Name: chromosome screening of human preimplantation embryos by using spent culture medium: sample collection and chromosomal ploidy analysis
Uploaded: 2021-09-07T14:00:00
Description: Watch this Scientific Journal Video about Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis at JoVE.com

The authors would like to thank Shiping Bo and Shujie Ma for their assistance in NGS data analysis. Funding: this work was supported by the National Key Research and Development Program (Grant No. 2018YFC1003100).

Ethical permission was acquired from the Ethics Committee of Peking University Third Hospital.
1. Preparation
NOTE: The required materials and equipment are listed in Table of Materials.
<ol>
	<li>Reagents
	<ol>
		<li>Prewarm and equilibrate (balanced) 20-30 &#181;L of gamete medium/fertilization medium and cleavage/blastocyst-stage culture medium (covered with mineral oil) and hyaluronidase (in a tightly capped tube) at 37 &#176;C, 5% CO2</s...

<ol>
	<li>Barlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+pregnancy+loss+and+obstetrical+risk+after+in-vitro+fertilization+and+embryo+replacement.">Early pregnancy loss and obstetrical risk after in-vitro fertilization and embryo replacement.</a> Human Reproduction. 3 (5), 671-675 (1988).</li><li>Munne, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+abnormalities+and+their+relationship+to+morphology+and+development+of+human+embryos.">Chromosome abnormalities and their relationship to morphology and development of human embryos.</a> Reproductive BioMedicine Online. 12 (2), 234-253 (2006).</li><li>Harton, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+effect+of+maternal+age+on+implantation+after+preimplantation+genetic+diagnosis+with+array+comparative+genomic+hybridization.">Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization.</a> Fertility and Sterility. 100 (6), 1695-1703 (2013).</li><li>Hodes-Wertz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+recurrent+miscarriage+is+caused+mostly+by+aneuploid+embryos.">Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos.</a> Fertility and Sterility. 98 (3), 675-680 (2012).</li><li>Keltz, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+genetic+screening+(PGS)+with+Comparative+genomic+hybridization+(CGH)+following+day+3+single+cell+blastomere+biopsy+markedly+improves+IVF+outcomes+while+lowering+multiple+pregnancies+and+miscarriages.">Preimplantation genetic screening (PGS) with Comparative genomic hybridization (CGH) following day 3 single cell blastomere biopsy markedly improves IVF outcomes while lowering multiple pregnancies and miscarriages.</a> Journal of Assisted Reproduction and Genetics. 30 (10), 1333-1339 (2013).</li><li>Scott, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastocyst+biopsy+with+comprehensive+chromosome+screening+and+fresh+embryo+transfer+significantly+increases+in+vitro+fertilization+implantation+and+delivery+rates:+a+randomized+controlled+trial.">Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.</a> Fertility and Sterility. 100 (3), 697-703 (2013).</li><li>Forman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fertilization+with+single+euploid+blastocyst+transfer:+a+randomized+controlled+trial.">In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial.</a> Fertility and Sterility. 100 (1), 100-107 (2013).</li><li>Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+single+blastocysts+for+fresh+transfer+via+standard+morphology+assessment+alone+and+with+array+CGH+for+good+prognosis+IVF+patients:+results+from+a+randomized+pilot+study.">Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study.</a> Molecular Cytogenetics. 5 (1), 24 (2012).</li><li>Cimadomo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Biopsy+on+Human+Embryo+Developmental+Potential+during+Preimplantation+Genetic+Diagnosis.">The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis.</a> BioMed Research International. 2016, 7193075 (2016).</li><li>Scott, R. T., Upham, K. M., Forman, E. J., Zhao, T., Treff, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage-stage+biopsy+significantly+impairs+human+embryonic+implantation+potential+while+blastocyst+biopsy+does+not:+a+randomized+and+paired+clinical+trial.">Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial.</a> Fertility and Sterility. 100 (3), 624-630 (2013).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastomere+biopsy+influences+epigenetic+reprogramming+during+early+embryo+development,+which+impacts+neural+development+and+function+in+resulting+mice.">Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice.</a> Cellular and Molecular Life Sciences. 71 (9), 1761-1774 (2014).</li><li>Zhao, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+epigenetic+modification+in+murine+brain+tissues+of+offspring+from+preimplantation+genetic+diagnosis+blastomere+biopsies.">Aberrant epigenetic modification in murine brain tissues of offspring from preimplantation genetic diagnosis blastomere biopsies.</a> Biology of Reproduction. 89 (5), 117 (2013).</li><li>Zeng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+genetic+diagnosis+(PGD)+influences+adrenal+development+and+response+to+cold+stress+in+resulting+mice.">Preimplantation genetic diagnosis (PGD) influences adrenal development and response to cold stress in resulting mice.</a> Cell and Tissue Research. 354 (3), 729-741 (2013).</li><li>Palini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+DNA+in+human+blastocoele+fluid.">Genomic DNA in human blastocoele fluid.</a> Reproductive BioMedicine Online. 26 (6), 603-610 (2013).</li><li>Gianaroli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastocentesis:+a+source+of+DNA+for+preimplantation+genetic+testing.+Results+from+a+pilot+study.">Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study.</a> Fertility and Sterility. 102 (6), 1692-1699 (2014).