Genetic analysis method and genetic determination method

Abstract

The present invention addresses the problem of providing a technique for detecting whether or not an egg, sperm, or fertilized egg sample has been misidentified at the stage when pregnancy is established by assisted reproductive therapy. A solution to the above problem is a genetic analysis method including comparing the following genotypes (A) and / or (B) with genotypes (C).　(A) Genotypes at a plurality of polymorphic loci of a mother undergoing assisted reproductive therapy.　(B) Genotypes at a plurality of polymorphic loci of a father undergoing assisted reproductive therapy.　(C) Genotypes at a plurality of polymorphic loci of circulating cell-free nucleic acids or a fetus, the genotypes being obtained by analyzing a circulating cell-free nucleic acid sample collected from a woman who became pregnant through assisted reproductive therapy.

Description

Genetic analysis method and determination method 【0001】 The present invention relates to a technique for detecting misidentification of samples in assisted reproductive medicine based on genetic information. 【0002】 Representative infertility treatments include artificial insemination and in vitro fertilization, and the number of procedures performed is increasing year by year. Worldwide, more than 27,000 assisted reproductive medicine procedures such as artificial insemination are performed every day in complex processes, so it is undeniable that misidentification of sperm, eggs, or fertilized eggs may occur in any of the processes. To date, there have been about 150 cases where mistakes such as misidentification due to assisted reproductive medicine have become a topic, but almost all people do not undergo DNA testing after receiving assisted reproductive medicine, so the actual number of mistakes that occur may be higher than this. 【0003】 As a measure to prevent misidentification of samples in assisted reproductive medicine, for example, an individual management system using an individual identification code or an IC tag as disclosed in Patent Document 1 is generally used. 【0004】 Japanese Unexamined Patent Application Publication No. 2018 - 042693 【0005】 The technique described in Patent Document 1 can suppress the incidence of misidentification of samples of eggs, sperm, and fertilized eggs, but it cannot detect cases where a mistake has occurred and pregnancy has occurred. Verification of misidentification of samples due to human error during current assisted reproductive medicine can only be carried out by performing maternal - child DNA testing or paternal - child DNA testing after the child is born, and new testing techniques that can eliminate anxiety during pregnancy are needed. 【0006】 An object of the present invention is to provide a technique for detecting whether there has been misidentification of a sample of an egg, sperm, or fertilized egg at the stage of pregnancy resulting from assisted reproductive medicine. 【0007】The present invention, which solves the above problems, is as follows: [1] A genetic analysis method comprising comparing the following genotypes of (A) and / or (B) and (C): (A) Genotypes at multiple polymorphic loci of a mother who is the subject of assisted reproductive technology. (B) Genotypes at multiple polymorphic loci of a father who is the subject of assisted reproductive technology. (C) Genotypes at multiple polymorphic loci of a circulating cell-free nucleic acid or fetus obtained by analyzing a circulating cell-free nucleic acid sample taken from a woman who became pregnant through assisted reproductive technology. 【0008】 [2] The genetic analysis method according to [1], comprising detecting a mismatch genotype in the circulating cell-free nucleic acid or the polymorphic locus of the fetus that does not appear when the mother and father are the biological parents of the fetus. 【0009】[3] When the mother who is the subject of assisted reproductive technology and the woman who became pregnant through assisted reproductive technology are the same person, when the mother's genotype at a certain polymorphic locus is homozygous, when the father's genotype at the same polymorphic locus is homozygous with the mother's, but the genotype of the circulating cell-free nucleic acid is heterozygous, and / or when the father's genotype at the same polymorphic locus is homozygous with the mother's, but the genotype of the circulating cell-free nucleic acid is homozygous with the mother's, the genotype at the same polymorphic locus of the circulating cell-free nucleic acid is evaluated as a mismatch genotype, when the mother who is the subject of assisted reproductive technology and the woman who became pregnant through assisted reproductive technology are different people, when the woman's genotype at a certain polymorphic locus is homozygous, The genetic analysis method described in [2], wherein, in cases where the genotype of the mother and / or father at the same polymorphic locus is homozygous for a different type from the woman, but the genotype of the circulating cell-free nucleic acid is homozygous for the same type as the woman, and / or, when the genotype of the woman at a certain polymorphic locus is homozygous, in cases where the genotype of the mother and the father at the same polymorphic locus is homozygous for the same type as the woman, but the genotype of the circulating cell-free nucleic acid is heterozygous, the genotype of the circulating cell-free nucleic acid at the same polymorphic locus is evaluated as a mismatch genotype. 【0010】 [4] The genetic analysis method according to [3], wherein, if the mother who is the subject of the assisted reproductive technology described above and the woman who became pregnant through the assisted reproductive technology described above are the same person, the method includes comparing the genotypes of (A) to (C) described above, or comparing the genotypes of (B) and (C) described above, for polymorphic loci that are homozygous in the mother, to detect the mismatch genotype; and if the mother who is the subject of the assisted reproductive technology described above and the woman who became pregnant through the assisted reproductive technology described above are different people, the method includes comparing the genotypes of (A) to (C) described above, the genotypes of (A) and (C) described above, or the genotypes of (B) and (C) described above, for polymorphic loci that are homozygous in the woman, to detect the mismatch genotype. 【0011】 [5] A genetic analysis method according to any one of [2] to [4], comprising calculating a quantitative variable of the polymorphic locus in which the mismatched genotype was detected. 【0012】 [6] The genetic analysis method according to [5], which includes determining that a sample mix-up occurred in the assisted reproductive technology if the quantitative variable is equal to or greater than a predetermined reference value. 【0013】 [7] A genetic analysis method according to any one of paragraphs [1] to [6], which includes performing a paternity test between the mother and / or the father and the fetus. 【0014】 [8] The genetic analysis method described in [7], wherein the paternity test includes analyzing whether a biological parent-child relationship exists between the fetus and either the mother or the father, with the mother or father designated as the pseudomother or pseudofather, or analyzing whether a biological parent-child relationship exists between the fetus and the mother and / or father, with the fetus designated as the pseudochild. 【0015】 [9] The genetic analysis method according to [7] or [8], wherein the paternity test includes determining whether or not there is a biological parent-child relationship between the father and the fetus by comparing two types of genotypes: the father and the circulating cell-free nucleic acid or the fetus, or determining whether or not there is a biological parent-child relationship between the mother and / or the father and the fetus by comparing three types of genotypes: the mother, the father and the fetus. 【0016】

[10] A genetic analysis method according to any one of paragraphs [7] to [9], which includes determining that a sample mix-up occurred in the assisted reproductive technology when the results of the paternity test negate the existence of a biological parent-child relationship between the mother and / or the father and the fetus. 【0017】

[11] A genetic analysis method according to any one of [2] to [6], comprising calculating a quantitative variable of the polymorphic locus in which the mismatched genotype is detected, and / or a quantitative variable that reflects the total DNA concentration and / or the concentration of circulating cell-free fetal nucleic acid in the circulating cell-free nucleic acid sample, wherein, based on the result of comparing the quantitative variable with a predetermined reference value, it is determined whether or not to perform a paternity test between the mother and / or the father and the fetus in accordance with a predetermined rule. 【0018】

[12] The genetic analysis method according to

[11] , wherein the rule is either Rule 1 or Rule 2 below. (Rule 1) If the quantitative variable is less than the reference value, the paternity test is performed, and if the quantitative variable is equal to or greater than the reference value, the analysis is terminated without performing the paternity test. (Rule 2) If the quantitative variable is less than the reference value, the analysis is terminated without performing the paternity test, and if the quantitative variable is equal to or greater than the reference value, the paternity test is performed. 【0019】

[13] A determination method comprising calculating a quantitative variable that reflects the total DNA concentration and / or the concentration of circulating cell-free fetal nucleic acid in the circulating cell-free nucleic acid sample, and determining whether or not to perform the genetic analysis method described in any one of [1] to

[10] according to the result of comparing the quantitative variable with a predetermined reference value, in accordance with the predetermined rule 3. (Rule 3) If the quantitative variable is less than the reference value, the genetic analysis method is not performed and the process is terminated; if the quantitative variable is equal to or greater than the reference value, the genetic analysis method is performed. 【0020】

