Use of ERAP2 detection reagent in preparation of product for predicting prognosis of poi

Abstract

The present invention relates to the technical field of medical detection. Disclosed is a use of an ERAP2 detection reagent in the preparation of a product for predicting the prognosis of POI. Also disclosed in the present invention is a use of ERAP2 as a biomarker in the preparation of a product for predicting the prognosis of POI. The prognosis of POI is intermittent recovery of ovarian function and / or a pregnancy outcome. In the present invention, it has been found by means of experimental studies that a serum ERAP2 level has a significant association with the prognosis of a POI patient, is an independent influencing factor, and has relatively high specificity and sensitivity. Therefore, ERAP2 can serve as a biomarker for predicting the prognosis of POI. The present invention provides a theoretical basis and clinical practice guidance for the prognosis of POI, and has important application value.

Description

Use of reagent for detecting ERAP2 in preparation of product for predicting POI prognosis TECHNICAL FIELD

[0001] The present application relates to the use of a reagent for detecting ERAP2 in the preparation of a product for predicting the prognosis of POI, belonging to the field of medical detection technology. BACKGROUND

[0002] Premature ovarian insufficiency (POI) is a common clinical female reproductive aging disease, which refers to the decline of ovarian function in women before the age of 40, mainly manifested as oligomenorrhea or amenorrhea, elevated follicle-stimulating hormone (FSH) levels (> 25 IU / L), and decreased estradiol (E2) levels. Its incidence is about 3.7%. POI is highly heterogeneous in clinical and etiology, lacks reliable disease progression warning indicators, and is difficult to treat clinically, and there is no effective measure to improve ovarian function and restore fertility. In addition, POI increases the risk of chronic diseases such as cardiovascular disease and osteoporosis, and increases the all-cause mortality and cancer mortality, seriously affecting the physical and mental health of women. Unlike natural menopause, about 50% of POI patients have intermittent follicular development and ovulation, and 5% to 10% of patients can still naturally conceive after being diagnosed for many years. Therefore, screening biomarkers for predicting ovarian function recovery or clinical pregnancy in POI patients can help clinicians better identify and focus on POI patients and provide early intervention and fertility counseling, helping them achieve their fertility desires.

[0003] Ovarian reserve mainly includes the number and quality of oocytes, which determines the reproductive potential of women and thus determines the reproductive life and menopausal age of women. The evaluation of ovarian reserve has become a commonly used detection method for assisting POI prediction, diagnosis and prognosis. At present, the widely used ovarian reserve indicators in the clinic mainly include FSH, E2, anti-müllerian hormone (AMH), antral follicle count (AFC), inhibin B (INHB), etc. Among them, AMH is a sensitive indicator reflecting ovarian reserve and ovarian responsiveness, and can better predict live birth in people with low ovarian reserve, but its predictive advantage mainly exists in the early stage of ovarian function decline. For most POI patients who come to the clinic when they are in a state of almost ovarian failure, the serum AMH level has been significantly reduced and reached a plateau or is not measurable, and its predictive value for pregnancy or live birth is limited. Therefore, it is of great significance to screen and identify sensitive indicators that can help monitor and predict the prognosis of POI women.

[0004] Endoplasmic reticulum aminopeptidase-2 (ERAP2) is a zinc metallo aminopeptidase of the M1 protease family, the amino acid sequence is shown as SEQ ID NO. 1, as follows:

[0005] MFHSSAMVNSHRKPMFNIHRGFYCLTAILPQICICSQFSVPSSYHFTEDPGAFPVATNGERFPWQELRLPSVVIPLHYDLFVHPNLTSLDFVASEKIEVLVSNATQFIILHSKDLEITNATLQSEEDSRYMKPGKELKVLSYPAHEQIALLVPEKLTPHLKYYVAMDFQAKLGDGFEGFYKSTYRTLGGETRILAVTDFEPTQARMAFPCFDEPLFKANFSIKIRRESRHIALSNMPKVKTIELEGGLLEDHFETTVKMSTYLVAYIVCDFHSLSGFTSSGVKVSIYASPDKRNQTHYALQASLKLLDFYEKYFDIYYPLSKLDLIAIPDFAPGAMENWGLITYRETSLLFDPKTSSASDKLWVTRVIAHELAHQWFGNLVTMEWWNDIWLKEGFAKYMELIAVNATYPELQFDDYFLNVCFEVITKDSLNSSRPISKPAETPTQIQEMFDEVSYNKGACILNMLKDFLGEEKFQKGIIQYLKKFSYRNAKNDDLWSSLSNSCLESDFTSGGVCHSDPKMTSNMLAFLGENAEVKEMMTTWTLQKGIPLLVVKQDGCSLRLQQERFLQGVFQEDPEWRALQERYLWHIPLTYSTSSSNVIHRHILKSKTDTLDLPEKTSWVKFNVDSNGYYIVHYEGHGWDQLITQLNQNHTLLRPKDRVGLIHDVFQLVGAGRLTLDKALDMTYYLQHETSSPALLEGLSYLESFYHMMDRRNISDISENLKRYLLQYFKPVIDRQSWSDKGSVWDRMLRSALLKLACDLNHAPCIQKAAELFSQWMESSGKLNIPTDVLKIVYSVGAQTTAGWNYLLEQYELSMSSAEQNKILYALSTSKHQEKLLKLIELGMEGKVIKTQNLAALLHAIARRPKGQQLAWDFVRENWTHLLKKFDLGSYDIRMIISGTTAHFSSKDKLQEVKLFFESLEAQGSHLDIFQTVLETITKNIKWLEKNLPTLRTWLMVNT.

[0006] ERAP2 is involved in the final trimming of major histocompatibility complex class I (MHC-I) peptide. ERAP2 can be secreted by peripheral blood mononuclear cells under the stimulation of interferon gamma (IFN-gamma) and lipopolysaccharide (LPS), and plays a key role in antigen presentation and cytotoxic T cell activation, and the expression level is positively correlated with the proportion of CD8, NK and other immune cell infiltration. ERAP2 is reported to be related to the prognosis of squamous cell lung cancer and the abortion outcome of in vitro fertilization-embryo transfer (IVF-ET). The ELISA kit for detecting ERAP2 is a commercial product, which is composed of enzyme-coated plates, standard products, detection reagent A (detection antibody), diluent A (detection antibody diluent), detection reagent B (horseradish peroxidase labeled avidin), diluent B (horseradish peroxidase labeled avidin diluent), sample diluent, concentrated washing solution, TMB substrate solution, stop solution, and plate sealing film, which is used for scientific research of serum, plasma, tissue homogenate, cell culture supernatant and other samples. There is no report on the association between ERAP2 and POI. SUMMARY

[0007] In view of the above prior art, the application provides the use of a reagent for detecting ERAP2 in the preparation of a product for predicting the prognosis of POI, belonging to the technical field of medical detection.

[0008] The application is realized by the following technical solutions:

[0009] The use of a reagent for detecting ERAP2 in the preparation of a product for predicting the prognosis of POI.

[0010] Further, the prognosis of POI is intermittent ovarian function recovery and / or pregnancy outcome.

[0011] Further, the reagent for detecting ERAP2 is a reagent for detecting serum ERAP2 level, and the cutoff value of serum ERAP2 level is 5.535 ng / mL.

[0012] Further, the reagent for detecting ERAP2 is an ELISA kit.

[0013] The use of ERAP2 as a biomarker in the preparation of a product for predicting the prognosis of POI.

[0014] Further, the prognosis of POI is intermittent ovarian function recovery and / or pregnancy outcome.

[0015] The present application is found through experimental research that the serum ERAP2 level has significant relevance with the prognosis of POI patients (including intermittent ovarian function recovery and pregnancy outcome), and is an independent influencing factor, with high specificity and sensitivity; therefore, ERAP2 can be used as a biomarker for predicting the prognosis of POI, and the expression level of ERAP2 in serum can be detected, and the result can be used as one of the bases for predicting the prognosis of POI, providing guidance for clinical intervention, especially assisted reproductive technology (ART) assisted pregnancy. The present application shows good performance in predicting the intermittent ovarian function recovery and pregnancy outcome of POI patients, provides a theoretical basis and clinical practice guidance for POI prognosis, and has important application value.

[0016] Various terms and phrases used in the present application have the general meanings known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1: Discrimination of ERAP2 on intermittent ovarian function recovery.

[0018] Figure 2: Discrimination of AFC on intermittent ovarian function recovery.

[0019] Figure 3: Discrimination of ERAP2 and AFC combined on intermittent ovarian function recovery.

[0020] Figure 4: Discrimination of ERAP2 on clinical pregnancy.

[0021] Figure 5: Discrimination of AFC on clinical pregnancy.

[0022] Figure 6: Discrimination of ERAP2 and AFC combined on clinical pregnancy. DETAILED DESCRIPTION

[0023] The present application will be further described below in conjunction with examples. However, the scope of the present application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present application without departing from the spirit and scope of the present application.

[0024] The instruments, reagents, materials, etc. involved in the following examples, if not specifically stated, are conventional instruments, reagents, materials, etc. already existing in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, if not specifically stated, are conventional experimental methods, detection methods, etc. already existing in the prior art.

[0025] The ELISA kit used in the present application for detecting ERAP2 is ERAP2 ELISA kit (E15293h) produced by Wuhan Yibo Company, which can be purchased through conventional commercial channels, and can detect the level of ERAP2 in serum, plasma, tissue homogenate, cell culture supernatant, etc.

[0026] Example 1 Research on ERAP2 as a biomarker for predicting the prognosis of POI

[0027] The present application obtains the ovarian transcriptome data of young women (20-30 years old) and old women (50-60 years old) from the GTEx database, performs mixed linear model (MLM) analysis based on the random forest algorithm and the XGBoost algorithm, and screens and identifies a new biomarker-ERAP2 related to ovarian aging according to the gene expression difference and the change of the candidate gene with age. It is found through verification that the serum ERAP2 level of POI patients is significantly higher than that of normal women, and the ERAP2 level is positively correlated with the FSH level and negatively correlated with the AMH, AFC and testosterone (T) levels, indicating that the increase of serum ERAP2 level is related to the severity of ovarian aging disease in POI patients.

[0028] In order to explore whether ERAP2 is helpful for predicting the clinical prognosis of POI patients, 30 POI patients were prospectively included, and whether they had intermittent ovarian function recovery and clinical pregnancy was followed up for 3 years.

[0029] The inclusion criteria of POI patients are based on the diagnostic criteria formulated by the European Society of Human Reproduction and Embryology (ESHRE) in 2016. The inclusion criteria are as follows: ① age < 40 years old; ② FSH > 25 IU / L (2 times, interval > 4 weeks); ③ oligomenorrhea or amenorrhea for more than 4 months; ④ no endocrine abnormality diseases such as polycystic ovary syndrome, hyperprolactinemia and hyperandrogenemia; ⑤ no estrogen and progesterone drugs were taken in the past 3 months.

[0030] When the patient comes to the hospital for diagnosis of POI, the patient's blood sample is collected, the peripheral blood specimen is taken in a coagulation tube, centrifuged at 4°C (2500 rpm, 10 min), and the upper serum sample is carefully aspirated for standby. The collection of blood samples has obtained the informed consent of the patient and passed the discussion and approval of the ethics committee.

[0031] The FSH, E2, T, AMH, INHB and other indicators are routinely detected on the 2nd to 4th day of the menstrual cycle. The patient is given a gynecological ultrasound examination, and the AFC is defined as the number of bilateral follicles (diameter 2-10 mm) in the early follicular phase. The ELISA kit for detecting ERAP2 is used to detect the level of ERAP2 in the serum.

[0032] Follow-up standard: follow-up once every 3-6 months, a total of 3 years. Two weeks before the start of follow-up, every patient was informed by telephone or WeChat and invited to come to the Shandong University Reproductive Hospital for follow-up screening. Each follow-up investigation included a standardized personal face-to-face interview questionnaire, mainly including menstrual conditions, estrogen and progesterone medication, infertility treatment history and pregnancy, etc. The follow-up process was guided by trained and certified researchers. In order to reduce the loss rate, patients who could not participate in the face-to-face interview questionnaire were interviewed by telephone.

[0033] Intermittent ovarian function recovery criteria: POI patients with one or more of the following conditions were considered to have intermittent ovarian function recovery: ① natural pregnancy or pregnancy through self-ovum in vitro fertilization; ② without hormone replacement therapy (HRT), menstrual cycle recovery (at least two consecutive uterine bleeding within 4 months); ③ without HRT treatment, FSH < 15 IU / L.

[0034] Among the 30 POI patients, 10 patients (33.33%) had intermittent ovarian function recovery within 3 years, and 4 patients (13.33%) successfully conceived. According to the intermittent ovarian function recovery, the POI patients were divided into two groups (non-recovery group, recovery group); According to the pregnancy, the POI patients were divided into two groups (non-pregnancy group, pregnancy group). The indicators of each group were statistically analyzed, and the comparison results of each indicator between groups are shown in Tables 1 and 2.

[0035]

[0036] As shown in Tables 1 and 2, the ERAP2 level of patients with intermittent ovarian function recovery (4.25±1.67 ng / mL) was significantly lower than that of patients without recovery (6.40±2.28 ng / mL), and the ERAP2 level of patients with successful pregnancy (3.43±1.54 ng / mL) was significantly lower than that of patients without pregnancy (6.03±2.23 ng / mL).

[0037] In order to evaluate the importance of each indicator to intermittent ovarian function recovery, single factor Cox regression was performed on each indicator, and variables with P<0.1 were included in the stepwise regression model for analysis. The results are shown in Table 3, and it is found that ERAP2 and AFC are independent influencing factors of intermittent ovarian function recovery.

[0038] Similarly, in order to evaluate the importance of each indicator to the prognosis of pregnancy outcome, single factor Cox regression was performed on each indicator, and variables with P<0.1 were included in the stepwise regression model for analysis. The results are shown in Table 4, and it is found that ERAP2 is an independent influencing factor of clinical pregnancy.

[0039] Example 2 Verification of ERAP2 as a biomarker for predicting the prognosis of POI.

[0040] To further verify the role of ERAP2 in the clinical prognosis of POI patients, 30 POI patients (as a verification set) were additionally included for verification. The inclusion criteria, detection process, follow-up criteria, etc. were the same as in Example 1.

[0041] Among the 30 POI patients in the verification set, 9 patients (30.00%) had intermittent ovarian function recovery within 3 years, and 7 patients (23.33%) had clinical pregnancy. The grouping and statistical analysis were the same as in Example 1. The comparison results of various indicators between groups in the verification set are shown in Tables 5 and 6.

[0042] As can be seen from Tables 5 and 6, the ERAP2 level of patients with intermittent ovarian function recovery (4.28 ± 1.84 ng / mL) was significantly lower than that of patients without recovery (6.55 ± 2.18 ng / mL), and the ERAP2 level of patients with successful pregnancy (4.06 ± 1.88 ng / mL) was significantly lower than that of patients without pregnancy (6.42 ± 2.17 ng / mL).

[0043] To verify the importance of each indicator to intermittent ovarian function recovery, single factor Cox regression was performed on each indicator, and variables with P<0.1 were included in the stepwise regression model for analysis. The results are shown in Table 7, which verifies that ERAP2 and AFC are independent influencing factors for intermittent ovarian function recovery. The discrimination degree of ERAP2 for intermittent ovarian function recovery is shown in Figure 1 (AUC=0.762), the discrimination degree of AFC for intermittent ovarian function recovery is shown in Figure 2 (AUC=0.697), and the discrimination degree of ERAP2 and AFC combined for intermittent ovarian function recovery is shown in Figure 3 (AUC=0.873). The prediction performance of ERAP2 and AFC for intermittent ovarian function recovery is shown in Table 8. Compared with using only the AFC indicator to predict the outcome, the addition of ERAP2 significantly improves the discrimination degree for intermittent ovarian function recovery in POI patients (from 0.697 to 0.873, P=0.041).

[0044]

[0045] Similarly, in order to verify the importance of each index on the prognosis of pregnancy outcome, single factor Cox regression was performed on each index, and variables with P<0.1 were included in the stepwise regression model for analysis. The results are shown in Table 9. ERAP2 and AFC were finally verified as independent influencing factors of clinical pregnancy. The discrimination degree of ERAP2 on clinical pregnancy is shown in Figure 4 (AUC=0.764), the discrimination degree of AFC on clinical pregnancy is shown in Figure 5 (AUC=0.693), and the discrimination degree of ERAP2 and AFC combined on clinical pregnancy is shown in Figure 6 (AUC=0.888). The prediction performance of ERAP2 on clinical pregnancy is shown in Table 10. Compared with the use of AFC index alone to predict the outcome, the addition of ERAP2 significantly improved the discrimination degree of clinical pregnancy outcome in POI patients (from 0.693 to 0.888, P=0.035).

[0046]

[0047] Conclusion: The detection of ERAP2 level in serum can be used as a basis for predicting the prognosis of POI. The cutoff value is 5.535 ng / mL. For intermittent ovarian function recovery, the sensitivity is 77.78% and the specificity is 66.67%; for pregnancy outcome, the sensitivity is 85.71% and the specificity is 65.22%. The sensitivity is high and the specificity is good. ERAP2 can be used as a biomarker to predict the prognosis of POI.

[0048] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure disclosed herein. Modifications obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. Use of an agent that detects ERAP2 in the manufacture of a product for predicting the prognosis of a POI, characterized in that: The POI prognosis is intermittent recovery of ovarian function and / or pregnancy outcome.

2. Use according to claim 1, characterized in that: The reagent for detecting ERAP2 is a reagent for detecting serum ERAP2 level.

3. Use according to claim 1 or 2, characterized in that: The reagent for detecting ERAP2 is an ELISA kit.

4. Use of ERAP2 as a biomarker in the manufacture of a product for predicting the prognosis of a POI, characterized in that: The POI prognosis is intermittent recovery of ovarian function and / or pregnancy outcome.

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