Non-invasive method for detecting endometrial receptivity

Abstract

The application belongs to the field of life science, and relates to a non-invasive endometrial receptivity detection method. The detection method detects the methylation state of free DNA in a donor peripheral blood sample to determine whether the endometrial receptivity window of the donor is deviated. The detection method provided by the application takes free DNA in peripheral blood as a detection object, determines the endometrial receptivity of the donor by detecting the methylation state of a specific region of the free DNA, is used for assisting the in-vitro fertilization process, does not need to biopsy endometrial tissue, reduces the physical and mental burden of the patient, and realizes non-invasive detection. The detection route is simple, the operation is simple, the cost is lower, the clinical detection efficiency can be significantly improved, and the application range is wider.

Description

Technical Field

[0001] This application belongs to the field of life sciences and relates to a non-invasive method for detecting endometrial receptivity. Background Technology

[0002] Assisted reproductive technology (ART) is an effective clinical technique for solving infertility. According to statistics from *Reproductive Biology and Endocrinology*, more than 20% of infertile couples currently require ART treatment. Statistical data shows that the clinical pregnancy rate of ART has hovered around 30% for many years, and more than 70% of infertile couples need to undergo multiple embryo transfers to achieve pregnancy. Recurrent implantation failure (RIF) has become a bottleneck problem in the clinical application of ART. The incidence of RIF in ART is approximately 10%-20%, causing heavy psychological and financial burdens for infertile couples, and even leading to the termination of treatment.

[0003] There are two main factors for successful embryo implantation: an embryo with high developmental potential and a normal uterine lining suitable for implantation. Currently, morphological screening, as well as preimplantation genetic and epigenetic screening techniques, are used clinically to select embryos with high developmental potential. However, there is currently a lack of effective methods for testing endometrial receptivity.

[0004] The menstrual cycle of women of reproductive age is generally 28-30 days. The endometrium is only receptive to embryo implantation during a specific period within the menstrual cycle, generally considered to be the mid-secretory phase (D20-24, i.e., +7 to +11 post-ovulation), known as the implantation window. Once this period is exceeded, the endometrial implantation window closes, rejecting embryo implantation. Therefore, establishing methods for assessing endometrial receptivity and determining the implantation window is of significant clinical importance for improving the birth rate of assisted reproductive technologies.

[0005] Currently, the commonly used clinical methods for assessing endometrial receptivity mainly include the following:

[0006] 1. Morphological assessment

[0007] This method primarily assesses receptivity by observing the "appearance characteristics" of the endometrium using ultrasound. The main indicators include endometrial thickness, endometrial type, and endometrial blood flow. Generally, an endometrial thickness ≥7-8 mm before implantation is considered more conducive to implantation. Furthermore, based on ultrasound echogenicity, the endometrium is classified into three types: Type A (proliferative phase, clear three-line sign), Type B (transitional phase, blurred three-line sign), and Type C (secretory phase, homogeneous hyperechoic). Type A or Type B is generally preferred. Finally, receptivity is assessed by observing the blood flow resistance (RI) and pulsatility index (PI) of the uterine artery and spiral artery; low resistance blood flow indicates good blood supply and is more conducive to receptivity.

[0008] 2. Molecular biological detection methods

[0009] This method primarily determines whether the endometrium is in the "implantation window" (a critical period for embryo implantation) by detecting the expression of "receptivity-related genes / proteins" in the endometrial tissue. The main detection method is endometrial receptivity microarray (ERA). The detection process requires obtaining a small amount of endometrial tissue through hysteroscopy or curettage to detect the expression patterns of 238 receptivity-related genes and analyze whether the endometrium is currently in the "receptivity phase," "non-receptivity phase," or "transitional phase."

[0010] However, current methods for testing endometrial receptivity have significant limitations. Among them:

[0011] Morphological assessment is highly subjective, and different doctors may have different judgments on the clarity of the "three lines sign" and the "thickness threshold." Furthermore, morphological assessment only reflects "morphology" and does not represent "function." Some women may have adequate endometrial thickness and morphology, but may still experience implantation failure due to receptivity defects at the molecular level. In addition, blood flow assessment has poor stability; blood flow resistance may vary in the same person at different times due to emotional and hormonal fluctuations.

[0012] Molecular biology testing methods are invasive procedures that require sampling through the uterine cavity, which may lead to minor bleeding, infection risks, and temporary effects on the endometrial environment. Furthermore, they are subject to timeliness and individual variability; the implantation window may shift due to individual hormone levels and diseases (such as polycystic ovary syndrome). ERA results only reflect the state at the time of sampling and cannot fully predict subsequent cycles.

[0013] Therefore, there is an urgent clinical need for an effective method to detect endometrial receptivity. Summary of the Invention

[0014] Based on this, one or more embodiments of this application provide a non-invasive method for detecting endometrial receptivity. This includes the following technical solutions:

[0015] One or more embodiments of this application provide a non-invasive method for detecting endometrial receptivity, wherein the detection method determines whether the endometrial receptivity window of the donor has shifted by detecting the methylation status of cell-free DNA in a donor peripheral blood sample.

[0016] In some embodiments of this application, the detection method includes the following steps:

[0017] Detecting the methylation status of the time-specific promoter regions in the free DNA; and,

[0018] Based on the obtained test results, determine whether the endometrial receptivity window of the donor has shifted;

[0019] The period-specific promoter regions include the promoter regions of the following genes: PSRC1, PER3, MFSD2A, SLC2A5, ACOT11, IGSF9, TP53I3, MRPL37, DISP3, MCOLLN2, KCNK1, PPM1B, L1TD1, LDLRAD2, SPAG17, VRK2, PROC, TYW3, YIPF1, TADA1, SMYD5, HDAC4, S100A6, FAM163A, ARF4, ERCC3, CCDC36, TMEFF2, KIF3C, FAM162A, HHATL, FAM107A, SCN5A, CCRL2, USP13, CA CNA2D2, MBD4, SEPT11, UCN2, ARHGAP24, NIT2, RGS12, SNX24, USP19, CAMK4, FGFR4, PAPD4, RIPPLY2, PARL, HTR4, PDLIM7, AEBP1, STK31, FBXO4, TMEM242, CCDC146, FKBP6, CCDC180, OOEP, CALU, C7orf55, SCARA5, STRBP, ZC3H12D, MAL2, ENG, CA13, PKN3, SFT2D1, PDCL, EHMT1, BAALC, SARDH, RHBDD2, STXBP1, L HPP, RGS3, COX7B, FAM131B, LC25A25, FDX1, RALGDS, PDZD4, RPP38, GPSM1, FLI1, ZNF75D, WAC, OR51B5, SPANXB1, RILPL2, IDH3G, TFAM, WDR74, CELF2, GCS H, SLK, CACUL1, RERG, DHTKD1, PPP1R27, RASSF7, SYT7, ITGB7, TCF7L2, ZNF559, AP2A2, ANKRD49, RNF31, IFITM10, ZNF177, BCL9L, RASSF8, CCPG1, C1QTNF 4. IER2, HHIPL1, HTR2A, ABCA3, HOXC6, ZNF546, SETD6, RP11-310K10, ALG1, ARHGAP9, MED25, ANKRD40, PRSS22, MT1F, SPATA13, BFSP1, TCF4, TNFRSF12A, CNP, TM9SF2, SALL4, RFX1, ZC3H7A, HAMP, PRPF39, MRPL39, ILVBL, METTL16, TGIF2, PYGL, C21orf33, ZNF667, CDRT15, ​​C20orf100, OTUB2, ADARB1, CPNE1,DUSP14, TRAPPC2B, NIPA1, RNF185, SULF2, CFAP53, TMEM74B, TK2, MGAT3, ABCG1, SERPINB5, SYNJ1, CACNB1, WNT7B, COL6A2, D NMT1, CLIC6, P3H4, TRABD, ARVCF, CERS1, PPIL2, PITPNC1, BBC3, PITPNB, ASXL1, SH3BP1, THOP1, NOSIP, ADM2, SPATA2 and CDC37. ,

