A method for scRRBS analysis of embryo culture medium

Abstract

This invention provides a method for single-cell reduced-representation bifulfite sequencing (scRRBS) of embryo culture medium. Using medical waste (blastocyst culture medium) from in vitro fertilization (IVF) procedures as raw material, this invention allows for simultaneous dual analysis of embryonic chromosomal aneuploidy and DNA methylation status. It assesses embryonic developmental potential from a novel perspective, considering epigenetics and the embryo's response to its culture environment, providing a new reference for selecting the "right" embryos in assisted reproduction and strongly supporting improvements in the success rate of IVF cycles.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and molecular cell biology, specifically to a method for single-cell reduced-representation bifulfitesequencing (scRRBS) of embryo culture medium, which utilizes epigenetic analysis techniques to analyze the embryo chromosome status and DNA methylation status in blastocyst culture medium. Background Technology

[0002] Since the birth of Louise Brown, the first "test-tube baby," in July 1978, and the Nobel Prize in Physiology or Medicine awarded to its pioneer Robert G. Edwards in 2010, millions of test-tube babies have been born worldwide in the past four decades, and the term "test-tube baby" has become widely known. Although with the rapid development of technology, in-vitro fertilization (IVF) technology has gone through first and second generations, and is now in its third generation, the overall success rate still does not meet people's expectations, currently generally around 40%.

[0003] In vitro fertilization (IVF) involves multiple disciplines, including obstetrics and gynecology, andrology, reproductive physiology, genetics, embryology, and developmental biology, and also relies heavily on laboratory techniques and clinical practice. An IVF cycle includes comprehensive examinations of both parents, ovulation induction to obtain healthy eggs, sperm retrieval, in-vitro fertilization, fertilized egg and embryo culture, selection of a suitable embryo for transfer, and a series of prenatal monitoring tests after the transfer until the birth of a healthy baby. During this approximately year-long cycle, many factors influence the embryo's developmental potential, which is a crucial factor in the success of the cycle.

[0004] Currently, the primary standard for selecting embryos and assessing their developmental potential is the morphological standard. While this standard is not entirely uniform, it is widely accepted by medical professionals and patients. With advancements in technology and deeper clinical analysis, researchers have discovered that the main cause of most miscarriages is chromosomal aneuploidy in the embryo. Morphologically normal embryos may not necessarily have normal chromosomal euploidy, a significant reason for implantation failure. Various aneuploidy screening (PGS) methods have emerged to address this, selecting embryos with normal chromosomes for implantation by screening a subset of embryonic cells to improve success rates.

[0005] However, even with PGS technology, some "normal" high-quality embryos still fail to implant. This is because after assisted reproductive technology (ART) in vitro fertilization, embryos are cultured in vitro for 3-5 days. During this period, from the fertilized egg to the morula or blastocyst stage, development occurs outside the mother's body, making the embryonic development heavily influenced by the culture environment. In the early stages of embryonic development, the embryo's epigenetic status undergoes dramatic changes. These changes can be mutated by external environmental influences, affecting the embryo's developmental potential. This can lead to reduced developmental potential in some morphologically sound embryos with normal chromosomes, resulting in implantation failure due to epigenetic interference. Currently, preimplantation genetic screening (PGS) does not analyze the embryo's epigenetic status.

[0006] Meanwhile, the mainstream PGS currently uses trophoblast cell biopsy at the blastocyst stage. Although this biopsy has not been proven to be harmful to the embryo and IVF, it requires a high level of skill and improper operation can lead to consequences such as embryo arrest.

[0007] CN105368936A uses waste liquid after embryo culture as material in an attempt to achieve non-invasive detection, but it still only analyzes the chromosome status.

[0008] In summary, current mainstream techniques for evaluating embryonic developmental potential have the following shortcomings:

[0009] Biopsy sampling requires a high level of skill in handling the embryo. Even if performed correctly, it can still cause some damage to the embryo. If an error is made, it can harm the embryo and lead to serious consequences.

[0010] The trophoblast cells may exhibit chimerism, and the few cells taken (3-5 cells) may not fully represent the embryo, leading to misjudgment.

[0011] Assessing solely based on chromosome status ignores crucial epigenetic changes in early embryonic development and fails to provide a more comprehensive reference for evaluating embryonic developmental potential.

