitomycin for 2.5 h and cultured in DMEM with 0.1 mM β-mercaptoethanol, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 0.1 mM MEM non-essential amino-acid, 20 ng/mL rh-bFGF, 40 ng/mL rh-LIF, 0.03 mM adenosine, 0.03 mM guanosine, 0.03 mM cytidine, 0.03 mM uridine, 0.01 mM thymidine, and 15% FBS. The culture was maintained by changing the medium every day after initiation of ICM attachment onto the feeder layer. Any ES-like colonies were individually picked off the feed layer, dissected with 0.25% trypsin-0.02% EDTA for 3–5 min and reseeded on to new STO feed layer. Out of 140 blastocysts (25, in vivo; 55, IVF; 60, parthenotes) used, attaching rates of the ICMs onto the feeder layer were 88% (22/25, in vivo), 56.4% (31/55, IVF), and 58.3% (35/60, parthenotes). A total of 15 primary ES-like colonies was formed in in vivo (3, 12%), IVF (5, 9.1%), and parthenote (7, 11.7%). However, only one ES cell line from in vivo blastocyst was established, which was confirmed as positive by AP activity (Promega, Madison, WI, USA), and was maintained through four passages. In conclusion, for establishment of an ES cell line in pig, the in vivo blastocyst method is superior to currently available methods utilizing IVF or parthenotes. This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

88 Evaluation of embryo transfer results using embryos cryopreserved in ethylene glycol for 8 years or in glycerol for 30 years

2019 ◽  
Vol 31 (1) ◽  
pp. 170
Author(s):  
C. Acevedo ◽  
S. Romo ◽  
C. López ◽  
A. Cortes-Mcnealy ◽  
M. I. Cruz-González ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Pregnancy Rate ◽  
Liquid Nitrogen ◽  
Embryo Transfer ◽  
Animal Health ◽  
Pregnancy Rates ◽  
Future Research ◽  
Embryo Viability ◽  
Exact Test ◽  
Significant Difference

Various permeating cryoprotectants, such as glycerol and ethylene glycol, have been used in the cryopreservation of embryos to help maintain cellular viability during indefinite and prolonged periods of storage in liquid nitrogen. The objective of this study was to compare the efficiency of glycerol (G) and ethylene glycol (EG) after storage in liquid nitrogen for a considerable period of time before transfer. The work was carried out in Palenque, Chiapas, Mexico. A total of 50 embryos were transferred, 24 Brahman (G) cryopreserved in the 1990s and 26 Brangus (EG) cryopreserved in 2010. Synchronous recipients were selected based on 3 characteristics: body condition (5-7, scale of 1-9), reproductive health, and multiparity. Recipient cows (n=62) were synchronized using a FTET protocol as follows. On Day 0, cows received a progesterone intravaginal device (CIDR) and 2mg of oestradiol benzoate IM. On day 8, the CIDR was removed and all cows received 25mg of dinoprost tromethamine (Lutalyse, Pfizer Animal Health, Montreal, Quebec, Canada), 200IU of eCG, and 0.5mg oestradiol cipionate IM. Day 10 was considered the day of oestrus and embryos were transferred (n=50) to the ipsilateral uterine horn of those recipients with a corpus luteum greater than 1.5cm in diameter on Day 17. The G embryos were produced with 4 bulls whereas the EG embryos were produced with 6 different bulls. The G straws were thawed for 12s in the air plus 12s in 20°C water. Embryos were immersed for 8min in a thawing solution containing 1.0M sucrose (ViGRO One-Step) and then transferred to holding medium (ViGRO Holding) for rehydration before loading into straws for embryo transfer. The EG embryos were thawed by allowing the straws to stand in air for 10s and then immersing them in a 30°C water bath for 10s and were transferred immediately. Pregnancy diagnosis 35 days after the transfer revealed 19 pregnancies of 50 embryos transferred (38%), distributed as 46% embryos in EG (12 pregnant of 26 transferred) and 29% embryos in G (7 pregnant of 24). A Fisher’s exact test was performed showing that no significant difference existed between groups (P&gt;0.05). There was no effect of bull on pregnancy rates, and Brahman breed results by individual bull were 5 pregnancies of 13 (38%), 2 of 6 (33%), 0 of 4 (0%), and 0 of 1 (0%) for bulls I to IV, respectively. Pregnancy rate by Brangus bulls were 6 pregnancies of 7 (86%), 2 of 3 (67%), 2 of 4 (50%), 2 of 4 (50%), 0 of 4 (0%), and 0 of 3 (0%) for bulls 1 to 6, respectively. It is important to remember that the embryos cryopreserved in G remained in the nitrogen tank for more than 30 years, whereas the embryos cryopreserved in EG remained stored in liquid nitrogen for less than 10 years. Although pregnancy rate was numerically lower with Brahman embryos stored in G, pregnancy rates were considered acceptable considering the length of storage. Future research is needed with greater numbers and different breeds to determine whether G or EG will consistently produce higher embryo viability and pregnancies after storage for considerable periods before transfer.