[0020] In some embodiments of this application, the sampling time range of the peripheral blood sample includes days 1 to 11 after the donor ovulates.

[0021] In some embodiments of this application, determining whether the endometrial receptivity window of the donor has shifted based on the obtained detection results includes:

[0022] If the test results meet the conditions shown in (A1) to (A5), then the endometrial receptivity window of the donor has not shifted; if the test results do not meet any of the conditions shown in (A1) to (A5), then the endometrial receptivity window of the donor has shifted.

[0023] (A1) The methylation level of the promoter regions of the following genes was relatively higher on day 3 post-ovulation (PO3) than on day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11).

[0024] PSRC1, IGSF9, KCNK1, VRK2, SMYD5, ERCC3, HHATL, CACNA2D2, NIT2, FGFR4, PDLIM7, CCDC146, C7orf55, ENG, EHMT1, LHPP , FDX1, FLI1, RILPL2, GCSH, PPP1R27, ZNF559, ZNF177, IER2, ZNF546, MED25, BFSP1, SALL4, MRPL39, C21orf33 and ADARB1;

[0025] (A2) The methylation level of the promoter regions of the following genes was higher on day 5 post-ovulation (PO5) than on day 3 post-ovulation (PO3), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11):

[0026] PER3, TP53I3, PPM1B, PROC, HDAC4, CCDC36, FAM107A, MBD4, RGS12, PAPD4, AEBP1, FKBP6, SCARA5, CA13, BAALC, RGS3, RALGDS, ZNF75D, IDH 3G, SLK, RASSF7, AP2A2, BCL9L, HHIPL1, SETD6, ANKRD40, TCF4, RFX1, ILVBL, ZNF667, CPNE1, SULF2, ABCG1, COL6A2, ARVCF, PITPNB and ADM2;

[0027] (A3) The methylation level of the promoter regions of the following genes was higher on day 7 post-ovulation (PO7) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11):

[0028] MFSD2A, MRPL37, L1TD1, TYW3, S100A6, TMEFF2, SCN5A, SEPT11, SNX24, RIPPLY2, STK31, CCDC180, STRBP, PKN3, SARDH, COX7B, PDZD4, WAC, TFAM, CACUL1, SYT7, ANKRD49 , RASSF8, HTR2A, RP11-310K10, PRSS22, TNFRSF12A, ZC3H7A, METTL16, CDRT15, ​​DUSP14, CFAP53, SERPINB5, DNMT1, CERS1, ASXL1, SPATA2, RNF185, MGAT3, WNT7B and TRABD;

[0029] (A4) The methylation level of the promoter regions of the following genes was higher on day 9 post-ovulation (PO9) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), and day 11 post-ovulation (PO11):

[0030] SLC2A5, DISP3, LDLRAD2, YIPF1, FAM163A, KIF3C, CCRL2, UCN2, USP19, PARL, FBXO4, OOEP, ZC3H12D, SFT2D1, RHBD D2, FAM131B, RPP38, OR51B5, WDR74, RERG, ITGB7, RNF31, CCPG1, ABCA3, ALG1, MT1F, CNP, HAMP, TGIF2, C20orf100;

[0031] (A5) The methylation level of the promoter regions of the following genes was higher on day 11 post-ovulation (PO11) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), and day 9 post-ovulation (PO9):

[0032] ACOT11, MCOLN2, SPAG17, TADA1, ARF4, FAM162A, USP13, ARHGAP24, CAMK4, HTR4, TMEM242, CALU, MAL2, PDCL, STXBP1, SLC25A25, GPSM1, SPANXB1, CELF2, DHTKD1, TCF7L2, IFITM10, C 1QTNF4, HOXC6, ARHGAP9, SPATA13, TM9SF2, PRPF39, PYGL, OTUB2, NIPA1, TK2, CACNB1, P3H4, PITPNC1, THOP1, CDC37, BBC3, NOSIP, TRAPPC2B, TMEM74B, SYNJ1, CLIC6, PPIL2 and SH3BP1.

[0033] In some embodiments of this application, if it is determined that the endometrial receptivity window of the donor has shifted, the detection method further includes determining, based on the correspondence between the obtained detection results and the conditions shown in (A1) to (A5), the actual day after ovulation of the donor's endometrium.

[0034] In some embodiments of this application, the period-specific promoter region includes a region 0.5kb~2.5kb upstream to 0.5kb~2.5kb downstream of the transcription start site of the gene.

[0035] In some embodiments of this application, the donor includes a patient receiving assisted reproductive technology treatment.

[0036] In some embodiments of this application, the detection includes the following steps: constructing a sequencing library with the free DNA and sequencing.

[0037] In some embodiments of this application, sequencing is performed using NGS sequencing technology.

[0038] In some embodiments of this application, the sequencing library is constructed under conditions where the concentration of the free DNA is 0.04 ng / μL to 0.4 ng / μL.

[0039] Compared with traditional technologies, this application has the following advantages:

[0040] This application provides a detection method that uses cell-free DNA in peripheral blood as the detection target. By detecting the methylation status of specific regions of the cell-free DNA, the receptivity of the donor's endometrium is determined. This method can be used in assisted reproductive technology without the need for endometrial tissue biopsy, reducing the psychological and physical burden on patients and achieving non-invasive testing. The detection route is simple, the operation is straightforward, and the cost is lower, significantly improving clinical testing efficiency and broadening its application range. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 The diagram shows the process for screening feature variables in this application.

