A complex family pre-embryo implantation gene detection method and system

Abstract

This invention relates to the field of genetic testing technology, and discloses a method and system for preimplantation genetic testing of blastocysts cultured to 5-6 days in in vitro pedigrees based on third-generation sequencing. The method includes: performing third-generation sequencing on probands or carriers to construct a first SNP set containing pathogenic haplotypes; performing whole-genome amplification and second-generation sequencing on biopsy samples of blastocysts cultured to 5-6 days in vitro, performing aneuploidy detection, and constructing a second SNP set; matching the two SNP sets to screen for effective homozygous sites in blastocysts cultured to 5-6 days in vitro, classifying them based on linkage with pathogenic variants and calculating mutation scores to determine the pathogenic variant carrier status of blastocysts cultured to 5-6 days in vitro; and finally, comprehensively assessing the suitability of blastocysts cultured to 5-6 days in vitro for transplantation by combining chromosome ploidy and pathogenic variant carrier status. This invention effectively solves the testing problem in families with missing samples, optimizes the diagnostic process, and significantly reduces computational difficulty and testing costs, making it particularly suitable for preimplantation genetic testing of blastocysts cultured to 5-6 days in in vitro pedigrees in complex families.

Description

Technical Field

[0001] This invention relates to the field of medical data processing technology, and in particular to a method and system for preimplantation genetic testing of embryos in complex families. Background Technology

[0002] According to the latest statistics from the Online Mendelian Inheritance in Man (OMIM) database, there are over 7,000 known monogenic diseases, affecting approximately 400 million people worldwide. These diseases are serious and often lack effective treatments, placing a heavy burden on families and society. Preimplantation genetic testing for monogenic diseases (PGT-M) is a primary prevention method to block their transmission. Currently, the mainstream clinical PGT-M strategy mainly relies on linkage analysis of single nucleotide polymorphism (SNP) sites detected by next-generation sequencing (NGS) technology. This method constructs haplotypes using the genetic information of core family members (usually the couple and the proband), effectively overcoming the risk of allele decoupling associated with whole genome amplification (WGA) and ensuring diagnostic accuracy.

[0003] However, existing conventional technologies still have significant limitations and application bottlenecks when faced with complex clinical genetic contexts. First, for families with newly discovered mutations or missing pedigree members, conventional linkage analysis cannot be performed due to the lack of key parental genetic information required to construct haplotypes. Although gametes (such as sperm, polar bodies) or discarded embryos can be used for auxiliary analysis in clinical practice, this not only leads to cumbersome and prolonged testing procedures but also involves the consumption of precious reproductive resources, which is often unacceptable for elderly patients or those with diminished ovarian reserve. Second, some pathogenic genes have extremely complex genomic structures, such as those with high homologous pseudogene interference (e.g., PKD1 gene), large copy number variations (e.g., deletion or duplication of DMD gene), or located in tandem repeat regions (e.g., D4Z4). Currently attempted targeted long-read sequencing methods based on polymerase chain reaction (PCR) amplification often fail due to difficulties in primer design, uneven amplification efficiency, or non-specific amplification, lacking clinical universality.

[0004] The development of third-generation sequencing (TGS) technology has provided new opportunities for haplotype construction and characterization of complex genomic regions. Compared with the short-read sequencing (hundreds of bp) of NGS, TGS can generate reads longer than tens of kb, spanning complex repetitive regions and covering complete breakpoints of large structural variations, thus significantly improving the sensitivity and accuracy of structural variation detection. TGS can also read long fragments containing multiple linkage sites at once, resulting in more continuous and longer haplotype blocks, making allele-specific genotyping more accurate and containing richer information. Although TGS has significant advantages in variation detection and haplotype genotyping, its application in the PGT process is currently very complex, requiring TGS testing on multiple family members, which increases the economic burden and time costs. Summary of the Invention

[0005] The purpose of this invention is to provide a method and system for preimplantation genetic testing (PGT) of blastocysts cultured to 5-6 days in complex families. This method overcomes, to some extent, the problems of existing technologies. After identifying the pathogenic mutation, it eliminates the need for genome sequencing of other family members; only the proband or carrier needs to undergo TGS testing to complete the accurate PGT process. DNA samples from the proband or carrier are extracted for TGS library construction and sequencing. The data is processed to obtain BAM and VCF files with haplotype identifiers, identifying the haplotype containing the pathogenic mutation and screening for heterozygous SNP sites. DNA samples from blastocysts cultured to 5-6 days are extracted for NGS library construction and sequencing. The sequencing data is processed to obtain VCF datasets of blastocysts cultured to 5-6 days, and effective homozygous SNP sites are screened. The mutation fraction (MF) of the haplotype block containing the pathogenic mutation (within 1 Mb upstream and downstream) is calculated to determine whether the blastocysts cultured to 5-6 days are suitable for transplantation, thus improving efficiency and reducing costs.

