97 Loss of aggregation capacity of bovine invitro-produced embryos and blastocyst-derived trophoblasts from Day 6 of development

2020 ◽  
Vol 32 (2) ◽  
pp. 174
Author(s):  
V. Alberio ◽  
M. Yauri Felipe ◽  
D. Salamone
Keyword(s):  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Culture Conditions ◽  
Sodium Pyruvate ◽  
Trophoblastic Cells ◽  
Stereoscopic Observation ◽  
Fetal Bovine ◽  
Humidified Air ◽  
Oviductal Fluid

Embryo aggregation consists of placing more than one zona-free (ZF) embryo in contact during development to obtain a unique structure. It has been reported in different species that aggregated cloned embryos show certain benefits compared with nonaggregated embryos. One way to obtain these benefits in IVF embryos would be to generate a transient chimera by the introduction of trophoblastic cells. Bovine trophoblastic cells can be obtained by embryo bisection of blastocysts, cutting asymmetrically to use trophoblasts (Tr) for aggregation and leaving aside the portion that contains the inner cell mass (ICM). Taking all this into account, the objectives of this work are to study the aggregation of Tr at different days of development and to determine the appropriate time of aggregation. To this aim, cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10µgmL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. We performed IVF with 16×106 spermatozoa per mL for 5h. Afterwards, presumptive zygotes were cultured in synthetic oviductal fluid (SOF) for 7 days in a humidified atmosphere at 38.5°C, 5% O2, 5% CO2, and 90% N2. In Experiment 1, embryo bisection of Day 7 blastocysts was performed manually under stereoscopic observation with a microblade to obtain Tr. These were aggregated, with the bisected part containing the ICM (n=22) or with ZF embryos of Days 4 (n=23), 5(n=25), or 6 (n=22) and blastocysts (n=25), and placed in microwells in a 100-μL SOF drop covered by mineral oil (Gambini et al. 2012 Biol. Reprod. 87, 15; https://doi.org/10.1095/biolreprod.112.098855). In Experiment 2, ZF synchronous whole embryos were aggregated in microwells at different developmental days: Day 3 (n=18), 4 (n=18), 5 (n=47), 6 (n=48), and 7 (n=45). In both experiments, aggregation was assessed at Day 8. In Experiment 1, no aggregation was observed between the Tr and the embryos or the bisected ICM. Experiment 2 showed embryo aggregation on Days 3 (55%), 4 (27%), and 5 (61%), whereas on Days 6 and 7 no aggregation was observed. According to these results, we can conclude that, in our culture conditions, Tr obtained by blastocyst bisection have no capacity for aggregation. Day 6 and 7 whole ZF embryos also do not aggregate. As a general conclusion, there is a period from Days 0-5 of the invitro development of bovine embryos in which aggregation is possible. Aggregation of blastocyst-derived Tr to cloned or high-value IVF embryos, aiming for quality improvement, is not an effective strategy.

scholarly journals 2POSTHATCHING DEVELOPMENT SYSTEM: A NOVEL IN VITRO CULTURE OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 123 ◽  
Author(s):  
D.O. Brandão ◽  
G. Vajta ◽  
P. Maddox-Hyttel ◽  
D. Stringfellow ◽  
P. Lövendahl ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture System ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Fetal Bovine Serum ◽  
Cell Mass ◽  
Embryo Morphology ◽  
Fetal Bovine

Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683–700). On Day 8, degenerated embryos were removed from each well and 400L of modified culture medium (SOFaaci plus 0.5% glucose and 10% fetal bovine serum) were added. At Day 11, hatched blastocysts were selected by scoring them as Quality 1 (Q1: >1.0mm, clear trophoblast, compact inner cell mass), Quality 2 (Q2: 0.5mm, dark spots in the trophoblast, less compact inner cell mass), or Quality 3 (Q3: <0.5mm, many dark spots in the trophoblast, spread inner cell mass). The resulting 304 blastocysts in 12 replicates were then loaded into 15mm×1.2 gel tunnels of 2.4% agarose in PBS, supplemented with either 5% (Agar5) or 10% (Agar10) fetal bovine serum, covered with the modified culture medium, and then incubated at 38.5°C in 5% CO2, 5% O2, 90% N2. Embryo morphology and length were evaluated using a stereomicroscope on Days 12, 13, 14 and 15. On Day 14, 75 embryos were removed, biopsed (1mm) for sex determination of each embryo, and processed for light and transmission electron microscopy. Qualitative and quantitative data were analyzed by χ2 test and GLM procedure of SAS, respectively, with P level of 0.05. A total of 170 embryos (56% of total) initiated elongation. This percentage was higher (LSmeansSD, n=12; P<0.05) in Agar10 v. Agar5 in both Q1 (889 v. 637), Q2 (667 v. 485) and Q3 embryos (529 v. 278). Mean embryo length (mm; LSmeansSEM) on Day 13 was higher (P<0.05) in Q1 (2.10.2, n=49) and Q2 (1.71.4, n=98) than Q3 (1.20.3, n=23). On Day 14, Q1 embryos (3.50.2) were longer (P<0.01) than Q2 and Q3 embryos (2.70.1 and 2.00.3). On Day 15, Q1, Q2 and Q3 embryos (4.40.5, n=24, 4.00.3, n=45 and 2.90.6, n=14, respectively) had similar length, probably influenced by the low number of Q3 embryos. The percentage of males was higher (P<0.001) in Q1 (95%; n=40), but similar in Q2 (39%; n=26) and Q3 (71%; n=7). Light microscopy confirmed hypoblast and epiblast formation. Ultrastructural analysis revealed that the latter had penetrated the trophoblast (Rauber’s layer), forming an embryonic disc including many degenerative cells. In conclusion, this culture system represents the first model for rapid growth, elongation, and initial differentiation of bovine posthatching embryos.

Berberine alleviates LPS-induced apoptosis, oxidation, and skewed lineages during mouse preimplantation development

2022 ◽  
Author(s):  
Xiaosu Miao ◽  
Wei Cui
Keyword(s):  
Inner Cell Mass ◽  
Polycystic Ovary ◽  
Cell Mass ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Embryonic Cells ◽  
Preimplantation Embryo Development ◽  
New Treatment ◽  
Induced Apoptosis

Abstract Female infertility is a heterogeneous disorder with a variety of complex causes, including inflammation and oxidative stress, which are also closely associated with the pathogenesis of Polycystic Ovary Syndrome (PCOS). As a new treatment for PCOS, berberine (BER), a natural compound from Berberis, has been clinically applied recently. However, the mechanisms underlying the association between BER and embryogenesis are still largely unknown. In this study, effects of BER on preimplantation development was evaluated by using both normal and inflammatory culture conditions induced by lipopolysaccharide (LPS) in the mouse. Our data first suggest that BER itself (25 nM) does not affect embryo quality or future developmental potency, moreover, it can effectively alleviate LPS-induced embryonic damage by mitigating apoptosis via ROS−/caspase-3-dependent pathways and by suppressing pro-inflammatory cytokines via inhibition of NF-κB signaling pathway during preimplantation embryo development. In addition, skewed cell lineage specification in inner cell mass (ICM) and primitive endoderm (PE) caused by LPS can also be successfully rescued with BER. In summary, these findings for the first time demonstrate the non-toxicity of low doses of BER and its anti-apoptotic and anti-oxidative properties on embryonic cells during mammalian preimplantation development.

scholarly journals Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Young-Ho Choi ◽  
Pablo Ross ◽  
Isabel C Velez ◽  
B Macías-García ◽  
Fernando L Riera ◽  
...  
Keyword(s):  
High Glucose ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Primitive Endoderm ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Effect Of Glucose

Equine embryos developin vitroin the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31–46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively forPOU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did.GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation inin vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

From fibroblasts and stem cells: implications for cell therapies and somatic cloning

2005 ◽  
Vol 17 (2) ◽  
pp. 125 ◽  
Author(s):  
Wilfried A. Kues ◽  
Joseph W. Carnwath ◽  
Heiner Niemann
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Human Embryos ◽  
Somatic Stem Cells ◽  
Primary Fibroblast ◽  
Cell Therapies