[0043] Figure 2a and Figure 2b The results of cluster analysis on the feature variables are shown.

[0044] Figures 3 to 14 The results shown are those of exemplary patients one through eleven. Detailed Implementation

[0045] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0047] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0048] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0049] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0050] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0051] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0052] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0053] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0054] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0055] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0056] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0057] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0058] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0059] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0060] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0061] This application provides a non-invasive method for detecting endometrial receptivity. The method determines whether the endometrial receptivity window of the donor has shifted by detecting the methylation status of cell-free DNA in a donor peripheral blood sample.

[0062] In some embodiments of this application, the detection method includes the following steps:

[0063] Detecting the methylation status of the time-specific promoter regions in the free DNA; and,

[0064] Based on the obtained test results, determine whether the endometrial receptivity window of the donor has shifted;

[0065] The period-specific promoter regions include the promoter regions of the following genes: PSRC1, PER3, MFSD2A, SLC2A5, ACOT11, IGSF9, TP53I3, MRPL37, DISP3, MCOLLN2, KCNK1, PPM1B, L1TD1, LDLRAD2, SPAG17, VRK2, PROC, TYW3, YIPF1, TADA1, SMYD5, HDAC4, S100A6, FAM163A, ARF4, ERCC3, CCDC36, TMEFF2, KIF3C, FAM162A, HHATL, FAM107A, SCN5A, CCRL2, USP13, CA CNA2D2, MBD4, SEPT11, UCN2, ARHGAP24, NIT2, RGS12, SNX24, USP19, CAMK4, FGFR4, PAPD4, RIPPLY2, PARL, HTR4, PDLIM7, AEBP1, STK31, FBXO4, TMEM242, CCDC146, FKBP6, CCDC180, OOEP, CALU, C7orf55, SCARA5, STRBP, ZC3H12D, MAL2, ENG, CA13, PKN3, SFT2D1, PDCL, EHMT1, BAALC, SARDH, RHBDD2, STXBP1, L HPP, RGS3, COX7B, FAM131B, LC25A25, FDX1, RALGDS, PDZD4, RPP38, GPSM1, FLI1, ZNF75D, WAC, OR51B5, SPANXB1, RILPL2, IDH3G, TFAM, WDR74, CELF2, GCS H, SLK, CACUL1, RERG, DHTKD1, PPP1R27, RASSF7, SYT7, ITGB7, TCF7L2, ZNF559, AP2A2, ANKRD49, RNF31, IFITM10, ZNF177, BCL9L, RASSF8, CCPG1, C1QTNF 4. IER2, HHIPL1, HTR2A, ABCA3, HOXC6, ZNF546, SETD6, RP11-310K10, ALG1, ARHGAP9, MED25, ANKRD40, PRSS22, MT1F, SPATA13, BFSP1, TCF4, TNFRSF12A, CNP, TM9SF2, SALL4, RFX1, ZC3H7A, HAMP, PRPF39, MRPL39, ILVBL, METTL16, TGIF2, PYGL, C21orf33, ZNF667, CDRT15, ​​C20orf100, OTUB2, ADARB1, CPNE1,DUSP14, TRAPPC2B, NIPA1, RNF185, SULF2, CFAP53, TMEM74B, TK2, MGAT3, ABCG1, SERPINB5, SYNJ1, CACNB1, WNT7B, COL6A2, D NMT1, CLIC6, P3H4, TRABD, ARVCF, CERS1, PPIL2, PITPNC1, BBC3, PITPNB, ASXL1, SH3BP1, THOP1, NOSIP, ADM2, SPATA2 and CDC37. ,

[0066] In some embodiments of this application, the sampling time range of the peripheral blood sample includes days 1 to 11 after the donor ovulates.

[0067] In some embodiments of this application, determining whether the endometrial receptivity window of the donor has shifted based on the obtained detection results includes:

[0068] If the test results meet the conditions shown in (A1) to (A5), then the endometrial receptivity window of the donor has not shifted; if the test results do not meet any of the conditions shown in (A1) to (A5), then the endometrial receptivity window of the donor has shifted.

[0069] (A1) The methylation level of the promoter regions of the following genes was relatively higher on day 3 post-ovulation (PO3) than on day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11):

[0070] PSRC1, IGSF9, KCNK1, VRK2, SMYD5, ERCC3, HHATL, CACNA2D2, NIT2, FGFR4, PDLIM7, CCDC146, C7orf55, ENG, EHMT1, LHPP , FDX1, FLI1, RILPL2, GCSH, PPP1R27, ZNF559, ZNF177, IER2, ZNF546, MED25, BFSP1, SALL4, MRPL39, C21orf33 and ADARB1;

[0071] (A2) The methylation level of the promoter regions of the following genes was higher on day 5 post-ovulation (PO5) than on day 3 post-ovulation (PO3), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11):

[0072] PER3, TP53I3, PPM1B, PROC, HDAC4, CCDC36, FAM107A, MBD4, RGS12, PAPD4, AEBP1, FKBP6, SCARA5, CA13, BAALC, RGS3, RALGDS, ZNF75D, IDH 3G, SLK, RASSF7, AP2A2, BCL9L, HHIPL1, SETD6, ANKRD40, TCF4, RFX1, ILVBL, ZNF667, CPNE1, SULF2, ABCG1, COL6A2, ARVCF, PITPNB and ADM2;

[0073] (A3) The methylation level of the promoter regions of the following genes was higher on day 7 post-ovulation (PO7) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11):

[0074] MFSD2A, MRPL37, L1TD1, TYW3, S100A6, TMEFF2, SCN5A, SEPT11, SNX24, RIPPLY2, STK31, CCDC180, STRBP, PKN3, SARDH, COX7B, PDZD4, WAC, TFAM, CACUL1, SYT7, ANKRD49 , RASSF8, HTR2A, RP11-310K10, PRSS22, TNFRSF12A, ZC3H7A, METTL16, CDRT15, ​​DUSP14, CFAP53, SERPINB5, DNMT1, CERS1, ASXL1, SPATA2, RNF185, MGAT3, WNT7B and TRABD;

[0075] (A4) The methylation level of the promoter regions of the following genes was higher on day 9 post-ovulation (PO9) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), and day 11 post-ovulation (PO11):

[0076] SLC2A5, DISP3, LDLRAD2, YIPF1, FAM163A, KIF3C, CCRL2, UCN2, USP19, PARL, FBXO4, OOEP, ZC3H12D, SFT2D1, RHBD D2, FAM131B, RPP38, OR51B5, WDR74, RERG, ITGB7, RNF31, CCPG1, ABCA3, ALG1, MT1F, CNP, HAMP, TGIF2, C20orf100;

[0077] (A5) The methylation level of the promoter regions of the following genes was higher on day 11 post-ovulation (PO11) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), and day 9 post-ovulation (PO9):

[0078] ACOT11, MCOLN2, SPAG17, TADA1, ARF4, FAM162A, USP13, ARHGAP24, CAMK4, HTR4, TMEM242, CALU, MAL2, PDCL, STXBP1, SLC25A25, GPSM1, SPANXB1, CELF2, DHTKD1, TCF7L2, IFITM10, C 1QTNF4, HOXC6, ARHGAP9, SPATA13, TM9SF2, PRPF39, PYGL, OTUB2, NIPA1, TK2, CACNB1, P3H4, PITPNC1, THOP1, CDC37, BBC3, NOSIP, TRAPPC2B, TMEM74B, SYNJ1, CLIC6, PPIL2 and SH3BP1.