[0006] To achieve this objective, the present invention adopts the following technical solution: According to one aspect of the present invention, a method for preimplantation genetic testing of complex family blastocysts cultured for 5-6 days in vitro for non-therapeutic and non-diagnostic purposes is provided. The method includes: performing TGS on probands or carriers, and constructing a first SNP set containing pathogenic variants and haplotype information after data processing; next, performing NGS on blastocysts cultured for 5-6 days in vitro, performing aneuploidy detection after data processing, and constructing a second SNP set containing genotype information of blastocysts cultured for 5-6 days in vitro; matching the first SNP set with the second SNP set, screening for effective heterozygous sites in the first SNP set and effective homozygous sites in the second SNP set, and calculating MF scores to determine whether blastocysts cultured for 5-6 days in vitro carry pathogenic variants; and comprehensively evaluating whether blastocysts cultured for 5-6 days in vitro are suitable for transplantation by combining the aneuploidy detection results and the pathogenic variant carrying status.

[0007] According to another aspect of the present invention, a preimplantation genetic testing system for blastocysts cultured in vitro to 5-6 days in complex families is provided. This system includes a haplotype construction module, an embryo analysis module, and a genetic diagnosis module. The haplotype construction module comprises, in sequence, a proband or carrier tissue collection unit, a DNA extraction and library construction unit, a third-generation sequencing unit, and a haplotype analysis unit, configured to construct a first SNP set containing haplotype information of pathogenic variants based on third-generation sequencing data from proband or carrier samples. The embryo analysis module comprises, in sequence, an embryo biopsy unit, a DNA extraction, amplification, and library construction unit, a second-generation sequencing unit, and an embryo data processing unit, configured to perform aneuploidy detection based on second-generation sequencing data from embryo samples and construct a second SNP set containing genotype information of blastocysts cultured in vitro to 5-6 days. The genetic testing result integration module is connected to the haplotype construction module and the embryo analysis module. It includes a variation score calculation unit and a test result output unit. It is configured to match the first SNP set and the second SNP set, screen the effective heterozygous sites of the first SNP set and the effective homozygous sites of the second SNP set and calculate the MF score. It combines the aneuploidy test results to output an embryo transfer evaluation report for blastocysts cultured in vitro for 5-6 days.

[0008] On the other hand, the embryos described in this invention are all blastocyst stage embryos cultured in vitro for 5-6 days.

[0009] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: First, this invention provides a linkage analysis method based on heterozygous matching between probands or carriers and homozygous matching of embryos. It eliminates the need for samples from parents or other family members; only third-generation sequencing data from the proband or carrier are required to construct a haplotype backbone. By anchoring and matching effective heterozygous SNPs from the proband or carrier with effective homozygous SNPs from the embryo, the genetic origin of blastocysts cultured to day 5-6 can be accurately inferred. This method effectively solves the problem of missing samples in complex families, achieving high-precision PGT linkage analysis with only a single sample.

[0010] Second, this invention employs a combined approach of proband or carrier third-generation sequencing and embryo second-generation sequencing, avoiding the expensive third-generation sequencing of batches of blastocysts cultured to 5-6 days in vitro. Simultaneously, by utilizing a homozygous site screening algorithm, it simplifies the traditionally complex family comparison process, significantly reducing the economic cost and bioinformatics computational load of a single testing cycle. Attached Figure Description

[0011] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This invention provides a flowchart of a method for preimplantation genetic testing of blastocysts cultured in vitro for 5-6 days in complex families. Figure 2 The genetic pedigree of family 6 in this embodiment of the invention is shown, along with the results of pathogenic variation detection in blastocysts cultured in vitro for 5-6 days. Figure 3 The diagram shows a structural block diagram of a preimplantation genetic testing system for blastocysts cultured in vitro for 5-6 days in complex families, provided by an embodiment of the present invention. Detailed Implementation

[0012] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0013] Example 1: Preimplantation genetic testing method for blastocyst stage embryos cultured in vitro to 5-6 days in complex families The following is combined with Figure 1 This invention describes a method for preimplantation genetic testing of complex family pedigrees cultured in vitro to the blastocyst stage at 5-6 days for non-diagnostic and non-therapeutic purposes, according to an exemplary embodiment of the present invention.