Ratio and number of inner cell mass and trophoblast cells of in vitro and in vivo produced porcine embryos

1995 ◽  
Vol 43 (1) ◽  
pp. 304 ◽  
Author(s):  
D. Rath ◽  
H. Niemann ◽  
T. Tao ◽  
M. Boerjan
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblast Cells

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

P–530 The use of wide thresholds for detecting intermediate chromosomal CNV up to 80% doesn’t improve PGT-A ability to discriminate true mosaic from uniformly aneuploid embryos

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
L Girardi ◽  
M Serdaroğulları ◽  
C Patassini ◽  
S Caroselli ◽  
M Costa ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
False Negative ◽  
Cell Mass ◽  
Categorical Variables ◽  
P Value ◽  
Sequencing Data ◽  
Preimplantation Genetic Testing ◽  
Exact Test ◽  
Institutional Review ◽  
Wide Range

Abstract Study question What is the effect of varying diagnostic thresholds on the accuracy of Next Generation Sequencing (NGS)-based preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer When single trophectoderm biopsies are tested, the employment of 80% upper threshold increases mosaic calls and false negative aneuploidy results compared to more stringent thresholds. What is known already Trophectoderm (TE) biopsy coupled with NGS-based PGT-A technologies are able to accurately predict Inner Cell Mass’ (ICM) constitution when uniform whole chromosome aneuploidies are considered. However, minor technical and biological inconsistencies in NGS procedures and biopsy specimens can result in subtle variability in analytical results. In this context, the stringency of thresholds employed for diagnostic calls can lead to incorrect classification of uniformly aneuploid embryos into the mosaic category, ultimately affecting PGT-A accuracy. In this study, we evaluated the diagnostic predictivity of different aneuploidy classification criteria by employing blinded analysis of chromosome copy number values (CNV) in multifocal blastocyst biopsies. Study design, size, duration The accuracy of different aneuploidy diagnostic cut-offs was assessed comparing chromosomal CNV in intra-blastocysts multifocal biopsies. Enrolled embryos were donated for research between June and September 2020. The Institutional Review Board at the Near East University approved the study (project: YDU/20l9/70–849). Embryos diagnosed with uniform chromosomal alterations (single or multiple) in their clinical TE biopsy (n = 27) were disaggregated into 5 portions: the ICM and 4 TE biopsies. Overall, 135 specimens were collected and analysed. Participants/materials, setting, methods Twenty-seven donated blastocysts were warmed and disaggregated in TE biopsies and ICM (n = 135 biopsies). PGT-A analysis was performed using Ion ReproSeq PGS kit and Ion S5 sequencer (ThermoFisher). Sequencing data were blindly analysed with Ion-Reporter software. Intra-blastocyst comparison of raw NGS data was performed employing different thresholds commonly used for aneuploidy classification. CNV for each chromosome were reported as aneuploid according to 70% or 80% thresholds. Categorical variables were compared using Fisher’s exact test. Main results and the role of chance In this study, a total of 50 aneuploid patterns in 27 disaggregated embryos were explored. Single TE biopsy results were considered as true positive when they displayed the same alteration detected in the ICM at levels above the 70% or 80% thresholds. Alternatively, alterations detected in the euploid or mosaic range were considered as false negative aneuploidy results. When the 70% threshold was applied, aneuploidy findings were confirmed in 94.5% of TE biopsies analyzed (n = 189/200; 95%CI=90.37–37.22), while 5.5% showed a mosaic profile (50–70%) but uniformly abnormal ICM. Positive (PPV) and negative predictive value (NPV) per chromosome were 100.0% (n = 189/189; 95%CI=98.07–100.00) and 99.5% (n = 2192/2203; 95%CI=99.11–99.75) respectively. When the upper cut-off was experimentally placed at 80% of abnormal cells, a significant decrease (p-value=0.0097) in the percentage of confirmed aneuploid calls was observed (86.5%; n = 173/200; 95%CI=80.97–90.91), resulting in mosaicism overcalling, especially in the high range (50–80%). Less stringent thresholds led to extremely high PPV (100.0%; n = 173/173; 95%CI=97.89–100.00), while NPV decreased to 98.8% (n = 2192/2219; 95%CI=98.30–99.23). Furthermore, no additional true mosaic patterns were identified with the use of wide range thresholds for aneuploidy classification. Limitations, reasons for caution This approach involved the analysis of aneuploidy CNV thresholds at the embryo level and lacked from genotyping-based confirmation analysis. Moreover, aneuploid embryos with known meiotic partial deletion/duplication were not included. Wider implications of the findings: The use of wide thresholds for detecting intermediate chromosomal CNV up to 80% doesn’t improve PGT-A ability to discriminate true mosaic from uniformly aneuploid embryos, lowering overall diagnostic accuracy. Hence, a proportion of the embryos diagnosed as mosaic using wide calling thresholds may actually be uniformly aneuploid and inadvertently transferred. Trial registration number N/A