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
D. B. Koo ◽  
J. S. Park ◽  
K. K. Lee ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Developmental Rate ◽  
Treated Group ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Frozen Semen ◽  
Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

186 LASER CAPTURE MICRODISSECTION FOR GENE EXPRESSION ANALYSIS OF INNER CELL MASS AND TROPHOBLAST FROM BOVINE BLASTOCYSTS

2011 ◽  
Vol 23 (1) ◽  
pp. 194
Author(s):  
M. Filliers ◽  
W. de Spiegelaere ◽  
L. J. Peelman ◽  
K. Goossens ◽  
C. Burvenich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Serial Sections ◽  
Trophoblastic Cells ◽  
Keratin 18 ◽  
Laser Capture

Isolation of pure inner cell mass (ICM) and trophoblast samples from a single blastocyst is necessary to obtain accurate information on the transcriptome of these cells. Microsurgical techniques have been described to separate the ICM and trophoblast, but unfortunately, contamination of the ICM cell population with trophoblastic cells is inevitable with these methods. Alternatively, immunosurgery has been described as a valuable technique to obtain a pure ICM sample, although this technique seems to alter the normal gene expression pattern. Laser capture microdissection (LCM) provides the possibility of isolating small tissue fractions from heterogeneous tissue sections, without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. In this study, a protocol is described for the application of LCM to isolate homogeneous ICM and trophoblast samples from single bovine blastocysts for downstream gene expression analysis. The absence of contaminating trophoblastic fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered a trophoblast-specific marker in bovine blastocysts. Expanded blastocysts were produced by routine in vitro methods described by (Vandaele et al. 2010 Reproduction 139, 505–511) and fixed in a modified methacarn solution for 24 h. After fixation, the blastocysts were embedded in RNase-free soluble agarose 2%, processed in an STP 420D Tissue Processor, embedded in paraffin, cut in serial sections, and adhered to glass slides, followed by deparaffinization in xylene and staining of the sections with 0.1% cresyl violet in a 85% ethanol solution. Laser capture microdissection was performed as described previously by (De Spiegelaere et al. 2008 Anal. Biochem. 382, 72–74). The ICM was isolated by placing the same cap over 3 to 4 serial sections of one blastocyst. Subsequently, the same procedure was performed with a second cap to isolate the trophoblast. Total RNA was isolated from the LCM-derived ICM and trophoblast on the caps and converted into cDNA. Gene-specific primers for KRT18 (5′-GCAGACCGCTGAGATAGGA-3′ and 5′-GCATATCGGGCCTCCACTT-3′) and for 18S rRNA, a commonly used reference gene (5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′), were used and PCR was carried out. Expression of the control gene 18S rRNA was readily detectable in all cell samples. Keratin 18 was detectable in LCM-derived trophoblast, but was absent in the LCM-derived ICM cells, indicative of the successful isolation of ICM cells without contaminating trophoblastic cells. This study demonstrates a novel approach for the application of LCM on small tissue samples that are difficult to handle and which can be used for molecular analysis of specific cell lineages within embryos of different species. Supported by the Fund for Scientific Research–Flanders, Belgium, aspirant 1.1.477.07N00.

102. THE EFFECT OF INSULIN ON EMBRYONIC STEM CELL PROGENITOR CELLS IN THE MOUSE BLASTOCYST

2009 ◽  
Vol 21 (9) ◽  
pp. 21
Author(s):  
J. M. Campbell ◽  
I. Vassiliev ◽  
M. B. Nottle ◽  
M. Lane
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Human Embryos ◽  
Cell Stage ◽  
Stage Of Development

