Deformity sperm detection panel, detection kit and application thereof

Abstract

The invention belongs to the technical field of assisted reproduction polygene detection, and discloses a malformation sperm detection panel, a detection kit and application thereof, and the detection panel comprises mutant genes related to malformation of the head, neck and tail of sperms for detection, copy number variation genes and rearrangement event and deletion genes. By adopting the detection panel or the detection kit provided by the invention, accurate sequencing can be realized. By detecting the abnormal condition of the gene contained in the application as a reference for clinical diagnosis and genetic analysis of the teratospermia, optimization of an adjuvant therapy scheme is facilitated, fertilization abnormality is avoided or reduced, the clinical outcome of assisted reproduction is improved, and the risk of offspring heredity is blocked. The method has important guiding significance for clinical diagnosis, genetic analysis, fertilization failure risk assessment, in-vitro fertilization clinical outcome prediction, offspring genetic risk blocking propagation and potential personalized intervention measures of the patients with the abnormal sperms and the patients with high abnormal sperm rate to be treated by the assisted reproductive technology.

Description

Technical Field

[0001] The present application relates to the technical field of multi-gene detection in assisted reproduction, and in particular to a teratozoospermia detection panel, a detection kit and applications thereof. Background Art

[0002] Infertility is a major issue in human reproductive health. Approximately 10-15% of couples worldwide experience infertility during pregnancy, with male infertility accounting for 50%. Teratozoospermia is a major cause of male infertility. Teratozoospermia refers to a sperm abnormality ratio exceeding the normal range (typically, the diagnosis is made when the proportion of sperm with normal morphology is less than 4%). Teratozoospermia is primarily classified based on the location and characteristics of the sperm morphological abnormalities, primarily manifesting as abnormalities of the head, neck, and tail. The sperm head, a key part of the uterus that carries genetic material, is the most common site of abnormalities, including large, small, tapered, amorphous, double, and vacuolated heads. The neck connects the head and tail and is responsible for transmitting motility signals. Deformities include curved, thickened, thin, and broken necks. The tail is the driving force behind sperm movement, and abnormalities can severely impact motility. These abnormalities include short, long, curly, double, multiple, broken, and multiple morphological abnormalities of the sperm tail (MMAF). Sperm morphological abnormalities greatly damage male fertility, leading to decreased sperm motility, decreased fertilization rate and affecting fertility.

[0003] Teratozoospermia, characterized by a high proportion of abnormal sperm in semen, is a common cause of fertilization failure and poor clinical outcomes. Clinically, selecting normal sperm for intracytoplasmic sperm injection (ICSI) is an important treatment option for patients with teratozoospermia. However, 2%-3% of ICSI cycles still result in fertilization failure, low fertilization rates, and poor assisted reproductive clinical outcomes due to sperm abnormalities. In the absence of an accurate diagnosis, doctors have no predictive information for male patients with teratozoospermia other than morphological analysis, and cannot accurately predict prognosis or provide interventional treatment. They can only rely on repeated symptomatic treatment, which does not address the fundamental problem. Patients who successfully undergo assisted reproduction also worry about the health and safety of their offspring, and whether there is a risk of inheritance to their offspring. This places tremendous psychological, physical, and financial pressure on patients seeking assisted reproductive treatment, potentially leading to negative emotions such as anxiety and depression.

[0004] With the advancement of gene sequencing technology and the deepening of reproductive genetics research, high-risk genes for teratozoospermia have been increasingly recognized and discovered. However, this research has largely focused on basic research and morphological analysis, while clinical genetic testing and evaluation for teratozoospermia are still limited. Therefore, there is an urgent need for a genetic testing panel and method that can cover the majority of genes with clinical diagnostic and therapeutic significance for teratozoospermia. This approach can help target mutant genes associated with teratozoospermia, optimize assisted reproductive treatment options, avoid or reduce fertilization abnormalities, improve clinical outcomes of assisted reproduction, and mitigate genetic risks to offspring. Summary of the Invention

[0005] The purpose of this application is to provide a teratozoospermia gene detection panel, detection kit, and their applications to efficiently detect gene mutations with clinical diagnostic and therapeutic significance for teratozoospermia. The specific technical solution is as follows:

[0006] A first aspect of the present application provides a teratozoospermia detection panel based on high-throughput sequencing, wherein the detection panel includes teratozoospermia-related mutant genes, genes with copy number variations, and genes with rearrangement events and deletions for detection.

[0007] In some embodiments of the present application, the mutant gene includes the gene:

[0008] <h2 style=";text-align:left;direction:ltr">ACTL7A、ACTL7B、ADAD2、ADCY10、ADGB、AGTPBP1、AK7、AKAP3、AKAP4、AMZ2、ANKRD31、ARL2BP、ARMC2、AR MC4、ATG7、ATP2B4、AU040320、AURKC、AXDND1、BRDT、BRWD1、BSCL2、CALICIN、C2CD6、C7orf61、CATSPER 1、CATSPER 2、CATSPER 3、CC2D1B、CCDC103、CCDC113、CCDC114、CCDC136、CCDC146、CCDC151、CCDC181、CCDC188、CCDC189、CCDC34、CCDC38、CCDC39 、CCDC40、CCDC62、CCDC65、CCIN、CCNB3、CCNO、CDY1、CEP78、CEP112、CEP128、CEP135、CEP250、CEP290、CEP350、CFAP206、CF AP251、CFAP298、CFAP300、CFAP43、CFAP44、CFAP47、CFAP52、CFAP53、CFAP54、CFAP57、CFAP58、CFAP61、CFAP65、CFAP69、CF AP70、CFAP74、CFAP91、CHPT1、CTE1 / DRC5、CNTROB、CSNK2A2、DHC1、DNAAF4、DNAF1、DNAF2、DNAF3、DNAF4、DNAF5、DNAF6、DNAF 7、DNAH1、DNAH10、DNAH11、DNAH12、DNAH17、DNAH2、DNAH3、DNAH5、DNAH6、DNAH7、DNAH8、DNAH9、DNAI1、DNAI2、DNAJB13、DNA JB13a、DNALI1、DNHD1、DRC1、DRC4、DPY19L2、DZIP1、FBXO43、FADS2、FAM71D、FSIP2、GALNT3、GANLNTL5、GAPDS、GAS2L2、GAS8、 GBA2、GGN、GOPC、GM130、HRB、HOOK1、HSP90B1、HYDIN、IFT74、KATNAL2、KIAA1210、LRRC46、LRRC56、LRRC6、MAATS1、MDC1、MC IDAS、MEIKIN、MFSD14A、MKRN2、MNS1、NDUFA13、NHE8、NME8、NPHP4、NUP210L、OAZ3、ODAD3、ODF1、ODF2、PACRG、PDCL2、PICK1、PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSP H4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPAT C1L, SPATA16, SPATA20, SPATA6, SPEM1, SPINK2, SPEF2, SPPL2C, SSP411, SSMEM1, STK33, STK36, SUN5, TBC1D21, TCTE1, TDRD9, TENT5D, TNP2, T PTE2, TSSK3, TSGA10, TTC12, TTC21A, TTC25, TTC29, VDAC2, VDAC3, VPS13B, VPS54, WDR12, WDR19, WDR63, WDR66, ZCWPW1, ZMYND15, ZPBP, ZPBP1. ,

[0009] In some embodiments of the present application, the gene with copy number variation includes the gene:

[0010] <h2 style=";text-align:left;direction:ltr">ACTL7A、ACTL7B、ADAD2、ADCY10、ADGB、AGTPBP1、AK7、AKAP3、AKAP4、AMZ2、ANKRD31、ARL2BP、ARMC2、AR MC4、ATG7、ATP2B4、AU040320、AURKC、AXDND1、BRDT、BRWD1、BSCL2、CALICIN、C2CD6、C7orf61、CATSPER 1、CATSPER 2、CATSPER 3、CC2D1B、CCDC103、CCDC113、CCDC114、CCDC136、CCDC146、CCDC151、CCDC181、CCDC188、CCDC189、CCDC34、CCDC38、CCDC39 、CCDC40、CCDC62、CCDC65、CCIN、CCNB3、CCNO、CDY1、CEP78、CEP112、CEP128、CEP135、CEP250、CEP290、CEP350、CFAP206、CF AP251、CFAP298、CFAP300、CFAP43、CFAP44、CFAP47、CFAP52、CFAP53、CFAP54、CFAP57、CFAP58、CFAP61、CFAP65、CFAP69、CF AP70、CFAP74、CFAP91、CHPT1、CTE1 / DRC5、CNTROB、CSNK2A2、DHC1、DNAAF4、DNAF1、DNAF2、DNAF3、DNAF4、DNAF5、DNAF6、DNAF 7、DNAH1、DNAH10、DNAH11、DNAH12、DNAH17、DNAH2、DNAH3、DNAH5、DNAH6、DNAH7、DNAH8、DNAH9、DNAI1、DNAI2、DNAJB13、DNA JB13a、DNALI1、DNHD1、DRC1、DRC4、DPY19L2、DZIP1、FBXO43、FADS2、FAM71D、FSIP2、GALNT3、GANLNTL5、GAPDS、GAS2L2、GAS8、 GBA2、GGN、GOPC、GM130、HRB、HOOK1、HSP90B1、HYDIN、IFT74、KATNAL2、KIAA1210、LRRC46、LRRC56、LRRC6、MAATS1、MDC1、MC IDAS、MEIKIN、MFSD14A、MKRN2、MNS1、NDUFA13、NHE8、NME8、NPHP4、NUP210L、OAZ3、ODAD3、ODF1、ODF2、PACRG、PDCL2、PICK1、PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSP H4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPAT C1L, SPATA16, SPATA20, SPATA6, SPEM1, SPINK2, SPEF2, SPPL2C, SSP411, SSMEM1, STK33, STK36, SUN5, TBC1D21, TCTE1, TDRD9, TENT5D, TNP2, T PTE2, TSSK3, TSGA10, TTC12, TTC21A, TTC25, TTC29, VDAC2, VDAC3, VPS13B, VPS54, WDR12, WDR19, WDR63, WDR66, ZCWPW1, ZMYND15, ZPBP, ZPBP1. ,

[0011] In some embodiments of the present application, the genes that undergo rearrangement events and deletions include genes:

[0012] <h2 style=";text-align:left;direction:ltr">ACTL7A、ACTL7B、ADAD2、ADCY10、ADGB、AGTPBP1、AK7、AKAP3、AKAP4、AMZ2、ANKRD31、ARL2BP、ARMC2、AR MC4、ATG7、ATP2B4、AU040320、AURKC、AXDND1、BRDT、BRWD1、BSCL2、CALICIN、C2CD6、C7orf61、CATSPER 1、CATSPER 2、CATSPER 3、CC2D1B、CCDC103、CCDC113、CCDC114、CCDC136、CCDC146、CCDC151、CCDC181、CCDC188、CCDC189、CCDC34、CCDC38、CCDC39 、CCDC40、CCDC62、CCDC65、CCIN、CCNB3、CCNO、CDY1、CEP78、CEP112、CEP128、CEP135、CEP250、CEP290、CEP350、CFAP206、CF AP251、CFAP298、CFAP300、CFAP43、CFAP44、CFAP47、CFAP52、CFAP53、CFAP54、CFAP57、CFAP58、CFAP61、CFAP65、CFAP69、CF AP70、CFAP74、CFAP91、CHPT1、CTE1 / DRC5、CNTROB、CSNK2A2、DHC1、DNAAF4、DNAF1、DNAF2、DNAF3、DNAF4、DNAF5、DNAF6、DNAF 7、DNAH1、DNAH10、DNAH11、DNAH12、DNAH17、DNAH2、DNAH3、DNAH5、DNAH6、DNAH7、DNAH8、DNAH9、DNAI1、DNAI2、DNAJB13、DNA JB13a、DNALI1、DNHD1、DRC1、DRC4、DPY19L2、DZIP1、FBXO43、FADS2、FAM71D、FSIP2、GALNT3、GANLNTL5、GAPDS、GAS2L2、GAS8、 GBA2、GGN、GOPC、GM130、HRB、HOOK1、HSP90B1、HYDIN、IFT74、KATNAL2、KIAA1210、LRRC46、LRRC56、LRRC6、MAATS1、MDC1、MC IDAS、MEIKIN、MFSD14A、MKRN2、MNS1、NDUFA13、NHE8、NME8、NPHP4、NUP210L、OAZ3、ODAD3、ODF1、ODF2、PACRG、PDCL2、PICK1、PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSP H4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPAT C1L, SPATA16, SPATA20, SPATA6, SPEM1, SPINK2, SPEF2, SPPL2C, SSP411, SSMEM1, STK33, STK36, SUN5, TBC1D21, TCTE1, TDRD9, TENT5D, TNP2, T PTE2, TSSK3, TSGA10, TTC12, TTC21A, TTC25, TTC29, VDAC2, VDAC3, VPS13B, VPS54, WDR12, WDR19, WDR63, WDR66, ZCWPW1, ZMYND15, ZPBP, ZPBP1. ,

[0013] In some embodiments of the present application, the mutation types of the mutant gene include genomic structural variation, single nucleotide variation, frameshift mutation, point mutation, deletion, insertion and fusion.

[0014] The second aspect of the present application provides a teratozoospermia detection kit based on high-throughput sequencing, wherein the detection kit includes detection probes and detection reagents; the detection probes are targeted at mutant genes, genes with copy number variations, and genes with rearrangement events and deletions in the detection panel described in the first aspect of the present application.

[0015] The third aspect of the present application provides a use of the detection panel described in the first aspect of the present application or the detection kit described in the second aspect of the present application in preparing a device for detecting teratozoospermia.

[0016] A fourth aspect of the present application provides a device for detecting teratozoospermia, comprising:

[0017] The sequencing module is used to extract and perform high-throughput sequencing on the DNA sample to be tested to obtain sequencing results;

[0018] A verification module, configured to process the high-throughput sequencing results and compare them with the detection panel described in the first aspect of this application to obtain the mutation information;

[0019] The evaluation module is used to conduct literature verification and comparison of the mutation information, verify the pathogenic mutation sites in the literature and the case evaluation in the literature; for newly detected mutation sites and / or mutation sites that are assessed as having unclear pathogenicity by the mutation software variation classification, the software is used to accurately construct and analyze the three-dimensional structural model of the entire gene protein, and molecular modeling analysis is used to evaluate the possible impact of gene mutations on the secondary and tertiary structures of the protein to determine whether they are new pathogenic mutation sites.

[0020] In some embodiments of the present application, the software is Alpha Fold-3 software.

[0021] In some embodiments of the present application, the device for detecting teratozoospermia further includes an intervention module for analyzing the information obtained by the verification module and / or the evaluation module and providing clinical recommendations, including optimizing treatment plans, implementing precision treatments, and personalized intervention strategies. For example, after the pathogenic mutation is identified, timely genetic intervention can be performed for carriers of the pathogenic gene in combination with family screening and preimplantation genetic diagnosis (PGT-M) services to avoid and reduce the risk of inheritance to offspring.

[0022] Beneficial effects of this application:

[0023] (1) The detection panel or detection kit provided in this application can be used to efficiently detect gene mutations that are of clinical diagnostic and therapeutic significance for teratozoospermia; the detection probes of the multi-gene panel constructed by the gene set provided in this application can achieve accurate sequencing, and by detecting the mutations of the genes listed in this application, it can be used as a reference for clinical diagnosis, helping doctors to have a deeper understanding of the genetic conditions of patients with teratozoospermia and male patients who are planning to undergo assisted reproductive technology cycles, and also helping doctors to design assisted treatment cycles and optimize treatment plans in a targeted manner; this has important guiding significance for the clinical diagnosis, accurate treatment and potential personalized intervention measures of patients with teratozoospermia and patients with high sperm deformity rates who are planning to undergo assisted reproductive technology treatment.

[0024] (2) The device for evaluating teratozoospermia provided in this application has constructed multiple gene sets and includes a multi-gene detection panel, which includes genes and gene mutations that have been clinically found to be associated with sperm head, neck and tail deformities, as well as genes and gene mutations that have been found in knockout mice in basic research but have not yet been found in human genes. It can more efficiently detect gene mutations that have clinical diagnostic and therapeutic significance for teratozoospermia; whether it is for the diagnosis of teratozoospermia or the subsequent exploration of new therapeutic targets for teratozoospermia gene mutations, it can provide useful genetic information.

[0025] (3) Based on the gene set and intervention measures provided in this application, this application not only detects high-risk mutant genes in a targeted manner, allowing patients to understand the status of their own genetic mutations, but also recommends treatment and intervention measures, which not only reduces the trial-and-error costs of patients' assisted reproductive cycles, but also provides clinicians with a basis for genetic analysis of mutant genes, genetic intervention, risk blocking, optimization of assisted reproductive technology programs, and personalized treatment; in addition, the recommended intervention measures can also help patients improve fertilization rates and improve assisted reproductive clinical outcomes.

[0026] Of course, it is not necessary to achieve all the advantages described above at the same time when implementing any product or method of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0028] Figure 1 This is a protein 3D structure comparison diagram generated using the "Alpha Fold-3" software for the newly-occurring pathogenic mutation of the SUN5 gene in the teratozoospermia patient in the example, where A represents the normal SUN5 protein 3D configuration and B represents the SUN5 mutant c.381del (p.V128Sfs*7) protein 3D configuration.

[0029] Figure 2 The figures are diagrams of sperm morphology abnormalities in patients with teratozoospermia in the examples, wherein A indicates that the sperm head of patient A is abnormally morphologically manifested as headless sperm (SUN5 mutation was detected during testing); B indicates that the sperm head of patient B is abnormally morphologically manifested as round-headed sperm (DPY19L2 mutation was detected during testing); C indicates that the sperm tail of patient C is abnormally morphologically manifested as short-tailed sperm (DNAH1 mutation was detected during testing); D indicates that the sperm tail of patient D is abnormally morphologically manifested as polycystic sperm (AURKC mutation was detected during testing).

[0030] Figure 3 These are electron micrographs of sperm head and neck morphology abnormalities in patients with head and neck teratozoospermia in the examples, wherein A represents an electron micrograph of normal sperm head morphology in the control group; B represents an electron micrograph of sperm head morphology abnormalities in patient B, which appear as round-headed sperm; C represents an electron micrograph of sperm head and neck morphology abnormalities in patient C, which appear as headless sperm; and D represents an electron micrograph of sperm head morphology abnormalities in patient D, which appear as multiple morphological abnormalities in the sperm head.

[0031] Figure 4These are electron micrographs of the sperm tail axoneme morphology of patients with tail teratozoospermia in the examples, wherein A represents an electron micrograph of the normal structure of the axoneme "9+2" of a normal sperm tail; B represents an electron micrograph of the abnormal morphology of the axoneme "9+2" of the sperm tail of patient B, which manifests as a deformed sperm tail; C represents the disordered morphology of the axoneme "9+2" of the sperm tail of patient C, which manifests as a short-tailed sperm; and D represents the disordered morphology of the axoneme "9+2" of the sperm tail of patient D, which manifests as multiple morphological abnormalities of the sperm tail. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present application and the accompanying drawings to clearly and completely describe the technical solutions in this application. Obviously, the embodiments described are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0033] In order to achieve accurate detection of risk genes for patients with teratozoospermia and provide more effective information for assisted reproductive diagnosis. On the one hand, this application collects genes and gene mutations associated with the clinical phenotype of teratozoospermia. The collection channels include patients with a sperm deformity rate of 100%, patients with recurrent sperm deformities, and patients with sperm deformities leading to fertilization failure. The relevant genes obtained by sequencing, the relevant genes disclosed in the collection database, and the relevant genes disclosed in the literature; the information collected from the three aspects and their related genes are merged and de-redundant, and the standard gene names are determined by HGNCapprovedOfficial Symbol to form a gene set; on the other hand, gene variation data from multiple platforms such as TCGA database, MSKCC-IMPACT, ClinVar, LitVar and Varsome database are selected. Based on high-throughput sequencing technology, the important exon regions and some intron regions of the relevant genes are enriched by exon sequencing / whole genome sequencing or biotin probe hybridization method, and high-depth sequencing is performed to discover mutant genes, copy number variation genes, or rearrangement events and deleted genes with clear clinical relevance to teratozoospermia.

[0034] A first aspect of the present application provides a teratozoospermia detection panel based on high-throughput sequencing, wherein the detection panel includes teratozoospermia-related mutant genes, genes with copy number variations, and genes with rearrangement events and deletions for detection.

[0035] In some embodiments of the present application, the mutant gene includes the gene:

[0036] <h2 style=";text-align:left;direction:ltr">ACTL7A、ACTL7B、ADAD2、ADCY10、ADGB、AGTPBP1、AK7、AKAP3、AKAP4、AMZ2、ANKRD31、ARL2BP、ARMC2、AR MC4、ATG7、ATP2B4、AU040320、AURKC、AXDND1、BRDT、BRWD1、BSCL2、CALICIN、C2CD6、C7orf61、CATSPER 1、CATSPER 2、CATSPER 3、CC2D1B、CCDC103、CCDC113、CCDC114、CCDC136、CCDC146、CCDC151、CCDC181、CCDC188、CCDC189、CCDC34、CCDC38、CCDC39 、CCDC40、CCDC62、CCDC65、CCIN、CCNB3、CCNO、CDY1、CEP78、CEP112、CEP128、CEP135、CEP250、CEP290、CEP350、CFAP206、CF AP251、CFAP298、CFAP300、CFAP43、CFAP44、CFAP47、CFAP52、CFAP53、CFAP54、CFAP57、CFAP58、CFAP61、CFAP65、CFAP69、CF AP70、CFAP74、CFAP91、CHPT1、CTE1 / DRC5、CNTROB、CSNK2A2、DHC1、DNAAF4、DNAF1、DNAF2、DNAF3、DNAF4、DNAF5、DNAF6、DNAF 7、DNAH1、DNAH10、DNAH11、DNAH12、DNAH17、DNAH2、DNAH3、DNAH5、DNAH6、DNAH7、DNAH8、DNAH9、DNAI1、DNAI2、DNAJB13、DNA JB13a、DNALI1、DNHD1、DRC1、DRC4、DPY19L2、DZIP1、FBXO43、FADS2、FAM71D、FSIP2、GALNT3、GANLNTL5、GAPDS、GAS2L2、GAS8、 GBA2、GGN、GOPC、GM130、HRB、HOOK1、HSP90B1、HYDIN、IFT74、KATNAL2、KIAA1210、LRRC46、LRRC56、LRRC6、MAATS1、MDC1、MC IDAS、MEIKIN、MFSD14A、MKRN2、MNS1、NDUFA13、NHE8、NME8、NPHP4、NUP210L、OAZ3、ODAD3、ODF1、ODF2、PACRG、PDCL2、PICK1、PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSP H4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPAT C1L, SPATA16, SPATA20, SPATA6, SPEM1, SPINK2, SPEF2, SPPL2C, SSP411, SSMEM1, STK33, STK36, SUN5, TBC1D21, TCTE1, TDRD9, TENT5D, TNP2, T PTE2, TSSK3, TSGA10, TTC12, TTC21A, TTC25, TTC29, VDAC2, VDAC3, VPS13B, VPS54, WDR12, WDR19, WDR63, WDR66, ZCWPW1, ZMYND15, ZPBP, ZPBP1. ,

[0037] In some embodiments of the present application, the gene with copy number variation includes the gene:

[0038] <h2 style=";text-align:left;direction:ltr">ACTL7A、ACTL7B、ADAD2、ADCY10、ADGB、AGTPBP1、AK7、AKAP3、AKAP4、AMZ2、ANKRD31、ARL2BP、ARMC2、AR MC4、ATG7、ATP2B4、AU040320、AURKC、AXDND1、BRDT、BRWD1、BSCL2、CALICIN、C2CD6、C7orf61、CATSPER 1、CATSPER 2、CATSPER 3、CC2D1B、CCDC103、CCDC113、CCDC114、CCDC136、CCDC146、CCDC151、CCDC181、CCDC188、CCDC189、CCDC34、CCDC38、CCDC39 、CCDC40、CCDC62、CCDC65、CCIN、CCNB3、CCNO、CDY1、CEP78、CEP112、CEP128、CEP135、CEP250、CEP290、CEP350、CFAP206、CF AP251、CFAP298、CFAP300、CFAP43、CFAP44、CFAP47、CFAP52、CFAP53、CFAP54、CFAP57、CFAP58、CFAP61、CFAP65、CFAP69、CF AP70、CFAP74、CFAP91、CHPT1、CTE1 / DRC5、CNTROB、CSNK2A2、DHC1、DNAAF4、DNAF1、DNAF2、DNAF3、DNAF4、DNAF5、DNAF6、DNAF 7、DNAH1、DNAH10、DNAH11、DNAH12、DNAH17、DNAH2、DNAH3、DNAH5、DNAH6、DNAH7、DNAH8、DNAH9、DNAI1、DNAI2、DNAJB13、DNA JB13a、DNALI1、DNHD1、DRC1、DRC4、DPY19L2、DZIP1、FBXO43、FADS2、FAM71D、FSIP2、GALNT3、GANLNTL5、GAPDS、GAS2L2、GAS8、 GBA2、GGN、GOPC、GM130、HRB、HOOK1、HSP90B1、HYDIN、IFT74、KATNAL2、KIAA1210、LRRC46、LRRC56、LRRC6、MAATS1、MDC1、MC IDAS、MEIKIN、MFSD14A、MKRN2、MNS1、NDUFA13、NHE8、NME8、NPHP4、NUP210L、OAZ3、ODAD3、ODF1、ODF2、PACRG、PDCL2、PICK1、PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSP H4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPAT C1L, SPATA16, SPATA20, SPATA6, SPEM1, SPINK2, SPEF2, SPPL2C, SSP411, SSMEM1, STK33, STK36, SUN5, TBC1D21, TCTE1, TDRD9, TENT5D, TNP2, T PTE2, TSSK3, TSGA10, TTC12, TTC21A, TTC25, TTC29, VDAC2, VDAC3, VPS13B, VPS54, WDR12, WDR19, WDR63, WDR66, ZCWPW1, ZMYND15, ZPBP, ZPBP1. ,

[0039] In some embodiments of the present application, the genes that undergo rearrangement events and deletions include genes:

[0040] <h2 style=";text-align:left;direction:ltr">ACTL7A、ACTL7B、ADAD2、ADCY10、ADGB、AGTPBP1、AK7、AKAP3、AKAP4、AMZ2、ANKRD31、ARL2BP、ARMC2、AR MC4、ATG7、ATP2B4、AU040320、AURKC、AXDND1、BRDT、BRWD1、BSCL2、CALICIN、C2CD6、C7orf61、CATSPER 1、CATSPER 2、CATSPER 3、CC2D1B、CCDC103、CCDC113、CCDC114、CCDC136、CCDC146、CCDC151、CCDC181、CCDC188、CCDC189、CCDC34、CCDC38、CCDC39 、CCDC40、CCDC62、CCDC65、CCIN、CCNB3、CCNO、CDY1、CEP78、CEP112、CEP128、CEP135、CEP250、CEP290、CEP350、CFAP206、CF AP251、CFAP298、CFAP300、CFAP43、CFAP44、CFAP47、CFAP52、CFAP53、CFAP54、CFAP57、CFAP58、CFAP61、CFAP65、CFAP69、CF AP70、CFAP74、CFAP91、CHPT1、CTE1 / DRC5、CNTROB、CSNK2A2、DHC1、DNAAF4、DNAF1、DNAF2、DNAF3、DNAF4、DNAF5、DNAF6、DNAF 7、DNAH1、DNAH10、DNAH11、DNAH12、DNAH17、DNAH2、DNAH3、DNAH5、DNAH6、DNAH7、DNAH8、DNAH9、DNAI1、DNAI2、DNAJB13、DNA JB13a、DNALI1、DNHD1、DRC1、DRC4、DPY19L2、DZIP1、FBXO43、FADS2、FAM71D、FSIP2、GALNT3、GANLNTL5、GAPDS、GAS2L2、GAS8、 GBA2、GGN、GOPC、GM130、HRB、HOOK1、HSP90B1、HYDIN、IFT74、KATNAL2、KIAA1210、LRRC46、LRRC56、LRRC6、MAATS1、MDC1、MC IDAS、MEIKIN、MFSD14A、MKRN2、MNS1、NDUFA13、NHE8、NME8、NPHP4、NUP210L、OAZ3、ODAD3、ODF1、ODF2、PACRG、PDCL2、PICK1、PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSP H4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPAT C1L, SPATA16, SPATA20, SPATA6, SPEM1, SPINK2, SPEF2, SPPL2C, SSP411, SSMEM1, STK33, STK36, SUN5, TBC1D21, TCTE1, TDRD9, TENT5D, TNP2, T PTE2, TSSK3, TSGA10, TTC12, TTC21A, TTC25, TTC29, VDAC2, VDAC3, VPS13B, VPS54, WDR12, WDR19, WDR63, WDR66, ZCWPW1, ZMYND15, ZPBP, ZPBP1. ,

[0041] In some embodiments of the present application, the mutation types of the mutant gene include genomic structural variation, single nucleotide variation, frameshift mutation, point mutation, deletion, insertion and fusion.

[0042] In some embodiments of the present application, the high-throughput sequencing includes exon sequencing or whole genome sequencing.

[0043] The second aspect of the present application provides a teratozoospermia detection kit based on high-throughput sequencing, wherein the detection kit includes detection probes and detection reagents; the detection probes are targeted at mutant genes, genes with copy number variations, and genes with rearrangement events and deletions in the detection panel described in the first aspect of the present application.

[0044] In some embodiments of the present application, the detection probe is an RNA probe.

[0045] The third aspect of the present application provides a use of the detection panel described in the first aspect of the present application or the detection kit described in the second aspect of the present application in preparing a device for detecting teratozoospermia.

[0046] A fourth aspect of the present application provides a device for detecting teratozoospermia, comprising:

[0047] The sequencing module is used to extract and perform high-throughput sequencing on the DNA sample to be tested to obtain sequencing results;

[0048] A verification module, configured to process the high-throughput sequencing results and compare them with the detection panel described in the first aspect of this application to obtain the mutation information;

[0049] The evaluation module is used to conduct literature verification and comparison of the mutation information, verify the pathogenic mutation sites in the literature and the case evaluation in the literature; for newly detected mutation sites and / or mutation sites that are assessed as having unclear pathogenicity by the mutation software variation classification, the software is used to accurately construct and analyze the three-dimensional structural model of the entire gene protein, and molecular modeling analysis is used to evaluate the possible impact of gene mutations on the secondary and tertiary structures of the protein to determine whether they are new pathogenic mutation sites.

[0050] In some embodiments of the present application, the software is Alpha Fold-3 software.

[0051] In some embodiments of the present application, the device for detecting teratozoospermia further includes: an intervention module for analyzing the information obtained by the verification module and / or the evaluation module, and providing clinical recommendations, including optimizing treatment plans, performing precision treatment, and personalized intervention strategies.

[0052] The present application is based on high-throughput sequencing technology, and has established a teratozoospermia detection device (model), and genetic screening analysis is performed on patients with teratozoospermia and patients with a high rate of teratozoospermia who are about to enter an assisted reproductive cycle, so as to reduce and avoid the probability of fertilization failure during traditional test-tube babies and improve clinical pregnancy rates. This model will help to help patients diagnose early and clarify the cause of the disease, avoid repeated invalid attempts at fertilization failure and / or excessive low fertilization rates. At the same time, it provides a basis for clinical decision-making for clinicians, facilitates clinicians to determine the best treatment plan and the best fertilization method, and then improves fertilization rate, transplantation rate, clinical pregnancy rate and optimizes assisted reproductive technology. This has an important role in the clinical diagnosis, prevention and treatment of patients with a high rate of sperm deformity who are about to enter an assisted reproductive cycle and patients with repeated teratozoospermia, and is also of great significance for the selection of assisted reproductive treatment options, genetic counseling, genetic intervention, precision treatment and the offspring inheritance of blocking risk genes.

[0053] A fifth aspect of the present application provides a method for detecting teratozoospermia risk genes, comprising the steps of:

[0054] S1: Extract genomic DNA of the sample to be tested;

[0055] S2: Construction of the whole genome DNA library of the sample to be tested;

[0056] S3: Construction of detection probes for the detection panel;

[0057] S4: Panel probe (detection probe) hybridizes with DNA library for capture and high-throughput sequencing;

[0058] S5: Perform biological information analysis on the sequencing results to obtain the mutation results of the sample to be tested;

[0059] S6: Comparing the mutation results of the sample to be tested with the detection panel to obtain the gene mutation results of the sample to be tested; performing pathogenicity analysis in combination with clinical information to obtain a final diagnosis;

[0060] S7: Intervention (precision treatment) measures after the final diagnosis of teratozoospermia.

[0061] In some embodiments of the present application, S1 includes: extracting approximately 2 mL of peripheral blood from the patient or scraping tissue DNA with an oral swab, extracting genomic DNA using the QIAamp whole blood DNA extraction kit (Qiagen, Germany) according to its instructions, or scraping tissue DNA with an oral swab for genomic DNA extraction; then performing quality inspection, and the quality inspection standards are based on the standards for library construction of Beijing BGI Genomics Co., Ltd.

[0062] In some embodiments of the present application, S2 includes: taking genomic DNA that has passed quality inspection and using the KAPA library construction kit to prepare a library, as follows:

[0063] (1) The extracted DNA was fragmented into 100-700 bp fragments using a Covaris S2 ultrasonicator (Covaris, USA);

[0064] (2) End repair and product purification: Prepare the reaction system on ice (the amplification primers can be obtained according to the gene sequence using primer design software), add primers and DNA to the reaction system, mix thoroughly, centrifuge, and place in a PCR instrument for PCR amplification; then purify the obtained PCR product with magnetic beads to obtain a purified product; wherein, the volume ratio of magnetic beads to sample is 1.5:1;

[0065] (3) Library amplification PCR and product purification: After the purified product was subjected to library quality inspection, the whole genome library DNA was obtained. The quality inspection standard was based on the library construction standards of Beijing BGI Genomics Co., Ltd. At the same time, the Nanodrop 2000 sample quantification instrument (Thermo Fisher Scientific, Inc., USA) and the Agilent 2100 bioanalyzer (Agilent Technologies, Inc., USA) were used for quality control to prepare the whole genome library. After the library preparation was completed, it was stored at -20°C.

[0066] In some embodiments of the present application, S3 includes: (1) selection of target regions for genetic testing: based on the gene set obtained above, select SNPs and In Dels sites with a frequency of ≤0.02 or no occurrence in the 1000 Genomes, ESP6500si, Ex AC_ALL, and Ex AC_EAS population databases; retain non-synonymous mutations in exon regions (synonymous mutations reported in the literature are retained); c HGMD database literature reports; the number of site mutations is >5, and the mutation frequency is >0.3;

[0067] (2) Panel probe design: Based on the human genome, a database of probe design regions for capture probe design is formed; based on the position information of each target region in the generated bed file, the corresponding probe design region of each target region is searched in the database; probes are generated according to the probe design region and probe design parameters; preferably, the design coverage of the generated probes is weighted, and the design coverage weight is based on the depth of each probe design region predicted by the human whole genome sequencing data. Specifically, the depth predicted by the sequencing data of each probe design region is quantified and marked, and the probes of the probe design region with a depth above or below the average are weighted so that the final designed probes can uniformly capture each target region; for example, the weight of the probe design region below the average depth is increased so that the probe can also have a better capture ability and effect on the low-depth region, thereby achieving the effect of uniform capture of each target region;

[0068] (3) Probe sensitivity assessment: For important sites and all target regions, align them with the selected probe design region and calculate the coverage of each probe for the important sites and the coverage of the entire target region. The calculation formula is: coverage = number of reads on the alignment / number of target sequencing reads. Probes with coverage of important sites ≥99% and coverage of the entire target region ≥90% are selected for panel synthesis. If the probe coverage does not meet the above requirements, reselect a suitable probe near the probe design region.

[0069] (4) Panel synthesis: Add fixed amplification sequences to both ends of the designed probes, synthesize single-stranded DNA, and PCR amplify, combine and make a panel.

[0070] In some embodiments of the present application, S4 includes: using the prepared panel probe to hybridize with the quality-inspected qualified whole-genome library DNA under certain conditions, covalently binding the biotin-labeled probe with streptavidin-modified magnetic beads to capture the target gene, and finally using a magnetic stand to adsorb the magnetic beads carrying the target gene, eluting and purifying, enriching the target gene, and capturing the coding exon region of the gene related to teratozoospermia; using the Illumina Nextseq 500 second-generation sequencer to perform double-end sequencing on the captured region with a read length of 150bp; wherein, the name and corresponding accession number of the gene captured by the probe are as shown in NCBI (https: / / www.ncbi.nlm.nih.gov / ).

[0071] In some embodiments of the present application, S5 includes: the sequencing results are fastq files, and the sequencing results are analyzed using the algorithm for obtaining gene mutations published by Broad to obtain the results of gene mutations and annotate them; the main steps include: quality control of fastq files, genome alignment, analysis of somatic mutations and germline mutations, and annotation.

[0072] (1) The software used included: fastqc and fastx_toolkit for quality control; bwa for alignment, gatk and mutect2 for obtaining somatic mutations; HaplotypeCaller for obtaining germline mutations; and ANNOVAR for annotation.

[0073] (2) The reference databases are: normal population databases: 1000 Genomes, ESP6500si, Ex AC_ALL, ExAC_EAS population database, DGV, db SNP, etc.; disease databases: OMIM, HGMD (Professional Edition), Clivar, Bcipher, Cosmic, etc.; c-protein function prediction databases: SIFT, Poly Phen2, Mutation Taster, GERP++, REVEL, etc.

[0074] In some embodiments of the present application, S6 includes: analyzing data according to the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) standards and guidelines for interpreting genetic variants published in Genetics in Medicine in May 2015; the standards state that when using high-throughput sequencing to detect pathogenic genes for Mendelian genetic diseases, sequence variants are classified into five categories: benign, likely benign, uncertain significance, likely pathogenic, and pathogenic; then, for each sequence variant, integrating existing research evidence to classify the variant, wherein each variant, pathogenic research evidence, and evidence classification are provided; synthesizing primers based on the DNA fragments to be sequenced, amplifying the fragments using the polymerase chain reaction (PCR) method, sequencing the fragments using the Sanger sequencing method using an ABI3730xl sequencer (Applied Biosystems, USA), and comparing the sequencing results with the reference sequence using "Mutation Surveyor" software.

[0075] It should be noted that when using the teratozoospermia detection panel provided in this application for genetic testing, conventional high-throughput sequencing methods such as whole genome sequencing and whole exome sequencing can also be used.

[0076] In some embodiments of the present application, in S7, the intervention module can provide intervention (precision treatment) measures after the final diagnosis of teratozoospermia gene mutation.

[0077] Basic and clinical studies have found that mutations in some genes (such as ACTL7A, DPY19L2, and DNAH17) lead to sperm head deformities, which often lead to fertilization failure due to the inability to activate the oocyte. To address this problem, an increasing number of studies have confirmed that artificial oocyte activation (AOA) can significantly improve the fertilization rate and clinical outcomes of some patients with teratozoospermia. Currently, assisted reproductive laboratories use artificial activators (such as A23187, ionomycin, and strontium chloride) to activate oocytes to improve fertilization rates and clinical outcomes. Personalized AOA programs guided by genetic testing can significantly improve the reproductive outcomes of male infertility patients caused by gene mutations, providing precision treatment for reproductive medicine.

[0078] The following examples are given to further illustrate the embodiments of the present application. Various tests and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are based on mass.

[0079] Example

[0080] All 80 patients included in this example were informed and agreed to the study.

[0081] S1: Extraction of genomic DNA samples

[0082] After obtaining the patient's consent, approximately 2 mL of peripheral blood was drawn from each patient, and genomic DNA was extracted using the QIAamp whole blood DNA extraction kit (Qiagen, Germany) according to its instructions; then, the library constructed by Beijing BGI Genomics Co., Ltd. was used for quality control.

[0083] S2: DNA sample library preparation

[0084] The genomic DNA that passed the quality inspection was prepared using the KAPA library construction kit: (1) First, ultrasonic fragmentation was performed using a Covaris S2 ultrasonic instrument (Covaris, USA) to break the extracted DNA into 100-700bp; 400ng of DNA was taken, diluted to 50μL with nuclease-free water, transferred to a 0.5mL Eppendorf LoBind Tube, mixed thoroughly, centrifuged briefly, and placed on ice for use. (2) End repair with "A" and product purification: The reaction system was prepared, mixed thoroughly, centrifuged, and placed in a PCR instrument for PCR amplification; the PCR product was purified using magnetic beads, with the volume of magnetic beads to sample being 1.5:1. (3) After the purified product was subjected to library quality inspection, the whole genome library DNA was obtained, and the quality inspection standard was the same as the library construction standard of Beijing BGI Genomics Co., Ltd. Quality control was performed using a Nanodrop 2000 sample quantification instrument (Thermo Fisher Scientific, Inc., USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA). Whole-genome libraries were prepared and stored at −20°C.

[0085] S3: Panel probe hybridization capture with DNA library and high-throughput sequencing

[0086] Using Panel probes (specifically targeting ACTL7A, ACTL7B, ADAD2, ADCY10, ADGB, AGTPBP1, AK7, AKAP3, AKAP4, AMZ2, ANKRD31, ARL2BP, ARMC2, ARMC4, ATG7, ATP2B4, AU040320, AURKC, AXDND1, BRDT, BRWD1, BSCL2, CALICIN, C2CD6, C7orf61, CATSPER 1, CATSPER 2, CATSPER 3, CC2D1B, CCDC103, CCDC113, CCDC114, CCDC136, CCDC146, CCDC151, CCDC181, CCDC188, CCDC189, CCDC34, CCDC38, CCDC39, CCDC40, CCDC62, CCDC65, CCIN, CCNB3, CCNO, CDY1, CEP78, CEP112, CEP128, CEP135, CEP250, CEP290, CEP350, CFAP206, CFAP25I, CFAP298, CFAP300, CFAP43, CFAP44, CFAP47, CFAP52, CFAP53, CFAP54, CFAP57, CFAP58, CFAP61, CFAP65, CFAP69, CFAP70, CFAP74, CFAP91, CHPT1, CTE1 / DRC5, CNTROB, CSNK2A2, DHC1, DNAAF4, DNAF1, DNAF2, DNAF3, DNAF4, DNAF5, DNAF6, DNAF7, DNAH1, DNAH10, DNAH11, DNAH12, DNAH17, DNAH2, DNAH3, DNAH5, DNAH6, DNAH7, DNAH8, DNAH9, DNAI1, DNAI2, DNAJB13, DNAJB13a, DNALI1, DNHD1, DRC1, DRC4, DPY19L2, DZIP1, FBXO43, FADS2, FAM71D, FSIP2, GALNT3, GALNTL5, GAPDS, GAS2L2, GAS8, GBA2, GGN, GOPC, GM130, HRB, HOOK1, HSP90B1, HYDIN, IFT74, KATNAL2, KIAA1210, LRRC46, LRRC56, LRRC6, MAATS1, MDC1, MCIDAS, MEIKIN, MFSD14A, MKRN2, MNS1, NDUFA13, NHE8, NME8, NPHP4, NUP210L, OAZ3, ODAD3, ODF1, ODF2 It should be noted that there seems to be a possible error in the original text where "CFAP25I" might be "CFAP251" in the English translation. This has been corrected in the translation above.PACRG, PDCL2, PICK1, PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSPH4A, RSPH9, SEPT IN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPATC1L, SPATA16, SPATA20, SPATA6, SPEM1, SPINK2, SPEF2, SPPL2C, SSP411, SSMEM1, STK33, STK36, SUN5, TBC1D21, TCTE1, TDRD9, TENT5D, TNP2, TPTE2, TSSK3, TSGA10, TTC12, TTC21A, TTC25, TTC29, VDAC2, VDAC3, VPS13B, VPS54, WDR12, WDR19, WDR63, WDR66, ZCWPW1, ZMYND15, ZPBP, ZPBP1) were hybridized with the quality-tested whole-genome library DNA under certain conditions, and the biotin-labeled probe was covalently bound to the streptavidin-modified magnetic beads to capture the target gene. Finally, the magnetic beads carrying the target gene were adsorbed on a magnetic stand, eluted and purified, and the target gene was enriched to capture the coding exon region of the gene related to teratozoospermia. The specific steps are as follows:

[0087] (1) Take a portion of the RNA probe library and dilute it to 100 ng / μL with 1×TE (pH 8.0). This method can produce probes that can be applied to 200-300 samples, greatly reducing the cost of sequencing.

[0088] (2) Hybrid capture: Mix 95 μL of Block and a total of not less than 500 ng of DNA library in a volume of about 5 μL, then centrifuge, mark it as "A", place it on a PCR instrument, cover it with a hot cover, and incubate at 95℃ for 5 minutes; maintain at 65℃. Prepare the Mix reaction system in a PCR reaction tube, mark it as "B", and when the temperature of the PCR instrument drops to 65℃ for 2.5 minutes, place the "B" tube on the PCR instrument and incubate it with a hot cover. After placing the PCR tube "B" in the PCR instrument for 2.5 minutes, draw 13 μL of Hyb Buffer from the "hyb buffer" and transfer it to the "A" sample, draw 6 μL from the "B" well and transfer it to the "A" sample, gently pipette 10 times, mix thoroughly to avoid generating a lot of bubbles, apply a film, cover the PCR instrument with a hot cover, and incubate at 65℃ overnight for 24 hours. Then prepare the capture magnetic beads. Each sample requires 3 x 165 μL of high-stringency buffer. Aliquot the high-stringency buffer into a 96-well plate. Turn on the dryer and adjust the temperature to 65°C. Once the temperature stabilizes at 65°C, place the plate in the dryer to preheat. Then, capture the target region DNA library and complete the DNA library enrichment (post-PCR reaction). The library is then pooled and sent for sequencing.

[0089] Tables 1 and 2 list the genes, mutation sites and phenotypes associated with sperm head, neck and tail deformities, respectively.

[0090] Table 1 Genes, mutation sites and phenotypes associated with sperm head and neck deformities

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Note: “-” means not shown

[0097] Table 2 Genes, mutation sites and phenotypes associated with sperm tail deformity

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] Note: “-” means not shown

[0104] (3) The captured regions were sequenced using an Illumina Nextseq 500 second-generation sequencer with a read length of 150 bp. The names and accession numbers of the genes captured by the probes are shown in NCBI (https: / / www.ncbi.nlm.nih.gov / ).

[0105] S4: Perform biological information analysis on the sequencing results to obtain sample mutation results

[0106] The sequencing results are generated as fastq files. We analyze the results using a Broad-published algorithm for detecting gene mutations, obtaining and annotating the gene mutations. The main steps include quality control of the fastq files, genome alignment, analysis of somatic and germline mutations, and annotation.

[0107] (1) The software used included: fastqc and fastx_toolkit for quality control; bwa for alignment, gatk and mutect2 for obtaining somatic mutations; HaplotypeCaller for obtaining germline mutations; and ANNOVAR for annotation.

[0108] (2) The reference databases are: normal population databases: 1000 Genomes, ESP6500si, Ex AC_ALL, ExAC_EAS population database, DGV, db SNP, etc.; disease databases: OMIM, HGMD (Professional Edition), Clivar, Bcipher, Cosmic, etc.; c-protein function prediction databases: SIFT, Poly Phen2, Mutation Taster, GERP++, REVEL, etc.

[0109] S5: Gene mutation annotation results for all DNA samples are shown in Tables 3 and 4. Tables 3 and 4 show the detection results of gene mutations in the patient DNA in the embodiment, which are the specific genetic variations of mutations related to the gene list listed in this application in the results of the bioinformatics analysis described above after the patient's DNA sample is sequenced.

[0110] Table 3 Detection results of genes in patients with sperm head and neck deformities in the embodiment

[0111]

[0112]

[0113] Table 4 Detection results of sperm tail deformity patient genes in the embodiment

[0114]

[0115]

[0116]

[0117] Figure 1 This is a comparison of the protein 3D structures of the newly discovered pathogenic mutations in the SUN5 gene in patients with teratozoospermia described in the Examples, generated using Alpha Fold-3 software. A represents the 3D structure of the normal SUN5 protein, and B represents the 3D structure of the SUN5 mutant c.381del (p.V128Sfs*7). As can be seen from the figure, compared with the normal SUN5 protein 3D structure, the SUN5 mutant protein 3D structure is simpler, lacking a portion of the amino acid sequence and corresponding structure (indicated by the arrow), indicating that the gene's amino acid sequence is terminated at position 128, resulting in truncation. The amino acids and structure of some functional regions are missing, leading to protein and functional abnormalities. Clinically, this manifests as headless sperm and abnormalities in the ultrastructure of the sperm head and neck.

[0118] Figure 2 Figure 1 shows abnormal sperm morphology in patients with teratozoospermia in the examples. A indicates abnormal sperm head morphology in patient A, manifesting as headless sperm (SUN5 mutation detected); B indicates abnormal sperm head morphology in patient B, manifesting as round-headed sperm (DPY19L2 mutation detected); C indicates abnormal sperm tail morphology in patient C, manifesting as short-tailed sperm (DNAH1 mutation detected); and D indicates abnormal sperm tail morphology in patient D, manifesting as polycystic sperm (AURKC mutation detected). Clinically, this condition manifests as fertilization failure or severely reduced fertilization rate, leading to poor IVF (in vitro fertilization) outcomes.

[0119] Figure 3 These are electron microscopic images of abnormal sperm head and neck morphology in patients with head and neck teratozoospermia in the examples, wherein A represents an electron microscopic image of normal sperm head morphology in the control image; B represents an electron microscopic image of abnormal sperm head morphology in patient B, which appears as round-headed sperm; C represents an image of abnormal sperm head and neck morphology in patient C, which appears as acephalospermia; and D represents an image of abnormal sperm head morphology in patient D, which appears as multiple morphological abnormalities in the sperm head.

[0120] Figure 4These are electron micrographs of the sperm tail axoneme morphology of patients with tail teratozoospermia in the examples, wherein A represents an electron micrograph of the normal structure of the axoneme "9+2" of a normal sperm tail; B represents an electron micrograph of the abnormal morphology of the axoneme "9+2" of the sperm tail of patient B, which manifests as a deformed sperm tail; C represents the disordered morphology of the axoneme "9+2" of the sperm tail of patient C, which manifests as a short-tailed sperm; and D represents the disordered morphology of the axoneme "9+2" of the sperm tail of patient D, which manifests as multiple morphological abnormalities of the sperm tail.

[0121] S6: Recommended interventions for patients with teratozoospermia after final diagnosis

[0122] This application combines clinical information to analyze and predict the pathogenicity of teratozoospermia mutation genes and obtains gene mutations that are ultimately diagnosed as pathogenic, such as the gene mutations shown in Tables 1 and 2 (e.g., DPY19L2 and other gene mutations). Intervention measures such as oocyte activation (AOA) can be used for precise treatment to prevent fertilization failure and / or low fertilization rate caused by teratozoospermia. The intervention measures, using ionomycin as an example, are as follows:

[0123] (1) Place the oocyte after ICSI fertilization into the first well of the prepared activation culture dish; the first well of the activation culture dish contains AOA activation solution, that is, the final concentration of calcium ion carrier is 5-15μM, and the final concentration of phosphatidylinositol-4,5-bisphosphate is 5-15μM.

[0124] (2) Place the activated culture dish in a 37°C, 6 vol% carbon dioxide incubator and incubate for 5 minutes.

[0125] (3) Remove the activated culture dish from the carbon dioxide incubator, use a sharp pipette to place the oocytes in the embryo culture medium in the second and third wells, wash them five times each, and then transfer them to the fourth well of the activated culture dish; place them in a 37°C, 6 vol% carbon dioxide incubator and culture for 20 minutes.

[0126] (4) Repeat steps (1) to (3) to complete the activation operation, and then transfer the oocyte into embryo culture medium and culture until pronuclear formation is observed.

[0127] By adopting the clinical evaluation model of teratozoospermia of this application, whole genome / whole exome sequencing or probe detection is performed on the sample, and the mutation status of all teratozoospermia genes is obtained after analysis. The genes in the gene set listed in this application are found, and the next step of analysis is carried out in combination with the clinical data to predict and reduce the probability of assisted reproductive fertilization failure in individuals. According to the gene set provided in this application, multi-gene panel probes are made and accurately sequenced. After obtaining the results, they are combined with the clinical information of the patient sample to provide a strong reference for the clinical diagnosis and evaluation of the patient, and also facilitate doctors to have a deeper understanding of the patient's gene mutation status, facilitate the formulation of treatment plans, and provide precise treatment and intervention measures.

[0128] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A teratozoospermia detection panel based on high-throughput sequencing, wherein: The detection panel includes teratozoospermia-related mutant genes, genes with copy number variations, and genes with rearrangement events and deletions for detection.

2. The detection panel according to claim 1, wherein: The mutant genes include genes: <h2 style=";text-align:left;direction:ltr">ACTL7A、ACTL7B、ADAD2、ADCY10、ADGB、AGTPBP1、AK7、AKAP3、AKAP4、AMZ2、ANKRD31、ARL2BP、ARMC2、AR MC4、ATG7、ATP2B4、AU040320、AURKC、AXDND1、BRDT、BRWD1、BSCL2、CALICIN、C2CD6、C7orf61、CATSPER 1、CATSPER 2、CATSPER 3、CC2D1B、CCDC103、CCDC113、CCDC114、CCDC136、CCDC146、CCDC151、CCDC181、CCDC188、CCDC189、CCDC34、CCDC38、CCDC39 、CCDC40、CCDC62、CCDC65、CCIN、CCNB3、CCNO、CDY1、CEP78、CEP112、CEP128、CEP135、CEP250、CEP290、CEP350、CFAP206、CF AP251、CFAP298、CFAP300、CFAP43、CFAP44、CFAP47、CFAP52、CFAP53、CFAP54、CFAP57、CFAP58、CFAP61、CFAP65、CFAP69、CF AP70、CFAP74、CFAP91、CHPT1、CTE1 / DRC5、CNTROB、CSNK2A2、DHC1、DNAAF4、DNAF1、DNAF2、DNAF3、DNAF4、DNAF5、DNAF6、DNAF 7、DNAH1、DNAH10、DNAH11、DNAH12、DNAH17、DNAH2、DNAH3、DNAH5、DNAH6、DNAH7、DNAH8、DNAH9、DNAI1、DNAI2、DNAJB13、DNA JB13a、DNALI1、DNHD1、DRC1、DRC4、DPY19L2、DZIP1、FBXO43、FADS2、FAM71D、FSIP2、GALNT3、GANLNTL5、GAPDS、GAS2L2、GAS8、 GBA2、GGN、GOPC、GM130、HRB、HOOK1、HSP90B1、HYDIN、IFT74、KATNAL2、KIAA1210、LRRC46、LRRC56、LRRC6、MAATS1、MDC1、MC IDAS、MEIKIN、MFSD14A、MKRN2、MNS1、NDUFA13、NHE8、NME8、NPHP4、NUP210L、OAZ3、ODAD3、ODF1、ODF2、PACRG、PDCL2、PICK1、PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSPH4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPAT C1L、SPATA16、SPATA20、SPATA6、SPEM1、SPINK2、SPEF2、SPPL2C、SSP411、SSMEM1、STK33、STK36、SUN5、TBC1D21、TCTE1、TDRD9、TENT5D、TNP2、TP TE2、TSSK3、TSGA10、TTC12、TTC21A、TTC25、TTC29、VDAC2、VDAC3、VPS13B、VPS54、WDR12、WDR19、WDR63、WDR66、ZCWPW1、ZMYND15、ZPBP、ZPBP1。 3. The detection panel according to claim 1, wherein: The genes with copy number variations include genes: <h2 style=";text-align:left;direction:ltr">ACTL7A、ACTL7B、ADAD2、ADCY10、ADGB、AGTPBP1、AK7、AKAP3、AKAP4、AMZ2、ANKRD31、ARL2BP、ARMC2、AR MC4、ATG7、ATP2B4、AU040320、AURKC、AXDND1、BRDT、BRWD1、BSCL2、CALICIN、C2CD6、C7orf61、CATSPER 1、CATSPER 2、CATSPER 3、CC2D1B、CCDC103、CCDC113、CCDC114、CCDC136、CCDC146、CCDC151、CCDC181、CCDC188、CCDC189、CCDC34、CCDC38、CCDC39 、CCDC40、CCDC62、CCDC65、CCIN、CCNB3、CCNO、CDY1、CEP78、CEP112、CEP128、CEP135、CEP250、CEP290、CEP350、CFAP206、CF AP251、CFAP298、CFAP300、CFAP43、CFAP44、CFAP47、CFAP52、CFAP53、CFAP54、CFAP57、CFAP58、CFAP61、CFAP65、CFAP69、CF AP70、CFAP74、CFAP91、CHPT1、CTE1 / DRC5、CNTROB、CSNK2A2、DHC1、DNAAF4、DNAF1、DNAF2、DNAF3、DNAF4、DNAF5、DNAF6、DNAF 7、DNAH1、DNAH10、DNAH11、DNAH12、DNAH17、DNAH2、DNAH3、DNAH5、DNAH6、DNAH7、DNAH8、DNAH9、DNAI1、DNAI2、DNAJB13、DNA JB13a、DNALI1、DNHD1、DRC1、DRC4、DPY19L2、DZIP1、FBXO43、FADS2、FAM71D、FSIP2、GALNT3、GANLNTL5、GAPDS、GAS2L2、GAS8、 GBA2、GGN、GOPC、GM130、HRB、HOOK1、HSP90B1、HYDIN、IFT74、KATNAL2、KIAA1210、LRRC46、LRRC56、LRRC6、MAATS1、MDC1、MC IDAS、MEIKIN、MFSD14A、MKRN2、MNS1、NDUFA13、NHE8、NME8、NPHP4、NUP210L、OAZ3、ODAD3、ODF1、ODF2、PACRG、PDCL2、PICK1、PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSPH4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, SPAG6, SPAT C1L、SPATA16、SPATA20、SPATA6、SPEM1、SPINK2、SPEF2、SPPL2C、SSP411、SSMEM1、STK33、STK36、SUN5、TBC1D21、TCTE1、TDRD9、TENT5D、TNP2、TP TE2、TSSK3、TSGA10、TTC12、TTC21A、TTC25、TTC29、VDAC2、VDAC3、VPS13B、VPS54、WDR12、WDR19、WDR63、WDR66、ZCWPW1、ZMYND15、ZPBP、ZPBP1。 4. The detection panel according to claim 1, wherein: The genes involved in the rearrangement and deletion events include: ACTL7A, ACTL7B, ADAD2, ADCY10, ADGB, AGTPBP1, AK7, AKAP3, AKAP4, AMZ2, ANKRD31, ARL2BP, ARMC2, ARMC4, ATG7, ATP2B4, AU040320, AURKC, AXDND1, BRDT, BRWD1, BSCL2, CALICIN, C2CD6, C7orf61, CATSPER 1, CATSPER 2, CATSPER 3. CC2D1B, CCDC103, CCDC113, CCDC114, CCDC136, CCDC146, CCDC151, CCDC181, CCDC188, CCDC189, CCDC34, CCDC38, CCDC 39. CCDC40, CCDC62, CCDC65, CCIN, CCNB3, CCNO, CDY1, CEP78, CEP112, CEP128, CEP135, CEP250, CEP290, CEP350, CFAP20 6. CFAP251, CFAP298, CFAP300, CFAP43, CFAP44, CFAP47, CFAP52, CFAP53, CFAP54, CFAP57, CFAP58, CFAP61, CFAP65, CFA P69, CFAP70, CFAP74, CFAP91, CHPT1, CTE1 / DRC5, CNTROB, CSNK2A2, DHC1, DNAAF4, DNAF1, DNAF2, DNAF3, DNAF4, DNAF5, D NAF6, DNAF7, DNAH1, DNAH10, DNAH11, DNAH12, DNAH17, DNAH2, DNAH3, DNAH5, DNAH6, DNAH7, DNAH8, DNAH9, DNAI1, DNAI2, DNAJB13, DNAJB13a, DNALI1, DNHD1, DRC1, DRC4, DPY19L2, DZIP1, FBXO43, FADS2, FAM71D, FSIP2, GALNT3, GALNTL5, GAPD S, GAS2L2, GAS8, GBA2, GGN, GOPC, GM130, HRB, HOOK1, HSP90B1, HYDIN, IFT74, KATNAL2, KIAA1210, LRRC46, LRRC56, LRRC 6. MAATS1, MDC1, MCIDAS, MEIKIN, MFSD14A, MKRN2, MNS1, NDUFA13, NHE8, NME8, NPHP4, NUP210L, OAZ3, ODAD3, ODF1, ODF2,PACRG, PDCL2, PICK1, PIWIL4, PKD1, PKD2, PMFBP1, PNLDC1, PPP1CC, PRM1, PRM2, PRSS21, PRSS50, PRSS55, QRICH2, RBMX, RIMBP3, RNF126, RNF220, RSPH1, RSPH3, RSPH4A, RSPH9, SEPTIN4, SEPTIN12, SEPTIN14, SETD2, SIRT1, SLC26A3, SLC26A8, SLC9C1, SLO3, SSFA2, SPAC1, SPACA1, SPAG1, SPAG17, S PAG6、SPATC1L、SPATA16、SPATA20、SPATA6、SPEM1、SPINK2、SPEF2、SPPL2C、SSP411、SSMEM1、STK33、STK36、SUN5、TBC1D21、TCTE1、TDRD9、TENT5D、TN P2、TPTE2、TSSK3、TSGA10、TTC12、TTC21A、TTC25、TTC29、VDAC2、VDAC3、VPS13B、VPS54、WDR12、WDR19、WDR63、WDR66、ZCWPW1、ZMYND15、ZPBP、ZPBP1。 5. The detection panel according to any one of claims 1 to 4, wherein: The mutation types of the mutant gene include genomic structural variation, single nucleotide variation, frameshift mutation, point mutation, deletion, insertion and fusion.

6. A teratozoospermia detection kit based on high-throughput sequencing, wherein: The detection kit comprises a detection probe and a detection reagent; the detection probe is directed against the mutated genes, genes with copy number variations, and genes with rearrangement events and deletions in the detection panel according to any one of claims 1 to 5.

7. Use of the detection panel according to any one of claims 1 to 5 or the detection kit according to claim 6 in preparing a device for detecting teratozoospermia.

8. A device for detecting teratozoospermia, wherein: include: The sequencing module is used to extract and perform high-throughput sequencing on the DNA sample to be tested to obtain sequencing results; a verification module, configured to process the high-throughput sequencing results and compare them with the detection panel of any one of claims 1 to 5 to obtain the mutation information; The evaluation module is used to conduct literature verification and comparison of the mutation information, verify the pathogenic mutation sites in the literature and the case evaluation in the literature; for newly detected mutation sites and / or mutation sites that are assessed as having unclear pathogenicity by the mutation software variation classification, the software is used to accurately construct and analyze the three-dimensional structural model of the entire gene protein, and molecular modeling analysis is used to evaluate the possible impact of the mutation on the secondary and tertiary structures of the protein to determine whether it is a new pathogenic mutation site.

9. The device according to claim 8, wherein The software is Alpha Fold-3 software.

10. The device according to claim 8 or 9, wherein Also includes: The intervention module is used to analyze the information obtained by the verification module and / or the evaluation module, and provide clinical recommendations, including optimizing treatment plans, conducting precision treatments, and personalized intervention strategies.

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