[14] If the mother who is the subject of the assisted reproductive technology described above and the woman who became pregnant through the assisted reproductive technology described above are different people, the genetic analysis method described in any one of paragraphs [1] to

[12] includes comparing the genotypes of (A), (B), and (C), or (A), (B), (C), and (D) below. (D) The genotypes of multiple polymorphic loci of the woman who became pregnant through the assisted reproductive technology described above. 【0021】According to the present invention, it is possible to detect a mix-up of egg, sperm, or embryo samples that occurred during the process of assisted reproductive technology at the stage when pregnancy is achieved through such technology. 【0022】 This figure shows the analysis flow for an embodiment (a) that includes only mismatch testing and an embodiment (b) that includes only paternity testing. This figure shows the analysis flow for an embodiment that includes paternity testing. (a) An embodiment in which a paternity test is performed as the first paternity test and a maternal test is performed as the second paternity test. (b) An embodiment in which a maternal test is performed as the first paternity test and a paternity test is performed as the second paternity test. (c) An embodiment in which a maternal test and / or a paternity test are performed. (a) This figure shows the analysis flow for an embodiment in which the decision on whether or not to perform paternity testing is made by a Rule 1 determination made after mismatch testing. (b) This figure shows the analysis flow for an embodiment in which the decision on whether or not to perform paternity testing is made by a Rule 2 determination made after mismatch testing. (a) This figure shows the analysis flow for an embodiment in which the decision on whether or not to perform mismatch testing is made by a Rule 3 determination made after genotyping. (b) This figure shows the analysis flow for an embodiment in which the decision on whether or not to perform paternity testing is made by a Rule 3 determination made after genotyping. 【0023】 The genetic analysis method of the present invention includes comparing the following genotypes: (A) Genotypes at multiple polymorphic loci of the mother who is the subject of assisted reproductive technology; (B) Genotypes at multiple polymorphic loci of the father who is the subject of assisted reproductive technology; (C) Genotypes at multiple polymorphic loci of the circulating cell-free nucleic acid or fetus, obtained by analyzing a circulating cell-free nucleic acid sample taken from a woman who became pregnant through assisted reproductive technology. 【0024】In this specification, "assisted reproductive technology" includes artificial insemination of husband (AIH), in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and infertility treatments using sperm or eggs produced by in vitro gametogenesis (IVG). Artificial insemination is a method of injecting washed and concentrated sperm from a partner into the uterus, timed to coincide with the woman's ovulation. In vitro fertilization is an infertility treatment in which eggs collected from the ovaries are fertilized with sperm outside the body, and the resulting embryos, cultured outside the body, are returned to the uterus. Intracytoplasmic sperm injection (ICSI) is an infertility treatment in which a single sperm is selected and directly injected into an egg using a thin glass tube under a microscope to fertilize it, and the resulting embryo, cultured outside the body, is then returned to the uterus. In vitro gamete formation is the process of producing germ cells such as sperm or eggs through in vitro culture, and is expected to be applied to infertility treatments caused by gamete deletion, etc. This invention can be applied without limitation to assisted reproductive technologies that include processes of manipulating eggs, sperm, or fertilized eggs outside the body. 【0025】 In this specification, "mother who is the subject of assisted reproductive technology" refers to a woman who will be the biological mother of a child born through assisted reproductive technology. In other words, "mother who is the subject of assisted reproductive technology" includes women who receive sperm injection into the uterus in artificial insemination, women who donate eggs in in vitro fertilization, women who donate eggs in intracytoplasmic sperm injection, and women who provide cells that will become egg cells in in vitro gamete formation. 【0026】 In this specification, "father subject to assisted reproductive technology" refers to a man who will be the biological father of a child born through assisted reproductive technology. In other words, "father subject to assisted reproductive technology" includes men who donate sperm in artificial insemination, men who donate sperm in in vitro fertilization, men who donate sperm in intracytoplasmic sperm injection, and men who donate cells that will become sperm in in vitro gamete formation. 【0027】In this specification, "a woman who became pregnant through assisted reproductive technology" may be the same person as "a mother who is the subject of assisted reproductive technology." In other words, "a woman who became pregnant through assisted reproductive technology" may be "a mother who became pregnant through assisted reproductive technology." 【0028】 If the "woman who became pregnant through assisted reproductive technology" is the same person as the "mother who is the subject of assisted reproductive technology," the present invention may be an embodiment in which the genotypes of (A) and (B) are compared with (C), or an embodiment in which the genotypes of (B) and (C) are compared. When the embodiment is in which the genotypes of (A) and (B) and (C) are compared, there are no particular restrictions on the timing of the analysis. Even when the embodiment is in which the genotypes of (B) and (C) are compared, there are no particular restrictions on the timing of the analysis, however, from the viewpoint of ease of distinguishing between nucleic acids derived from the woman who is pregnant with the fetus and nucleic acids derived from the fetus in the circulating cell-free nucleic acid sample, the woman who became pregnant through assisted reproductive technology (the mother who is the subject of assisted reproductive technology) is preferably 24 weeks pregnant or less, more preferably 20 weeks pregnant or less, and more preferably 18 weeks pregnant or less. 【0029】 Furthermore, in this specification, "mother who is the subject of assisted reproductive technology" and "woman who became pregnant through assisted reproductive technology" may be different people (so-called host mother). If "mother who is the subject of assisted reproductive technology" and "woman who became pregnant through assisted reproductive technology" are different people, then "woman who became pregnant through assisted reproductive technology" includes women who are the subject of in vitro fertilization or intracytoplasmic sperm injection, where the egg of "mother who is the subject of assisted reproductive technology" is fertilized with the sperm of "father who is the subject of assisted reproductive technology," and the resulting embryo, cultured in vitro, is returned to the uterus; or women who are the subject of in vitro fertilization, where the egg of "mother who is the subject of assisted reproductive technology" or an egg produced by in vitro gamete formation using the "mother who is the subject of assisted reproductive technology" or cells of "mother who is the subject of assisted reproductive technology" is fertilized with the sperm of "father who is the subject of assisted reproductive technology" or sperm produced by in vitro gamete formation using the "father who is the subject of assisted reproductive technology" or cells of "father who is the subject of assisted reproductive technology," and the resulting embryo, cultured in vitro, is returned to the uterus. 【0030】If the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people, the genetic analysis method of the present invention is more preferably one of the following forms: a form that includes comparing the genotypes of (A) and (C), a form that includes comparing the genotypes of (B) and (C), a form that includes comparing the genotypes of (A), (B), and (C), or a form that includes comparing the genotypes of (A), (B), (C), and the following (D). (D) Genotypes at multiple polymorphic loci of a woman who became pregnant through assisted reproductive technology. 【0031】 Furthermore, in the embodiment where the genotypes of (A), (B), (C), and (D) are compared, there are no particular restrictions on the timing of the analysis. Even in the embodiment where the genotypes of (A), (B), and (C) are compared, there are no particular restrictions, however, from the viewpoint of ease of distinguishing between nucleic acids derived from a woman pregnant with a fetus and nucleic acids derived from the fetus in a circulating cell-free nucleic acid sample, the woman who became pregnant through assisted reproductive technology is preferably 24 weeks pregnant or less, more preferably 20 weeks pregnant or less, and more preferably 18 weeks pregnant or less. 【0032】 The method for determining the genotype is not particularly limited and includes methods such as nucleotide sequence analysis, mass spectrometry, digital PCR, SNV microarrays, and real-time PCR. A specific method for nucleotide sequence analysis is next-generation sequencing (NGS). Furthermore, the polymorphic loci to be analyzed are not particularly limited, but single nucleotide polymorphisms (SNPs) are an example. Here, SNPs may be tetra-allelic SNPs (SNPs) where all four types of bases are observed, tri-allelic SNPs (SNPs) where three types of bases are observed, or biallelic SNPs (SNPs) where two types of bases are observed. This specification will describe biallelic SNPs (SNPs) in detail. The following embodiments are merely examples, and the single nucleotide polymorphism loci are not limited to biallelic SNPs (SNPs) in which two types of bases are observed. For example, STRs (Short Tandem Repeats) or polymorphism loci used for personal identification may also be used. 【0033】There are no particular restrictions on the method for determining the genotype of the mother (A), the father (B), and the woman (D). Examples of methods for determining genotypes include analyzing the genomic DNA of oral mucosal cells collected from the oral cavity of the mother, father, or woman. Naturally, there are no particular restrictions on the site from which cells or samples are collected to obtain the genomic DNA. 