[0012] Therefore, there is an urgent need for a new, non-invasive method that can simultaneously provide multi-angle references for detecting embryonic developmental potential. Summary of the Invention

[0013] This invention represents a novel improvement over existing detection technologies by introducing an epigenetic analysis method. It utilizes blastocyst culture medium to simultaneously analyze the chromosomal and methylation status of the embryo. This approach is not only non-invasive to the embryo and simple to obtain samples, but also provides a multi-faceted assessment of the embryo's developmental potential, making it safer and more reliable.

[0014] This invention provides a novel method for single-cell reduced-representation bifulfite sequencing (scRRBS) of embryo culture medium, comprising the following steps:

[0015] (1) Obtaining non-invasive samples: Fertilized eggs were obtained by intracytoplasmic sperm injection (ICSI). After 3 days of culture in blastocyst medium, the embryos were transferred to blastocyst medium and cultured until day 5 until they developed into blastocysts. The blastocysts were artificially shrunken to expel the fluid from the blastocyst cavity. The blastocysts were then transferred to new culture medium or frozen. All remaining culture medium was transferred to PCR tubes for later use.

[0016] (2) Construction of scRRBS sequencing library: According to the volume of culture medium sample, add lysis buffer and lysis enzyme in proportion, mix and incubate to inactivate lysis enzyme, and perform MspI digestion, end repair and A addition, adapter ligation and loop opening treatment, CT transformation and purification and recovery, PCR amplification, purification and second amplification, and recovery of target product.

[0017] (3) Library sequencing: Next-generation sequencing or nucleic acid chip, immunoblotting and other technologies are used to analyze the sequencing results, analyze the embryonic chromosome status and DNA methylation profile, and assess the embryonic developmental potential.

[0018] In step (1), it is crucial to completely remove the granulosa cells to ensure that there is no interference from DNA other than that from the embryo in the culture medium. When transferring the embryo to the blastocyst culture on day 3, it is necessary to wash it several times with blastocyst culture medium to remove DNA interference from non-embryo sources as thoroughly as possible. During blastocyst culture, gallbladder embryo culture is carried out, with each blastocyst culture droplet being between 5-50 μl in size and each culture medium sample being collected in a volume between 5-50 μl.

[0019] In step (2), the main components of the lysis buffer include 2-200mM Tris-EDTA, 1-50mM KCl, and 0.1wt%-5wt% of surfactants such as Triton X-100, SDS, Tween-20, and NP40.

[0020] The lysin is selected from one or more of proteinase K, Qiagen Protease, pepsin, papain, trypsin, and lysozyme, with an enzyme concentration of 1-30 μg / ml. The incubation temperature is 37-65℃ for 30 min to 12 h, and the inactivation temperature is not exceeding 80℃ for 10-45 min.

[0021] In the Msp I enzyme digestion system, Tango buffer is preferred. The amount of unmethylated λDNA added as reference DNA is about 1% of the DNA in the sample. The digestion temperature is 37℃ and the time is 1-6 hours. The inactivation temperature is 60-80℃ and the time is 10-30 minutes.

[0022] End repair and A addition use DNA polymerase Klenow fragment, Tango buffer is preferred, reaction temperature is 37℃, repair time is 20-60 min, and inactivation is performed at 60-80℃ for 5-30 min.

[0023] Adapter ligation uses methylated adapters and T4 DNA ligase. The preferred treatment process is as follows: pre-ligation at 16°C for 30 min, followed by ligation at 4°C overnight or for at least 8 hours, and finally treatment at 50-75°C for 20 min to inactivate the enzyme.

[0024] In one particular implementation, the connector is a hairpin structure sequence to ensure the stability of the connector and facilitate connection with the target fragment. There is a special U base in the loop structure of the hairpin, which can serve as a substrate for the USER enzyme. Therefore, the loop structure of the connector can be opened by treating it with the USER enzyme at 37°C for 20-50 min (preferably 30 min).

[0025] For CT conversion using bisulfite, the Invitrogen MethylCode Kit is preferred, and the kit instructions should be followed.

[0026] In one specific implementation, PCR amplification uses one or both of the following: KAPA HiFi U+ master Mix, Phusion high-fidelity (HF) PCR master Mix with HF buffer. The first amplification program is as follows: 98°C, 2 min; 98°C, 20 s, 60°C, 30 s, 72°C, 60 s, 6-12 cycles; 72°C, 5 min; store at 4°C.

