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 improve the rate of pregnancy ^5,6,7,8. However, PGS requires complex technical expertise, which requires specific training and experience. The invasive embryo biopsy procedure could also potentially cause damage to the embryos ^9,10. Although long-term biosafety issue of embryo biopsy has not been evaluated thoroughly in human yet, animal studies have shown its negative influences on embryo development ^11,12.

It was previously reported that trace amount of DNA materials are secreted into the culture medium during embryo development, and efforts have been made to perform comprehensive chromosome screening (CCS) using spent embryo culture medium ^{13,14,15,16,17}. However, the detection rate and the accuracy of the tests have not met the requirement of extensive clinical use. We reported an improvement of using MALBAC for increasing the detection rate as well as the accuracy of the NICS test ¹⁸. Here we report a detailed protocol including spent media sample preparation, NGS preparation and data analysis. By carefully removing cumulus cells from the oocytes, we perform intracytoplasmic single sperm injection (ICSI) and blastocyst culture. We then collect the Day 3-Day 5/Day 6 spent medium for MALBAC WGA and NGS library preparation. By using the NICS technology, we streamline the WGA and NGS library preparation steps in about 3 h, which enables obtaining CCS results noninvasively in about 9 h.

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 cleavage medium plus 10% SPS (GM) by mixing 9 mL Quinn's cleavage medium and 1 mL SPS.
   3. Make blastocyst medium with 10% SPS (BM) by mixing 9 mL Quinn's blastocyst medium and 1 mL SPS.
   4. Equilibrate all the above medium and 10 mL m-HTF medium with HEPES within 37 °C, 5% CO₂, 5% O₂ for more than 6 h.
2. (Optional) Prepare denudation and transfer pipettes by pulling glass Pasteur pipettes to generate fire polished open fine tips. The internal diameter of is 135-150 µm for denudation pipettes and 180-200 µm for transfer pipettes.
   1. Prepare manipulation pipettes by pulling glass Pasteur pipettes to generate fine fire polished and sealed end tips as show in Figure 1.

2. Sample Collection: Protocol 1

1. Pretreatment of oocyte-corona-cumulus complex (OCCC) before digestion with hyaluronidase.
   NOTE: Here, ovarian stimulation was achieved with both Follicle Stimulating Hormone (FSH) and human menopause gonadotropin (hMG) preparations. When the lead follicle was >18 mm, 10.000 IU of chorionic gonadotropin (hCG) was used for final oocyte maturation. Oocytes retrieval was performed 36 h after trigger shot.
   1. Pick up oocytes and transfer to tissue culture dishes with 2.5 mL pre-warmed m-HTF covered with mineral oil. Carefully remove as many cumulus cells as possible without damaging these oocytes using 1 mL injection needle to cut out cumulus granulosa cells. Gently aspirate and release OCCCs in IM. Gently aspirate 2-3 times with transfer Pipette.
   2. Rapidly transfer these OCCCs to the central well of organ culture dish containing 1 mL IM using transfer Pipette. Incubate the oocytes in a 37 °C, 5% CO₂ and 5% O₂ incubator.
2. Digestion of OCCCs with hyaluronidase
   1. Add 1 mL 37 °C pre-warmed hyaluronidase (80 IU/mL) to the central well of organ culture dish containing OCCCs and mix thoroughly. Keep the final concentration of hyaluronidase at 40 IU/mL.
   2. Incubate the OCCCs on 37 °C thermal platform for 2 min. Observe the change under microscope every 30 s until only 1 or 2 layers of granulosa cells are left.
3. Denudation of granulosa cells
   1. Rapidly transfer the digested OCCCs to a 4-well IVF dish pre-filled with 0.5 mL GM covered by mineral oil. Observe the separated granulosa cells under the microscope.
   2. Gently aspirate and release the oocytes 5 times to remove the residual granulosa cells around the oocytes. Repeat this step with the remaining 3 wells to completely remove the granulosa cells.
4. Evaluation of the oocyte