[0014] In one embodiment, the present invention proposes a method and system for preimplantation genetic testing of embryos from complex families. Figure 1 The diagram illustrates a flowchart of a preimplantation genetic testing method for blastocyst stage embryos cultured in vitro to 5-6 days in a complex family, according to an embodiment of the present invention.

[0015] S101 involves extracting genomic DNA from proband or carrier samples from a family, purifying and repairing the DNA with magnetic beads, ligating sequencing adapters, and purifying the DNA again with magnetic beads to complete library construction and sequencing.

[0016] In a preferred embodiment of the present invention, the experimental samples were obtained from Peking University Third Hospital. After obtaining approval from the hospital's ethics committee and signing informed consent forms from the subjects (probands or carriers), peripheral venous blood or tissue samples were collected. Subsequently, pure genomic DNA was isolated using a commercially available genomic DNA extraction kit [Qiagen® Blood Genomic DNA Mini-Prep Kit (QIAGEN GmbH, Germany)], ensuring DNA purity (OD260 / 280 ≈ 1.8-2.0) and integrity (agarose gel electrophoresis showed no significant degradation).

[0017] Take an appropriate amount of qualified DNA sample, add magnetic beads for purification, and take an appropriate amount of sample for Qubit quantification; use DNA repair reagent to repair the damaged and terminally damaged DNA and prepare DNA fragments suitable for connecting sequencing adapters.

[0018] DNA was purified again with magnetic beads and further eluted with elution buffer. 1 µl of the eluted sample was quantified using a Qubit fluorometer. Sequencing adapters were ligated using salt-tolerant T4 DNA ligase. Magnetic beads were added for purification to remove unligated adapters and short fragments.

[0019] The purified library is loaded into a sequencing chip and installed into the corresponding sequencer (such as PromethION, R10.4.1 chip) for whole genome sequencing, completing the TGS whole genome library preparation and sequencing process.

[0020] S102. After the TGS data is taken off the machine, it is first processed to generate high-quality sequences, and then quality assessment and generation of BAM unaligned format files are performed. Then, through an integrated workflow, alignment, variant detection and haplotype analysis are performed, the information richness of the haplotype block where the pathogenic variant is located is assessed, and the first SNP set is obtained.

[0021] In a preferred embodiment of the present invention, for TGS data from ONT, the chopper tool is used to filter low-quality and short sequences, and the samtools tool is used to convert FASTQ format files into unaligned BAM format files; for TGS data from PacBio, the cccs tool is used to generate high-quality consistent sequences, generating unaligned BAM format files. The Nanoplot tool is used to evaluate the sequencing data quality, including the total number of bases produced, read length distribution, and read quality distribution.

[0022] Alignment was performed using the Epi2Me workflow wf-human-variation (with the reference genome set to hg38 or hs1), and SNP, SV, CNV, and STR variants were detected using the parameters "--cnv --sv --snp --str". Haplotype typing was performed using the "--phased" parameter, resulting in a BAM file, a VCF file with typing markers, and a GTF file containing the start and end positions of haplotype blocks.

[0023] The haplotype (hap1 or hap2) containing the pathogenic variant, along with the start and end positions of the haplotype block, can be visualized using a gene browser (such as IGV software). The length of the haplotype block containing the pathogenic variant can then be calculated. Genotype information of heterozygous SNPs within the haplotype block containing the pathogenic variant (the first SNP set) can be extracted using the bcftools tool. The first SNP set is then counted (if the haplotype block's start or end point is more than 1 Mb from the pathogenic variant, only heterozygous SNPs within 1 Mb upstream and downstream are included; if the haplotype block's start or end point is less than 1 Mb from the pathogenic variant, the haplotype block's start or end point is used as the start or end point for SNP inclusion). This allows for an assessment of the richness of information in the haplotype block containing the pathogenic variant, thereby evaluating the likelihood of successful PGT. The longer the haplotype block containing the pathogenic variant, the more heterozygous SNPs it covers, and the greater the likelihood of successful PGT; otherwise, the likelihood of successful PGT may be lower.

[0024] S103, after confirming the implementation of PGT, further procedures were performed including embryo biopsy and DNA extraction at the blastocyst stage (5-6 days after in vitro culture), and WGA and NGS testing.