【0034】 First, we will detail the genotyping of "mothers undergoing assisted reproductive technology (ART)," "fathers undergoing ART," and "women who have become pregnant through ART" using genomic DNA. For example, among two distinct alleles at a specific polymorphic locus, we will denote the allele with a relatively high signal intensity in the analysis of the genomic DNA sample as A, and the allele with a relatively low signal intensity as B. In this case, genotyping can be performed using the ratio (Fb) of the signal intensity indicating the presence of B to the sum of the signal intensities at the specific polymorphic locus as an indicator, according to the following criteria: Homozygous for AA: Fb ≤ default value 1 Heterozygous for AB: default value 2 ≤ Fb ≤ 50% Here, default values ​​1 and 2 can be appropriately set within the range that satisfies the following equation: 0% ≤ default value 1 ≤ default value 2 < 50% 【0035】 Based on the above, by collecting genomic DNA samples from a mother undergoing assisted reproductive technology (ART), a father undergoing ART, or a woman who became pregnant through ART, and using Fb as an indicator, the genotype of the mother undergoing ART, the father undergoing ART, or the woman who became pregnant through ART can be determined, for example, as shown in Table 1 below. 【0036】 【0037】Furthermore, if the "woman who became pregnant through assisted reproductive technology" is the same person as the "mother who was the subject of assisted reproductive technology," the mother's genotype in (A) can also be determined by indirect maternal genotyping based on a circulating cell-free nucleic acid sample. For example, among two distinct alleles at a particular polymorphic locus, the allele with a relatively high signal intensity in the analysis of the circulating cell-free nucleic acid sample is denoted as A, and the allele with a relatively low signal intensity is denoted as B. Maternal genotyping can be performed using the ratio (Fb) of the signal intensity indicating the presence of B to the sum of the signal intensities at the specific polymorphic locus, as an indicator, according to the following criteria: Homozygous for AA: Fb ≤ specified value 3 Heterozygous for AB: Specified value 4 ≤ Fb ≤ 50% Here, specified values ​​3 and 4 can be appropriately set within the range that satisfies the following equation: 0% ≤ specified value 3 ≤ specified value 4 < 50% 【0038】 Furthermore, the method of indirect maternal genotyping is not limited to the method described above. Any method that utilizes the fact that maternal nucleic acids are generally present in a larger proportion of fetal nucleic acids in a circulating cell-free nucleic acid sample to perform indirect maternal genotyping is included within the scope of the present invention. 【0039】 Furthermore, the polymorphic seating used for genotyping may be limited to polymorphic seating that meets certain conditions (for example, publication WO-A1-2022 / 131328). 【0040】 Furthermore, even when the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people, the "genotype of the woman who became pregnant through assisted reproductive technology" can be determined by indirect female genotyping, using the explanation for the case where the "woman who became pregnant through assisted reproductive technology" is the same person as the "mother who is the subject of assisted reproductive technology." 【0041】The method for identifying the genotype at the polymorphic locus of the fetus (genotyping) in (C) circulating cell-free nucleic acid is not particularly limited. The circulating cell-free nucleic acid sample contains nucleic acids from a woman who is pregnant with the fetus and nucleic acids from the fetus. Therefore, when a circulating cell-free nucleic acid sample taken from a pregnant woman in the early stages of pregnancy is analyzed and the ratio of signals indicating the presence of a specific allele to the sum of signal intensities derived from a specific polymorphic locus is bloted, more than 80% of the signals will be distributed in one of the following ranges: 0-25%, 35-65%, or 75-100%. It may be assumed that there is no contamination from other people's DNA from the outside. Here, when performing fetal genotyping based on the circulating cell-free nucleic acid sample, first the genotype of the woman who is pregnant with the fetus is identified by genotyping the woman. Then, fetal genotyping can be performed by subtracting the information of the woman's genotype from the data obtained from the analysis of the circulating cell-free nucleic acid sample. Furthermore, the method for identifying the genotype in circulating cell-free nucleic acid or fetal polymorphic loci in (C) can be used in the same way even if the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people, or if the "genotype of the woman who became pregnant through assisted reproductive technology" is the same person as the "mother who is the subject of assisted reproductive technology". 【0042】 For example, among two distinct alleles at a particular polymorphic locus, the allele with a relatively high signal intensity in the analysis of a circulating cell-free nucleic acid sample will be denoted as A, and the allele with a relatively low signal intensity will be denoted as B. Genotyping of circulating cell-free nucleic acid or fetuses can be performed using the ratio (Fb) of the signal intensity indicating the presence of B to the sum of the signal intensities at the specific polymorphic locus, as an indicator, according to the following criteria: Homozygous for AA: Fb ≤ Normal value 5 Heterozygous for AB: Normal value 6 ≤ Fb ≤ Normal value 7 Here, the normal values ​​5 to 7 can be appropriately set within the range that satisfies the following equation: 0% ≤ Normal value 5 ≤ Normal value 6 ≤ Normal value 7 ≤ 50% 【0043】Based on the above-described content, a cell-free circulating nucleic acid sample can be collected from a woman who became pregnant through assisted reproductive medicine, and using Fb as an index, for example, as shown in Table 2 below, the genotype of the cell-free circulating nucleic acid and the genotype of the woman who became pregnant through assisted reproductive medicine can be determined. In the present specification, when the genotype of the cell-free circulating nucleic acid is heterozygous (described as Ab in Table 2), it means that at the same polymorphic locus, the genotype of the woman who became pregnant through assisted reproductive medicine (when the "woman who became pregnant through assisted reproductive medicine" is the same person as the "mother who is the subject of assisted reproductive medicine", the "mother who is the subject of assisted reproductive medicine"; when the "woman who became pregnant through assisted reproductive medicine" is a different person from the "mother who is the subject of assisted reproductive medicine", the "woman who became pregnant through assisted reproductive medicine") is homozygous (described as AA in Table 2), and a valid minor allele b derived from a fetus of a different type from the woman or the mother (hereinafter, in all tables, the minor allele is denoted in lowercase) is detected (if necessary, the case of 1 ≤ Fb < 20 in Table 2, Ab in Tables 2 to 17). Note that in Table 2, the "mother who is the subject of assisted reproductive medicine" and the "woman who became pregnant through assisted reproductive medicine" may be different persons, or the "mother who is the subject of assisted reproductive medicine" and the "woman who became pregnant through assisted reproductive medicine" may be the same person. 【0044】 【0045】 Also, attention may be paid to the difference in the DNA methylation profiles of the mother and the fetus, and a method of distinguishing and specifying the genotype of the fetus from that of the mother may be adopted (for example, Japanese Patent Application Laid-Open No. 2012-502631, etc.). 【0046】 There is no limitation as long as the number of polymorphic loci to be analyzed is plural. For example, 10 or more, or 100 or more, or 1000 or more can be exemplified. 【0047】In a preferred embodiment of the present invention, it includes analyzing the existence or non-existence of a biological parent-child relationship between a mother and / or father who are the subjects of assisted reproductive medicine and a fetus. If there is no mix-up of samples of eggs, sperm, or fertilized eggs during the process of assisted reproductive medicine, the biological parent-child relationship between the mother and / or father and the fetus should be affirmed. In other words, if the biological parent-child relationship between the mother and / or father and the fetus is negated, it can be determined that there is a high possibility of a sample mix-up during the process of assisted reproductive medicine. 【0048】 When analyzing the existence or non-existence of a biological parent-child relationship between a mother and / or father who are the subjects of assisted reproductive medicine and a fetus, it may also be an embodiment to analyze the existence or non-existence of a biological parent-child relationship between either the mother or the father, respectively regarded as a putative mother or a putative father, and the fetus. 【0049】 Also, when analyzing the existence or non-existence of a biological parent-child relationship between a mother and / or father who are the subjects of assisted reproductive medicine and a fetus, it may also be an embodiment to analyze the existence or non-existence of a biological parent-child relationship between the mother and / or father and the fetus, regarding the fetus as a putative child. 【0050】 In this specification, specific embodiments of comparing the genotypes of (A) and / or (B) with (C) will be described. Also, in this specification, embodiments of comparing the genotypes of (A), (B), (C), and (D) will also be described. These two embodiments are respectively referred to as "mismatch identification" and "parentage testing". 【0051】 Hereinafter, "mismatch identification" and "parentage testing" will be described in detail using tables. In this specification, in "mismatch identification" and "parentage testing", when "the mother who is the subject of assisted reproductive medicine" and "the woman who became pregnant through assisted reproductive medicine" are the same person, polymorphic loci that are homozygous at least in the mother will be described in detail. Also, when "the mother who is the subject of assisted reproductive medicine" and "the woman who became pregnant through assisted reproductive medicine" are different persons, polymorphic loci that are homozygous at least in the woman will be described in detail. 