Non-invasive Chromosome Screening (NICS) of Human Preimplantation Embryos: Sample Collection and Chromosomal Ploidy Analysis by MALBAC-NGS

Summary

We report a protocol for chromosome screening of human embryos by using spent culture medium, which avoids embryo biopsy and enables reporting chromosome ploidy using next generation sequencing (NGS). We present the detailed procedure including the preparation of culture medium, whole genome amplification (WGA), NGS library preparation, and data analysis.

Abstract

Chromosomal abnormalities are common in human embryos and cause implantation failure, early pregnancy losses, and birth defects in practice of assisted reproductive technology (ART). Non-invasive chromosome screening (NICS) is an emerging technology that enables the selection of chromosomal-balanced embryos, without performing invasive embryo biopsy. Here we report the full protocol of NICS, which includes culture medium pretreatment, whole genome amplification (WGA) by multiple annealing and looping-based amplification cycles (MALBAC), library preparation for next generation sequencing (NGS) and NGS data analysis. To validate the reliability and efficiency of NICS, we have already performed NICS on 27 transfer cycles in 23 couples with balanced translocation, azoospermia, recurrent pregnancy loss (RPL), or recurrent implantation failure (RIF), 17 of them have achieved successful clinical pregnancies, and 9 among them have already obtained healthy live births. No pregnancy loss has been reported thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use.

Introduction

Assisted reproductive technologies (ART) have been increasingly used for treatment of infertility. However, the success rate of ART, such as in-vitro fertilization (IVF) has been limited and the pregnancy loss rate has been significantly higher than the normal population ¹. The main cause of these problems are chromosomal abnormalities, which commonly exist in preimplantation human embryos ². PGS came as an effective way to screen the embryos for chromosomal balance before implantation ^3,4. And some studies have proved PGS can reduce the rate of abortion and impr....

Protocol

Ethics statement: Institutional review board (IRB) approvals (Nanjing Jinlin: 2014NZKY-005; Wuxi Maternity: 2014-04-0515-02) were obtained. All of the embryos were voluntarily donated by patients, with informed consent obtained before performing the experiments on each embryo.

1. Preparation

1. Prepare the following reagents
   1. Prepare fertilization medium plus 10% serum protein substitute (SPS) (IM) by adding 1 mL SPS to 9 mL Quinn's fertilization medium.
   2. Make.......

Discussion

If the NICS results are contaminated with parental genetic material, then make sure all cumulus-corona radiata cells are removed and ensure that ICSI is performed for fertilization. Avoid inappropriate storage of the culture medium or template preparation processes that can potentially degrade DNA. Decontaminate the workspace thoroughly by DNase and RNase decontamination reagents. To avoid the contamination from other embryos, always culture one embryo in single droplet of medium to avoid cross-contamination from Day 3. .......

Materials

Name	Company	Catalog Number	Comments
Hyaluronidase solution, 80 U/mL	SAGE	ART4007-A	Digest oocyte-corona-cumulus complex
Quinn's Advantage m-HTF Medium with HEPES	SAGE	ART-1023	For embryo clutrure
Quinn's Advantage Fertilization Medium	SAGE	ART-1020	For oocyte and sperm fertilization
Quinn's Advantage Cleavage Medium	SAGE	ART-1026	For embryo cleavage stage culture
Quinn's Advantage Blastocyst Medium	SAGE	ART-1029	For embryo blastocyst stage culture
Quinn's Advantage SPS Serum protein Substitute Kit	SAGE	ART-3010	To denude the oocyte
Quinn's Advantage Tissue culture mineral oil	SAGE	ART-4008P	To cover the culture medium
STRIPPER TIPS	ORIGIO	MXL3-IND-135	For denudating granulosa cells
Pasteur pipettes	ORIGIO	PP-9-1000	For IVF laboratory
ZILOS-tk Laser System	Hamilton Thorne	CLASS 1 laser	For artificial blastocoele collapse
ICSI	ORIGIO	MPH-35-35	For ICSI
HOLDNIG	ORIGIO	MPH-MED-35	For ICSI
9"IVF Pasteur Pipette	Oirgio	MXL3-IND-135	For embryo tansfer
microscope	OLYMPUS	1X71	For embryo observation
incubator	Labotect	Inkubator C16	For embryo culture
Vitrification kit	KITAZATO BioPharma	VT101	For embryo vitrification
ES (Vitrification kit)	KITAZATO BioPharma	Reagent inVitrification kit	For embryo vitrification
VS (Vitrification kit)	KITAZATO BioPharma	Reagent inVitrification kit	For embryo vitrification
Cryotop open systerm	KITAZATO BioPharma	81110	For embryo vitrification
BD Falcon Tissue culture Dishes, Sterile	BD Bioscience	353002	For embryo culture
BD Falcon Tissue culture Dishes（Easy Grip）, Sterile	BD Bioscience	353001	For embryo culture
BD Falcon Organ Culture Dish, Sterile	BD Bioscience	363037	For embryo culture
Nunc IVF 4-Well Dish	Thermo Scientific	144444	For embryo washing and blastocyst culture
Vitrification Cryotop Open systerm	KIZTAZATO	81111	For embryo vitrification
NICSInst library preparation kit	Yikon Genomics	KT1000800324	Whole genome amplification and library construction
MT Enzyme Mix	Yikon Genomics	Reagent in NICSInst library preparation kit	For culture medium pre-treatment
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
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
Barcode Primer1-48	Yikon Genomics	Reagent in NICSInst library preparation kit	For library amplificaton
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
CMPure Magbeads	Yikon Genomics	Reagent in NICSInst library preparation kit	For library purification
Distill water	Yikon Genomics	Reagent in NICSInst library preparation kit	To dissolve DNA
NICSInst Sample Prep Station	Yikon Genomics	ME1001003	Amplificate DNA
Illumina MiSeq System	Illumina	SY-410-1001	For library sequencing
Vortexer	Qilinbeier	DNYS8	Sample mix
Mini-centrifuge	ESSENSCIEN	ELF6	For separation
Magnetic Stand	DynaMagTM-2	12321D	For library purification
100 % ethanol	Sinopharm Chemical	10009218	For DNA library purification
Qubit 3.0 Fluorometer	Thermo Scientific	Q33216	For library quantification
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	For sample transfer
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.