</li><li>Stigliani, S., Anserini, P., Venturini, P. L., Scaruffi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+content+in+embryo+culture+medium+is+significantly+associated+with+human+embryo+fragmentation.">Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.</a> Human Reproduction. 28 (10), 2652-2660 (2013).</li><li>Stigliani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+in+Day+3+embryo+culture+medium+is+a+novel,+non-invasive+biomarker+of+blastocyst+potential+and+implantation+outcome.">Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome.</a> Molecular Human Reproduction. 20 (12), 1238-1246 (2014).</li><li>Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium-Based+Noninvasive+Preimplantation+Genetic+Diagnosis+for+Human+&#945;-Thalassemias-SEA.">Medium-Based Noninvasive Preimplantation Genetic Diagnosis for Human &#945;-Thalassemias-SEA.</a> Medicine. 94 (12), e669 (2015).</li><li>Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+chromosome+screening+of+human+embryos+by+genome+sequencing+of+embryo+culture+medium+for+in+vitro+fertilization.">Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.</a> Proceedings of the National Academy of Sciences. 113 (42), 11907-11912 (2016).</li><li>Capalbo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+efficacy+of+blastocoel+fluid+and+spent+media+as+sources+of+DNA+for+preimplantation+genetic+testing+in+standard+clinical+conditions.">Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions.</a> Fertility and Sterility. 110 (5), 870-879 (2018).</li><li>Tobler, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastocoel+fluid+from+differentiated+blastocysts+harbors+embryonic+genomic+material+capable+of+a+whole-genome+deoxyribonucleic+acid+amplification+and+comprehensive+chromosome+microarray+analysis.">Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis.</a> Fertility and Sterility. 104 (2), 418-425 (2015).</li><li>Magli, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+genetic+testing:+polar+bodies,+blastomeres,+trophectoderm+cells,+or+blastocoelic+fluid?.">Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid?.</a> Fertility and Sterility. 105 (3), 676-683 (2016).</li><li>Kuznyetsov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+novel+non-invasive+preimplantation+genetic+screening+approach.">Evaluation of a novel non-invasive preimplantation genetic screening approach.</a> PLoS One. 13 (5), e0197262 (2018).</li><li>Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+Genetic+Screening+with+Spent+Culture+Medium/Blastocoel+Fluid+for+in+Vitro+Fertilization.">Preimplantation Genetic Screening with Spent Culture Medium/Blastocoel Fluid for in Vitro Fertilization.</a> Scientific Reports. 8 (1), 9275 (2018).</li><li>Jiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+preimplantation+genetic+testing+using+blastocyst+culture+medium.">Minimally invasive preimplantation genetic testing using blastocyst culture medium.</a> Human Reproduction. 34 (7), 1369-1379 (2019).</li><li>Palermo, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Births+after+intracytoplasmic+injection+of+sperm+obtained+by+testicular+extraction+from+men+with+nonmosaic+Klinefelter's+syndrome.">Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter's syndrome.</a> New England Journal of Medicine. 338 (9), 588-590 (1998).</li><li>Alpha Scientists in Reproductive, M., &#38; Embryology, E. S. I. G. o. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Istanbul+consensus+workshop+on+embryo+assessment:+proceedings+of+an+expert+meeting.">The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting.</a> Human Reproduction. 26 (6), 1270-1283 (2011).</li><li>. Qubit dsDNA HS Assay Kit Available from: <a target="_blank" href="https://www.thermofisher.com/order/catalog/product/Q32851?ICID=search-product">https://www.thermofisher.com/order/catalog/product/Q32851?ICID=search-product</a> (2015)</li><li>. Miseq system use guide Available from: <a target="_blank" href="https://support.illumina.com/downloads/miseq_system">https://support.illumina.com/downloads/miseq_system</a> (2016)</li><li>Lane, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+to+detect+aneuploidy+from+cell+free+DNA+collected+from+media+is+dependent+on+the+stage+of+development+of+the+embryo.">Ability to detect aneuploidy from cell free DNA collected from media is dependent on the stage of development of the embryo.</a> Fertility and Sterility. 108 (3), (2017).</li><li>Rubio, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+prospective+study+of+concordance+between+embryonic+cell-free+DNA+and+trophectoderm+biopsies+from+1301+human+blastocysts.">Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts.</a> American Journal of Obstetrics and Gynecology. 223 (5), 751-751 (2020).</li><li>Rubio, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+cell-free+DNA+versus+trophectoderm+biopsy+for+aneuploidy+testing:+concordance+rate+and+clinical+implications.">Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.</a> Fertility and Sterility. 112 (3), 510-519 (2019).</li><li>Lledo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistent+results+of+non-invasive+PGT-A+of+human+embryos+using+two+different+techniques+for+chromosomal+analysis.">Consistent results of non-invasive PGT-A of human embryos using two different techniques for chromosomal analysis.</a> Reproductive BioMedicine Online. 42 (3), 555-563 (2021).</li><li>Kuznyetsov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+Invasive+Cell-Free+Human+Embryo+Aneuploidy+Testing+(miPGT-A)+Utilizing+Combined+Spent+Embryo+Culture+Medium+and+Blastocoel+Fluid+-Towards+Development+of+a+Clinical+Assay.">Minimally Invasive Cell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined Spent Embryo Culture Medium and Blastocoel Fluid -Towards Development of a Clinical Assay.</a> Scientific Reports. 10 (1), 7244 (2020).</li></ol>