   NOTE: Granulosa cells need to be completely removed to avoid maternal contamination from the NICS results. If granulosa cells are still attached to the oocyte, perform either of the following procedure for complete removal of these cells.
   1. Evaluate the removal completeness of granulosa cells using a 20×10 microscope. If granulosa cells are still attached to the oocyte, perform one of the following procedures for complete removal of these cells:
      1. Transfer the oocyte with residual granulosa cells to the central well of the organ culture dish. Digest for 30 s (step 2.2.1) and gently aspirate and release the oocyte to remove residual granulosa cells using denudation pipette.
      2. Transfer the oocyte with residual granular cells to the central well of tissue culture dish with 2 mL pre-warmed m-HTF without serum. Place SPS on the round end of the manipulation pipette and roll the oocytes back and forth using the pipette as shown in Figure 2 to completely remove granulosa cells around the oocyte.
   2. After performing ICSI¹⁹, transfer the oocytes into 30 µL GM micro-droplets (one oocyte to one micro-droplet) using transfer pipettes and incubate in a 37 °C, 5% CO₂ and 5% O₂ incubator. Record the day of ICSI as Day 0.
      1. Check the embryos at 8-9 am the next morning using 20×20 microscope. Score the embryos according to the Istanbul consensus workshop on embryo assessment¹⁶ on Day 1 for fertilization (about 18 h), Day 2 (about 45 h) and Day 3 (about 68 h) for embryo cleavage.
5. Wash the embryo.
   1. Prepare the 30 µL BM micro-droplets for each embryo covered with mineral oil in tissue culture dishes at 8-9 am of Day 3. Prepare another three 50 µL BM micro-droplets covered with mineral oil in new tissue culture dishes for washing. Incubate all micro-droplets at 37 °C before use.
   2. Transfer the Day 3 embryos into the BM micro-droplets. Gently aspirate and release the embryos 3 times in each droplet using denudation pipettes.
      NOTE: This procedure can also help remove the residual granular cells attached to the embryo.
6. Transfer each embryo into a single BM micro-droplet. Perform blastocyst embryo culture to Day 5/Day 6 at 37 °C, 5% CO₂, 5% O₂.
7. Select the Day 5 blastocysts for vitrification based on laboratory criteria.
   NOTE: This is when the embryos are fully expanded through to the hatched blastocyst with the grading of inner cell mass (ICM) and trophoblast reaching level B or above according to the Istanbul consensus workshop on blastocyst assessment ²⁰. The whole blastocysts were given a score from 1 to 6 to assess their degree of development. The blastocysts were graded in three ranks based on morphological appearance. For example, the ICM was graded as A (many tightly packed cells), B (several loosely grouped cells) or C (few cells), and the trophectoderm was graded as A (many cells forming a cohesive epithelium), B (fewer cells forming a loose epithelium) or C (very few large cells). The grade of selected embryo was 4-6 AA or AB or BA or BB or BC or CB for vitrification²⁰.
8. Perform vitrification at room temperature (RT).
   1. Gently adjust the ICM at a considerable distance from the targeted point of the 200 ms laser beam. Focus at the cell junction of the trophectoderm to generate a small hole to release the fluid from the blastocoel cavity.
   2. After 5-7 min, transfer the blastocyst (with a minimum volume of spent BM) using a transfer pipette to equilibrated solution (ES) (vitrification kit) for equilibration. To prevent cross contamination, transfer each embryo using a new disposable transfer pipette.
   3. After 15 min, place the embryo in vitrification solution (VS) for 1 min. Place the embryo in the vitrification device and immediately store it in liquid nitrogen.
9. For sample collection, transfer 15-20 µL of spent BM (step 2.8.2) from each cultured embryo into a RNase/DNase-free PCR tube containing 5 µL of cell lysis buffer.
   NOTE: The same amount of BM that was not used for embryo culture is collected as a negative control. All collected samples are frozen immediately in liquid nitrogen then stored at −80 °C until ready for NICS assay. Incubate early-stage blastocysts to Day 6 for sample collection.