Human ESCs are produced from embryos donated at the mid-stage of pre-implantation development. This cryostorage reduced viability. However, it has been shown that this can be improved by the addition of growth factors to culture medium. The aim of the present study was to examine whether the addition of insulin to embryo culture medium from the 8-cell stage of development increases the number of ES cell progenitor cells in the epiblast in a mouse model. In vivo produced mouse zygotes (C57Bl6 strain) were cultured in G1 medium for 48h to the 8-cell stage, followed by culture in G2 supplemented with insulin (0, 0.17, 1.7 and 1700pM) for 68h, at 37 o C , in 5% O2, 6%CO2, 89% N2 . The number of cells in the inner cell mass (ICM) and epiblast was determined by immunohistochemical staining for Oct4 and Nanog. ICM cells express Oct4, epiblast cells express both Oct4 and Nanog. The addition of insulin at the concentrations examined did not increase the ICM. However, at 1.7pM insulin increased the number of epiblast cells (6.6±0.5 cells vs 4.1±0.5, P=0.001) in the ICM, which increased the proportion of the ICM that was epiblast (38.9±3.7% compared to 25.8±3.4% in the control P=0.01). This indicates that the increase in the epiblast is brought about by a shift in cell fate as opposed to an increase in cell division. The effect of insulin on the proportion of cells in the epiblast was investigated using inhibitors of phosphoinositide3-kinase (PI3K) (LY294002, 50µM); one of insulin's main second messengers, and p53 (pifithrin-α, 30µg/ml); a pro-apoptotic protein inactivated by PI3K. Inhibition of PI3K eliminated the increase caused by insulin (4.5±0.3 cells versus 2.2±0.3 cells, P<0.001), while inhibition of p53 increased the epiblast cell number compared to the control (7.1±0.8 and 4.1±0.7 respectively P=0.001). This study shows that insulin increases epiblast cell number through the activation of PI3K and the inhibition of p53, and may be a strategy for improving ESC isolation from human embryos.

scholarly journals Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos

Reproduction ◽  
1995 ◽  
Vol 104 (2) ◽  
pp. 251-258 ◽  
Author(s):  
T. Tao ◽  
B. Reichelt ◽  
H. Niemann
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblastic Cells

scholarly journals A preliminary note upon the question of the nutrition of the early embryo, with special reference to the guinea-pig and man

1905 ◽  
Vol 76 (508) ◽  
pp. 164-165 ◽  
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Early Embryo ◽  
Uterine Horn ◽  
Dense Layer ◽  
Preliminary Note ◽  
Trophoblastic Cells ◽  
Decidual Cells ◽  
Uterine Secretion

During the progress of a research into the earliest implantation of the embryo of the guinea-pig, I have been particularly struck with the way in which the nutrition of the embryo is anticipated and provided for during the time it remains free in the uterine horn. The so-called yolk-granules of the ovum are obviously insufficient to provide for the growth of the embryo to the stage prior to differentiation of the inner cell-mass, to which it attains during the five or six days which elapse before it comes into contact with the maternal tissues. It is clear that it must derive nourishment from the medium in which it lies―the product of the secretion of the uterine or other glands, which, during the period of pro-œstrum, exhibit such marked activity. I suggest that this secretion, which consists of mucus and probably albumin, is assimilated by the embryo after having undergone a process of digestion, the result of a secretory activity on the part of the outermost cells of the embryo―the cells of the Trophoblast. This suggestion I base on my observations in the guinea-pig, where I am able to demonstrate a breaking-down of maternal cells before the Trophoblastic cells are in actual contact; likewise in human placentation where a more or less dense layer of fibrin and broken-down leucocytes and decidual cells, the result of Trophoblastic activity, affords a barrier interposed between the invading Trophoblastic cells and the Decidua. This layer I purpose naming the “Protective Layer.” Looked at from a comparative point of view, there is in all probability a close analogy between the uterine secretion of mammals, and the secretion of the oviducts of the lower vertebrata. In the case of birds the analogy is very striking, on account of the direct and important share in the nutrition of the embryo afforded by this secretion, commonly known as the white of the egg. In the case of the frog the ovum receives in its passage down the oviduct, corresponding to the uterine horn of the guinea-pig, a coating of mucus and probably albumin, comparable to the uterine secretion referred to above; when it reaches the water and becomes fertilised, this swells up by absorption, forming a gelatinous covering. The embryo for nutriment depends upon the yolk contained in the ovum before fertilisation, upon the covering of mucus and probably albumin, and lastly upon the water in which it lies. In certain mammals, as, for example, the rabbit and the mole, a distinct gelatinous envelope is described as surrounding the embryo before implantation occurs; this envelope is, I suggest, possibly due to some digestive action of the cells of the Trophoblast upon the adjacent medium, producing a form of coagulation.