【0052】<Mismatch Analysis> This embodiment includes detecting mismatch genotypes in circulating cell-free nucleic acids or the polymorphic locus of the fetus that do not appear when the mother and father are the biological parents of the fetus. In this embodiment, the "mother who is the subject of assisted reproductive technology" may be the same person as the "woman who became pregnant through assisted reproductive technology," or the "mother who is the subject of assisted reproductive technology" may be a different person from the "woman who became pregnant through assisted reproductive technology." 【0053】 First, using Tables 3 and 4, we will detail a preferred embodiment in which the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are the same person. 【0054】 Table 3 describes a mismatch test comparing the genotype of the father undergoing assisted reproductive technology (ART) with the genotype of the circulating cell-free nucleic acid (CRV) sample when the mother undergoing ART and the woman who became pregnant through ART are the same person. Table 3 shows the patterns of the genotype of the father undergoing ART and the circulating cell-free nucleic acid sample for polymorphic loci estimated to be homozygous for AA in the mother through indirect genotyping based on analysis of the circulating cell-free nucleic acid sample. Note that the cases that should be judged as mismatched genotypes shown in Table 3 are just examples. 【0055】 【0056】 Cases 1 and 2 show that the father's genotype at a certain polymorphic locus is homozygous for AA, the same as the mother's genotype. Of these, Case 2, in which the father's genotype at the same polymorphic locus is homozygous for AA, but the genotype of the circulating cell-free nucleic acid is heterozygous for Ab, represents a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. Cases 3 and 4 show that the father's genotype at a certain polymorphic locus is homozygous for BB, which is different from the mother's genotype. Case 3, in which the father's genotype at the same polymorphic locus is homozygous for BB, but the genotype of the circulating cell-free nucleic acid is homozygous for AA, which is different from the father's genotype, also represents a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. 【0057】 In a preferred embodiment of the present invention, the genotype at the same polymorphic locus of the circulating cell-free nucleic acid in cases 2 and / or 3 described above is evaluated as a mismatch genotype. This embodiment is applicable to any gestational age, but is preferably suitable for 4 weeks or more, and more preferably for 7 weeks or more. 【0058】 Next, Table 4 describes a mismatch test that compares the genotype of the mother undergoing assisted reproductive technology (ART) with the genotype of the father undergoing ART and the genotype of the circulating cell-free nucleic acid (CRV) sample when the mother undergoing ART and the woman who became pregnant through ART are the same person. Table 4 shows the patterns of the genotypes of the father undergoing ART and the circulating cell-free nucleic acid in cases 1 to 6 where the mother undergoing ART is homozygous for AA. Note that the cases to be determined as mismatched genotypes shown in Table 4 are just examples. Furthermore, the genotype of the mother undergoing ART in Table 4 may be determined either by genotyping based on the mother's genomic DNA or by indirect genotyping through the analysis of the circulating cell-free nucleic acid sample described above. 【0059】 【0060】 Note that while Table 4 limits the polymorphic loci to those where the mother's genotype is AA, the method for identifying the mother's genotype is not limited. It may be identified based on genomic DNA sampled from the mother's oral cells, for example. Furthermore, it is possible to identify polymorphic loci where the mother's genotype is homozygous for AA by analyzing circulating cell-free nucleic acids. 【0061】Cases 1 to 6 show that the mother's genotype is homozygous at a certain polymorphic locus. Of these, Case 2, in which the father's genotype is homozygous with the mother's at the same polymorphic locus, yet the genotype of the circulating cell-free nucleic acid is heterozygous, represents a fetal genotype that would not be conceivable if the mother and / or father were the biological parents of the fetus. Similarly, Case 3, in which the father's genotype is homozygous with the mother's at the same polymorphic locus, yet the genotype of the circulating cell-free nucleic acid is homozygous with the mother's, also represents a fetal genotype that would not be conceivable if the mother and / or father were the biological parents of the fetus. 【0062】 In a preferred embodiment of the present invention, the genotype at the same polymorphic locus of the circulating cell-free nucleic acid in cases 2 and / or 3 described above is evaluated as a mismatch genotype. This embodiment is applicable to any gestational age, but is preferably suitable for 4 weeks or more, and more preferably for 7 weeks or more. 【0063】 The present invention may also be an embodiment in which, when the "mother who is the subject of assisted reproductive technology" and the "woman who has become pregnant through assisted reproductive technology" are the same person, a comparison of the genotypes (A) to (C) above is performed for polymorphic loci that are homozygous in the mother, and the mismatch genotype is detected. 【0064】 Furthermore, in cases where the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are the same person, an embodiment may be provided in which the genotypes of (B) and (C) above are compared for polymorphic loci that are homozygous in the mother, and the mismatch genotype is detected. As mentioned in the explanation of Table 4, the "genotype of the mother who is the subject of assisted reproductive technology" may be determined either by genotyping based on the mother's genomic DNA or by indirect genotyping by analysis of the circulating cell-free nucleic acid sample described above. 【0065】Next, using Tables 5 to 8, we will describe in detail a preferred embodiment in the case where the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people. 【0066】 Table 5 describes a mismatch test that compares the genotype of the father undergoing assisted reproductive technology (ART) with the genotype of the circulating cell-free nucleic acid (CNU) sample when the mother undergoing ART is a different person from the woman who became pregnant through ART. Table 5 shows the patterns of CNU genotypes in cases 1-4 where the father undergoing ART is homozygous for the same AA genotype in the aforementioned woman, based on polymorphic loci estimated to be homozygous for AA by indirect genotyping through analysis of the CNU sample. Note that the cases that should be judged as mismatched genotypes shown in Table 5 are just examples. 【0067】 【0068】 Cases 1 to 4 show that the father's genotype is homozygous at a certain polymorphic locus. Of these, Case 3, in which the father's genotype is homozygous at the same polymorphic locus, but the genotype of the circulating cell-free nucleic acid is homozygous for a different type from the father, represents a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. 【0069】 In a preferred embodiment of the present invention, the genotype at the same polymorphic locus of the circulating cell-free nucleic acid in Case 3 described above is evaluated as a mismatch genotype. This embodiment is applicable to any gestational age, but is preferably suitable for 4 weeks or more, and more preferably for 7 weeks or more. 【0070】Next, in Table 6, we will explain a mismatch test that compares the genotype of the mother undergoing assisted reproductive technology (ART) with the genotype of the circulating cell-free nucleic acid (CNU) sample when the mother undergoing ART is a different person from the mother undergoing ART. Table 6 shows the patterns of the circulating cell-free nucleic acid genotype in cases 1 to 4, where the mother undergoing ART is homozygous for the same AA genotype at a polymorphic locus estimated to be homozygous for AA in the aforementioned woman by indirect genotyping through analysis of the circulating cell-free nucleic acid sample. Note that the cases to be determined as mismatched genotypes shown in Table 6 are just examples. 【0071】 【0072】 Cases 1 to 4 show that the mother's genotype is homozygous at a certain polymorphic locus. Of these, Case 3, in which the mother's genotype is homozygous at the same polymorphic locus, but the genotype of the circulating cell-free nucleic acid is homozygous for a different type from the mother, represents a fetal genotype that would not be conceivable if the mother were the biological parent of the fetus. 【0073】 In a preferred embodiment of the present invention, the genotype at the same polymorphic locus of the circulating cell-free nucleic acid in Case 3 described above is evaluated as a mismatch genotype. This embodiment is applicable to any gestational age, but is preferably suitable for 4 weeks or more, and more preferably for 7 weeks or more. 【0074】 Next, Table 7 describes a mismatch test that compares the genotype of the mother undergoing assisted reproductive technology (ART), the genotype of the father undergoing ART, and the genotype of the circulating cell-free nucleic acid (CRV) in cases where the mother undergoing ART and the woman who became pregnant through ART are different people. Table 7 shows the patterns of the genotype of the mother undergoing ART, the genotype of the father undergoing ART, and the genotype of the circulating cell-free nucleic acid in the polymorphic locus estimated to be homozygous for AA in the aforementioned woman by indirect genotyping through analysis of the circulating cell-free nucleic acid sample. Note that the cases that should be judged as mismatched genotypes shown in Table 7 are just examples. 