[0079] In some embodiments of this application, if it is determined that the endometrial receptivity window of the donor has shifted, the detection method further includes determining, based on the correspondence between the obtained detection results and the conditions shown in (A1) to (A5), the actual day after ovulation of the donor's endometrium.

[0080] Examples of endometrial receptivity window displacement include:

[0081] Example B1: The test result on day 3 after ovulation (PO3) does not match the DNA methylation characteristics corresponding to day 3 after ovulation (PO3), but matches the genome corresponding to day 5 after ovulation (PO5), day 7 after ovulation (PO7), day 9 after ovulation (PO9), or day 11 after ovulation (PO11).

[0082] Example B2: The results of cell-free DNA detection on day 5 post-ovulation (PO5) do not conform to the DNA methylation characteristics corresponding to day 5 post-ovulation (PO5), but conform to the DNA methylation characteristics corresponding to day 3 post-ovulation (PO3), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), or day 11 post-ovulation (PO11).

[0083] Example B3: The results of cell-free DNA detection on day 7 after ovulation (PO7) do not conform to the DNA methylation characteristics corresponding to day 7 after ovulation (PO7), but they do conform to the DNA methylation characteristics corresponding to day 3 after ovulation (PO3), day 5 after ovulation (PO5), day 9 after ovulation (PO9), or day 11 after ovulation (PO11).

[0084] Example B4: The results of cell-free DNA detection on day 9 post-ovulation (PO9) do not conform to the DNA methylation characteristics corresponding to day 9 post-ovulation (PO9), but they do conform to the DNA methylation characteristics corresponding to day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), or day 11 post-ovulation (PO11).

[0085] Example B4: The results of cell-free DNA detection on day 11 post-ovulation (PO11) do not conform to the DNA methylation characteristics corresponding to day 11 post-ovulation (PO11), but conform to the DNA methylation characteristics corresponding to day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), or day 9 post-ovulation (PO9).

[0086] In the above case, the day after ovulation can be determined by which feature (A1) to (A5) the obtained test result matches.

[0087] For example, if the test results show relatively high methylation levels in the period-specific promoter regions of the following genes (higher than data from other days), then it can be determined that it is day 3 post-ovulation: PSRC1, IGSF9, KCNK1, VRK2, SMYD5, ERCC3, HHATL, CACNA2D2, NIT2, FGFR4, PDLIM7, CCDC146, C7orf55, ENG, EHMT1, LHPP, FDX1, FLI1, RILPL2, GCSH, PPP1R27, ZNF559, ZNF177, IER2, ZNF546, MED25, BFSP1, SALL4, MRPL39, C21orf33, and ADARB1.

[0088] For example, if the test results show that the methylation level of the time-specific promoter regions of the following genes is relatively high (higher than the data for other days), then it can be determined that it is day 5 after ovulation: PER3, TP53I3, PPM1B, PROC, HDAC4, CCDC36, FAM107A, MBD4, RGS12, PAPD4, AEBP1, FKBP6, SCARA5, CA13, BAALC, RGS3, RALGDS, ZNF75D, IDH3G, SLK, RASSF7, AP2A2, BCL9L, HHIPL1, SETD6, ANKRD40, TCF4, RFX1, ILVBL, ZNF667, CPNE1, SULF2, ABCG1, COL6A2, ARVCF, PITPNB, and ADM2.

[0089] In some embodiments of this application, the period-specific promoter region includes a region 0.5kb~2.5kb upstream to 0.5kb~2.5kb downstream of the transcription start site of the gene.

[0090] In some embodiments of this application, the donor includes a patient receiving assisted reproductive technology treatment.

[0091] In some embodiments of this application, the detection includes the following steps: constructing a sequencing library with the free DNA and sequencing.

[0092] In some embodiments of this application, sequencing is performed using NGS sequencing technology.

[0093] In some embodiments of this application, the sequencing library is constructed under conditions where the concentration of the free DNA is 0.04 ng / μL to 0.4 ng / μL.

[0094] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0095] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0096] This application embodiment reflects the changes in the endometrium by detecting the methylation status of free DNA in the blood, in order to predict the implantation window of the endometrium.

[0097] I. Construction Method

[0098] 1. Sample preparation

[0099] Patient inclusion criteria: The samples were from patients who underwent assisted reproductive treatment, whose endometrial lining did not shift as detected, and who successfully conceived after embryo transfer within the theoretical window.

[0100] Method for determining the ovulation day: Starting from day 10-12 of menstruation, use a high-precision LH test strip to test urine every day. The day when the color of the test strip reaches or is darker than the control line is determined as the LH peak day, and LH+1 is the ovulation day.

[0101] 125 peripheral blood samples were collected from 25 patients (5 peripheral blood samples from each patient). The blood collection time points for each patient included five time points: day 3, 5, 7, 9, and 11 after ovulation (with the ovulation day as the baseline, recorded as day 0).

[0102] For each sample, this application follows the procedure below for testing.

[0103] (1) Collect 900 μL of peripheral blood using a purple blood collection tube; invert the tube several times and transfer it to a new 1.5 mL centrifuge tube;

[0104] (2) Place the 1.5 mL centrifuge tube in a small high-speed centrifuge and centrifuge at 13000 g for 15 min.

[0105] 2. Extraction of cell-free DNA

[0106] Cell-free DNA was extracted using automated equipment, and the extraction steps are as follows:

[0107] (1) Slowly peel off the sealing film of the deep hole plate to prevent the liquid on the film from splashing into other holes, and make marks in the blank positions on both sides of the deep hole plate.

[0108] (2) Remove the plasma from the centrifuge and arrange it in the order of sample addition. Be careful not to shake the sample violently.

[0109] (3) If the amount of plasma extracted is 400 μL, add 400 μL of plasma to the second / eighth column and leave the first / seventh column empty; if the amount of plasma extracted is 800 μL, add 400 μL of plasma to the first, second / seventh and eighth columns respectively; if it is less than 800 μL, add 400 μL of plasma to one column and make up the remaining 400 μL with PBS in the other column.

