Biomarker for determining pregnancy and method for determining using the same

Abstract

The present invention aims to provide a biomarker and a criterion for judging the same, which can judge a high-implantation / pregnancy-probability embryo, i.e., a high-fertility embryo, from an embryo obtained by embryo culture after in vitro fertilization in infertility treatment. A criterion is provided, by which the concentration of soluble CD163 present in follicular fluid collected at the same time as oocytes at the time of oocyte collection or in serum before oocyte collection in infertility treatment is measured, so that an oocyte suitable for implantation / pregnancy can be judged.

Description

Technical Field

[0001] This invention relates to a method for determining the fertility of an egg or embryo by using the concentration of soluble CD163 in human body fluids as an indicator. Background Technology

[0002] In 2017, the number of infertility treatment cycles in Japan exceeded 400,000, and the number of babies born exceeded 50,000. In the future, due to factors such as later marriage, the number of infertility treatments and babies born through in-vitro fertilization is expected to gradually increase (Non-Patent Literature 1).

[0003] Factors leading to infertility exist in both men and women. In men, these primarily include spermatogenesis disorders, obstruction of the vas deferens, and insufficient sperm motility. In women, besides aging, factors include gynecological diseases (endometriosis, uterine fibroids, etc.), endocrine disorders, and metabolic disorders. However, there are also cases where no abnormalities are observed in routine infertility examinations, and the cause remains unclear. When no obvious abnormalities are observed, it is generally believed that decreased egg quality leads to decreased fertility. Here, fertility refers to the ease of conception, the ability of an egg or the embryo formed after fertilization to complete the entire process of implantation and pregnancy. The quality of eggs obtained through egg retrieval cannot be determined; therefore, even with in vitro fertilization and transfer of good embryos obtained through embryo culture, the probability of implantation is only about 30% at most (Non-Patent Literature 1). To increase this implantation probability, multiple embryos obtained through in vitro fertilization are usually transferred; however, multiple pregnancies increase the physical burden, therefore, single embryo transfer, which in principle involves transferring only one embryo, has been disclosed (Non-Patent Literature 2).

[0004] Currently, embryo transfer includes split embryo transfer using split embryos and blastocyst transfer using embryos that have reached the blastocyst stage. As morphological methods for evaluating embryos during each transfer, the Veeck classification (Non-Patent Literature 3) and Gardner classification (Non-Patent Literature 4) are mainly used, respectively. Typically, after morphological evaluation of embryos obtained through in vitro fertilization, the embryo with the highest evaluation within the same cycle is transferred. However, even the highest-evaluated embryo may not necessarily achieve implantation / pregnancy, and vice versa; therefore, morphological embryo evaluation is not strictly correlated with actual embryo quality. In recent years, time-lapse culture has been developed to confirm the embryo's growth process. Methods have also been proposed that evaluate embryos based on their growth process rather than a final morphological judgment after culture (Non-Patent Literature 5), but without clear criteria for judgment. Therefore, to judge single embryo transfers with a high probability of implantation / pregnancy, biomarkers that can provide better objective judgment criteria by combining morphological evaluation are needed.

[0005] In recent years, there have been many reports on biomarker research for infertility treatment. Patent document 1 discloses MICA (MHC class I chain-associated protein A) as a biomarker for non-invasive evaluation of in vitro fertilization. Although it is proposed as a biomarker indicating insufficient implantation function, in vitro fertilization failure, or miscarriage when the MICA level in a human subject exceeds a threshold, it does not indicate the actual quality of the oocyte or embryo.

[0006] Patent Document 2 proposes soluble CD146 as a biomarker for pre-selecting embryos suitable for implantation in the uterus. A method for selecting suitable embryos for transfer using soluble CD146, which can be identified in embryo culture medium, is disclosed, but it does not involve human body fluids.

[0007] Patent document 3 discloses a genetic method for identifying oocytes capable of carrying a pregnancy. It discloses indicators that enable a viable pregnancy when the oocyte is transplanted by analyzing and evaluating gene clusters obtained from cumulus cells, but it does not conduct research at the protein level and does not use human body fluids as the subject.

[0008] The CD163 of interest in this invention is known to be a hemoglobin / haptoglobin scavenger receptor, belonging to the cysteine-rich superfamily of scavenger receptors, and is a type I transmembrane protein expressed in monocytes or macrophages. During macrophage activation, it is cleaved by metalloproteinases to produce soluble CD163, which circulates in the blood. Therefore, circulating soluble CD163 serves as a marker of macrophage activity, and in recent years it has been reported as a predictive marker for the therapeutic efficacy of nivolumab, a molecularly targeted therapy for malignant melanoma and advanced non-small cell lung cancer (Patent Document 4).

[0009] Existing technical documents

[0010] Patent documents

[0011] Patent Document 1: International Publication No. 2008 / 084105

[0012] Patent Document 2: International Publication No. 2016 / 170021

[0013] Patent Document 3: International Publication No. 2011 / 060080

[0014] Patent Document 4: International Publication No. 2018 / 003995

[0015] Non-patent literature

[0016] Non-patent document 1: Japan Society of Obstetrics and Gynecology 2018 ART Data Handbook

[0017] Non-patent literature 2: "Views on the Prevention of Multiple Pregnancy in Reproductive Assistance Medicine" published by the Japanese Society of Obstetrics and Gynecology in April 2011

[0018] Non-patent literature 3: Atlas of The Human Oocyte & Early Conceptus, 2, 1991

[0019] Non-patent literature 4: Gardner DK, et al., Fertil. Stellil., 73: 1155-1158, 2000

[0020] Non-patent literature 5: Pribenszky et al, Reprod. Biomed. Online, 35(5): 511-520, 2017 Summary of the Invention

[0021] The problem the invention aims to solve

[0022] The purpose of this invention is to provide biomarkers, methods for determining their use, and reagents for determination, which can be used in infertility treatment to identify embryos with high implantation / pregnancy probability, i.e., high fertility, from embryos obtained through embryo culture after in vitro fertilization.

[0023] Solution for solving the problem

[0024] The inventors of this application conducted in-depth research and discovered that by measuring the concentration of soluble CD163 in bodily fluids such as follicular fluid collected simultaneously with the eggs and serum before egg retrieval during in vitro fertilization, the fertility of the eggs or the embryos formed after fertilization can be determined, thus completing the following invention of this application.

[0025] That is, the present invention is as follows.

[0026] [1] A biomarker used to determine the fertility of an egg or an embryo formed after fertilization of an egg, which is composed of soluble CD163.

[0027] [2] Application of soluble CD163 as a biomarker for determining the fertility of an oocyte or an embryo formed after fertilization.

[0028] [3] A method for determining the fertility of an egg or an embryo formed after fertilization of an egg, which involves measuring the concentration of soluble CD163 in human body fluids and using the measured value to determine the fertility of the egg or an embryo formed after fertilization of an egg.

[0029] [4] According to the method described in [3], when the concentration of soluble CD163 is higher than the baseline value, it is determined that the fertility of the oocyte or the embryo formed after fertilization of the oocyte is high. The baseline value is the concentration of soluble CD163 in other human body fluids of the embryo formed after fertilization of the transplanted human oocyte but without establishing pregnancy.

[0030] [5] The method according to [3] or [4], wherein the aforementioned body fluid is follicular fluid, whole blood, serum, plasma or urine.

[0031] [6] The method according to any one of [3] to [5], wherein the method for determining the concentration of soluble CD163 is an immunological assay method.

[0032] [7] A determination reagent or determination kit for use in the method described in [6], characterized in that it contains an antibody that specifically recognizes soluble CD163.

[0033] [8] Use of antibodies that specifically recognize soluble CD163 in the manufacture of reagents or kits for determining pregnancy.

[0034] [9] Application of antibodies that specifically recognize soluble CD163 in determining pregnancy.

[0035]

[10] An antibody that specifically recognizes soluble CD163 is used to determine pregnancy.

[0036]

[11] Methods for treating infertility in patients or improving methods for establishing pregnancy based on in vitro fertilization, including:

[0037] (i) The procedure for determining the concentration of soluble CD163 in bodily fluids collected from a patient;

[0038] (ii) When the aforementioned measured value exceeds a preset benchmark value, the procedure of identifying oocytes collected from the patient on the same day as the aforementioned subject, or embryos formed after fertilization of those oocytes, as having a high fertility rate; and

[0039] (iii) The procedure of fertilizing the oocytes identified in (ii) to form an embryo or the identified embryo and then transferring it to the aforementioned patient.

[0040] The effects of the invention

[0041] According to the present invention, instead of relying solely on the morphological judgment based on physician experience, objective numerical values ​​can also be referenced to select suitable embryos for transplantation, which can greatly assist physicians in making embryo evaluation / selection judgments. Attached Figure Description

[0042] Figure 1Box plot showing the results of comparing the levels of soluble CD163 in follicular fluid containing oocytes that have been transferred to establish pregnancy with anti-CD163 antibody between follicular fluid containing oocytes that have not established pregnancy. P = 0.127 (Mann-Whitney test).

[0043] Figure 2 A graph showing the receiver operating characteristic (ROC) curve analysis results of soluble CD163 in follicular fluid containing oocytes that have been transferred to establish a pregnancy and in follicular fluid containing oocytes that have not established a pregnancy.

[0044] Figure 3 The results were obtained by immunoblotting of follicular fluid containing oocytes with confirmed pregnancies after embryo transfer and follicular fluid containing oocytes with uncertain pregnancies using anti-CD163 antibody. The bands in the confirmed pregnancy group were significantly darker than those in the uncertain pregnancy group.

[0045] Figure 4 A standard curve was prepared using the assay system of assay example 1 with recombinant CD163.

[0046] Figure 5 A box plot showing the results of comparing the pre-retrieval serum levels of soluble CD163 in patients who were able to retrieve oocytes for embryo transfer and establish pregnancy with those in patients who were unable to retrieve oocytes for establishing pregnancy. P = 0.0832 (Mann-Whitney test).

[0047] Figure 6 This is a graph showing the receiver operating characteristic (ROC) curve analysis results of soluble CD163 in the serum of patients who were able to collect oocytes for embryo transfer and establish pregnancy, and in the serum of patients who were unable to collect oocytes for establishing pregnancy.

[0048] Figure 7 This graph shows the results of comparing the median values ​​of soluble CD163 in the serum and follicular fluid of patients who were able to have oocytes collected for embryo transfer and establish pregnancy, with those in the serum and follicular fluid of patients who were unable to have oocytes collected for establishing pregnancy, using anti-CD163 antibodies. Detailed Implementation

[0049] The first aspect of this invention is a biomarker used in infertility treatment to determine the fertility of an egg or an embryo formed after fertilization. The biomarker of this invention consists of soluble CD163 present in human body fluids.

[0050] CD163, a transmembrane protein with a molecular weight of approximately 130–160 kDa, is expressed on monocytes and macrophages. After macrophages are activated by lipopolysaccharide (LPS) and other substances, they are cleaved from the membrane by metalloproteinases, thereby producing and releasing soluble CD163 into the body.

[0051] It is known that CD163 has four isoforms with different C-terminal sequences (Uniprotkb protein isoforms: Q86VB7-1, 2, 3, and 4, with amino acid sequences shown in SEQ ID NO: 1 to 4, respectively). These four isoforms are cleaved at specific positions to produce soluble CD163. In this case, CD163 isoforms 1, 2, and 3 produce the same soluble CD163. On the other hand, CD163 isoform 4 produces a soluble CD163 with a different sequence. Each soluble CD163 has a sequence corresponding to positions 42 to 578 and positions 580 to 1050 of SEQ ID NO: 1. The biomarker of this invention can be any one or both of the two soluble CD163 isoforms.

[0052] As illustrated in the examples described later, the concentration (also referred to as level) of soluble CD163 in the follicular fluid containing oocytes for which pregnancy has been established, or in the patient's serum immediately before oocyte retrieval, is significantly higher than the concentration of soluble CD163 in the follicular fluid containing oocytes for which pregnancy has not been established, or in the patient's serum immediately before oocyte retrieval. Therefore, soluble CD163 in the follicular fluid or in the serum immediately before oocyte retrieval can serve as an indicator for determining the fertility of an oocyte or the embryo formed after fertilization.

[0053] Another aspect of this method is the application of soluble CD163 as a biomarker for determining the fertility of an egg or an embryo formed after fertilization.

[0054] A second aspect of the present invention, based on the aforementioned insights, involves determining the concentration of soluble CD163 in human body fluids and using this measurement to determine the fertility of an oocyte or an embryo formed from its fertilization. The determination of the concentration of soluble CD163 in human body fluids is typically performed in vitro. This method allows for the selection of oocytes or embryos with high fertility. As a result, it provides a basis for selecting embryos suitable for uterine transfer, in addition to conventional blastocyst morphological evaluation based on microscopic observation, thereby increasing the probability of establishing pregnancy.

[0055] It should be noted that the method of the present invention includes the process up to the stage of determining pregnancy, but does not include the final determination of whether the embryo is suitable for transfer. Physicians can refer to the determination results obtained based on the method of the present invention to determine whether the embryo can be transferred to the uterus, select a suitable embryo for transfer, or formulate an infertility treatment plan.

[0056] In this instruction manual, "pregnancy-promoting" refers to the ability of an egg or an embryo formed from a fertilized egg to complete the entire process of implantation and pregnancy. By selecting embryos with high pregnancy-promoting potential before transfer to the uterus, the probability of establishing a pregnancy can be increased.

[0057] The level of soluble CD163 in a human subject can be investigated by measuring the soluble CD163 protein or fragments thereof in the sample. CD163 is a membrane-bound protein and therefore exists in body fluids in a fragmented, soluble form. The soluble CD163 protein or fragments thereof measured include those present in the subject in a form that has bound or associated with other proteins (e.g., those present in the subject as soluble CD163 molecules that have been completely or partially decomposed). Therefore, the term "soluble CD163" used as a biomarker in this invention includes the full-length soluble CD163 protein and its partial fragments present in the subject, as well as soluble CD163 protein and its partial fragments in a form that has bound or associated with other proteins or protein fragments.

[0058] In addition, the soluble CD163 measured in the method of the present invention can be any one or both of the aforementioned two types of soluble CD163.

[0059] The human subject refers to bodily fluids, such as follicular fluid, blood, serum, plasma, urine, etc. It is desirable to use follicular fluid, blood, serum, or plasma, and particularly, it is further desirable to use follicular fluid or serum. The subject used in the method of this invention refers to a subject collected from, and thus separated from, the subject.

[0060] The person collecting the specimen (the examinee) is usually a female patient who is suitable for in vitro fertilization in the treatment of infertility.

[0061] The collection of samples from the subject is usually performed on the same day as, or preferably simultaneously with, oocyte retrieval, which is done before ovulation. Specifically, collection is typically performed around day 10 to 14 from the start of menstruation. If the sample is follicular fluid, it is usually collected simultaneously with oocyte retrieval. If the sample is blood, serum, plasma, urine, etc., it is preferred to collect the sample just before oocyte retrieval, as this is believed to better reflect the condition of the retrieved oocytes.

[0062] The determination / judgment can be performed at any time before embryo transfer, such as after egg retrieval, after fertilization, or after separation to the blastocyst stage. Typically, the determination / judgment is performed when the physician assesses whether the embryo is suitable for transfer. Preferably, both the physician's morphological grading of the blastocyst under a microscope and the determination based on the method of this invention are obtained before the decision on whether to proceed with the transfer is made.

[0063] Furthermore, from the viewpoint of infertility treatment efficiency, it is preferable to perform the determination / assessment based on the method of the present invention early after egg retrieval. This is because by allowing eggs that have been determined to have a high fertility rate to enter the fertilization and culture processes, the likelihood of pregnancy can be increased.

[0064] Follicular fluid is present in mature follicles. It can be collected along with the eggs when using an oocyte retrieval needle from mature follicles that have undergone appropriate ovulation induction management. Follicular fluid is typically discarded during infertility treatment, therefore, the determination of this invention does not impose any further burden on the patient.

[0065] The method for determining soluble CD163 in human subjects is not particularly limited as long as it is a quantitative method such as immunoassay, liquid chromatography, electrophoresis, or mass spectrometry. Immunoassay does not require large instruments and equipment and is simple to operate, so it can be preferred for use in this invention.

[0066] Immunoassays are well-known in this field. Immunoassay methods can be classified based on the reaction mechanism, such as sandwich assays, competitive assays, agglutination assays, and Western blotting. They can also be classified based on the label, such as enzyme immunoassays, radioimmunoassays, and fluorescence immunoassays. In this invention, any immunoassay method capable of quantitative detection can be used. While not particularly limited, sandwich assays, such as sandwich ELISA, are preferred.

[0067] The method for manufacturing the antibodies used in this invention is not particularly limited. Typically, non-human animal polyclonal or monoclonal antibodies are prepared using non-human animals such as mice and rabbits. Furthermore, as mentioned above, the amino acid sequence of soluble CD163 and its encoding base sequence are well known; therefore, soluble CD163 antibodies that specifically recognize specific sites of soluble CD163 can be prepared using conventional methods such as the hybridoma method.

[0068] The antibodies used in this invention can be either polyclonal or monoclonal. Antiserum can also be used as a polyclonal antibody. In this invention, the term "polyclonal antibody" also includes the antiserum before purification. Alternatively, the antigen-binding fragment of the antibody can be used instead of the antibody itself. Hereinafter, unless the context clearly indicates otherwise, the term "antibody" also includes the antigen-binding fragment of the antibody. Polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments can all be prepared using well-known conventional methods.

[0069] Specifically, polyclonal antibodies recognizing specific sites of soluble CD163 can be obtained, for example, by mixing multiple monoclonal antibodies that specifically recognize that site. Alternatively, polypeptides containing that site of soluble CD163, soluble CD163 proteins, or polynucleotides encoding those sites, prepared by known methods such as chemical synthesis, can be used as immunogens. These immunogens, along with appropriate adjuvants, are used to immunize non-human animals. Antiserum is obtained from the blood of these animals, and the polyclonal antibodies (non-human animal anti-soluble CD163 polyclonal antibodies) in the antiserum are purified. To increase the antibody titer in the immunized animals, multiple immunizations are typically performed at intervals of several weeks. Purification of the antibodies in the antiserum can be performed using methods such as ammonium sulfate precipitation, fractionation using anion chromatography, or affinity column purification.

[0070] As an example of a known method for producing monoclonal antibodies, the hybridoma method can be cited. Specifically, for example, antibody-producing cells such as spleen cells or lymphocytes can be collected from non-human animals immunized as described above, and these cells can be fused with myeloma cells to prepare a hybridoma. Hybridomas that produce antibodies that bind to specific sites of soluble CD163 are selected, and the hybridomas are allowed to proliferate. Monoclonal antibodies that specifically recognize specific sites of soluble CD163 in non-human animals can be obtained from the culture supernatant.

[0071] "Antigen-binding fragments" refer, for example, to antibody fragments such as the Fab fragment or F(ab')2 fragment of an immunoglobulin that maintain the binding affinity (antigen-antibody reactivity) between the antibody and the corresponding antigen. It is well known that such antigen-binding fragments can also be used in immunoassays and are just as useful as the original antibody. As is known, Fab fragments and F(ab')2 fragments can be obtained by treating antibodies with proteases such as papain or pepsin. It should be noted that antigen-binding fragments are not limited to Fab fragments or F(ab')2 fragments; they can be any fragment that maintains binding affinity to the corresponding antigen, and can be fragments prepared using genetic engineering methods. Alternatively, antibodies obtained, for example, by expressing a single-chain variable region (scFv: single-chain fragment of variable region) in E. coli using genetic engineering methods can also be used. The method for producing scFv is also well-known. It involves extracting mRNA from hybridomas prepared as described above, preparing single-stranded cDNA, amplifying the immunoglobulin H and L chain genes via PCR using immunoglobulin H and L chain-specific primers, ligating these genes with adapters, conferring appropriate restriction enzyme sites, introducing the cDNA into a plasmid vector, transforming it into *E. coli*, and recovering the scFv from *E. coli*. Such scFv is also included in the category of "antigen-binding fragments".

[0072] Immunoassay methods are well-known techniques. To put it simply, for example, in the sandwich method, an antibody that binds to soluble CD163 is immobilized on a solid phase (immobilized antibody), reacts with the sample, and is washed as needed. Then, a labeled antibody is reacted and washed, and the labeled antibody bound to the solid phase is measured. The labeled antibody is obtained by labeling an antibody that binds to soluble CD163 at the same or different sites as the immobilized antibody.

[0073] The assay of labeled antibodies can be performed by measuring the signal from the labeled substance. The method for measuring the signal can be appropriately selected depending on the type of labeled substance. For example, in the case of enzyme labeling, a substrate such as a chromogenic substrate, fluorescent substrate, or luminescent substrate corresponding to the enzyme can be reacted with the enzyme, and the resulting colorimetric or luminescent signal can be measured using appropriate instruments such as a spectrophotometer or photometer, thereby determining the enzyme activity and measuring the analyte. For example, when using ALP as a labeled substance, a luminescent substrate such as 3-(4-methoxyspiro(1,2-dioxane-3,2'-tricyclo[3.3.1.13,7]decane)-4-yl)phenyl phosphate disodium salt (e.g., trade name AMPPD) can be used. In labeled antibodies, the labeled substance can bind directly to the antibody, or a specific binding molecule such as biotin or a hapten can bind to the antibody, and the chaperone molecule (streptavidin or hapten antibody, etc.) of the specific binding molecule bound to the labeled substance can react, thereby indirectly binding the labeled substance. For standard samples containing known concentrations of soluble CD163, immunoassay is performed using anti-soluble CD163 antibodies or their antigen-binding fragments. A standard curve is created by plotting the correlation between the amount of signal from the label and the concentration of soluble CD163 in the standard sample. The same procedure is performed on the analyte with unknown soluble CD163, and the amount of signal from the label is measured. Substituting the measured value into the standard curve allows for the quantification of soluble CD163 in the analyte.

[0074] Regarding whether the level of soluble CD163 in body fluids is high or low, a threshold value can be established by comparing it to a baseline value determined from the soluble CD163 levels in other body fluids containing oocytes from unconfirmed pregnancies. The baseline value could be, for example, the median concentration of soluble CD163 in the follicular fluids of multiple oocytes containing unconfirmed pregnancies. Furthermore, if the measured soluble CD163 levels in the follicular fluids obtained from oocyte retrieval of the test subject are higher than this baseline value, it can be considered as a high fertility level, i.e., high oocyte quality and a high probability of implantation / pregnancy. This threshold is set based on age (e.g., under 30, 30s, 40s, 50s, etc.).

[0075] In the method of the present invention, the oocyte or embryo for determining pregnancy is an oocyte collected on the day or at the time of collection of the subject to be tested, or an embryo formed after fertilization of the oocyte.

[0076] Furthermore, in the method of the present invention, the embryo for which pregnancy can be determined can be either a split embryo or a blastocyst, preferably a blastocyst.

[0077] The method for determining pregnancy according to the present invention can be used in infertility treatment. That is, according to the present invention, a method for treating infertility in patients or improving the method for establishing pregnancy based on in-vitro fertilization can be provided, comprising:

[0078] (i) The procedure for determining the concentration of soluble CD163 in bodily fluids collected from a patient;

[0079] (ii) When the aforementioned measured value exceeds the preset benchmark value, the procedure of identifying eggs collected from the patient on the same day as the aforementioned subject, or embryos formed after fertilization of such eggs, as having a high pregnancy rate; and

[0080] (iii) The procedure of fertilizing the oocytes identified in (ii) to form an embryo or the identified embryo and then transferring it to the aforementioned patient.

[0081] Furthermore, a third aspect of the present invention relates to reagents for a method of determining pregnancy containing an antibody that specifically recognizes soluble CD163.

[0082] The assay reagent may contain only antibodies or antigen-binding fragments that specifically recognize soluble CD163, or it may also contain other components that help stabilize these antibodies or their antigen-binding fragments. Furthermore, these antibodies or their antigen-binding fragments may be in the form of specifically binding molecules such as labeled substances or biotin, or immobilized on a solid phase such as a plate or particles.

[0083] A fourth aspect of the present invention can also be a pregnancy determination kit comprising the determination reagents of the present invention described above. This kit can also be an immunoassay kit, which may include other reagents required for the immunoassay. Other reagents required for the immunoassay are well known. For example, in addition to the determination reagents described above, the kit may also include a sample diluent, a washing buffer, and, if the labeling substance used for labeling the antibody is an enzyme, it may further include a substrate solution of that enzyme. Furthermore, the kit typically includes an instruction manual.

[0084] Another aspect of this approach is the application of antibodies that specifically recognize soluble CD163 in the manufacture of reagents for determining pregnancy.

[0085] In addition, another aspect is the application of antibodies that specifically recognize soluble CD163 in determining pregnancy.

[0086] On the other hand, there is an antibody that specifically recognizes soluble CD163 and is used to determine pregnancy.

[0087] Example

[0088] The present invention will be described in more detail below based on embodiments. Of course, the present invention is not limited to the embodiments described below.

[0089] <Example 1> Detection of soluble CD163 in follicular fluid using ELISA based on anti-CD163 antibody.

[0090] Follicular fluid samples containing oocytes with confirmed and uncertain pregnancies were collected simultaneously with oocyte retrieval from patients undergoing appropriate ovulation induction. The follicular fluid was stored at -80°C after retrieval. The culture records of the collected oocytes were compared with the patients' ability to conceive, thus dividing them into a confirmed pregnancy group and a uncertain pregnancy group. Thirteen samples of follicular fluid with confirmed pregnancies and six samples with uncertain pregnancies were used.

[0091] A commercially available anti-CD163 polyclonal antibody (R&D Systems) was used. This antibody was prepared by immunization with a sequence equivalent to Gly46-Ser1050 of sequence number 1, thus enabling the determination of soluble CD163. The antibody was diluted with carbonate buffer (pH 9.8) at a concentration of 50 ng / well and immobilized in MaxiSorp 96-well plates (Nunc). After reacting overnight at 4°C, the plates were washed three times with TBS-T (Tris-Buffered Saline containing 0.05% Tween 20), and 200 μL of TBS solution containing 3% bovine serum albumin (BSA) was added to each well. The plates were then incubated at room temperature for 2 hours.

[0092] After washing three times with TBS-T, 13 subjects in the confirmed follicular fluid group or 6 subjects in the non-confirmed follicular fluid group were diluted 4-fold with TBS-T solution containing 1% bovine serum albumin and added at 40 μL / well, and left at room temperature for 1 hour.

[0093] After washing three times with TBS-T, the purchased anti-CD163 monoclonal antibody (R&D Systems) was diluted to 1.0 μg / mL with TBS-T solution containing 1% bovine serum albumin, and added at 40 μL / well, incubated at room temperature for 1 hour. After washing three times with TBS-T, horseradish peroxidase (HRP)-labeled anti-mouse IgG (SIGMA) solution diluted 20,000 times with TBS-T solution containing 1% bovine serum albumin was added at 40 μL / well, incubated at room temperature for 1 hour. After washing four times with TBS-T, TMB Microwell Peroxidase Substrate (KPL) was added, and the reaction was stopped with 1 mol / L phosphate solution. The absorbance at 450 nm was measured using a microplate reader. It should be noted that, as shown in Reference Example 1, the assay system in Assay Example 1 can measure soluble CD163.

[0094] The results are shown in Figure 1 Compared with the non-confirmed pregnancy group, the soluble CD163 level in the follicular fluid of the confirmed pregnancy group was significantly higher. The median value was 119.9 ng / mL in the non-confirmed pregnancy group, while it was 213.5 ng / mL in the confirmed pregnancy group.

[0095] Table 1 and Figure 2 This study evaluated the ability of soluble CD163 in follicular fluid to establish pregnancy using ROC analysis. The results suggest a relationship between the concentration of soluble CD163 in follicular fluid as an independent variable and the outcome, such as the ability to establish pregnancy, as a dichotomous variable. The area under the ROC curve (AUC) was 0.73.

[0096] [Table 1]

[0097]

[0098] <Reference Example 1>

[0099] As the standard sample for the standard curve, recombinant human CD163 protein (recombinant CD163) manufactured by R&D Company was used. This recombinant CD163 was obtained from mouse melanoma cells, yielding a sequence equivalent to Gly46-Ser1050 of sequence number 1, which is also the sequence present in soluble CD163. The recombinant CD163 was adjusted with TBS-T solution containing 1% bovine serum albumin, and the assay was performed using the assay system of Assay Example 1 to construct a standard curve. The results are shown below. Figure 4 .

[0100] <Example 2> Detection of soluble CD163 using Western blotting with anti-CD163 antibody.

[0101] Follicular fluid samples containing oocytes with confirmed and uncertain pregnancies were collected simultaneously with oocyte retrieval from patients undergoing appropriate ovulation induction. The follicular fluid was stored at -80°C after retrieval. The culture records of the collected oocytes were compared with the patients' ability to conceive, thus dividing them into a confirmed pregnancy group and a uncertain pregnancy group. Two samples of follicular fluid with confirmed pregnancies and two samples of follicular fluid with uncertain pregnancies were used.

[0102] For the target follicular fluid, pretreatment was performed using High Select Top 14 Abundant Protein Depletion Mini Spin Columns (Thermo Scientific) according to the manufacturer's instructions. The protein concentration of the resulting eluent was determined, and the protein was loaded at 10 μg / lane onto a 5-20% gradient gel for SDS / PAGE. After electrophoresis, the protein was transferred to a PVDF membrane using a transfer device. The PVDF membrane with transferred protein was blocked for 1 hour at room temperature with TBST buffer containing 5% skim milk. After blocking, the anti-CD163 polyclonal antibody (R&Dsystem, the same antibody used in Example 1) was adjusted to 1.0 μg / mL in TBST buffer containing 5% skim milk, and the PVDF membrane was reacted at room temperature for 1 hour. After the reaction, the membrane was washed three times with TBST buffer. A horseradish peroxidase (HRP)-labeled anti-goat IgG (SIGMA) solution was prepared by diluting the solution 2000-fold with TBST buffer containing 5% skim milk. The membrane was then incubated at room temperature for 1 hour. After the reaction, the membrane was washed three times with TBST buffer, and then luminescent using an ECL Prime Western Blotting Detection Reagent (GEHEALTHCARE). Confirmation was performed using a CCD camera. Signal analysis of the detected bands was performed using a multi-gauge (Fujifilm) imager.

[0103] The results are shown in Figure 3 In the group with confirmed pregnancy, significantly deeper bands could be detected in the follicular fluid compared to the group with unconfirmed pregnancy.

[0104] The signal analysis results are shown in Table 2. The follicular fluid of the confirmed pregnancy group showed a significantly higher signal compared to that of the unconfirmed pregnancy group.

[0105] [Table 2]

[0106]

[0107] <Example 3> The soluble CD163 in serum collected just before oocyte retrieval was measured by ELISA using anti-CD163 antibody.

[0108] The patient serum used was collected from patients who underwent appropriate ovulation induction just before oocyte retrieval. The serum was stored at -80°C after recovery. The retrieved oocytes were used for in vitro fertilization, followed by embryo transfer to confirm pregnancy. Patients were divided into a group where oocytes capable of confirming pregnancy could be retrieved and a group where oocytes capable of confirming pregnancy could not be retrieved. Serum from 16 patients whose oocytes were capable of confirming pregnancy was used, and serum from 6 patients whose oocytes were not capable of confirming pregnancy was used.

[0109] Soluble CD163 in the test subjects was measured in the same manner as in Example 1.

[0110] The results are shown in Figure 5 Compared to the group where no oocytes were collected to establish pregnancy, the group where oocytes were collected to establish pregnancy had significantly higher serum soluble CD163 levels. The median value for the group where no oocytes were collected to establish pregnancy was 278.0 ng / mL, while the median value for the group with established pregnancy was 360.2 ng / mL.

[0111] Table 3 and Figure 6 This study evaluated the ability of serum soluble CD163 detection to establish pregnancy using ROC analysis. The results suggest a relationship between serum soluble CD163 concentration as an independent variable and the ability to establish pregnancy as a dichotomous variable. The area under the ROC curve was 0.75.

[0112] [Table 3]

[0113]

[0114] <Example 4> Using anti-CD163 antibody, soluble CD163 in serum and follicular fluid collected just before oocyte retrieval was measured by ELISA.

[0115] Follicular fluid samples, including oocytes with confirmed and unconfirmed pregnancies, were collected simultaneously with oocyte retrieval from patients undergoing appropriate ovulation induction. The follicular fluid was stored at -80°C after retrieval. Patient serum samples were collected from patients undergoing appropriate ovulation induction just before oocyte retrieval. The serum was stored at -80°C after retrieval. The retrieved oocytes were used for in vitro fertilization, followed by embryo transfer to confirm pregnancy. Patients were divided into a group where oocytes with confirmed pregnancies could be retrieved and a group where oocytes with confirmed pregnancies could not be retrieved. The following samples were used: 12 serum samples from patients with confirmed pregnancies, 12 follicular fluid samples from patients with confirmed pregnancies, 3 serum samples from patients whose oocytes could not be retrieved, and 3 follicular fluid samples from patients whose oocytes with unconfirmed pregnancies could not be retrieved.

[0116] Soluble CD163 in the test subjects was measured in the same manner as in Example 1.

[0117] The results are shown in Figure 7 Compared to the group where no oocytes were collected to confirm a pregnancy, the serum and follicular fluid samples from patients with confirmed pregnancies showed significantly higher levels of soluble CD163 in both follicular fluid and serum. The median serum level in the group where no oocytes were collected was 240.5 ng / mL, and the median follicular fluid level was 112.0 ng / mL, while the median serum level in the group with confirmed pregnancies was 360.2 ng / mL, and the median follicular fluid level was 198.4 ng / mL. sequence list <110> Yokohama City University (Public University Corporation) TOSOH corporation <120> Biomarkers for determining pregnancy and methods for determining pregnancy using them <130> 2200157-2161 <150> JP2021-000399 <151> 2021-01-05 <150> JP2021-139495 <151> 2021-08-30 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1156 <212> PRT <213> Homo sapiens <400> 1 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Thr Val Val 20 25 30 Leu Leu Leu Ser Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Asp 35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val 50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr 85 90 95 Ala Ile Lys Ala Pro Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg 100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp 115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln 130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asn Leu Glu Met Arg 145 150 155 160 Leu Thr Arg Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe 165 170 175 Gln Gly Arg Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asp His 180 185 190 Ala Ser Val Ile Cys Arg Gln Leu Glu Cys Gly Ser Ala Val Ser Phe 195 200 205 Ser Gly Ser Ser Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp 210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val 260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly 275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser Tyr Asp Ala Ala Val Ala Cys 290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Lys Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln 325 330 335 Gly His Glu Pro Ala Ile Trp Gln Cys Lys His His Glu Trp Gly Lys 340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly 355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly 370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala 420 425 430 Thr Asn Thr Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser 435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His 450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly 485 490 495 Asp Thr Trp Gly Ser Ile Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala 500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu 515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu 530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys 580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Gly Ala Trp Gly Ser Leu Cys 595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu 610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala Arg Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Ile Trp Arg His Met Phe His Cys Thr Gly Thr Glu 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys 660 665 670 Pro Ser Glu Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln 675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr 690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp 740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala 755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp 770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg 820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly 835 840 845 Thr Val Gly Lys Ser Ser Met Ser Glu Thr Thr Val Gly Val Val Cys 850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Pro Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys 900 905 910 Arg Leu Ala Ser Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys 915 920 925 Ile Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile 930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asp Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp 980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys 995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp Ala 1010 1015 1020 Ala Val Asn Cys Thr Asp Ile Ser Val Gln Lys Thr Pro Gln Lys Ala 1025 1030 1035 1040 Thr Thr Gly Arg Ser Ser Arg Gln Ser Ser Phe Ile Ala Val Gly Ile 1045 1050 1055 Leu Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu Phe Phe Leu Thr 1060 1065 1070 Lys Lys Arg Arg Gln Arg Gln Arg Leu Ala Val Ser Ser Arg Gly Glu 1075 1080 1085 Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu Asn 1090 1095 1100 Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Glu Asn Ser His Glu Ser 1105 1110 1115 1120 Ala Asp Phe Ser Ala Ala Glu Leu Ile Ser Val Ser Lys Phe Leu Pro 1125 1130 1135 Ile Ser Gly Met Glu Lys Glu Ala Ile Leu Ser His Thr Glu Lys Glu 1140 1145 1150 Asn Gly Asn Leu 1155 <210> 2 <211> 1161 <212> PRT <213> Homo sapiens <400> 2 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Thr Val Val 20 25 30 Leu Leu Leu Ser Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Asp 35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val 50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr 85 90 95 Ala Ile Lys Ala Pro Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg 100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp 115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln 130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asn Leu Glu Met Arg 145 150 155 160 Leu Thr Arg Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe 165 170 175 Gln Gly Arg Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asp His 180 185 190 Ala Ser Val Ile Cys Arg Gln Leu Glu Cys Gly Ser Ala Val Ser Phe 195 200 205 Ser Gly Ser Ser Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp 210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val 260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly 275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser Tyr Asp Ala Ala Val Ala Cys 290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Lys Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln 325 330 335 Gly His Glu Pro Ala Ile Trp Gln Cys Lys His His Glu Trp Gly Lys 340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly 355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly 370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala 420 425 430 Thr Asn Thr Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser 435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His 450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly 485 490 495 Asp Thr Trp Gly Ser Ile Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala 500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu 515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu 530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys 580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Gly Ala Trp Gly Ser Leu Cys 595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu 610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala Arg Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Ile Trp Arg His Met Phe His Cys Thr Gly Thr Glu 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys 660 665 670 Pro Ser Glu Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln 675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr 690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp 740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala 755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp 770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg 820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly 835 840 845 Thr Val Gly Lys Ser Ser Met Ser Glu Thr Thr Val Gly Val Val Cys 850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Pro Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys 900 905 910 Arg Leu Ala Ser Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys 915 920 925 Ile Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile 930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asp Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp 980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys 995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp Ala 1010 1015 1020 Ala Val Asn Cys Thr Asp Ile Ser Val Gln Lys Thr Pro Gln Lys Ala 1025 1030 1035 1040 Thr Thr Gly Arg Ser Ser Arg Gln Ser Ser Phe Ile Ala Val Gly Ile 1045 1050 1055 Leu Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu Phe Phe Leu Thr 1060 1065 1070 Lys Lys Arg Arg Gln Arg Gln Arg Leu Ala Val Ser Ser Arg Gly Glu 1075 1080 1085 Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu Asn 1090 1095 1100 Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Gly Leu Trp Val Leu Gly 1105 1110 1115 1120 Gly Ser Ile Ala Gln Gly Phe Arg Ser Val Ala Ala Val Glu Ala Gln 1125 1130 1135 Thr Phe Tyr Phe Asp Lys Gln Leu Lys Lys Ser Lys Asn Val Ile Gly 1140 1145 1150 Ser Leu Asp Ala Tyr Asn Gly Gln Glu 1155 1160 <210> 3 <211> 1121 <212> PRT <213> Homo sapiens <400> 3 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Thr Val Val 20 25 30 Leu Leu Leu Ser Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Asp 35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val 50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr 85 90 95 Ala Ile Lys Ala Pro Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg 100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp 115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln 130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asn Leu Glu Met Arg 145 150 155 160 Leu Thr Arg Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe 165 170 175 Gln Gly Arg Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asp His 180 185 190 Ala Ser Val Ile Cys Arg Gln Leu Glu Cys Gly Ser Ala Val Ser Phe 195 200 205 Ser Gly Ser Ser Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp 210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val 260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly 275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser Tyr Asp Ala Ala Val Ala Cys 290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Lys Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln 325 330 335 Gly His Glu Pro Ala Ile Trp Gln Cys Lys His His Glu Trp Gly Lys 340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly 355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly 370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala 420 425 430 Thr Asn Thr Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser 435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His 450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly 485 490 495 Asp Thr Trp Gly Ser Ile Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala 500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu 515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu 530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys 580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Gly Ala Trp Gly Ser Leu Cys 595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu 610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala Arg Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Ile Trp Arg His Met Phe His Cys Thr Gly Thr Glu 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys 660 665 670 Pro Ser Glu Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln 675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr 690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp 740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala 755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp 770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg 820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly 835 840 845 Thr Val Gly Lys Ser Ser Met Ser Glu Thr Thr Val Gly Val Val Cys 850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Pro Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys 900 905 910 Arg Leu Ala Ser Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys 915 920 925 Ile Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile 930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asp Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp 980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys 995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp Ala 1010 1015 1020 Ala Val Asn Cys Thr Asp Ile Ser Val Gln Lys Thr Pro Gln Lys Ala 1025 1030 1035 1040 Thr Thr Gly Arg Ser Ser Arg Gln Ser Ser Phe Ile Ala Val Gly Ile 1045 1050 1055 Leu Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu Phe Phe Leu Thr 1060 1065 1070 Lys Lys Arg Arg Gln Arg Gln Arg Leu Ala Val Ser Ser Arg Gly Glu 1075 1080 1085 Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu Asn 1090 1095 1100 Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Gly Gly His Ser Glu Pro 1105 1110 1115 1120 His <210> 4 <211> 1154 <212> PRT <213> Homo Sapiens <400> 4 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Thr Val Val 20 25 30 Leu Leu Leu Ser Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Asp 35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val 50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr 85 90 95 Ala Ile Lys Ala Pro Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg 100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp 115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln 130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asn Leu Glu Met Arg 145 150 155 160 Leu Thr Arg Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe 165 170 175 Gln Gly Arg Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asp His 180 185 190 Ala Ser Val Ile Cys Arg Gln Leu Glu Cys Gly Ser Ala Val Ser Phe 195 200 205 Ser Gly Ser Ser Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp 210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val 260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly 275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser Tyr Asp Ala Ala Val Ala Cys 290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Lys Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln 325 330 335 Gly His Glu Pro Ala Ile Trp Gln Cys Lys His His Glu Trp Gly Lys 340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly 355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly 370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala 420 425 430 Thr Asn Thr Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser 435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His 450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly 485 490 495 Asp Thr Trp Gly Ser Ile Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala 500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu 515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu 530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val 565 570 575 Cys Ser Ser Lys Thr Gln Lys Thr Ser Leu Ile Gly Ser Tyr Thr Val 580 585 590 Lys Gly Thr Gly Leu Gly Ser His Ser Cys Leu Phe Leu Lys Pro Cys 595 600 605 Leu Leu Pro Gly Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro 610 615 620 Cys Glu Gly Arg Val Glu Leu Lys Thr Leu Gly Ala Trp Gly Ser Leu 625 630 635 640 Cys Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln 645 650 655 Leu Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala Arg Phe Gly 660 665 670 Lys Gly Asn Gly Gln Ile Trp Arg His Met Phe His Cys Thr Gly Thr 675 680 685 Glu Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu 690 695 700 Cys Pro Ser Glu Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser 705 710 715 720 Gln Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro 725 730 735 Thr Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu 740 745 750 Arg Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr 755 760 765 His Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser 770 775 780 Asp Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn 785 790 795 800 Ala Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu 805 810 815 Asp Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His 820 825 830 Ser His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly 835 840 845 Val Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser 850 855 860 Arg Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp 865 870 875 880 Gly Thr Val Gly Lys Ser Ser Met Ser Glu Thr Thr Val Gly Val Val 885 890 895 Cys Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Pro Ala Ser 900 905 910 Leu Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys 915 920 925 Pro Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu 930 935 940 Lys Arg Leu Ala Ser Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn 945 950 955 960 Lys Ile Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu 965 970 975 Ile Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp 980 985 990 Leu Asp Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala 995 1000 1005 Leu Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile 1010 1015 1020 Trp Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp 1025 1030 1035 1040 Cys Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp 1045 1050 1055 Ala Ala Val Asn Cys Thr Asp Ile Ser Val Gln Lys Thr Pro Gln Lys 1060 1065 1070 Ala Thr Thr Gly Arg Ser Ser Arg Gln Ser Ser Phe Ile Ala Val Gly 1075 1080 1085 Ile Leu Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu Phe Phe Leu 1090 1095 1100 Thr Lys Lys Arg Arg Gln Arg Gln Arg Leu Ala Val Ser Ser Arg Gly 1105 1110 1115 1120 Glu Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu 1125 1130 1135 Asn Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Gly Gly His Ser Glu 1140 1145 1150 Pro His

Claims

1. The application of soluble CD163 as a biomarker for determining the fertility of an oocyte or the embryo formed after fertilization, among which, The soluble CD163 is the soluble CD163 present in the follicular fluid collected at the same time as the eggs during egg retrieval or in the serum of the person just before egg retrieval. The fertility refers to the degree to which the egg or the embryo formed after fertilization can complete the entire process of implantation and pregnancy. By selecting embryos with high fertility before transfer to the uterus, the probability of establishing pregnancy can be increased.

2. The application of reagents for determining the concentration of soluble CD163 in human body fluids in the manufacture of reagents for determining the fertility of an egg or an embryo formed after fertilization, wherein... The human fluid refers to follicular fluid collected simultaneously with the eggs during egg retrieval or human serum collected just before egg retrieval. Pregnancy refers to the ability of an egg or an embryo formed after fertilization to complete the entire process of implantation and pregnancy. By selecting embryos with high pregnancy before transfer to the uterus, the probability of establishing pregnancy can be increased.

3. The application according to claim 2, wherein, The judgment is made when the soluble CD163 concentration is higher than the baseline value, indicating that the egg or the embryo formed after fertilization of the egg has a high fertility. The baseline value is the soluble CD163 concentration in other human body fluids of an embryo formed after fertilization of a human egg but without established pregnancy.

4. The application according to claim 2 or 3, wherein, The concentration of soluble CD163 was determined using an immunological assay.

5. The application according to claim 4, characterized in that, The reagent used to determine the concentration of soluble CD163 in human body fluids contains an antibody that specifically recognizes soluble CD163.

6. The use of antibodies that specifically recognize soluble CD163 in the manufacture of reagents or kits for determining pregnancy, wherein, The soluble CD163 is the soluble CD163 present in the follicular fluid collected at the same time as the eggs during egg retrieval or in the serum of the person just before egg retrieval. The fertility refers to the degree to which the egg or the embryo formed after fertilization can complete the entire process of implantation and pregnancy. By selecting embryos with high fertility before transfer to the uterus, the probability of establishing pregnancy can be increased.

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