【0075】 【0076】Cases 2, 5, and 8, in which the female and the father are homozygous for different types at a certain polymorphic locus, yet the genotype of the circulating cell-free nucleic acid is homozygous for the female, indicate a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. Similarly, cases 4, 5, and 6, in which the female and the mother are homozygous for different types at a certain polymorphic locus, yet the genotype of the circulating cell-free nucleic acid is homozygous for the female, indicate a fetal genotype that would not be conceivable if the mother were the biological parent of the fetus. In other words, when the female's genotype is homozygous at a certain polymorphic locus, the mother's genotype at the same polymorphic locus is homozygous for the female, and the mother's genotype is homozygous for different types from the father's genotype, case 2, in which the genotype of the circulating cell-free nucleic acid is homozygous for the mother, indicates a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. Case 4, in which the genotype of the circulating cell-free nucleic acid is homozygous for the same type as the woman, even though the father's genotype is homozygous for the same type as the woman's at a certain polymorphic locus and the father's genotype is homozygous for a different type as the mother's at the same polymorphic locus, indicates a fetal genotype that would not be conceivable if the mother were the biological parent of the fetus. Furthermore, Case 5, in which the genotype of the circulating cell-free nucleic acid is homozygous for a different type as the mother, even though the genotype of the woman is homozygous for the same polymorphic locus and the mother's genotype is homozygous for the same type as the mother at the same polymorphic locus, indicates a fetal genotype that would not be conceivable if both the mother and father were the biological parents of the fetus. Furthermore, in case 6, when the female's genotype at a certain polymorphic locus is homozygous, the mother's genotype at the same polymorphic locus is homozygous for a different type from the female, and the genotype of the circulating cell-free nucleic acid is homozygous for a different type from the mother, the fetus's genotype is one that would not be conceivable if the mother were the biological parent of the fetus.Case 8, in which the female genotype at a certain polymorphic locus is homozygous, and the mother's genotype at the same polymorphic locus is heterozygous, and the father's genotype at the same polymorphic locus is homozygous for a different type from the female, yet the genotype of the circulating cell-free nucleic acid is homozygous for a different type from the father, represents a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. 【0077】 When the female's genotype at a certain polymorphic locus is homozygous, the case 10 in which the circulating cell-free nucleic acid genotype is heterozygous, even though the maternal and paternal genotypes at the same polymorphic locus are homozygous with the female's, indicates a fetal genotype that would not be conceivable if the maternal and / or paternal were the biological parents of the fetus. 【0078】 In a preferred embodiment of the present invention, the genotype at the same polymorphic locus of circulating cell-free nucleic acid in one or more cases selected from Cases 2, 4, 5, 6, 8, and 10 described above is evaluated as a mismatch genotype. Furthermore, this embodiment is applicable to any gestational week, but is preferably preferred for 4 weeks or more, and more preferably for 7 weeks or more. 【0079】Next, in Table 8, we will explain a mismatch test that compares the "genotype of the woman who became pregnant through assisted reproductive technology" with the "genotype of the mother who became pregnant through assisted reproductive technology" and the "genotype of the father who became pregnant through assisted reproductive technology" when the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people. Table 8 shows the patterns of the genotype of the woman who became pregnant through assisted reproductive technology, the genotype of the mother who became pregnant through assisted reproductive technology, the genotype of the father who became pregnant through assisted reproductive technology, and the genotype of the circulating cell-free nucleic acid in cases 1 to 18 where the woman who became pregnant through assisted reproductive technology is homozygous for AA. Note that the cases that should be judged as mismatched genotypes shown in Table 8 are just examples. In addition, the "genotype of the woman who became pregnant through assisted reproductive technology" in Table 8 may be either the genotype identified by genotyping based on the woman's genomic DNA or the genotype identified by indirect genotyping by analysis of the circulating cell-free nucleic acid sample described above. 【0080】 【0081】 In Table 8, we limit the polymorphism loci to those where the genotype of a woman who became pregnant through assisted reproductive technology is AA. However, the method for identifying the woman's genotype is not limited. It may be identified based on genomic DNA sampled from the woman's oral cells, for example. Furthermore, it is possible to identify polymorphism loci where the woman's genotype is homozygous for AA by analyzing circulating cell-free nucleic acids. 【0082】Cases 2, 5, and 8, in which the female and the father are homozygous for different types at a certain polymorphic locus, yet the genotype of the circulating cell-free nucleic acid is homozygous for the female, indicate a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. Similarly, cases 4, 5, and 6, in which the female and the mother are homozygous for different types at a certain polymorphic locus, yet the genotype of the circulating cell-free nucleic acid is homozygous for the female, indicate a fetal genotype that would not be conceivable if the mother were the biological parent of the fetus. In other words, when the female's genotype is homozygous at a certain polymorphic locus, the mother's genotype at the same polymorphic locus is homozygous for the female, and the mother's genotype is homozygous for different types from the father's genotype, case 2, in which the genotype of the circulating cell-free nucleic acid is homozygous for the mother, indicates a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. Case 4, in which the genotype of the circulating cell-free nucleic acid is homozygous for the same type as the woman, even though the father's genotype is homozygous for the same type as the woman's at a certain polymorphic locus and the father's genotype is homozygous for a different type as the mother's at the same polymorphic locus, indicates a fetal genotype that would not be conceivable if the mother were the biological parent of the fetus. Furthermore, Case 5, in which the genotype of the circulating cell-free nucleic acid is homozygous for a different type as the mother, even though the genotype of the woman is homozygous for the same polymorphic locus and the mother's genotype is homozygous for the same type as the mother at the same polymorphic locus, indicates a fetal genotype that would not be conceivable if both the mother and father were the biological parents of the fetus. Furthermore, in case 6, when the female's genotype at a certain polymorphic locus is homozygous, the mother's genotype at the same polymorphic locus is homozygous for a different type from the female, and the genotype of the circulating cell-free nucleic acid is homozygous for a different type from the mother, the fetus's genotype is one that would not be conceivable if the mother were the biological parent of the fetus.Case 8, in which the female genotype at a certain polymorphic locus is homozygous, and the mother's genotype at the same polymorphic locus is heterozygous, and the father's genotype at the same polymorphic locus is homozygous for a different type from the female, yet the genotype of the circulating cell-free nucleic acid is homozygous for a different type from the father, represents a fetal genotype that would not be conceivable if the father were the biological parent of the fetus. 【0083】 When the female's genotype at a certain polymorphic locus is homozygous, the case 10 in which the circulating cell-free nucleic acid genotype is heterozygous, even though the maternal and paternal genotypes at the same polymorphic locus are homozygous with the female's, indicates a fetal genotype that would not be conceivable if the maternal and / or paternal were the biological parents of the fetus. 【0084】 In a preferred embodiment of the present invention, the genotype at the same polymorphic locus of circulating cell-free nucleic acid in one or more cases selected from Cases 2, 4, 5, 6, 8, and 10 described above is evaluated as a mismatch genotype. This embodiment is applicable to any gestational age, but is preferably suitable for 4 weeks or more, and more preferably for 7 weeks or more. 【0085】 The present invention may also be an embodiment in which, in cases where "a mother who is the subject of assisted reproductive technology" and "a woman who has become pregnant through assisted reproductive technology" are different people, the genotypes of (A) and (C) above are compared for polymorphic loci that are homozygous in at least the woman, and the mismatch genotype is detected. 【0086】 The present invention may also be an embodiment in which, in cases where "a mother who is the subject of assisted reproductive technology" and "a woman who has become pregnant through assisted reproductive technology" are different people, the genotypes of (B) and (C) above are compared for polymorphic loci that are homozygous in at least the woman, and the mismatch genotype is detected. 【0087】The present invention may also be an embodiment in which, in cases where "a mother who is the subject of assisted reproductive technology" and "a woman who has become pregnant through assisted reproductive technology" are different people, the genotypes of (A), (B), and (C) above are compared for polymorphic loci that are homozygous in at least the woman, and the mismatch genotype is detected. 【0088】 The present invention may also be an embodiment in which, when the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people, the genotypes of (A), (B), (C), and (D) above are compared for polymorphic loci that are homozygous in at least the woman, and the mismatch genotype is detected. As mentioned in the explanation of Table 8, the "genotype of the woman who became pregnant through assisted reproductive technology" may be determined either by genotyping based on the woman's genomic DNA or by indirect genotyping by analysis of the circulating cell-free nucleic acid sample described above. 【0089】 One embodiment of the present invention includes calculating a quantitative variable for polymorphic loci in which mismatched genotypes are detected. The quantitative variable to be calculated is not particularly limited as long as it directly or indirectly reflects the amount of polymorphic loci in which mismatched genotypes are detected. For example, the number of polymorphic loci in which mismatched genotypes are detected may be calculated as the quantitative variable. Alternatively, the proportion of polymorphic loci in which mismatched genotypes are detected out of the total number of polymorphic loci analyzed may be calculated as the quantitative variable. 