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.

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