[0027] After the first amplification, the amplified product is preferably purified by 0.5-1.5 times the volume of XP magnetic beads, and the purified product is then used for the second amplification. The second amplification program is as follows: 98℃, 2 min; 98℃, 20 s, 60℃, 30 s, 72℃, 60 s, 15-21 cycles; 72℃, 5 min; store at 4℃.

[0028] The target DNA fragment, ranging from 200 to 700 bp, is recovered. In one specific implementation, recovery is achieved using 1.5-2% TAE or TBE agarose gel electrophoresis. Purification of the target DNA fragment can be performed using kits such as the QIAquick gel extraction kit, following the kit's instructions.

[0029] In a specific implementation, NGS sequencing is used in step (3). The main instruments are Illumina series such as XTEN, NEXT-seq, and Highseq-2500, as well as the corresponding reagents and consumables. The operation is carried out in accordance with the instrument and reagent consumable SOP.

[0030] Another aspect of the present invention provides a method for dual analysis of chromosomal aneuploidy and DNA methylation status in embryos, including performing single-cell simplified representative bisulfite sequencing on embryo culture medium using the method of the present invention.

[0031] In some specific implementations, the step of assessing the developmental potential of the embryo based on the analysis of chromosomal aneuploidy and DNA methylation status is also included.

[0032] This invention utilizes medical waste (blastocyst culture medium) from in vitro fertilization (IVF) procedures as raw material to simultaneously perform dual analysis of embryonic chromosomal aneuploidy and DNA methylation status. The analysis results can be used for various purposes, including preimplantation genetic screening (PGS) and laboratory studies on embryonic development. Compared to the currently mainstream trophoblast cell biopsy, it offers the advantage of being non-invasive and safe for the embryo. Compared to the non-invasive PGS analysis using culture medium for chromosomal status (chromosomal aneuploidy) currently under development, it provides the same chromosomal aneuploidy detection and analysis while also analyzing DNA methylation. This allows for a novel assessment of embryonic developmental potential from an epigenetic perspective and considering the embryo's response to its culture environment. This provides a new reference for selecting the "right" embryo in assisted reproduction and strongly supports improving the success rate of IVF cycles. Attached Figure Description

[0033] Figure 1 shows the chromosome aneuploidy analysis of the trophoblast (TE) of two embryos, A and B, using sequencing data obtained from conventional PGS experimental procedures.

[0034] Figure 2 shows the chromosome aneuploidy analysis of the culture medium (SM) of two embryos, A and B, obtained using the scRRBS experimental procedure.

[0035] Figure 3 shows the methylation profile analysis of the trophoblast (TE) and corresponding culture medium (SM) of two embryos, A and B, obtained using the scRRBS experimental procedure (only a portion of chromosome 5 is shown). SD is the reference methylation profile. Detailed Implementation

[0036] The solutions in the embodiments of the present invention will be clearly and completely described below, but the present invention is not limited thereto. The described embodiments are only some embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the protection scope of the present invention. Similarly, the accompanying drawings of the embodiments are only some embodiments of the present invention. Other drawings obtained by those skilled in the art based on these drawings are also within the protection scope of the present invention.

[0037] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0038] Two intracytoplasmic sperm injection (ICSI) embryo samples (denoted as sample A and sample B) were collected. PGS and scRRBS sequencing were performed on the corresponding blastocyst trophoblast biopsy cells and blastocyst culture medium to analyze chromosome status and DNA methylation status. The specific procedures are as follows:

[0039] 1. Sample Acquisition:

[0040] Select qualified MII stage mature oocytes, remove granulosa cells attached to the oocytes as completely as possible, select healthy sperm, and obtain fertilized eggs by intracytoplasmic sperm injection. Culture the embryos in G1 medium (blastocyst medium, William A Cook Austrialia Pty Ltd) for 3 days until the blastocyst stage. Wash the embryos several times in fresh G2 medium (blastocyst medium, William A Cook Austrialia Pty Ltd) to remove any possible residual granulosa cells, and then transfer them into 10-20 μl of fresh G2 medium microdrops, with one embryo cultured per microdrop.

[0041] Cultured in G2 medium until day 5, the blastocysts are stimulated under a microscope to shrink for 5-15 minutes. The culture medium (10-20 μl) is carefully collected into a PCR tube. The blastocysts are then transferred to a new culture medium. After taking the trophoblast cells for control biopsy, they are transferred to a new culture medium or put into the freezing process.