Modifications and troubleshooting
If the NICS results are contaminated with parental genetic materials, make sure all cumulus-corona radiata cells are removed and make sure ICSI is performed for fertilization. Improper medium storage or template preparation processes are avoided, which may degrade DNA. The working space was purified thoroughly with DNase and RNase decontamination reagents. To avoid contamination from other embryos, one embryo was always cultured in a single dr...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62619">Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis</a>. The Protocol and Representaive Results sections were&#160;updated.
In the Protocol, step 3.8.2 was updated from:
After logging into the system, click Create Submission under the NICS tab. Then, select the sequencing platform, choose ChromInst for the reagent, enter the project information in the box under Project ID, set the analysis preferences and upload the files. Once all sequencing files are successfully uploaded, click Submit to start the analysis (Figure 3A).
to:
After logging into the system, click Create Submission under the NICS-A tab. Then, choose NGS for the platform, select corporation, choose ChromInst for the reagent, enter the project information in the box under Project ID, set the analysis preferences and upload the files. Once all sequencing files are successfully uploaded, click Submit to start the analysis (Figure 3A).
In the Representative Results, Figure 3 was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62619/62619fig03.jpg" /> 
Figure 3. Data Analysis. (A) The page of Create Submission. There are different options for the user application. For sequencing platform, users can choose Illumina or Ion Torrent. For analysis criterion, there are two length detection resolution for selection, the whole chromosome and whole arm level. The users also can choose whether the mosaicism or gender information is reported. Finished the above parameter setting,click on the box under File upload and choose the appropriate sequencing files to upload. For Illumina, choose the files with an extension of fastq.gz. For Ion Torrent platform, choose files with an extension of bam. Click Submit to start the analysis after successfully upload. (B) The view of summary table. The summary table consists of following information: Sample Name: The name of each NICS sample is listed; Data QC: Indicates whether the sequencing file passes the QC for NICS analysis; Conclusion: Indicates whether the NICS analysis is normal or abnormal, &#34;N/A&#34; indicates no conclusive result is available; Gender: If the user chooses to report the sex information, this column will appear in the summary table; Karyotype: Shows the analysis results; CNV plot (Whole Genome): View the CNV profiles of all chromosomes; CNV plot (By Chromosome): View the CNV profiles of each chromosome. (C) The Save Report Page. Click Export report button next to the Summary of Results. Select the information you want to show on the final report and click Export. Select Save File in the appearing dialog window and then click OK. The reports will be saved to the Download folder of the computer. <a href="https://www.jove.com/files/ftp_upload/62619/62619fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62619/62619fig3v2.jpg" /> 
Figure 3. Data Analysis. (A) There are different options for the user application. For sequencing platform corporation, users can choose Illumina, Ion Torrent or MGI. The users can choose whether the gender information is reported. Finished the above parameter setting, click on the box under File upload and choose the appropriate sequencing files to upload. For Illumina, choose the files with an extension of fastq.gz. Click Submit&#160;to start the analysis after successfully upload. (B) The view of summary table. The summary table consists of following information: Sample Name: The name of each NICS sample is listed; Data QC: Indicates whether the sequencing file passes the QC for NICS analysis; AI Rating: The rating (A, B or C) for each NICS sample; AI_Rating Interpretation: Evaluation of embryo implantation potential; AI Grading: The score for each NICS sample; CNV plot (Whole Genome): View the CNV profiles of all chromosomes; (C) The Save Report Page. Click Export report&#160;button next to the Summary of Results. Select the information you want to show on the final report and click Export. The reports will be saved to the Download&#160;folder of your computer. <a href="https://www.jove.com/files/ftp_upload/62619/62619fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62619">Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis</a>. The Protocol and Representaive Results sections were&#160;updated.