[0025] In a preferred embodiment of the present invention, the experimental samples were obtained from the Reproductive Medicine Center of Peking University Third Hospital. After obtaining approval from the hospital's ethics committee and obtaining informed consent from the patients, mature MII-stage oocytes were collected from female patients participating in the PGT cycle. Subsequently, the oocytes were fertilized with intracytoplasmic sperm injection (ICSI) and cultured in vitro for 5-6 days to the blastocyst stage. Three to five trophoblast cells were biopsied from each blastocyst. DNA extraction was performed using the same steps as in S101.

[0026] Whole genome amplification was performed on biopsied trophoblast cells using a commercial amplification kit [MALBAC® Single Cell Whole Genome Amplification Kit (EconGene)] strictly following the instructions. Sequencing libraries were constructed from the whole genome amplification products using a commercial library construction kit [NEBNext® Ultra™ II DNA Library Preparation Kit (New England Biolabs, USA)], and sequencing was performed on a next-generation sequencer (such as Illumina HiSeq X Ten) at approximately 2× coverage depth.

[0027] S104 performs quality control, alignment, and deduplication on the NGS data after it is processed, performs aneuploidy detection, and performs local realignment, correction, and mutation detection. It further filters to obtain high-quality SNP loci (second SNP set) and constructs the second SNP set genotype and deep dataset of embryos cultured to blastocyst stage 5-6 days in vitro.

[0028] In a preferred embodiment of the present invention, the initial raw FASTQ file from NGS is quality controlled using the trim_galore tool (parameters --quality 20 --phred33 --stringency 3), removing adapters, primers, and low-quality sequences to evaluate and generate high-quality sequence data. Then, using a human reference genome (e.g., hg38), sequence alignment is performed using the BWA tool, and PCR duplicate reads are further removed using the Picard tool to obtain a BAM format file after whole-genome alignment.

[0029] All chromosomes were divided into multiple windows with a unit size of 1 Mb, and the read depth of all windows for each chromosome was calculated using the readCounter tool. The raw sequencing depth was normalized to a sample average depth using the HMMcopy package in R, and the normalized depth factor was calculated. Deletions or duplications ≥10 Mb with a chimerism rate ≥30% were detected to assess chromosome ploidy in blastocysts cultured to day 5-6.

[0030] Based on the BAM format file obtained after whole-genome alignment, the RealignerTargetCreator tool in GATK was used to perform local realignment on the chromosomes containing pathogenic variants. IndelRealigner was used to optimize the alignment accuracy of the indels, and BaseRecalibrator was used for base quality correction, resulting in a locally aligned and optimized BAM format file. The HaplotypeCaller tool in GATK was then used for variant detection, yielding a VCF format file containing a dataset of variant sites from all embryonic samples.

[0031] SNP loci were further extracted using the SelectVariants tool in GATK, and low-quality SNP loci were removed using the VariantFiltration tool (parameters --filterExpression "DP<10 || QD<2.0 || FS>60.0 || MQ<40.0 || MQRankSum<-12.5 || ReadPosRankSum<-8.0"), resulting in a second SNP set. Genotypic and depth information of the SNPs were then extracted using the bcftools tool to construct embryonic SNP genotypic and depth datasets.

[0032] S105: TGS analysis of probands or carriers yields the locations of all loci in the first SNP set, and NGS analysis of blastocysts cultured to 5-6 days in vitro matches the second SNP set. Effective homozygous loci are then screened in blastocysts cultured to 5-6 days in vitro. Homozygous loci are classified according to their linkage with pathogenic variants, and MF scores are calculated to determine whether blastocysts cultured to 5-6 days in vitro carry pathogenic variants.

[0033] In a preferred embodiment of the present invention, based on the second SNP set (obtained in process S102), information on homozygous sites with a depth greater than or equal to 3 in blastocysts cultured to 5-6 days in vitro (obtained in process S104) is identified. If the bases at the corresponding site in the blastocysts cultured to 5-6 days in vitro are identical to the bases linked to the mutant haplotype of the proband or carrier, it is determined to be a mutant supporting site (MSS); if the bases are identical to the bases linked to the wild-type haplotype, it is determined to be a wild-type supporting site (WSS); if neither of the above conditions is met, it is determined to be an unknown site (US).