【0090】 The quantitative variables of polymorphic loci in which mismatched genotypes are detected can be used as indicators to determine whether or not a sample mix-up has occurred in assisted reproductive technology. Specifically, an embodiment may include determining that a sample mix-up has occurred in assisted reproductive technology if the quantitative variable is above a predetermined reference value. The predetermined reference value can be set arbitrarily and is not particularly limited. 【0091】Furthermore, the calculation of quantitative variables for polymorphic loci where mismatched genotypes are detected is not particularly limited, regardless of whether the "mother undergoing assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are the same person, or whether the "mother undergoing assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people. 【0092】 <Paternity Testing> This embodiment includes conducting paternity testing between the mother and / or father who are subjects of assisted reproductive technology and the fetus. If the paternity test denies a biological parent-child relationship between the mother and / or father and the fetus, it can be determined that there is a high probability that a mix-up of egg, sperm, or embryo samples occurred during the process of assisted reproductive technology. 【0093】 The method of performing paternity testing is not particularly limited. In paternity testing, the terms "pseudo-father," "pseudo-mother," and "pseudo-child" are used to refer to hypothetical roles established to verify biological and genetic relationships. A pseudo-father refers to a person assumed to be the father, and paternity testing verifies the possibility that this person is the biological father of the child. A pseudo-mother refers to a person assumed to be the mother, and similarly, it verifies whether this person is the biological mother. A pseudo-child refers to a person assumed to be the child, and in this case, it verifies whether this person is the child of a particular parent. In the paternity testing of this invention, verification may be performed in any of the following patterns. 【0094】 - Analyze the existence of a biological parent-child relationship between the fetus produced by assisted reproductive technology and the fetus, treating the mother or father who is the subject of assisted reproductive technology as a pseudo-mother or pseudo-father, respectively. - Analyze the existence of a biological parent-child relationship between the fetus produced by assisted reproductive technology and the mother and / or father who are the subject of assisted reproductive technology, treating the fetus as a pseudo-child. 【0095】In the following explanation, where "pseudo-father" is simply written, it indicates a method of verifying whether the father, who is the subject of assisted reproductive technology (ART), is the biological father of the fetus, assuming the mother is the biological mother of the fetus. Similarly, where "pseudo-mother" is simply written, it indicates a method of verifying whether the mother, who is the subject of ART, is the biological mother of the fetus, assuming the father is the biological father of the fetus. 【0096】 Paternity testing can be performed using any method. For example, the existing NIPPT (Non-Invasive Prenatal Paternity Test) can be applied to perform biological paternity testing between the father and the fetus. Known NIPPT methods include single hypothesis rejection tests, maximum likelihood estimates, or maximum posterior probability methods to determine the probability of a biological parent-child relationship (e.g., Japanese Patent Publication No. 2014-502845), and these can be applied without restriction. Furthermore, even if only paternity testing is performed and a blood relationship between the father and the fetus is confirmed, the mother may still be recognized as the biological parent. Alternatively, even if only maternal testing is performed and a blood relationship between the mother and the fetus is confirmed, the father may still be recognized as the biological parent. 【0097】If the "woman who became pregnant through assisted reproductive technology" and the "mother who is the subject of assisted reproductive technology" are the same person, or if the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people, the paternity test may be implemented in which the existence of a biological parent-child relationship between the father and the fetus is determined by comparing two types of genotypes: the father and the circulating cell-free nucleic acid or the fetus. If the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people, the paternity test may be implemented in which the existence of a biological parent-child relationship between the mother and the fetus is determined by comparing two types of genotypes: the mother and the circulating cell-free nucleic acid or the fetus. Furthermore, if the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are the same person, or if the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people, the paternity test may be implemented in which the existence of a biological parent-child relationship between the mother and / or the father and the fetus is determined by comparing three types of genotypes: the mother, the father, and the circulating cell-free nucleic acid or the fetus. And if the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people, the paternity test may be implemented in which the existence of a biological parent-child relationship between the mother and / or the father and the fetus is determined by comparing four types of genotypes: the woman, the mother, the father, and the circulating cell-free nucleic acid or the fetus. 【0098】 By calculating a Paternity Index (PI) for each of several single nucleotide polymorphism loci and multiplying the PIs together to obtain a Combined Paternity Index (CPI), it is also possible to determine whether or not a parent-child relationship exists between the suspected father and the fetus. 【0099】 By calculating a Maternity Index (MI) for each of the multiple single nucleotide polymorphism loci and multiplying the MIs together to obtain a Combined Maternity Index (CMI), it is also possible to determine whether or not a parent-child relationship exists between the suspected mother and the fetus. 【0100】The existence or non-existence of a parentage relationship can be determined by calculating the CMI or CPI, or by calculating both the CMI and CPI. In other words, one form of determining the existence or non-existence of a parentage relationship is to determine that if CPI and / or CMI < A, then biological parentage is denied; if A < CPI and / or CMI < B, then biological parentage is undeterminable; and if B < CPI and / or CMI, then biological parentage is affirmed. Here, the specific values ​​of A and B can be set appropriately within the range that satisfies the relationship A < B. 【0101】 The value of A can be set to any value, preferably 0.1 or less, more preferably 0.0001 or less, and more preferably 0.000001 or less. The value of B can be set to any value, preferably 100 or more, more preferably 10000 or more, and more preferably 1000000 or more. 【0102】 The following describes in detail four embodiments for calculating PI or MI, with reference to Tables 9 to 17. In Tables 9 to 17, k0 is the false negative rate, kb is the false positive rate, a is the frequency of occurrence of A, and b is the frequency of occurrence of B. 【0103】 The value of kb can be set to any value, preferably 0.1 or less, more preferably 0.0001 or less, and more preferably 0.000001 or less. Similarly, the value of k0 can be set to any value, preferably 0.1 or less, more preferably 0.0001 or less, and more preferably 0.000001 or less. 【0104】 <Two-person examination (1)> In this embodiment, the "woman who became pregnant through assisted reproductive technology" and the "mother who is the subject of assisted reproductive technology" are the same person, and the PI is calculated according to Table 9 based on the pseudo-paternal genotype and the genotype of the circulating cell-free nucleic acid at polymorphic loci that are estimated to be homozygous for AA in the mother by indirect genotyping through analysis of the circulating cell-free nucleic acid sample. 【0105】 【0106】 <Two-Person Test (2)> In this embodiment, the "woman who became pregnant through assisted reproductive technology" and the "mother who is the subject of assisted reproductive technology" are different people. Based on the genotype of the suspected father and the genotype of the circulating cell-free nucleic acid at the polymorphic locus estimated to be homozygous for AA in the woman by indirect genotyping through analysis of the circulating cell-free nucleic acid sample, the PI is calculated according to Table 10. 【0107】 【0108】 <Two-Person Test (3)> In this embodiment, the "woman who became pregnant through assisted reproductive technology" and the "mother who is the subject of assisted reproductive technology" are different people. MI is calculated according to Table 11 based on the genotype of the pseudo-mother and the genotype of the circulating cell-free nucleic acid at the polymorphic locus estimated to be homozygous for AA in the woman by indirect genotyping through analysis of the circulating cell-free nucleic acid sample. 【0109】 【0110】 <3-Person Test (1)> In this embodiment, the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are the same person, and the PI is calculated according to Table 12 based on the genotypes of the mother, the suspected father, and the circulating cell-free nucleic acid. Note that the genotype of the mother who is the subject of assisted reproductive technology in Table 12 may be a genotype identified by genotyping based on the mother's genomic DNA, or a genotype identified by indirect genotyping by analysis of the circulating cell-free nucleic acid sample. 【0111】 【0112】 <3-person examination (2)> In this embodiment, the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are the same person, and MI is calculated according to Table 13 based on the genotypes of the pseudomother, the father, and circulating cell-free nucleic acid. 【0113】 【0114】<3-Person Test (3)> In this embodiment, the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people. Based on the genotypes of the mother, the suspected father, and the circulating cellular nucleic acid at the polymorphic locus estimated to be homozygous for AA in the woman by indirect genotyping through analysis of the circulating cellular nucleic acid sample, the PI is calculated according to Table 14. 