[0110] (4) Add 20 μL of magnetic beads and 10 μL of proteinase K to the first, second or seventh and eighth columns of the deep well plate. If the amount of plasma extracted is 400 μL, only the magnetic beads and proteinase K need to be added to the second or eighth column.

[0111] (5) Place the deep well plate with the sample added on the automated extractor, insert the double-sided magnetic stirring sleeve, place it firmly and not loose, and close the instrument door; select the program: extraction program group 2---CFDM-800 / 400 (select the program according to the amount of plasma to be extracted, select CFDM-800 for 800 μL of plasma), and start the program.

[0112] (6) When the procedure is finished, remove the deep well plate and collect the DNA from column 6 / 12 into the labeled 1.5 mL centrifuge tubes in sequence. Label the tubes and dispose of the waste liquid in the deep well plate according to laboratory regulations.

[0113] 3. Nucleic acid quality control

[0114] Take 3 μL of the extracted DNA for Qubit 4.0 dsDNA concentration determination. When extracting 800 μL of plasma, the final concentration is usually between 0.04-0.4 ng / μL.

[0115] 4. Library Construction

[0116] (1) DNA fragmentation: Take 50 μL of free DNA into a PCR tube, vortex to mix, and then centrifuge briefly.

[0117] (2) End repair with A: Add end repair with A enzyme and end repair with A buffer to the PCR tube containing the cell sample, mix well, and incubate in a PCR instrument. PCR program settings: 20℃, 30min; 65℃, 30min; 4℃, ∞.

[0118] (3) Adding adapters: Add methylated adapters, ligase mixture and reaction enhancer to the end-repair A-addition product, mix well and incubate in a PCR instrument. PCR program settings: 20℃, 15min; 4℃, ∞.

[0119] (4) Purification of ligation products:

[0120] Add magnetic beads to the ligation product, mix well and let stand for 5 minutes;

[0121] Place the PCR tube in the magnetic rack, let it stand for 2 minutes, and then discard the liquid;

[0122] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, and then discard the liquid.

[0123] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, discard the liquid, and then open the cap to air dry for 5 minutes.

[0124] Add the elution buffer to the tube, mix well, and let stand for 5 minutes.

[0125] (5) Place the PCR tube in the magnetic rack, let it stand for 2 minutes, and then transfer the liquid to a new PCR tube.

[0126] (6) TET2 oxidation: Add TET2 enzyme, reaction buffer, reaction enhancement solution and reaction supplement to the purified library on ice, vortex mix, add iron solution, vortex mix again and incubate at 37°C for 1 hour.

[0127] (7) Oxidation termination: Immediately after the reaction is complete, remove the PCR tube and place it on ice. Add the stop solution, mix well, and then place it in the PCR instrument for incubation at 37°C for 30 min.

[0128] (8) Purification of oxidation products:

[0129] Add magnetic beads to the oxidation product, mix well and let stand for 5 minutes;

[0130] Place the PCR tube in the magnetic rack, let it stand for 2 minutes, and then discard the liquid;

[0131] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, and then discard the liquid.

[0132] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, discard the liquid, and then open the cap to air dry for 5 minutes.

[0133] Add the elution buffer to the tube, mix well, and let stand for 5 minutes;

[0134] Place the PCR tube in the magnetic rack, let it stand for 2 minutes, and then transfer the liquid to a new PCR tube.

[0135] (9) DNA denaturation: Add 0.1 mol of NaOH to the PCR tube, and then place the sample in a PCR instrument preheated to 50°C for 10 min. After incubation, place it on ice immediately.

[0136] (10) Deamination reaction: Add deaminase, deamination buffer and reaction supplement to PCR tube on ice, vortex to mix, and then incubate at 37°C for 3 hours in a PCR instrument.

[0137] (11) Purification of deamination products:

[0138] Add magnetic beads to the deamination product, mix well and let stand for 5 minutes;

[0139] Place the PCR tube in the magnetic rack, let it stand for 2 minutes, and then discard the liquid;

[0140] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, and then discard the liquid.

[0141] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, discard the liquid, and then open the cap to air dry for 5 minutes.

[0142] Add the elution buffer to the tube, mix well, and let stand for 5 minutes;

[0143] Place the PCR tube in the magnetic rack, let it stand for 2 minutes, and then transfer the liquid to a new PCR tube.

[0144] (12) PCR amplification

[0145] Add the amplification primer and PCR enzyme mixture to the PCR tube, mix well, and then place it in the PCR instrument for amplification. The PCR program settings are: 98°C, 45s; (98°C, 15s; 65°C, 30s; 72°C, 30s) × 15; 72°C, 1min; 4°C, ∞.

[0146] (13) Purification of amplification products

[0147] Add magnetic beads to the amplification product, mix well and let stand for 5 minutes;

[0148] Place the PCR tube in the magnetic rack, let it stand for 2 minutes, and then discard the liquid;

[0149] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, and then discard the liquid.

[0150] Add freshly prepared 80% ethanol to the tube, let it stand for 30 seconds, discard the liquid, and then open the cap to air dry for 5 minutes.

[0151] Add the elution buffer to the tube, mix well, and let stand for 5 minutes;

[0152] Place the PCR tube in a magnetic rack, let it stand for 2 minutes, and then transfer the liquid to a new centrifuge tube.

[0153] (13) Library quality control: The library was quantified using a qPCR instrument and the size of the library fragments was determined using an Agilent 2100.

[0154] (14) Library sequencing: After the library passes the quality inspection, the NGS sequencing platform is used to sequence the library.

[0155] 5. Data Analysis

[0156] The whole-genome methylation sequencing data obtained after sequencing were analyzed according to the following data analysis workflow, the specific process of which is as follows:

[0157] Data quality control was performed on the raw whole-genome methylation sequencing data to remove adapter sequences and low-quality bases. Then, genome alignment was performed, and the aligned data were merged to remove PCR repetitive sequences.

[0158] For cytosine sites on the genome, the methylation level (ML) of each site is calculated by counting how much thymine (T) and cytosine (C) cover that site. The calculation formula is as follows:

[0159]

[0160] ML i : Methylation level of the i-th cytosine site;

[0161] C i The number of cytosine bases covering the i-th CG site;

[0162] T i The number of thymine bases covering the i-th CG site;

[0163] For calculating the DNA methylation level of a specific region of the genome, such as a promoter region or an imprinted gene control region, the total number of thymine (T) and cytosine (C) covering all cytosine sites in that region is counted, and the methylation level of each region is calculated using the formula mentioned above.