[0034] After assessing all available sites, the MSS (M), WSS (W), and US (U) counts were calculated for each embryo. The MF score was calculated using the following formula: For the sake of conservatism, a blastocyst cultured to day 5-6 is considered to carry a pathogenic variant only when the MF score is greater than 60%; a blastocyst cultured to day 5-6 is considered not to carry a pathogenic variant when the MF score is less than 40%; and a blastocyst cultured to day 5-6 has a MF score between 40% and 60%, making it impossible to determine whether it carries a pathogenic variant. For example, in pedigree 6, the MF score of blastocyst E1 cultured to day 5-6 is 88.2%, indicating that this embryo carries a pathogenic variant; the MF score of blastocyst E3 cultured to day 5-6 is 5.4%, indicating that this embryo does not carry a pathogenic variant. The results of this method for all embryos in this family are consistent with the conventional method for determining whether blastocysts cultured to day 5-6 carry a pathogenic variant. Figure 2 ) S106. Based on the chromosome ploidy results of the blastocyst stage embryos cultured in vitro to day 5-6 obtained in S104 and the pathogenic variant carrier status obtained in S105, a comprehensive evaluation is made to determine whether the embryos can be used for embryo transfer.

[0035] In a preferred embodiment of the present invention, if the chromosome ploidy result of the blastocyst stage embryo cultured to 5-6 days in vitro obtained in S104 is normal, and S105 shows that the blastocyst stage embryo cultured to 5-6 days in vitro does not carry the pathogenic variant, it can be considered suitable for transplantation; if the chromosome ploidy result of the blastocyst stage embryo cultured to 5-6 days in vitro obtained in S104 is normal, and S105 shows that it is impossible to determine whether it carries the pathogenic variant, it is necessary to further supplement the proband or carrier's spouse with approximately 2× coverage depth NGS testing, and use all SNPs (including heterozygous and homozygous) upstream and downstream of the pathogenic variant in the blastocyst stage embryo cultured to 5-6 days in vitro to perform linkage analysis to further determine whether it carries the pathogenic variant; if the chromosome ploidy result of the blastocyst stage embryo cultured to 5-6 days in vitro is abnormal or carries the pathogenic variant, it is considered unsuitable for transplantation, or further genetic counseling is required.

[0036] In this embodiment, 82 blastocysts cultured in vitro to 5-6 days were tested from 16 families, and it was found that 89% of the embryos did not require NGS data from the proband or carrier's spouse for PGT.

[0037] Example 2: Preimplantation genetic testing system for blastocysts cultured in vitro to 5-6 days in complex families In one implementation, such as Figure 3 As shown, the present invention also provides a preimplantation genetic testing system for blastocyst-stage embryos cultured in vitro for 5-6 days in complex families, comprising: The haplotype construction module 301 is used to collect proband or carrier samples, extract sample DNA, construct third-generation sequencing libraries, perform third-generation sequencing, and construct haplotypes (first SNP set) containing pathogenic variants and heterozygous SNP site information based on the sequencing data. Embryo analysis module 302 is used to obtain biopsy cell samples from blastocyst embryos cultured in vitro to 5-6 days, perform DNA extraction and whole genome amplification (WGA), construct a second-generation sequencing library, perform second-generation sequencing, and perform aneuploidy detection (PGT-A) based on the sequencing data, as well as construct a second SNP set containing genotype information of blastocyst embryos cultured in vitro to 5-6 days. The genetic testing result integration module 303 is used to match the first SNP set with the second SNP set, screen out effective homozygous SNP sites in blastocysts cultured to 5-6 days in vitro, perform linkage analysis with heterozygous SNP sites in probands or carriers, calculate the MF score of the pathogenic haplotype block, and finally output an evaluation report for blastocyst transfer cultured to 5-6 days in vitro by combining the aneuploidy detection results.

[0038] The detection system provided in the above embodiments of the present invention and the preimplantation genetic detection method for blastocysts cultured in vitro for 5-6 days in complex families provided in the embodiments of the present invention are based on the same inventive concept and have the same beneficial effects as the methods used, run or implemented by their stored applications.

[0039] The various embodiments in this invention are described in a related manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the embodiment evaluating a preimplantation genetic testing system for blastocysts cultured in vitro to 5-6 days in complex families is described simply because it is substantially similar to the example of a preimplantation genetic testing method for blastocysts cultured in vitro to 5-6 days in complex families described above. Relevant details can be found in the description of the example of a preimplantation genetic testing method for blastocysts cultured in vitro to 5-6 days in complex families described above.