P–131 Significance of the phenomenon of blastomere exclusion from compaction: Its relation to irregular cleavage, blastocyst development rate, and pregnancy rate

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Watanabe ◽  
M Tomida ◽  
S Suzuki ◽  
Y Matsuda ◽  
K Yoshikai ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Chromosome Analysis ◽  
Development Rate ◽  
Pregnancy Rates ◽  
Blastocyst Transfer ◽  
Blastocyst Development ◽  
Miscarriage Rate ◽  
Single Blastocyst Transfer ◽  
Significant Difference ◽  
Irregular Cleavage

Abstract Study question When does blastomere exclusion from compaction increase and what effect does it have on the embryo? Summary answer More blastomere were excluded from compaction in embryos with irregular cleavage, resulting in lower blastocyst development rates, but no decrease in pregnancy rates at transfer. What is known already It has been reported that many of the chromosome analysis results of blastomere excluded from compaction were aneuploid, and pointed out that this exclusion may be related to the repair of blastocyst euploidy, but the effect of the number of excluded blastomere has not been reported. Study design, size, duration This is a retrospective study of 578 embryos that developed into morula with time-lapse monitoring by EmbryoScope (Vitrolife) in 2018–2019. Participants/materials, setting, methods The target embryos were classified into two groups: embryos with normal first and second cleavage (normal cleavage group) and embryos with irregular cleavage (dynamics of one cell dividing into three or more cells), called “direct cleavage”, at either cleavage (DC group), and the number of blastomere excluded from compaction during morula formation was recorded and compared. The blastocyst development rate and single blastocyst transfer pregnancy rates of the two groups were compared. Main results and the role of chance There are 286 in the normal cleavage group and 292 in the DC group. The mean number of excluded blastomere was 0.76 and 3.55, respectively, which was significantly higher in the DC group (P &lt; 0.01). Good blastocyst (Gardner classification 4 or higher) development rate was 84.5% (239/283) and 65.8% (181/275), respectively, and high grade blastocyst (Gardner classification BB or higher) development rate was 43.9% (105/239) and 14.9% (27/181) of them, both significantly higher in the normal cleavage group (P &lt; 0.01). The single blastocyst transfer pregnancy rates were 31.6% (25/79) and 32.4% (11/34), and the miscarriage rates were 24.0% (6/25) and 27.3% (3/11), respectively, neither was there a significant difference between the two groups. So, direct cleavage increased the number of blastomere excluded from compaction, decreased the rate of morula to good blastocyst development and reduced blastocyst grade, but did not affect blastocyst transfer pregnancy rate and miscarriage rate. Limitations, reasons for caution Please note that all target embryos must have developed into morula or larger (embryos that did not develop into morula will not be included in the study). Wider implications of the findings: Severe chromosomal aberrant blastomeres formed by direct cleavage were excluded from compaction, and the blastocyst development rate decreased due to a decrease in the amount of viable cells, but it is suggested that this blastomere exclusion mechanism is not related to euploidy after blastocyst development. Trial registration number Not applicable

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