Erratum: Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

Assisted reproductive technologies (ARTs) have been increasingly used for the treatment of infertility. However, the success rate of ART, such as IVF, has been limited, and the pregnancy loss rate is significantly higher than that of the normal population1. The main cause of these problems is chromosomal abnormalities, which commonly exist in preimplantation human embryos2. PGT-A is an effective method of screening embryos for chromosomal balance before implantation3,4. Some studies have proven that PGT-A can reduce the rate of abortion and improve the rate of pregnancy5,6,7,8. However, PGT-A requires complex technical expertise that requires specific training and experience. The invasive embryo biopsy procedure could also potentially cause damage to the embryos9. Studies have shown that blastomere biopsy can hinder subsequent development, and the number of biopsied TEs may affect the implantation rates10. Although the long-term biosafety issue of embryo biopsy has not yet been evaluated thoroughly in humans, animal studies have shown its negative influences on embryo development11,12,13.
Previous reports indicated that trace amounts of DNA materials were secreted into the culture medium during embryo development, and efforts have been made to perform comprehensive chromosome screening (CCS) using spent embryo culture medium14,15,16,17,18. However, the detection rates and the accuracy of the tests have not met the requirements for extensive clinical use. The present study reported an improvement in the NICS assay for increasing the detection rates as well as the accuracy of the NICS test19. In recent years, blastocoele fluid (BF) has been studied as an analytical sample of minimally invasive PGT-A. However, the proportion of successful genome-wide amplification and detectable DNA in blastocyst fluid samples ranges from 34.8% to 82%20,21,22. The volume of BF reported in various studies ranges from 0.3 nL to 1 &#181;L. In view of the low amount of DNA in BF, it is possible to increase the amount of cell-free DNA by mixing blastocyst fluid and culture medium to improve the success rate and consistency of detection. Kuznyetsov et al.23 and Li et al.24 treated the zona pellucida with a laser and released blastocyst fluid into the culture medium to improve the total amount of embryonic DNA, and the amplification rate of the combined medium/BF samples after WGA was 100% and 97.5%, respectively. Jiao et al.25 also obtained a 100% amplification success rate by using the same method.
The present study reports a detailed protocol that includes spent media sample preparation, NGS preparation and data analysis. By carefully removing cumulus cells from oocytes, the present study performed intracytoplasmic single sperm injection (ICSI) and blastocyst culture. The, day 4-day 5/day 6 spent medium was collected for WGA and NGS library preparation. By using NICS technology, the present study streamlined the WGA and NGS library preparation steps in approximately 3 h and obtained CCS results noninvasively in approximately 9 h.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL EP tube, 0.2 mL PCR tube</td>
			<td>Axygen</td>
			<td>MCT-150-C, PCR-02-C</td>
			<td>DNase/RNase free, Low Binding PCR tubes and 1.5 mL micro-centrifuge tubes are recommended.</td>
		</tr>
		<tr>
			<td>10 &#181;L, 200 &#181;L, 1000 &#181;L DNase /RNase Free Tips</td>
			<td>Axygen</td>
			<td>T-300-R-S, T-200-Y-R-S, T-1000-B-R-S</td>
			<td>This can be replaced by other brand/For sample transfer</td>
		</tr>
		<tr>
			<td>100 % ethanol</td>
			<td>Sinopharm Chemical</td>
			<td>10009218</td>
			<td>This can be replaced by other brand/For DNA library purification</td>
		</tr>
		<tr>
			<td>Barcode Primer1-48</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For library amplificaton</td>
		</tr>
		<tr>
			<td>BD Falcon Organ Culture Dish, Sterile</td>
			<td>BD Bioscience</td>
			<td>363037</td>
			<td>This can be replaced by other brand/For embryo culture</td>
		</tr>
		<tr>
			<td>BD Falcon Tissue culture Dishes (Easy Grip) , Sterile</td>
			<td>BD Bioscience</td>
			<td>353001</td>
			<td>This can be replaced by other brand/For embryo culture</td>
		</tr>
		<tr>
			<td>BD Falcon Tissue culture Dishes, Sterile</td>
			<td>BD Bioscience</td>
			<td>353002</td>
			<td>This can be replaced by other brand/For embryo culture</td>
		</tr>
		<tr>
			<td>Cell Lysis Buffer</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For culture medium pre-treatment</td>
		</tr>
		<tr>
			<td>Cell Lysis Enzyme</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For culture medium pre-treatment</td>
		</tr>
		<tr>
			<td>ChromGo software</td>
			<td>Yikon Genomics</td>
			<td></td>
			<td>Data analysis</td>
		</tr>
		<tr>
			<td>CMPure Magbeads</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For library purification</td>
		</tr>
		<tr>
			<td>Cryotop open systerm</td>
			<td>&#160;KITAZATO BioPharma</td>