【0115】 【0116】 <3-Person Test (4)> In this embodiment, the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people. Based on the genotypes of the pseudomother, the father, and the circulating cellular nucleic acid at the polymorphic locus estimated to be homozygous for AA in the woman by indirect genotyping through analysis of the circulating cellular nucleic acid sample, MI is calculated according to Table 15. 【0117】 【0118】 While there are no particular restrictions on the timing of analysis in the three-person test, from the viewpoint of ease of distinguishing between nucleic acids derived from women pregnant with a fetus and nucleic acids derived from the fetus in the circulating cell-free nucleic acid sample, it is preferable to analyze at 24 weeks of gestation or earlier, more preferably at 20 weeks of gestation or earlier, and more preferably at 18 weeks of gestation or earlier. 【0119】 <Four-Person Test (1)> In this embodiment, the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people. Based on the genotypes of the woman, the mother, the suspected father, and the circulating cell-free nucleic acid, the PI is calculated according to Table 16. The woman's genotype may be a genotype identified by genotyping based on the woman's genomic DNA, or it may be a genotype identified by indirect genotyping through analysis of the circulating cell-free nucleic acid sample. 【0120】 【0121】<Four-Person Test (2)> In this embodiment, the "mother who is the subject of assisted reproductive technology" and the "woman who became pregnant through assisted reproductive technology" are different people. MI is calculated according to Table 17 based on the genotypes of the woman, the pseudomother, the father, and the circulating cell-free nucleic acid. The genotype of the woman may be a genotype identified by genotyping based on the woman's genomic DNA, or a genotype identified by indirect genotyping through analysis of the circulating cell-free nucleic acid sample. 【0122】 【0123】 If the results of a paternity test deny the existence of a biological parent-child relationship between the mother and / or the father and the fetus, it can be determined that there was a mix-up of egg, sperm, or embryo samples at some point during the assisted reproductive technology process. 【0124】 The paternity test in this invention may produce not only two results, "affirmative" and "negative," regarding biological parentage, but also a result of "undeterminable." The determination of "affirmative," "negative," and "undeterminable" can be made using known methods of setting a determination threshold in the POM or POP as described above. The method of setting the determination threshold is not limited. 【0125】 <Analysis Flow> The specific analysis flow will be explained below with reference to Figures 1 to 4. 【0126】 The present invention may also be an embodiment in which, after genotyping to identify the genotypes of (A) the mother and / or (B) the father and (C) the circulating cell-free nucleic acid or fetus, only mismatch testing is performed to determine whether or not there was a mix-up of samples during the process of assisted reproductive technology (Figure 1(a)). 【0127】 Furthermore, the present invention may also be an embodiment in which, after genotyping to identify the genotypes of (A) the mother and / or (B) the father and (C) the circulating cell-free nucleic acid or fetus, only paternity testing is performed to determine whether or not there was a mix-up of samples during the process of assisted reproductive technology (Figure 1(b)). 【0128】Embodiments including paternity testing will be described in more detail. The present invention may also include embodiments that include a first-stage paternity test and a second-stage paternity test. An embodiment may be adopted in which a paternity test is performed as the first-stage paternity test and a maternal test as the second-stage paternity test (Figure 2(a)), or an embodiment may be adopted in which a maternal test is performed as the first-stage paternity test and a paternity test as the second-stage paternity test (Figure 2(b)). Furthermore, an embodiment including paternity testing may be an embodiment in which a maternal test and / or a paternity test are performed (Figure 2(c)). 【0129】 Regardless of the results of the first paternity test, a second paternity test may be conducted. Alternatively, conditions for conducting a second paternity test may be set based on the results of the first paternity test. 【0130】 Furthermore, paternity testing may consist of only a father-child test, or only a mother-child test, or both. 【0131】 For paternity testing, the existing NIPPT method may be used to determine the probability of a biological parent-child relationship existing, using a single hypothesis rejection test, maximum likelihood estimation, or maximum posterior probability method. Alternatively, one or more tests selected from the two-person tests (1), two-person tests (2), two-person tests (3), three-person tests (1), three-person tests (2), three-person tests (3), three-person tests (4), four-person tests (1), and four-person tests (2) detailed above may be performed. Furthermore, any combination of these may be included. 【0132】 For example, if biological paternity is confirmed in the first paternity test, a second paternity test may be conducted to re-examine whether or not there was a mix-up of samples during the assisted reproductive technology process, thus performing a double-check. In this embodiment, if biological paternity is denied in the first paternity test, the analysis may be terminated by determining that there is a high probability of a mix-up of samples during the assisted reproductive technology process, without conducting a second paternity test. 【0133】Furthermore, the embodiment may include both mismatch testing and paternity testing. The embodiment may also include performing paternity testing regardless of the results of the mismatch testing. Additionally, conditions for performing paternity testing may be set based on the results of the mismatch testing or the accuracy of the data obtained during the genotyping process. 【0134】 For example, quantitative variables are calculated for polymorphic loci where mismatched genotypes are detected, and / or quantitative variables reflecting the total DNA concentration and / or the concentration of circulating cell-free fetal nucleic acid in the circulating cell-free nucleic acid sample. 【0135】 The quantitative variables of polymorphic loci in which mismatched genotypes were detected are as described above. For the sake of clarity, in this specification, the quantitative variables of polymorphic loci in which mismatched genotypes were detected may also be referred to as mismatch quantitative variables. 【0136】 The total DNA concentration in a circulating cell-free nucleic acid sample refers to the concentration of all DNA contained in the sample, including maternal DNA and fetal DNA. Next, we will explain the quantitative variables that reflect the concentration of circulating cell-free fetal nucleic acid in a circulating cell-free nucleic acid sample. These quantitative variables may be the concentration of circulating cell-free fetal nucleic acid itself, or the ratio of circulating cell-free fetal nucleic acid to maternal nucleic acid. These can be easily calculated from the intensity of the signal indicating the presence of maternal alleles and the intensity of the signal indicating the presence of alleles contained in circulating cell-free fetal nucleic acid, which can be obtained when analyzing a circulating cell-free nucleic acid sample. For convenience of explanation, the quantitative variables that reflect the total DNA concentration and / or the concentration of circulating cell-free fetal nucleic acid in a circulating cell-free nucleic acid sample are sometimes called concentration-related quantitative variables. 【0137】 In this embodiment, a decision may be made whether or not to perform a paternity test between the mother and / or the father and the fetus, in accordance with predetermined rules, based on the results of comparing these mismatch quantitative variables and / or concentration-related quantitative variables with predetermined reference values ​​(Figure 3). 【0138】Specifically, the method may involve comparing a mismatch quantitative variable and / or a concentration-related quantitative variable with a predetermined reference value, and determining whether or not to perform a paternity test between the mother and / or the father and the fetus in accordance with Rule 1 (Figure 3(a)). (Rule 1) If the quantitative variable is less than the reference value, the paternity test is performed. If the quantitative variable is equal to or greater than the reference value, the analysis is terminated without performing the paternity test. 【0139】 Furthermore, an embodiment may be provided in which a mismatch quantitative variable and / or a concentration-related quantitative variable are compared with a predetermined reference value, and in accordance with Rule 2, a decision is made as to whether or not to perform a paternity test between the mother and / or the father and the fetus (Figure 3(b)). (Rule 2) If the quantitative variable is less than the reference value, the analysis is terminated without performing the paternity test, and if the quantitative variable is equal to or greater than the reference value, the paternity test is performed. 【0140】 Furthermore, depending on the concentration-related quantitative variable, the method may be configured to determine whether or not to perform a genetic analysis method according to predetermined criteria (Figure 4). Specifically, depending on the result of comparing the concentration-related quantitative variable with predetermined criteria, the method may be configured to determine whether or not to perform a mismatch test (Figure 4(a)) and / or a parentage test (Figure 4(b)) according to predetermined Rule 3. (Rule 3) If the quantitative variable is less than the criteria, the genetic analysis method is not performed and the process is terminated. If the quantitative variable is equal to or greater than the criteria, the genetic analysis method is performed. 【0141】 An embodiment may also be provided in which the rule 3 determination step shown in Figure 4 is inserted after the genotyping step in the flow shown in Figure 2 or Figure 3. 【0142】 This invention can be applied to techniques for determining whether or not egg, sperm, or embryo samples have been mixed up during the process of assisted reproductive technology.