[0164] This application first defines the promoter region of a gene as a 2kb region upstream and downstream of the transcription start site. Then, this application uses the average methylation level of at least 10 CpG sites covered by this region as the level of the feature; otherwise, the level of this region is considered as missing. Initial feature screening requires coverage of 90% of all clinical samples. Subsequently, the differences in methylation levels of the feature within and between groups are considered across five time periods: day 3 (PO3), day 5 (PO5), day 7 (PO7), day 9 (PO9), and day 11 (PO11) post-ovulation. For the next step of feature selection, the 90th percentile empirical quantile interval of methylation levels within the sample (within the group) at the same time period must be less than 0.6, and the standard deviation within the group must be less than 0.2. Finally, the average methylation level (AML) of the selected feature across different time periods (between groups) must be greater than 0.12. This application analyzes the DNA methylation changes of the promoter region at different time points, retains the time-specific promoter regions, and defines the time-specific promoter region as a characteristic variable of that time period. For example... Figure 1 As shown.

[0165] Using the above analysis strategy, this application obtained a total of 184 time-specific variables, as shown in Table 1.

[0166] Table 1

[0167]

[0168] Based on these characteristics, cluster analysis revealed that samples from different time periods can significantly cluster together. Furthermore, combining 2D and 3D visualizations, there are clear boundaries between PO (Post-Ovulation Day) 3, PO5, and PO7. While there is some overlap between PO9 and PO11, the clustering trend is also evident. Figure 2a and Figure 2b As shown above, the results demonstrate that the methylation characteristics of cell-free DNA in peripheral blood can distinguish different phases of the menstrual cycle. PO3, PO5, PO7, PO9, and PO11 exhibit the following clustering trends:

[0169] (A1) The methylation level of the promoter regions of the following genes was relatively higher on day 3 post-ovulation (PO3) than on day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11):

[0170] PSRC1, IGSF9, KCNK1, VRK2, SMYD5, ERCC3, HHATL, CACNA2D2, NIT2, FGFR4, PDLIM7, CCDC146, C7orf55, ENG, EHMT1, LHPP , FDX1, FLI1, RILPL2, GCSH, PPP1R27, ZNF559, ZNF177, IER2, ZNF546, MED25, BFSP1, SALL4, MRPL39, C21orf33 and ADARB1;

[0171] (A2) The methylation level of the promoter regions of the following genes was higher on day 5 post-ovulation (PO5) than on day 3 post-ovulation (PO3), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11):

[0172] PER3, TP53I3, PPM1B, PROC, HDAC4, CCDC36, FAM107A, MBD4, RGS12, PAPD4, AEBP1, FKBP6, SCARA5, CA13, BAALC, RGS3, RALGDS, ZNF75D, IDH 3G, SLK, RASSF7, AP2A2, BCL9L, HHIPL1, SETD6, ANKRD40, TCF4, RFX1, ILVBL, ZNF667, CPNE1, SULF2, ABCG1, COL6A2, ARVCF, PITPNB and ADM2;

[0173] (A3) The methylation level of the promoter regions of the following genes was higher on day 7 post-ovulation (PO7) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11):

[0174] MFSD2A, MRPL37, L1TD1, TYW3, S100A6, TMEFF2, SCN5A, SEPT11, SNX24, RIPPLY2, STK31, CCDC180, STRBP, PKN3, SARDH, COX7B, PDZD4, WAC, TFAM, CACUL1, SYT7, ANKRD49 , RASSF8, HTR2A, RP11-310K10, PRSS22, TNFRSF12A, ZC3H7A, METTL16, CDRT15, ​​DUSP14, CFAP53, SERPINB5, DNMT1, CERS1, ASXL1, SPATA2, RNF185, MGAT3, WNT7B and TRABD;

[0175] (A4) The methylation level of the promoter regions of the following genes was higher on day 9 post-ovulation (PO9) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), and day 11 post-ovulation (PO11):

[0176] SLC2A5, DISP3, LDLRAD2, YIPF1, FAM163A, KIF3C, CCRL2, UCN2, USP19, PARL, FBXO4, OOEP, ZC3H12D, SFT2D1, RHBD D2, FAM131B, RPP38, OR51B5, WDR74, RERG, ITGB7, RNF31, CCPG1, ABCA3, ALG1, MT1F, CNP, HAMP, TGIF2, C20orf100;

[0177] (A5) The methylation level of the promoter regions of the following genes was higher on day 11 post-ovulation (PO11) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), and day 9 post-ovulation (PO9):

[0178] ACOT11, MCOLN2, SPAG17, TADA1, ARF4, FAM162A, USP13, ARHGAP24, CAMK4, HTR4, TMEM242, CALU, MAL2, PDCL, STXBP1, SLC25A25, GPSM1, SPANXB1, CELF2, DHTKD1, TCF7L2, IFITM10, C 1QTNF4, HOXC6, ARHGAP9, SPATA13, TM9SF2, PRPF39, PYGL, OTUB2, NIPA1, TK2, CACNB1, P3H4, PITPNC1, THOP1, CDC37, BBC3, NOSIP, TRAPPC2B, TMEM74B, SYNJ1, CLIC6, PPIL2 and SH3BP1.

[0179] Based on the above trends, the following criteria are established for predicting whether the endometrial receptivity window has shifted:

[0180] If the test results meet the conditions shown in (A1) to (A5), then the endometrial receptivity window of the donor has not shifted;

[0181] If the test results do not meet any of the conditions shown in (A1) to (A5), then the endometrial receptivity window of the donor is shifted.

[0182] II. Verification

[0183] To verify the reliability of this technique, this application collected 60 peripheral blood samples from 12 patients. Clinical examination revealed no endometrial displacement in 6 patients, while clinical examination showed endometrial cycle irregularities in the other 6 patients. For each patient's sample, this application processed the samples according to the aforementioned method and performed bioinformatics analysis. The results are as follows:

[0184] Patient 1 (normal endometrium): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned method, and the test results on days 3, 5, 7, 9, and 11 corresponded to (A1) to (A5) under section one, respectively. Results are as follows... Figure 3 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample at each time point using this technique. The results show that the predicted endometrial cycle time points and the actual endometrial cycle time points are completely consistent.

[0185] Patient 2 (normal endometrium): Peripheral blood samples were collected from this patient at three time points: day 7, day 9, and day 11 after ovulation. Analysis was performed using the aforementioned method, and the test results on days 7, 9, and 11 corresponded to (A3) to (A5) under section I, respectively. Results are as follows... Figure 4 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample at each time point using this technique. The results show that the predicted endometrial cycle time points and the actual endometrial cycle time points are completely consistent.

[0186] Patient 3 (normal endometrium): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned method, and the test results on days 3, 5, 7, 9, and 11 corresponded to (A1) to (A5) under section one, respectively. The results are as follows... Figure 5 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample at each time point using this technique. The results show that the predicted endometrial cycle time points and the actual endometrial cycle time points are completely consistent.

[0187] Patient 4 (normal endometrium): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned method, and the test results on days 3, 5, 7, 9, and 11 corresponded to (A1) to (A5) under section one, respectively. The results are as follows... Figure 6 As shown, the X-axis represents the blood collection time point for the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample at each time point using this technique. We can see that the predicted endometrial cycle time points perfectly match the actual endometrial cycle time points.

[0188] Patient 5 (normal endometrium): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned method, and the test results on days 3, 5, 7, 9, and 11 corresponded to (A1) to (A5) under section one, respectively. Results are as follows... Figure 7 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample at each time point using this technique. The results show that the predicted endometrial cycle time points and the actual endometrial cycle time points are completely consistent.

[0189] Patient Six (normal endometrium): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned method, and the test results on days 3, 5, 7, 9, and 11 corresponded to (A1) to (A5) under section one, respectively. The results are as follows... Figure 8 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample at each time point using this technique. The results show that the predicted endometrial cycle time points and the actual endometrial cycle time points are completely consistent.

[0190] Patient 7 (Endometrial Abnormalities): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned methods, and the results are as follows: Figure 9 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample using this technique. The results showed that the DNA methylation characteristics of the endometrial tissue on day 5 post-ovulation were consistent with those on day 3, while the DNA methylation characteristics on days 7 and 11 were consistent with those on day 9. This result indicates that the patient's endometrial cycle was disordered, and the actual clinical test results confirmed that the patient did indeed have endometrial abnormalities.

[0191] Patient 8 (Endometrial Abnormalities): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned methods, and the results are as follows: Figure 10 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample using this technique. The results showed that the DNA methylation characteristics of the endometrial lining on day 5 after ovulation were consistent with those on day 7, while the DNA methylation characteristics on day 7 were consistent with those on day 9, and the DNA methylation characteristics on day 11 were consistent with those on day 7. These results indicate that the patient's endometrial cycle was disordered, and the actual clinical test results confirmed that the patient did indeed have endometrial abnormalities.

[0192] Patient Nine (Endometrial Abnormalities): Peripheral blood samples were collected from this patient at three time points: 3rd, 5th, and 7th time points after ovulation. Analysis was performed using the aforementioned methods, and the results are as follows: Figure 11As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample using this technique. The results showed that the DNA methylation characteristics of the endometrial tissue on day 5 post-ovulation were consistent with those on day 3, and the DNA methylation characteristics on day 7 were consistent with those on day 9. This result indicates that the patient's endometrial cycle was disordered, and the actual clinical test results confirmed that the patient did indeed have endometrial abnormalities.

[0193] Patient 10 (Endometrial Abnormality): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned methods, and the results are as follows: Figure 12 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample using this technique. The results showed that the DNA methylation characteristics of the endometrial tissue on day 3 post-ovulation were consistent with those on day 5; the DNA methylation characteristics on day 5 were consistent with those on day 3; the DNA methylation characteristics on day 7 were consistent with those on day 9; the DNA methylation characteristics on day 9 were consistent with those on day 7; and the DNA methylation characteristics on day 11 were consistent with those on day 5. These results indicate that the patient's endometrial cycle was disordered, and the actual clinical testing confirmed that the patient did indeed have endometrial abnormalities.

[0194] Patient 11 (Endometrial Abnormalities): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned methods, and the results are as follows: Figure 13 As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample using this technique. The results showed that the DNA methylation characteristics of the endometrial tissue on day 7 post-ovulation were consistent with those on day 5, while the DNA methylation characteristics on day 9 were consistent with those on day 11, and those on day 11 were consistent with those on day 5. These results indicate an irregular endometrial cycle in the patient, and the actual clinical testing confirmed the presence of endometrial abnormalities.

[0195] Patient 12 (abnormal endometrium): Peripheral blood samples were collected from this patient at five time points: days 3, 5, 7, 9, and 11 after ovulation. Analysis was performed using the aforementioned methods, and the results are as follows: Figure 14As shown, the X-axis represents the blood collection time point of the patient, and the Y-axis represents the predicted endometrial cycle time point after analysis of the sample using this technique. The results showed that the DNA methylation characteristics of the endometrial tissue on day 3 post-ovulation were consistent with those on day 7; the DNA methylation characteristics on day 5 were consistent with those on day 3; the DNA methylation characteristics on day 7 were consistent with those on day 5; and the DNA methylation characteristics on day 9 were consistent with those on day 5. These results indicate an irregular endometrial cycle in the patient, and the actual clinical testing confirmed that the patient did indeed have endometrial abnormalities.

[0196] Clinical validation data shows that for samples with normal endometrium, the molecular characteristics at specific time points analyzed using this technique correspond to the actual endometrial cycle time points. However, for abnormal endometrial samples, the molecular characteristics at specific time points differ significantly from the actual endometrial cycle time points. This result directly indicates that this technique can be effectively used to determine whether a patient's endometrial cycle is abnormal, providing support for further clinical testing.

[0197] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0198] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A non-invasive method for detecting endometrial receptivity, characterized in that, The detection method determines whether the endometrial receptivity window of the donor has shifted by detecting the methylation status of cell-free DNA in the donor's peripheral blood sample.

2. The non-invasive method for detecting endometrial receptivity according to claim 1, characterized in that, The detection method includes the following steps: Detecting the methylation status of the time-specific promoter regions in the free DNA; and, Based on the obtained test results, determine whether the endometrial receptivity window of the donor has shifted; The period-specific promoter regions include the promoter regions of the following genes: PSRC1, PER3, MFSD2A, SLC2A5, ACOT11, IGSF9, TP53I3, MRPL37, DISP3, MCOLLN2, KCNK1, PPM1B, L1TD1, LDLRAD2, SPAG17, VRK2, PROC, TYW3, YIPF1, TADA1, SMYD5, HDAC4, S100A6, FAM163A, ARF4, ERCC3, CCDC36, TMEFF2, KIF3C, FAM162A, HHATL, FAM107A, SCN5A, CCRL2, USP13, CA CNA2D2, MBD4, SEPT11, UCN2, ARHGAP24, NIT2, RGS12, SNX24, USP19, CAMK4, FGFR4, PAPD4, RIPPLY2, PARL, HTR4, PDLIM7, AEBP1, STK31, FBXO4, TMEM242, CCDC146, FKBP6, CCDC180, OOEP, CALU, C7orf55, SCARA5, STRBP, ZC3H12D, MAL2, ENG, CA13, PKN3, SFT2D1, PDCL, EHMT1, BAALC, SARDH, RHBDD2, STXBP1, L HPP, RGS3, COX7B, FAM131B, LC25A25, FDX1, RALGDS, PDZD4, RPP38, GPSM1, FLI1, ZNF75D, WAC, OR51B5, SPANXB1, RILPL2, IDH3G, TFAM, WDR74, CELF2, GCS H, SLK, CACUL1, RERG, DHTKD1, PPP1R27, RASSF7, SYT7, ITGB7, TCF7L2, ZNF559, AP2A2, ANKRD49, RNF31, IFITM10, ZNF177, BCL9L, RASSF8, CCPG1, C1QTNF 4. IER2, HHIPL1, HTR2A, ABCA3, HOXC6, ZNF546, SETD6, RP11-310K10, ALG1, ARHGAP9, MED25, ANKRD40, PRSS22, MT1F, SPATA13, BFSP1, TCF4, TNFRSF12A, CNP, TM9SF2, SALL4, RFX1, ZC3H7A, HAMP, PRPF39, MRPL39, ILVBL, METTL16, TGIF2, PYGL, C21orf33, ZNF667, CDRT15, ​​C20orf100, OTUB2, ADARB1, CPNE1,DUSP14, TRAPPC2B, NIPA1, RNF185, SULF2, CFAP53, TMEM74B, TK2, MGAT3, ABCG1, SERPINB5, SYNJ1, CACNB1, WNT7B, COL6A2, DNMT1, CLIC6, P3H4, TRABD, ARVCF, CERS1, PPIL2, PITPNC1, BBC3, PITPNB, ASXL1, SH3BP1, THOP1, NOSIP, ADM2, SPATA2 and CDC37.