			<td>81110</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>Distill water</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>To dissolve DNA</td>
		</tr>
		<tr>
			<td>ES (Vitrification kit)</td>
			<td>&#160;KITAZATO BioPharma</td>
			<td>Reagent inVitrification kit</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>HOLDNIG</td>
			<td>ORIGIO</td>
			<td>MPH-MED-35</td>
			<td>This can be replaced by other 
			brand/For ICSI</td>
		</tr>
		<tr>
			<td>Hyaluronidase solution, 80 U/mL</td>
			<td>SAGE</td>
			<td>ART4007-A</td>
			<td>This can be replaced by other brand/Digest oocyte-corona-cumulus complex</td>
		</tr>
		<tr>
			<td>ICSI</td>
			<td>ORIGIO</td>
			<td>MPH-35-35</td>
			<td>This can be replaced by other brand/For ICSI</td>
		</tr>
		<tr>
			<td>Illumina MiSeq&#174; System</td>
			<td>Illumina</td>
			<td>SY-410-1001</td>
			<td>For library sequencing</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Labotect</td>
			<td>Inkubator C16</td>
			<td>This can be replaced by other brand/For embryo culture</td>
		</tr>
		<tr>
			<td>Library buffer</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For library amplificaton</td>
		</tr>
		<tr>
			<td>Library Enzyme Mix</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For library amplificaton</td>
		</tr>
		<tr>
			<td>Magnetic Stand</td>
			<td>DynaMagTM-2</td>
			<td>12321D</td>
			<td>For library purification</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>OLYMPUS</td>
			<td>1X71</td>
			<td>This can be replaced by other brand/For embryo observation</td>
		</tr>
		<tr>
			<td>Mini-centrifuge</td>
			<td>ESSENSCIEN</td>
			<td>ELF6</td>
			<td>For separation</td>
		</tr>
		<tr>
			<td>MT Enzyme Mix</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>For culture medium pre-treatment</td>
		</tr>
		<tr>
			<td>NICSInst library preparation kit</td>
			<td>Yikon Genomics</td>
			<td>KT1000800324</td>
			<td>Whole genome amplification and library construction</td>
		</tr>
		<tr>
			<td>NICSInst Sample Prep Station</td>
			<td>Yikon Genomics</td>
			<td>&#160;ME1001003</td>
			<td>Amplificate DNA</td>
		</tr>
		<tr>
			<td>Nunc IVF 4-Well Dish</td>
			<td>Thermo Scientific</td>
			<td>144444</td>
			<td>This can be replaced by other brand/For embryo washing and blastocyst culture</td>
		</tr>
		<tr>
			<td>Pasteur Pipette</td>
			<td>Oirgio</td>
			<td>&#160;MXL3-IND-135</td>
			<td>This can be replaced by other brand/For embryo tansfer</td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>ORIGIO</td>
			<td>PP-9-1000</td>
			<td>This can be replaced by other brand/For IVF laboratory</td>
		</tr>
		<tr>
			<td>Pre-Lib Buffer</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>Pre-library preparation</td>
		</tr>
		<tr>
			<td>Pre-Lib Enzyme</td>
			<td>Yikon Genomics</td>
			<td>Reagent in NICSInst library preparation kit</td>
			<td>Pre-library preparation</td>
		</tr>
		<tr>
			<td>Qubit&#174; 3.0 Fluorometer</td>
			<td>Thermo Scientific</td>
			<td>Q33216</td>
			<td>For library quantification</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage Blastocyst Medium</td>
			<td>SAGE</td>
			<td>ART-1029</td>
			<td>For embryo blastocyst stage culture</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage Cleavage Medium</td>
			<td>SAGE</td>
			<td>ART-1026</td>
			<td>This can be replaced by other brand/For embryo cleavage stage culture</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage Fertilization Medium</td>
			<td>SAGE</td>
			<td>ART-1020</td>
			<td>This can be replaced by other brand/For oocyte and sperm fertilization</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage m-HTF Medium with HEPES</td>
			<td>SAGE</td>
			<td>ART-1023</td>
			<td>This can be replaced by other brand/For embryo clutrure</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage SPS Serum protein Substitute Kit</td>
			<td>SAGE</td>
			<td>ART-3010</td>
			<td>This can be replaced by other brand/To denude the oocyte</td>
		</tr>
		<tr>
			<td>Quinn&#39;s Advantage Tissue culture mineral oil</td>
			<td>SAGE</td>
			<td>ART-4008P</td>
			<td>This can be replaced by other brand/To cover the culture medium</td>
		</tr>
		<tr>
			<td>STRIPPER TIPS</td>
			<td>ORIGIO</td>
			<td>MXL3-IND-135</td>
			<td>This can be replaced by other brand/For denudating granulosa cells</td>
		</tr>
		<tr>
			<td>Vitrification Cryotop Open systerm</td>
			<td>KIZTAZATO</td>
			<td>81111</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>Vitrification kit</td>
			<td>&#160;KITAZATO BioPharma</td>
			<td>VT101</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Qilinbeier</td>
			<td>DNYS8</td>
			<td>Sample mix</td>
		</tr>
		<tr>
			<td>VS (Vitrification kit)</td>
			<td>&#160;KITAZATO BioPharma</td>
			<td>Reagent inVitrification kit</td>
			<td>This can be replaced by other brand/For embryo vitrification</td>
		</tr>
		<tr>
			<td>ZILOS-tk Laser System</td>
			<td>Hamilton Thorne</td>
			<td>CLASS 1 laser</td>
			<td>This can be replaced by other brand/For artificial blastocoele collapse</td>
		</tr>
	</tbody>
</table>