[0040] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preimplantation genetic testing of complex family blastocysts cultured for 5-6 days in vitro for non-therapeutic and non-diagnostic purposes, the method comprising: Step (1): Perform TGS (third-generation sequencing) on ​​probands or carriers, and construct the first SNP set containing pathogenic variants and haplotype information after data processing; Step (2): Next, NGS (next-generation sequencing) was performed on blastocysts cultured to 5-6 days in vitro. After data processing, aneuploidy was detected, and a second SNP set containing genotype information of blastocysts cultured to 5-6 days in vitro was constructed. Step (3): Match the first SNP set with the second SNP set, screen the effective heterozygous sites of the first SNP set and the effective homozygous sites of the second SNP set and calculate the MF score, so as to determine whether the blastocyst stage embryos cultured in vitro for 5-6 days carry pathogenic variants. Step (4): Combining the aneuploidy detection results and the pathogenic mutation carrier status, comprehensively evaluate whether the blastocyst stage embryos cultured in vitro for 5-6 days are suitable for transplantation.

2. The method according to claim 1, characterized in that, The specific steps (1) are as follows: S101 involves extracting genomic DNA from proband or carrier samples from a family, purifying and repairing the DNA with magnetic beads, ligating sequencing adapters, and purifying the DNA with magnetic beads again to complete library construction and sequencing. S102. After the TGS data is taken off the machine, it is first processed to generate high-quality sequences, and then quality assessment and generation of BAM unaligned format files are performed. Then, through an integrated workflow, alignment, variant detection and haplotype analysis are performed, the information richness of the haplotype block where the pathogenic variant is located is assessed, and the first SNP set is obtained.

3. The method according to claim 2, characterized in that, Step (2) specifically involves: S103, after confirming the implementation of PGT, further embryo biopsy and DNA extraction were performed on blastocysts cultured in vitro for 5-6 days, followed by WGA and NGS testing. S104 performs quality control, alignment, and deduplication on the NGS data after it is processed, performs aneuploidy detection, and performs local re-alignment, correction, and mutation detection. It further filters to obtain high-quality SNP loci, i.e., the second SNP set, and constructs the second SNP set genotype and deep dataset of embryos cultured in vitro to the blastocyst stage at 5-6 days.

4. The method according to claim 1, characterized in that, Step (3) specifically involves: S105: TGS analysis of probands or carriers yields the locations of all loci in the first SNP set, and NGS analysis of blastocysts cultured to 5-6 days in vitro matches the second SNP set. Valid homozygous loci are then screened in blastocysts cultured to 5-6 days in vitro. Based on their linkage to pathogenic variants, homozygous loci in blastocysts cultured to 5-6 days in vitro are classified, MF scores are calculated, and it is determined whether blastocysts cultured to 5-6 days in vitro carry pathogenic variants.

5. The method according to claim 1, characterized in that, Step (4) specifically involves: S106. Based on the chromosome ploidy results of the blastocyst stage embryos cultured in vitro for 5-6 days obtained in S104 according to claim 3 and the pathogenic variant carrier status obtained in S105 according to claim 4, a comprehensive evaluation is made as to whether the embryos can be used for embryo transfer.

6. The method according to any one of claims 1 and 4, characterized in that: The formula for calculating MF scores is: Where M represents the number of mutant supporting sites in each blastocyst stage embryo cultured to 5-6 days in vitro, W represents the number of wild-type supporting sites (W), and U represents the number of unknown sites.

7. A preimplantation genetic testing system for blastocysts in complex families cultured in vitro for 5-6 days, the system comprising a haplotype construction module, an embryo analysis module, and a genetic testing result integration module.

8. The detection system according to claim 7, characterized in that, The haplotype construction module includes a proband or carrier tissue collection unit, a DNA extraction and library construction unit, a third-generation sequencing unit, and a haplotype analysis unit connected in sequence. It is configured to construct a first SNP set containing haplotype information of pathogenic variants based on the third-generation sequencing data of proband or carrier samples.

9. The detection system according to claim 7, characterized in that, The embryo analysis module includes an embryo biopsy unit, a DNA extraction, amplification and library construction unit, a next-generation sequencing unit and an embryo data processing unit connected in sequence. It is configured to perform aneuploidy detection based on next-generation sequencing data of blastocyst embryos cultured in vitro to 5-6 days and to construct a second SNP set containing genotype information of blastocyst embryos cultured in vitro to 5-6 days.

10. The detection system according to claim 7, characterized in that, The genetic testing result integration module is connected to the haplotype construction module and the embryo analysis module. It includes a variation score calculation unit and a test result output unit. It is configured to match the first SNP set and the second SNP set, screen the effective heterozygous sites of the first SNP set and the effective homozygous sites of the second SNP set and calculate the MF score. It combines the aneuploidy test results to output an embryo transfer evaluation report for blastocysts cultured in vitro for 5-6 days.

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