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

Summary

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.

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 implantation^3,4. Some studies have proven that PGT-A can reduce the rate of abortion and improve the rate of pr....

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.

1. Reagents
   1. 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.
   2. Prewarm hyaluronidase to 37 °C on a working surface in a fume ho....

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 o.......

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.......

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