chromosome screening of human preimplantation embryos by using spent culture medium: sample collection and chromosomal ploidy analysis

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage that forms the placenta. This method, however, requires specialized skills, is invasive, and suffers from false positives and negatives because the chromosome numbers in the TE and the inner cell mass (ICM), which develops into the fetus, are not always the same. NICS, a technology requiring sequencing of DNA that released into the culture medium from both TE and ICM, may offer a way out to these problems but has previously been shown to have limited efficacy. The present study reports the full protocol of NICS, which includes culture medium sampling methods, whole genome amplification (WGA) and library preparation, and NGS data analysis by analysis software. Considering the different cryopreservation times in different embryo laboratories, embryologists have two methods for collecting embryo culture medium that can be selected according to the actual conditions of the IVF laboratory.

The present study applied the proposed method to a patient. IRB approval and informed consent were obtained before the application of NICS analysis. The present study obtained 6 blastocysts from patients and performed NICS on all 6 embryos on day 4 to day 5 medium. Chromosome abnormalities caused by the parents&#39; balanced translocation were detected in five of chromosomes with the NICS assay; therefore, they could not be used for transfer (Figure 4A-E). The NICS results o...

Watch this Scientific Journal Video about Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis at JoVE.com

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage ...

The protocol is to normalize the cultured medium collection and the chromosomal ploidy analysis based on non-invasive chromosome screening NICS of human pre-implantation embryos. The main advantage of NICS is easy to operate rather than TiDieR micro-manipulation, time-saving, and effectively reduces the invasive biopsy sample loss. The sampling method is very flexible.Here we offer you two different options, one for routine sequential embryo culture, and another for one step. These are the main colleagues of our team, Dr.Yaxin Yao and Dr.Xiaohui Zhu are mainly responsible for library preparation and the NICS regeneration sequencing. Dr.Jialin Jia and I complete embryology work and sample collection.Professor Liu is our group's leader. She is also the corresponding author of this article. To begin, prepare the reagents and tools.Prewarm and equilibrate 20 to 30 microliters of culture medium and hyaluronidase before use. Then place the culture medium and hyaluronidase on a working surface in a fume hood. Rapidly transfer the digested OCCCs in the culture dish for oocyte handling and cover with mineral oil in each well.Gently aspirate and release the oocytes five to 10 times to remove residual granulosa cells around the oocytes. Repeat this step in the remaining three wells to completely remove the granulosa cells. Evaluate the completeness of granulosa cell removal using a microscope.Then perform intracytoplasmic sperm injection, also called ICSI and transfer the embryo to the culture medium. Prepare 20 to 30 microliters of the blastocyst culture medium microdroplets for each embryo. Cover the embryos with mineral oil in a tissue culture dish on day two and incubate at 37 degrees Celsius, 5%carbon dioxide and 5%oxygen.After transferring the third-day embryo into the washing microdroplet, gently aspirate and release the embryo in the microdroplet in the three droplets serially using the pipettes. Then transfer the embryo to the blastocyst culture medium. Prepare the washing microdroplets and culture microdroplets.On the afternoon of the fourth day, transfer the embryo into pre-warmed microdroplets of culture medium and gently wash each embryo serially in three microdroplets with the pipettes. Transfer each embryo into a unique pre-warmed single microdroplet of culture medium for sample collection. Culture the blastocyst embryo before cryo-preservation.For performing the vitrification before embryo cryo-preservation, prepare the culture microdroplets and transfer the embryo into pre-warmed microdroplets of 10 to 15 microliters culture medium. And gently wash each embryo serially in three microdroplets by pipetting on the morning of the fifth day. As previously demonstrated, transfer each embryo into a unique pre-warmed single microdroplet of culture medium for sample collection.And culture the blastocyst embryo at 37 degrees Celsius and 5%carbon dioxide before cryo-preservation. Next, gently adjust the ICM at a considerable distance from the targeted point of the laser