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.
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Method Article
Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis
* These authors contributed equally
In This Article
Erratum Notice
Important: There has been an erratum issued for this article. View Erratum Notice
Summary
Abstract
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.
Introduction
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 pr....
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Protocol
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.
- Reagents
- Prewarm and equilibrate (balanced) 20-30 µL of gamete medium/fertilization medium and cleavage/blastocyst-stage culture medium (covered with mineral oil) and hyaluronidase (in a tightly capped tube) at 37 °C, 5% CO2 and 5% O2 in a Tri-gas incubator overnight before use.
- Prewarm hyaluronidase to 37 °C on a working surface in a fume ho....
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Results
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' 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 of th.......
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Discussion
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.......
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Disclosures
Yaxin Yao, Jieliang Ma, Jing Wang and Sijia Lu are employees of Yikon Genomics Co., Ltd.
Acknowledgements
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).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 1.5 mL EP tube, 0.2 mL PCR tube | Axygen | MCT-150-C, PCR-02-C | DNase/RNase free, Low Binding PCR tubes and 1.5 mL micro-centrifuge tubes are recommended. |
| 10 µL, 200 µL, 1000 µL DNase /RNase Free Tips | Axygen | T-300-R-S, T-200-Y-R-S, T-1000-B-R-S | This can be replaced by other brand/For sample transfer |
| 100 % ethanol | Sinopharm Chemical | 10009218 | This can be replaced by other brand/For DNA library purification |
| Barcode Primer1-48 | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| BD Falcon Organ Culture Dish, Sterile | BD Bioscience | 363037 | This can be replaced by other brand/For embryo culture |
| BD Falcon Tissue culture Dishes (Easy Grip) , Sterile | BD Bioscience | 353001 | This can be replaced by other brand/For embryo culture |
| BD Falcon Tissue culture Dishes, Sterile | BD Bioscience | 353002 | This can be replaced by other brand/For embryo culture |
| Cell Lysis Buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| Cell Lysis Enzyme | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| ChromGo software | Yikon Genomics | Data analysis | |
| CMPure Magbeads | Yikon Genomics | Reagent in NICSInst library preparation kit | For library purification |
| Cryotop open systerm | KITAZATO BioPharma | 81110 | This can be replaced by other brand/For embryo vitrification |
| Distill water | Yikon Genomics | Reagent in NICSInst library preparation kit | To dissolve DNA |
| ES (Vitrification kit) | KITAZATO BioPharma | Reagent inVitrification kit | This can be replaced by other brand/For embryo vitrification |
| HOLDNIG | ORIGIO | MPH-MED-35 | This can be replaced by other brand/For ICSI |
| Hyaluronidase solution, 80 U/mL | SAGE | ART4007-A | This can be replaced by other brand/Digest oocyte-corona-cumulus complex |
| ICSI | ORIGIO | MPH-35-35 | This can be replaced by other brand/For ICSI |
| Illumina MiSeq® System | Illumina | SY-410-1001 | For library sequencing |
| Incubator | Labotect | Inkubator C16 | This can be replaced by other brand/For embryo culture |
| Library buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| Library Enzyme Mix | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| Magnetic Stand | DynaMagTM-2 | 12321D | For library purification |
| Microscope | OLYMPUS | 1X71 | This can be replaced by other brand/For embryo observation |
| Mini-centrifuge | ESSENSCIEN | ELF6 | For separation |
| MT Enzyme Mix | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| NICSInst library preparation kit | Yikon Genomics | KT1000800324 | Whole genome amplification and library construction |
| NICSInst Sample Prep Station | Yikon Genomics | ME1001003 | Amplificate DNA |
| Nunc IVF 4-Well Dish | Thermo Scientific | 144444 | This can be replaced by other brand/For embryo washing and blastocyst culture |
| Pasteur Pipette | Oirgio | MXL3-IND-135 | This can be replaced by other brand/For embryo tansfer |
| Pasteur pipettes | ORIGIO | PP-9-1000 | This can be replaced by other brand/For IVF laboratory |
| Pre-Lib Buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | Pre-library preparation |
| Pre-Lib Enzyme | Yikon Genomics | Reagent in NICSInst library preparation kit | Pre-library preparation |
| Qubit® 3.0 Fluorometer | Thermo Scientific | Q33216 | For library quantification |
| Quinn's Advantage Blastocyst Medium | SAGE | ART-1029 | For embryo blastocyst stage culture |
| Quinn's Advantage Cleavage Medium | SAGE | ART-1026 | This can be replaced by other brand/For embryo cleavage stage culture |
| Quinn's Advantage Fertilization Medium | SAGE | ART-1020 | This can be replaced by other brand/For oocyte and sperm fertilization |
| Quinn's Advantage m-HTF Medium with HEPES | SAGE | ART-1023 | This can be replaced by other brand/For embryo clutrure |
| Quinn's Advantage SPS Serum protein Substitute Kit | SAGE | ART-3010 | This can be replaced by other brand/To denude the oocyte |
| Quinn's Advantage Tissue culture mineral oil | SAGE | ART-4008P | This can be replaced by other brand/To cover the culture medium |
| STRIPPER TIPS | ORIGIO | MXL3-IND-135 | This can be replaced by other brand/For denudating granulosa cells |
| Vitrification Cryotop Open systerm | KIZTAZATO | 81111 | This can be replaced by other brand/For embryo vitrification |
| Vitrification kit | KITAZATO BioPharma | VT101 | This can be replaced by other brand/For embryo vitrification |
| Vortexer | Qilinbeier | DNYS8 | Sample mix |
| VS (Vitrification kit) | KITAZATO BioPharma | Reagent inVitrification kit | This can be replaced by other brand/For embryo vitrification |
| ZILOS-tk Laser System | Hamilton Thorne | CLASS 1 laser | This can be replaced by other brand/For artificial blastocoele collapse |
References
- Barlow, P. Early pregnancy loss and obstetrical risk after in-vitro fertilization and embryo replacement. Human Reproduction. 3 (5), 671-675 (1988).
- Munne, S. Chromosome abnormalities and their relationshi....
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Reprints and Permissions
Erratum
Formal Correction: Erratum: Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis
Posted by JoVE Editors on 10/01/2021. Citeable Link.
An erratum was issued for: Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis. The Protocol and Representaive Results sections were 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:
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, "N/A" 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. Please click here to view a larger version of this figure.
to:
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 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 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 folder of your computer. Please click here to view a larger version of this figure.