Claims

1. A genetic analysis method comprising comparing the following genotypes (A) and / or (B) and (C): (A) Genotypes at multiple polymorphic loci of the mother undergoing assisted reproductive technology. (B) Genotypes at multiple polymorphic loci of the father undergoing assisted reproductive technology. (C) Genotypes at multiple polymorphic loci of the circulating cell-free nucleic acid or fetus, obtained by analyzing a circulating cell-free nucleic acid sample taken from a woman who became pregnant through assisted reproductive technology.

2. The genetic analysis method according to claim 1, comprising detecting a mismatch genotype in the circulating cell-free nucleic acid or the polymorphic locus of the fetus that does not appear when the mother and father are the biological parents of the fetus.

3. When the mother who is the subject of assisted reproductive technology and the woman who became pregnant through assisted reproductive technology are the same person, when the mother's genotype at a certain polymorphic locus is homozygous, when the father's genotype at the same polymorphic locus is homozygous with the mother's, but the genotype of the circulating cell-free nucleic acid is heterozygous, and / or when the father's genotype at the same polymorphic locus is homozygous with the mother's, but the genotype of the circulating cell-free nucleic acid is homozygous with the mother's, the genotype at the same polymorphic locus of the circulating cell-free nucleic acid is evaluated as a mismatch genotype. When the mother who is the subject of assisted reproductive technology and the woman who became pregnant through assisted reproductive technology are different people, when the woman's genotype at a certain polymorphic locus is homozygous, A genetic analysis method according to claim 2, wherein the genotype of the circulating cell-free nucleic acid is homozygous with the female despite the maternal and / or paternal genotypes at the same polymorphic locus being heterozygous with the female, and / or, when the female genotype at a certain polymorphic locus is homozygous, the genotype of the circulating cell-free nucleic acid at the same polymorphic locus is evaluated as a mismatch genotype in cases where the maternal and paternal genotypes at the same polymorphic locus are homozygous with the female, but the genotype of the circulating cell-free nucleic acid is heterozygous.

4. The genetic analysis method according to claim 3, wherein, if the mother who is the subject of the assisted reproductive technology described above and the woman who became pregnant through the assisted reproductive technology described above are the same person, the method includes comparing the genotypes of (A) to (C) or the genotypes of (B) and (C) for at least the homozygous polymorphic loci in the mother to detect the mismatch genotype; and if the mother who is the subject of the assisted reproductive technology described above and the woman who became pregnant through the assisted reproductive technology described above are different people, the method includes comparing the genotypes of (A) to (C) or the genotypes of (A) and (C) or the genotypes of (B) and (C) for at least the homozygous polymorphic loci in the woman to detect the mismatch genotype.

5. The genetic analysis method according to claim 2, comprising calculating a quantitative variable of the polymorphic locus in which the mismatched genotype was detected.

6. The genetic analysis method according to claim 5, further comprising determining that a sample mix-up occurred in the assisted reproductive technology if the quantitative variable is equal to or greater than a predetermined reference value.

7. The genetic analysis method according to claim 1, comprising performing a paternity test between the mother and / or the father and the fetus.

8. The genetic analysis method according to claim 7, wherein the paternity test includes analyzing whether a biological parent-child relationship exists between the fetus and either the mother or the father, with the mother or father designated as the pseudomother or pseudofather, or analyzing whether a biological parent-child relationship exists between the fetus and the mother and / or father, with the fetus designated as the pseudochild.

9. The genetic analysis method according to claim 7, wherein the paternity test includes determining whether or not there is a biological parent-child relationship between the father and the fetus by comparing two types of genotypes: the father and the circulating cell-free nucleic acid or the fetus, or determining whether or not there is a biological parent-child relationship between the mother and / or the father and the fetus by comparing three types of genotypes: the mother, the father and the fetus.

10. The genetic analysis method according to claim 7, which includes determining that a sample mix-up occurred in the assisted reproductive technology when the results of the paternity test negate the existence of a biological parent-child relationship between the mother and / or the father and the fetus.

11. A genetic analysis method according to claim 2, comprising calculating a quantitative variable of the polymorphic locus in which the mismatched genotype is detected, and / or a quantitative variable that reflects the total DNA concentration and / or the concentration of circulating cell-free fetal nucleic acid in the circulating cell-free nucleic acid sample, wherein, based on the results of comparing the quantitative variable with a predetermined reference value, it is determined whether or not to perform a paternity test between the mother and / or the father and the fetus, according to a predetermined rule.

12. The genetic analysis method according to claim 11, wherein the rule is rule 1 or rule 2 below. (Rule 1) If the quantitative variable is less than the standard value, the paternity test is performed; if the quantitative variable is equal to or greater than the standard value, the analysis is terminated without performing the paternity test. (Rule 2) If the quantitative variable is less than the standard value, the analysis is terminated without performing the paternity test; if the quantitative variable is equal to or greater than the standard value, the paternity test is performed.

13. A determination method comprising calculating a quantitative variable that reflects the total DNA concentration and / or the concentration of circulating cell-free fetal nucleic acid in the circulating cell-free nucleic acid sample, and determining whether or not to perform the genetic analysis method described in any one of claims 1 to 10, in accordance with a predetermined rule 3, based on the result of comparing the quantitative variable with a predetermined reference value. (Rule 3) If the quantitative variable is less than the reference value, the genetic analysis method is not performed and the process is terminated. If the quantitative variable is equal to or greater than the reference value, the genetic analysis method is performed.

14. If the mother who is the subject of the assisted reproductive technology described above and the woman who became pregnant through the assisted reproductive technology described above are different people, the genetic analysis method according to any one of claims 1 to 12, comprising comparing the genotypes of (A), (B), and (C), or (A), (B), (C), and (D) below. (D) Genotypes at multiple polymorphic loci of women who became pregnant through the aforementioned assisted reproductive technology.

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