beam which focuses on the cell junction of the trophectoderm to generate a small hole in the trophectoderm to release the fluid from the blastocoel cavity. To collect the sample, thaw the buffer in the kit at room temperature and centrifuge briefly.Then transfer the culture medium from each cultured embryo into an RNase-DNase-free PCR tube containing five microliters of the cell lysis buffer. Prepare the reagents and tools. After quantifying the genomic DNA diluted to a concentration of 50 picograms per microliter with the culture medium.After mixing thoroughly, centrifuge the tube briefly at 200 G for five seconds. After centrifuging the culture medium in a microcentrifuge for 30 to 60 seconds, pipette 10 microliters of culture medium from the bottom of the tube diluted positive control and fresh culture medium to a new 0.2 milliliter PCR tube. Add one microliter of MT enzyme mix and mix thoroughly by pipetting.Then centrifuge immediately for two to three seconds. Put the PCR tube in a pre-heated mixed sample prep station and run the lysis program. Once the pre-lib buffer is thawed and mixed, centrifuge it immediately for two to three seconds at 200 G.Prepare a master mix for the pre-library reaction by adding two microliters of pre-library enzyme mix to 60 microliters of pre-library buffer.Mix the reaction thoroughly and centrifuge briefly. Add 60 microliters of pre-library reaction mix into the pre-treated medium sample. After mixing it thoroughly by pipetting, centrifuge immediately for two to three seconds at 200 G.Place the PCR tube in the pre-heated sample prep station.Run the pre-library program and hold at four degrees Celsius. Prepare a master mix for the library reaction by adding 1.6 microliters of library enzyme mix to 60 microliters of library buffer. Mix the reaction thoroughly and centrifuge as demonstrated previously.Add 60 microliters of the library reaction mix and two microliters of the barcode primer to each pre-library product. Then mix the reaction thoroughly and centrifuge as demonstrated. After centrifugation, place the PCR tube in the thermal cycler.Run the library preparation program and then hold at four degrees Celsius. Prepare the reagents and tools. Add the prepared 1X prepared mega beads to the library sample.Quantify the purified libraries as described in the text manuscript and use 10 nanograms of each library sample for pooling. Refer to the sequencing user guide and perform data analysis after sequencing is completed. Enter the user's name and password on the login page for data analysis in ChromGo.Once logged in, click Create Submission under the NICS tab. Then choose NGS for the Platform, Illumina for Corporation, and NICSInst for the Reagent. Enter the project information in the box under Project ID.Set the analysis preferences and upload the files.Once all sequencing files are successfully uploaded, click Submit to start the analysis. The submitted projects are listed under the View Submissions tab. Click the Show button in the Report field to view the summary table of the NICS analysis.Click the Export Report button to save the reports. Six blastocysts were obtained from patients and NICS was performed on all six embryos from day four to day five medium. The chromosome abnormalities caused by parents'balanced translocation were detected in five of the chromosomes using the NICS assay and therefore, they were not used for transfer.The NICS results of the two embryos showed the same karyotype 45 XN and chromosome 18 deletion were both chromosome 18 deletions. The karyotype 46 XN short arm of the chromosome one region deletion was only the short arm of the chromosome region deletion. The NICS results showed karyotype 46 XN with the long arm of chromosome 18 region deletion and the short arm of chromosome one region duplication.Although the karyotypes were chromosome five duplications and showed eight mosaic differences, the NICS assay could screen all 24 chromosomes for aneuploidy. This process provides a new method for transferring single normal karyotype blastocysts. While attempting this procedure, it's important to remember to completely remove the cumular cells to avoid the maternal origin contamination and collect the culture medium and the expounded blastocyst stage when the DNA content is enough for amplification.By using NICS technology, the present study streamlined the WGA and the WGS library preparation steps in about three hours and opt in CCS result non-invasively in about nine hours. After its development, the technique provided clinician in ART a novel way to combine morphological assessment with NICS to transfer aploidy embryo with good morphology into the uterus might improve the ongoing pregnancy rates and live birth rates.