[0042] 2. Construction of scRRBS methylated library

[0043] Based on the volume of the culture medium sample, add 10× lysis buffer and 1 / 20 volume of protease to make a final concentration of 10 mM Tris-1 mM EDTA, 10 mM KCl, 1 mM CaCl2, 1% Triton-X 100, and 2 mg / ml protease. Mix well and centrifuge to collect at the bottom of the tube.

[0044] Place the sample in a PCR instrument, lyse the cells at 55°C for 4 hours to release DNA, and inactivate the protease at 75°C for 30 minutes.

[0045] Add MspⅠ, 10×Tango buffer, and unmethylated λDNA to the lysis products until the final concentration is approximately 1% of the estimated sample DNA volume, consisting of 5U of digested MspⅠ, 1×Tango buffer, and unmethylated λDNA. Place the sample in a PCR instrument at 37℃ for 4 hours and then at 80℃ for 20 minutes.

[0046] Add DNA polymerase Klenow fragment, 10×Tango buffer, and end-repair dNTP mix to the enzyme digestion product to final concentrations of 5U, 1×, and 40μM, respectively. Mix thoroughly, centrifuge briefly to collect at the bottom of the tube, and incubate at 37℃ for 40 minutes and 75℃ for 15 minutes on a PCR instrument.

[0047] Add T4 DNA ligase, 10×Tango buffer, ATP, and methylation adapter (NEBNextMultiplex Oligos for Illumina Methylated Adaptor Index Primers Ste1) to the repair product, mix well, and place on a PCR instrument at 16°C for 30 minutes, then at 4°C overnight or for at least 8 hours. After completion, inactivate the enzyme at 65°C for 20 minutes. Add USER enzyme and digest at 37°C for 30 minutes to break the circular structure.

[0048] Following the Invitrogen MethylCode Kit instructions, CT transformation was performed. In short, the bisulfite transformation mixture was added to the ligation product to a total volume of 150 μl, mixed thoroughly, and then briefly centrifuged to collect the product at the bottom of the tube. The product was denatured at 98°C for 10 minutes, followed by bisulfite transformation at 64°C for 2.5 hours, and then incubated at 4°C for 20 hours for further transformation. The transformation product was then centrifuged, washed, purified, and eluted. 20-30 μl of the eluent was used for amplification.

[0049] The transformation product was purified and used for the first PCR amplification. A 50 μl system was prepared by adding 25 μl of 2×Kapa HiFi U+master Mix, 0.5 μl each of 15 μM NEB Universal primer and Index primer, and 24 μl of purified product. Amplification was performed as follows: 98℃, 2 min; 98℃, 20 s, 60℃, 30 s, 72℃, 60 s, 6 cycles; 72℃, 5 min; stored at 4℃. The PCR product was purified using 0.8×XP magnetic beads and washed with 40 μl of water.

[0050] For the second PCR amplification, a 100 μl system was used, containing 50 μl of 2×Kapa HiFi U+master Mix, 5 μl each of 15 μM NEB Universal primer and Index primer, and 40 μl of purified product. The amplification reaction was performed as follows: 98℃, 2 min; 98℃, 20 s, 60℃, 30 s, 72℃, 60 s, 18 cycles; 72℃, 5 min; stored at 4℃.

[0051] The amplified products were subjected to 2% TAE agarose gel electrophoresis. Bands between 200-700 bp were excised under UV light, and the library DNA was purified and recovered using the QIAquick gel extraction kit. For detailed recovery procedures, please refer to the QIAquick gel extraction kit instruction manual.

[0052] After obtaining the library, X-TEN bidirectional 150-cycle sequencing was performed.

[0053] The obtained data were analyzed using a methylation analysis procedure to determine the DNA methylation status of the samples. Simultaneously, a general PGS analysis procedure was used to analyze the chromosomal aneuploidy status of the samples. From the PGS scatter plots in Figures 1 and 2, it can be seen that the PGS analysis results of the culture medium and feeder layer are highly consistent for both samples A and B. Therefore, both methods can be used to accurately determine the chromosomal status of the embryos. The methylation spectra of samples A and B were also compared with the corresponding embryo methylation spectra in the Human hg19 reference genome database (Figure 3). The figure only shows the local methylation status of chromosome 5, but it can still provide a preliminary analysis of its changes and offer a reference for assessing the embryo's developmental potential.

[0054] This invention is not limited to the preferred embodiments described above. Anyone can derive other forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. A method for performing simplified representative bisulfite sequencing (scRRBS) of single cells in embryo culture medium for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Obtaining non-invasive samples: Fertilized eggs were obtained by intracytoplasmic sperm injection (ICSI). After the fertilized eggs were cultured in blastocyst medium to the blastocyst stage, they were transferred to blastocyst medium to be cultured until the blastocyst mature stage. The blastocysts were artificially shrunken to expel the fluid in the blastocyst cavity. All the culture medium was transferred to PCR tubes for later use. (2) Constructing the scRRBS sequencing library of embryo culture medium: The culture medium sample from step (1) was subjected to lyase lysis, MspI digestion of the lysate, end repair and A addition, adapter ligation and adapter loop opening treatment, CT transformation and purification recovery, PCR amplification, purification and second amplification, and recovery of the target product. (3) Sequencing the scRRBS sequencing library constructed in step (2); (4) Perform dual analysis for non-diagnostic purposes, including: using a methylation analysis process to analyze the data to obtain the DNA methylation status of the sample; and using a general PGS analysis process to analyze the chromosome aneuploidy status of the sample. In step (2), during Msp I digestion, the amount of unmethylated λDNA added as reference DNA is approximately 1% of the DNA mass in the sample; methylated adapters are used for adapter ligation, and T4 DNA ligase is used for ligation. Pre-ligation is performed at 16°C for 30 min, followed by overnight ligation at 4°C or for at least 8 hours. After ligation, the adapter is treated at 50-75°C for 20 min to inactivate the enzyme; the adapter is a hairpin structure sequence with a U base in the loop structure of the hairpin. The loop structure of the adapter is opened by treatment with USER enzyme at 37°C for 20-50 min.

2. According to the method described in claim 1, in step (1), the method for obtaining fertilized eggs is as follows: select qualified MII stage mature oocytes, remove the granulosa cells attached to the oocytes as completely as possible, select healthy sperm, and obtain fertilized eggs by intracytoplasmic sperm injection.

3. According to the method of claim 1, in step (1), after the fertilized egg is cultured in G1 medium to the blastocyst stage, the embryo is washed several times in blastocyst medium to remove possible residual granulosa cells, and then transferred to 5-50 μl of fresh blastocyst medium microdrops, with one embryo cultured per microdrop.

4. The method according to claim 1, wherein, In step (2), the lyase is selected from one or more of proteinase K, Qiagen Protease, pepsin, papain, trypsin and lysozyme, and the enzyme concentration is 1-30 μg / ml; the lysis buffer includes 2-200 mM Tris-EDTA, 1-50 mM KCl and 0.1 wt%-5 wt% surfactant, wherein the surfactant is selected from one or more of Triton X-100, SDS, Tween-20 and NP40.

5. The method according to claim 4, wherein, The lyase is incubated at 37-65℃ for 30 min to 12 h, and inactivated at 80℃ for 10-45 min.

6. The method according to claim 1, wherein, In step (2), the Msp I digestion buffer is Tango buffer, the digestion temperature is 37℃, the time is 1-6 hours, the inactivation temperature is 60-80℃, and the time is 10-30 min.

7. The method according to claim 1, wherein, In step (2), the end repair and A addition were performed using the Klenow fragment of DNA polymerase, with Tango buffer as the buffer, at a reaction temperature of 37°C. The repair time was 20-60 min, and the DNA polymerase was inactivated at 60-80°C for 5-30 min.

8. The method according to claim 1, wherein, In step (2), bisulfite is used for CT conversion.

9. The method according to claim 1, wherein, Step (2) PCR amplification uses one or both of the following: KAPA HiFi U+masterMix and Phusion high-fidelity (HF) PCR master Mix with HF buffer.

10. The method according to claim 1, wherein, The PCR amplification program is as follows: 98℃, 2 min; 98℃, 20 s, 60℃, 30 s, 72℃, 60 s, 6-12 cycles; 72℃, 5 min; store at 4℃. The second amplification program is as follows: 98℃, 2 min; 98℃, 20 s, 60℃, 30 s, 72℃, 60 s, 15-21 cycles; 72℃, 5 min; store at 4℃.

11. The method according to claim 10, wherein, After PCR amplification, the amplification product was purified using 0.5-1.5 times the volume of XP magnetic beads.

12. The method according to claim 1, wherein, In step (2), the target DNA fragment between 200-700 bp is recovered.

13. The method according to claim 12, wherein, Recovery was achieved by gel cutting using 1.5-2% TAE or TBE agarose gel electrophoresis.

14. The method according to claim 1, wherein, NGS sequencing is used in step (3).

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