3. Library Construction: Protocol 2

1. Culture Medium Pre-treatment
   1. Dilute 1 µL positive control (10 ng human gDNA) in 199 µL fresh culture medium. Mix thoroughly and centrifuge the tube briefly (2000 x g for 5 s).
   2. Transfer 10 µL Day 5-Day 6 blastocyst culture medium, diluted positive control, and fresh culture medium to new 0.2 ml PCR tubes. Add 1 µL MT enzyme mix to each PCR tube. Mix thoroughly and centrifuge briefly.
   3. Place the PCR tubes in a pre-heated NICSInst sample prep station and run lysis program as below using the following cycle: 75 °C, 10 min; 95 °C, 4 min; 22 °C, forever.
      1. Click the "Lysis" icon to enter the setup screen. Select "Tube" for "Control mode", input 10 µL for "Sample volume", select "On" for "Hotlid control", enter 105 °C for the temperature, and select "No" for "Pause at the first seg". Click "OK" to proceed.
      2. Wait until "Remain time" shows "--:--:--"; indicating the end of the program. Click "Stop" to terminate the program.
         NOTE: Stop the program when the process is done. Proceed to the next step immediately.
2. Pre-Library Preparation
   1. Thaw the pre-library (pre-lib) buffer to RT. Mix thoroughly and centrifuge briefly.
   2. Prepare a master mix for pre-library reaction as follows: add 2 µL pre-lib enzyme mix to 60 µL pre-lib buffer. Mix thoroughly and centrifuge briefly.
   3. Add 60 µL pre-library reaction mix into each pre-treated medium sample from step 3.1.4. Mix thoroughly and centrifuge briefly.
   4. Put the PCR tube(s) from step 3.2.3 in the sample prep station and run the pre-library program as below using the following cycles: 95 °C, 2 min; (15 °C, 40 s; 22 °C, 40 s; 33 °C, 30 s; 65°C, 30 s; 72 °C, 40 s; 95 °C, 10 s; 63 °C, 10 s) × 12 cycles; 4 °C, forever.
      1. Click the "Pre_Lib" icon to enter the set-up screen. Select "Tube" for "Control mode", input 70 µL for "Sample volume", select "On" for "Hot lid control", enter 105 °C for the temperature, and select "No" for "Pause at the first seg". Click "OK" to proceed. Wait until "Remain time" shows "--:--:--". Click "Stop" to terminate the program.
         NOTE: Stop the program when the process is done. Proceed to the next step immediately.
3. Library Preparation
   1. Thaw the library buffer to RT. Mix thoroughly and centrifuge briefly.
   2. Prepare a master mix for library reaction as follows: add 1.6 µL library enzyme mix to 60 µL library buffer. Mix the reaction thoroughly and centrifuge briefly.
   3. Add 60 µL library reaction mix and 2 µL barcode primer to each pre-library product (step 3.2.3). Mix the reaction thoroughly and centrifuge briefly.
   4. Place the PCR tube(s) in the thermal cycler and run the library preparation program as below program using the following cycles: 94 °C, 30 s; (94 °C, 25 s; 62 °C, 30 s; 72 °C, 45 s) × 17 cycles; 4 °C, forever.
      1. Click the "Lib_Prep" icon to enter the set-up screen. Select "Tube" for "Control mode", input 130 µL for "Sample volume", select "On" for "Hotlid control", enter 105°C for corresponding temperature, and select "No" for "Pause at the first seg". Click "OK" to proceed. Wait until "Remain time" shows "--:--:--". Click "Stop" to terminate the program.
4. Library Purification
   1. Take out magnetic beads (see Table of Materials) from 2-8 °C at least 20 min before the purification step. Vortex and mix the beads for 20 s. Dispense enough beads for the purification step into a new 1.5 mL micro-centrifuge tube and warm beads to RT.
   2. Add 1 × magnetic beads to each library. Mix by pipetting up and down at least 10 times and incubate at RT for 5 min. Centrifuge briefly and place it on the magnetic stand.
      NOTE: For example, add 100 µL magnetic beads to 100 µL library sample.
   3. Wait for about 5 min until the solution becomes clear. While on the magnetic stand, carefully aspirate the solution and discard. Add 200 µL freshly prepared 80% ethanol to the tube. Incubate at RT for 30 s and carefully remove the supernatant. Repeat the wash step one more time.
   4. Remove as much ethanol as possible. Air dry the beads on the magnetic stand for about 5-10 min at RT. Remove the tube from the magnetic stand and add 17.5 µL elution buffer. Vortex to resuspend and centrifuge briefly. Incubate at RT for 5 min.
   5. Place the tube onto the magnetic stand and wait till the solution becomes clear. Carefully transfer 15 µL supernatant to a new tube.
5. Quantify purified libraries using a fluorometer according to the user guide of dsDNA HS assay kit²¹. The yield of libraries ranges from ~15 to 300 ng. Take 10 ng of each library sample for pooling.
6. Perform sequencing as per the system user guide ²².
   NOTE: Purified libraries sequence at single end 50 bp on the platform, yielding about 2 million reads for each sample. A 0.03 x sequencing depth is recommended.
7. Data Analysis
   1. Enter the users' Name and Password in the login page (Figure 3A). In the YKPGS_analysis_tool page, select the "data format", "sequencing method", and "the way of construction". Enter the "users' mail address" (Figure 3B).
   2. Function of YKPGS_analysis_tool kit
      1. Upload the data generated from the sequencing system.
         NOTE: The NICS CNV analysis is performed automatically by the YKPGS_analysis_toolkit software. After the analysis, the result will be sent to the email address the user has provided. The modules of YKPGS_analysis_tool kit (Figure 3C) are as follows: 1) dir.txt: A parameter file, consists of two lines: data direction and user email. It is required to re-set up parameters each time before running the software. 2) YKPGS_analysis_toolkit.bat: After setting up parameters in dir.txt, double click it to upload Fastq data and analysis. 3) reanalysis.bat: In case of an incomplete data upload due to problem like internet misconnection, double click reanalysis.bat to re-upload and analyze data. 4) Program files: Data upload tools.
   3. Operate the "YKPGS_analysis_toolkit"
      1. To edit "dir.txt" file, open and edit "dir.txt" file with notepad, enter the Miseq default output data direction in the first line. Enter the user's email address in the second line to receive the analysis result. Save the file.
      2. Data upload and analysis (A network > 1MB is advised)
         1. Double click the "YKPGS_analysis_toolkit" tool. The data will be uploaded and analyzed synchronously. The result will be sent to the user's email when the analysis completed.
            NOTE: If an incomplete report is received, double click Reanalysis.bat tool to restart the process.

Results

We applied the method on a patient with a balanced translocation. IRB approval and informed consents were obtained before applying the NICS assay on the patient. Karyotype analysis of the patient showed a balanced translocation (1; 18) (p13.3; q21). We obtained a total of six blastocysts from the patient and performed NICS on Day 3-Day 5 culture medium of all six embryos. Chromosome abnormalities caused by the parents' balanced translocation were detected in five of them with the NICS...

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.