Minimally invasive preimplantation genetic testing using blastocyst culture medium

2019 ◽  
Vol 34 (7) ◽  
pp. 1369-1379 ◽  
Author(s):  
Jiao Jiao ◽  
Bei Shi ◽  
Matthew Sagnelli ◽  
Dalei Yang ◽  
Yaxin Yao ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Minimally Invasive ◽  
Research And Development ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Development Program ◽  
Blastocyst Culture ◽  
Preimplantation Genetic Testing ◽  
Time Reduction

Abstract STUDY QUESTION Is minimally invasive chromosome screening (MICS) using blastocyst culture medium (BCM) sufficiently fast and accurate for preimplantation genetic testing (PGT) SUMMARY ANSWER A new assay for MICS, named MICS-Inst achieved high-resolution, comprehensive chromosome ploidy detection using BCM. WHAT IS KNOWN ALREADY BCM is a viable source of genomic DNA for use in PGT. STUDY DESIGN, SIZE, DURATION Forty-one vitrified blastocysts donated by 22 couples known to carry a chromosome rearrangement and 21 vitrified blastocysts donated from 8 couples with normal karyotypes were used in this study. Good-quality blastocysts, defined as Day 5 and Day 6 embryos ≥ BB (AA, AB, BA, BB) based on the Gardner system were used for analysis. Recruitment took place from May 2018 to August 2018. We performed PGT for structural rearrangements (PGT-SR) on 41 BCM, trophectoderm (TE) biopsy and blastocyst-stage embryo (BE) samples as well as PGT for aneuploidies (PGT-A) on 21 BCM, TE biopsy and BE samples. PARTICIPANTS/MATERIALS, SETTING, METHODS We made several significant modifications to the BCM composition (mixing blastocoel fluid and spent blastocyst medium) as well as the pre-existing multiple annealing and looping-based amplification cycles (MALBAC) techniques and library generation procedures. The design of a quasilinear preamplification (Pre-AMP) primer and AMP primers 1 and 2 enables the preparation of a next-generation sequencing library after the exponential amplification stage by introducing the Illumina P5 and P7 primers into the final products, which are then ready for sequencing. Sequencing was performed on the Illumina Hiseq 2500 platform with 2.0 Mb raw reads generated for each sample. MAIN RESULTS AND THE ROLE OF CHANCE For PGT-A, BCM and TE biopsy samples showed 90% and 86% clinical concordance with the corresponding BE samples, respectively. In addition, both BCM and TE biopsy samples showed 76% karyotype concordance with the corresponding BE samples. For PGT-SR, we successfully obtained ploidy information for all 23 chromosomes with the exception of any rearrangements involving the Y chromosome. Both BCM and TE biopsy samples showed 100% clinical concordance with the corresponding BE samples in detecting chromosomal rearrangements. BCM and TE biopsy samples showed 90% and 100% karyotype concordance with the corresponding BE samples, respectively. Additionally, no statistically significant differences were detected in the aforementioned values of the BCM and TE biopsy samples in either PGT-A or PGT-SR (P > 0.05). Moreover, we achieved accurate quantification of segmental abnormalities using BCM samples. In addition, MICS-Inst reduced the number of steps required for library preparation through the use of new primer designs, resulting in an overall time reduction of 7.5 h. This time reduction allows for the performance of fresh blastocyst transfers. LIMITATIONS, REASONS FOR CAUTION The main limitation is that BE, rather the inner cell mass, was used as the standard to evaluate the chromosome screening results. WIDER IMPLICATIONS OF THE FINDINGS These results show that MICS-Inst is effective in procedure and precision for PGT, and that it is possible to achieve fresh blastocyst transfer following PGT. The implications are significant, as these findings may lead to minimally invasive PGT methods in the future. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (No. 81671423 and No. 81402130), the National Key Research and Development Program of China (No. 2018YFC1003100), Liaoning Provincial Key Research and Development Program (No. 2018225090), the Fok Ying Tung Education Foundation (No. 151039) and Distinguished Talent Program of Shengjing Hospital (No. ME76). No competing interests declared.

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