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.