3. The non-invasive method for detecting endometrial receptivity according to claim 2, characterized in that, The peripheral blood samples were collected between days 1 and 11 after the donor ovulated.

4. The non-invasive method for detecting endometrial receptivity according to claim 3, characterized in that, Determining whether the endometrial receptivity window of the donor has shifted based on the obtained test results includes: If the test results meet the conditions shown in (A1) to (A5), then the endometrial receptivity window of the donor has not shifted; if the test results do not meet any of the conditions shown in (A1) to (A5), then the endometrial receptivity window of the donor has shifted. (A1) The methylation level of the promoter regions of the following genes was relatively higher on day 3 post-ovulation (PO3) than on day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11): PSRC1, IGSF9, KCNK1, VRK2, SMYD5, ERCC3, HHATL, CACNA2D2, NIT2, FGFR4, PDLIM7, CCDC146, C7orf55, ENG, EHMT1, LHPP , FDX1, FLI1, RILPL2, GCSH, PPP1R27, ZNF559, ZNF177, IER2, ZNF546, MED25, BFSP1, SALL4, MRPL39, C21orf33 and ADARB1; (A2) The methylation levels of the promoter regions of the following genes were higher on day 5 post-ovulation (PO5) than on day 3 post-ovulation (PO3), day 7 post-ovulation (PO7), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11): PER3, TP53I3, PPM1B, PROC, HDAC4, CCDC36, FAM107A, MBD4, RGS12, PAPD4, AEBP1, FKBP6, SCARA5, CA13, BAALC, RGS3, RALGDS, ZNF75D, IDH 3G, SLK, RASSF7, AP2A2, BCL9L, HHIPL1, SETD6, ANKRD40, TCF4, RFX1, ILVBL, ZNF667, CPNE1, SULF2, ABCG1, COL6A2, ARVCF, PITPNB and ADM2; (A3) The methylation level of the promoter regions of the following genes was higher on day 7 post-ovulation (PO7) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 9 post-ovulation (PO9), and day 11 post-ovulation (PO11): MFSD2A, MRPL37, L1TD1, TYW3, S100A6, TMEFF2, SCN5A, SEPT11, SNX24, RIPPLY2, STK31, CCDC180, STRBP, PKN3, SARDH, COX7B, PDZD4, WAC, TFAM, CACUL1, SYT7, ANKRD49 , RASSF8, HTR2A, RP11-310K10, PRSS22, TNFRSF12A, ZC3H7A, METTL16, CDRT15, ​​DUSP14, CFAP53, SERPINB5, DNMT1, CERS1, ASXL1, SPATA2, RNF185, MGAT3, WNT7B and TRABD; (A4) The methylation level of the promoter regions of the following genes was higher on day 9 post-ovulation (PO9) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), and day 11 post-ovulation (PO11): SLC2A5, DISP3, LDLRAD2, YIPF1, FAM163A, KIF3C, CCRL2, UCN2, USP19, PARL, FBXO4, OOEP, ZC3H12D, SFT2D1, RHBD D2, FAM131B, RPP38, OR51B5, WDR74, RERG, ITGB7, RNF31, CCPG1, ABCA3, ALG1, MT1F, CNP, HAMP, TGIF2, C20orf100; (A5) The methylation level of the promoter regions of the following genes was higher on day 11 post-ovulation (PO11) than on day 3 post-ovulation (PO3), day 5 post-ovulation (PO5), day 7 post-ovulation (PO7), and day 9 post-ovulation (PO9): ACOT11, MCOLN2, SPAG17, TADA1, ARF4, FAM162A, USP13, ARHGAP24, CAMK4, HTR4, TMEM242, CALU, MAL2, PDCL, STXBP1, SLC25A25, GPSM1, SPANXB1, CELF2, DHTKD1, TCF7L2, IFITM10, C 1QTNF4, HOXC6, ARHGAP9, SPATA13, TM9SF2, PRPF39, PYGL, OTUB2, NIPA1, TK2, CACNB1, P3H4, PITPNC1, THOP1, CDC37, BBC3, NOSIP, TRAPPC2B, TMEM74B, SYNJ1, CLIC6, PPIL2 and SH3BP1.

5. The non-invasive method for detecting endometrial receptivity according to claim 4, characterized in that, If it is determined that the endometrial receptivity window of the donor has shifted, the detection method further includes determining, based on the correspondence between the obtained detection results and the conditions shown in (A1) to (A5), the actual day after ovulation of the donor's endometrium.

6. The non-invasive method for detecting endometrial receptivity according to claim 1, characterized in that, The period-specific promoter region includes the region 0.5kb~2.5kb upstream to 0.5kb~2.5kb downstream of the transcription start site of the gene.

7. The non-invasive method for detecting endometrial receptivity according to any one of claims 1 to 6, characterized in that, The donors include patients who have received assisted reproductive technology treatment.

8. The non-invasive method for detecting endometrial receptivity according to any one of claims 1 to 6, characterized in that, The detection process includes the following steps: constructing a sequencing library using the free DNA, and sequencing.

9. The non-invasive method for detecting endometrial receptivity according to claim 8, characterized in that, The sequencing was performed using NGS sequencing technology.

10. The non-invasive method for detecting endometrial receptivity according to claim 8, characterized in that, The sequencing library was constructed under conditions where the concentration of the free DNA was 0.04 ng / μL to 0.4 ng / μL.

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