chromosome-screening-human-preimplantation-embryos-using-spent

Research

JoVE Journal

Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification (ADCI) and Dose Estimation

Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome assays is traditionally a manual, labor-intensive process not well suited to handle the volume of samples which may require examination in the wake of a mass casualty event. Automated Dicentric Chromosome Identifier and Dose Estimator (ADCI) software automates this process by examining sets of metaphase images using machine learning-based image processing techniques. The software selects appropriate images for analysis by removing unsuitable images, classifies each object as either a centromere-containing chromosome or non-chromosome, further distinguishes chromosomes as monocentric chromosomes (MCs) or dicentric chromosomes (DCs), determines DC frequency within a sample, and estimates biological radiation dose by comparing sample DC frequency with calibration curves computed using calibration samples. This protocol describes the usage of ADCI software. Typically, both calibration (known dose) and test (unknown dose) sets of metaphase images are imported to perform accurate dose estimation. Optimal images for analysis can be found automatically using preset image filters or can also be filtered through manual inspection. The software processes images within each sample and DC frequencies are computed at different levels of stringency for calling DCs, using a machine learning approach. Linear-quadratic calibration curves are generated based on DC frequencies in calibration samples exposed to known physical doses. Doses of test samples exposed to uncertain radiation levels are estimated from their DC frequencies using these calibration curves. Reports can be generated upon request and provide summary of results of one or more samples, of one or more calibration curves, or of dose estimation.

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification ADCI and Dose Estimation

The cytogenetic dicentric chromosome (DC) assay quantifies exposure to ionizing radiation. The Automated Dicentric Chromosome Identifier and Dose Estimator software accurately and rapidly estimates biological dose from DCs in metaphase cells. It distinguishes monocentric chromosomes and other objects from DCs, and estimates biological radiation dose from the frequency of DCs.

Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are two types of HM based on microscopic morphological evaluation, complete HM (CHM) and partial HM (PHM). These can be further subdivided based on the parental contribution to the molar genomes. Such characterization of HM, by morphology and genotype analyses, is crucial for patient management and for the fundamental understanding of this intriguing pathology. It is well documented that morphological analysis of HM is subject to wide interobserver variability and is not sufficient on its own to accurately classify HM into CHM and PHM and distinguish them from hydropic non-molar abortions. Genotyping analysis is mostly performed on DNA and tissues from formalin-fixed paraffin-embedded (FFPE) products of conception, which have less than optimal quality and may consequently lead to wrong conclusions. In this article, detailed protocols for multiplex genotyping and flow cytometry analyses of FFPE molar tissues are provided, along with the interpretation of the results of these methods, their troubleshooting, and integration with the morphological evaluation, p57KIP2 immunohistochemistry, and fluorescence in situ hybridization (FISH) to reach a correct and robust diagnosis. Here, the authors share the methods and lessons learned in the past 10 years from the analysis of approximately 400 products of conception.

Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results&#8217; interpretation and integration.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial patterns of gene expression. Embryoid bodies (EBs) derived from pluripotent stem cells have been a popular model to study the differentiation of the main three germ layers and to define regulatory circuits during cell fate specification. Although it is well-known that tissue-specific enhancers play an important role in these networks by interacting with promoters, assigning them to their relevant target genes still remains challenging. To make this possible, quantitative approaches are needed to study enhancer-promoter contacts and their dynamics during development. Here, we adapted a 4C method to define enhancers and their contacts with cognate promoters in the EB differentiation model. The method uses frequently cutting restriction enzymes, sonication, and a nested-ligation-mediated PCR protocol compatible with commercial DNA library preparation kits. Subsequently, the 4C libraries are subjected to high-throughput sequencing and analyzed bioinformatically, allowing detection and quantification of all sequences that have contacts with a chosen promoter. The resulting sequencing data can also be used to gain information about the dynamics of enhancer-promoter contacts during differentiation. The technique described for the EB differentiation model is easy to implement.

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture 4C

We report the application of quantitative chromosome conformation capture followed by high-throughput sequencing in embryoid bodies generated from embryonic stem cells. This technique allows to identify and quantitate the contacts between putative enhancers and promoter regions of a given gene during embryonic stem cell differentiation.

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial ...