Kit for predicting clinical pregnancy outcome of first-time frozen embryo transplantation

Abstract

The invention relates to the technical field of biology, and discloses a kit for predicting clinical pregnancy outcome of first-time frozen embryo transplantation. The kit comprises immune cells and immune factors in blood, the immune cells comprise CD19 < + > B cells, CD3 < + > T cells, CD4 < + > Th cells, CD8 < + > Ts cells, CD4 < + > / CD8 < + > ratio cells, CD56 < + > NK cells, CD68 < + > monocytes, CD86 < + > M1 sample monocytes, D206 < + > M2 sample monocytes and CD163 < + > M2 sample monocytes; the immune factors comprise IL-1beta, IL-2r, IL-6, IL-8 and TNF (Tumor Necrosis Factor)-alpha. According to the application, the importance of the peripheral blood CD163 + M2 sample mononuclear cell on predicting the continuous pregnancy of first embryo implantation of an FE T patient is confirmed, and the peripheral blood CD163 + M2 sample mononuclear cell is prompted to be possibly a biomarker of an FET period.

Description

Technical Field

[0001] This application relates to the field of biotechnology. More specifically, it relates to a kit for predicting the clinical pregnancy outcome of the first frozen embryo transfer. Background Art

[0002] Embryo transfer is a key step in achieving pregnancy during in vitro fertilization. The sufficient interaction between high-quality embryos and the uterine immune microenvironment is the basis for successful embryo implantation. It is speculated that patients with recurrent implantation failure (RIF) may not be able to recruit the lymphocytes required for embryo implantation on the decidual surface of the maternal-fetal interface. Immune cells in peripheral blood and uterine decidua during the implantation window period include uterine natural killer cells (uNK), macrophages, T cells, etc. These cells produce various cytokines, interleukins, and growth factors, which have a regulatory effect on endometrial thickness and endometrial receptivity.

[0003] As the second largest immune regulatory cell in the endometrium, macrophages play a key role in embryo recognition and successful implantation. Macrophages are mainly divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which secrete cytokines and then play different regulatory roles. The destruction of macrophages will reduce endometrial receptivity, leading to RIF. M1 macrophages promote embryo attachment to the decidua during the embryo implantation period through inflammatory cytokines. Another study shows that M2 macrophages are aggregated in the endometrium of mice during the implantation period, and the main phenotype of decidual macrophages in the early stage of human pregnancy is M2. However, the relationship between endometrial macrophages and pregnancy outcomes is currently unclear.

[0004] Does the imbalance of the immune microenvironment in patients who have transplanted high-quality embryos but still experienced pregnancy failure lead to embryo implantation failure, or is it the immune imbalance that causes the occurrence of embryo implantation failure? This study uses the peripheral blood immune cells and immune factors of patients undergoing the first frozen embryo transfer (FET) cycle to explore the role of systemic immune factors in embryo implantation. In the past, obtaining endometrial tissue during the embryo implantation window period was invasive, so synchronous embryo transfer could not be performed in the same cycle. Moreover, there has been no report on a kit for predicting the pregnancy outcome of the first FET patients from an immune perspective. Summary of the Invention

[0005] The object of the invention of the present application is to explore whether monocytes and other immune cells in peripheral blood are related to pregnancy outcomes during frozen embryo transfer. The present invention predicts FET pregnancy outcomes by detecting immune cell subsets and immune factors in biological samples and comparing them with controls. The present invention has also identified specific peripheral blood immune cell markers, which have high specificity and sensitivity in predicting FET clinical pregnancy outcomes.

[0006] To achieve the above object of the invention, the present application provides the following technical solutions:

[0007] In a first aspect, the present invention provides a kit for predicting the clinical pregnancy outcome of the first frozen embryo transfer, the kit comprising immune cells and immune factors in blood;

[0008] The immune cells include CD19+ B cells, CD3+ T cells, CD4+ Th cells, CD8+ Ts cells, CD4+ / CD8+ ratio, CD56+ NK cells, CD68+ monocytes, CD86+ M1-like monocytes, D206+ M2-like monocytes, CD163+ M2-like monocytes;

[0009] The immune factors include IL-1β, IL-2r, IL-6, IL-8 and TNF-α.

[0010] As a preferred example, the immune cell is selected from CD163+ M2-like monocytes.

[0011] In a second aspect, the present invention provides the use of the kit according to the first aspect, comprising the following steps:

[0012] On the day of blastocyst transfer during the FET cycle, peripheral blood of the patients was drawn, and lymphocyte flow cytometry sorting was performed and compared. According to the pregnancy outcome, a pregnancy group (n = 27 cases) and a non-pregnancy group (n = 23 cases) were divided. It was found that the peripheral blood CD163+ M2-like monocytes were significantly higher in the pregnancy group than in the non-pregnancy group (68.78 ± 5.02% vs 52.41 ± 6.27%, P = 0.0137). The abortion rate in the pregnancy group was 14.8%, and the continuous pregnancy rate was 63.0%. The percentage of peripheral blood CD163+ M2-like monocytes in those with continuous pregnancy was higher than that in the non-pregnancy subgroup (P = 0.029). The receiver operating characteristic curve (ROC) value of peripheral blood CD163+ M2-like monocytes in predicting pregnancy outcome was 0.671. According to the critical value of CD163+ M2-like monocytes in the ROC curve (40.6%), a further independent cohort study found that the clinical pregnancy rate of FET patients with peripheral blood CD163+ M2-like monocytes higher than the critical value was higher than that of FET patients with peripheral blood CD163+ M2-like monocytes lower than the critical value (6 cases in each group, 66.7% vs 16.7%, P = 0.01). In addition, CD163+ M2-like monocytes were significantly positively correlated with Th cells (r = 0.525, P = 0.01), and CD163+ M2-like monocytes were positively correlated with the CD4+ Th / CD8+ Ts ratio (r = 0.445, P = 0.02). These results indicate that peripheral blood CD163+ M2-like monocytes can be used as an indicator for predicting the pregnancy outcome of patients during the FET cycle.

[0013] In summary, the present application has the following beneficial effects:

[0014] The expression of peripheral blood CD163+ M2-like monocytes in the pregnancy group of women undergoing the first frozen embryo transfer was significantly higher than that in the non-pregnancy group (P = 0.0137), and the receiver operating characteristic curve (ROC) value of peripheral blood CD163+ M2-like monocytes in predicting pregnancy outcome was 0.671. According to the critical value of CD163+ M2-like monocytes in the ROC curve (40.6%), an independent cohort study further found that the clinical pregnancy rate of FET patients with peripheral blood CD163+ M2-like monocytes higher than the critical value was higher than that of FET patients with peripheral blood CD163+ M2-like monocytes lower than the critical value (P = 0.01). These results indicate that peripheral blood CD163+ M2-like monocytes can be used as an indicator for predicting the clinical pregnancy outcome of FET patients.

[0015] In summary, peripheral blood CD163+ M2-like monocytes are involved in regulating the immune balance of embryo implantation and can be used as an indicator for predicting the clinical pregnancy outcome of FET patients. Description of the Drawings

[0016] Figure 1A: Among patients who underwent the first frozen embryo transfer, the percentage of CD163+ M2-like monocytes in the peripheral blood of the continuous pregnancy group was higher than that in the non-pregnancy subgroup (P = 0.029). B: ROC curve of CD163+ M2-like monocytes predicting pregnancy outcome. C: The pregnancy rate of women with high expression of CD163+ M2-like monocytes was higher than that of women with low expression of CD163+ M2-like monocytes (66.7% vs 16.7%, P = 0.01). Detailed implementation manners

[0017] The present invention will be further described below in conjunction with the detailed implementation manners. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0018] Example 1

[0019] 1. Materials and methods

[0020] 1.1 Research subjects: Fifty patients who underwent frozen embryo transfer in the Reproductive Medicine Center of Zhongshan Hospital Affiliated to Fudan University were recruited from December 2022 to February 2023.

[0021] Inclusion criteria: ① Aged between 20 and 42 years old; ② Having high-quality blastocysts on the 5th or 6th day; ③ The endometrial thickness on the day of transplantation was greater than or equal to 7 mm. The judgment of high-quality blastocysts was based on Gardner grading, and the inner cell mass and trophoblast cells of the blastocysts were scored respectively. Those with a score of ≥ BB or above were high-quality blastocysts.

[0022] Exclusion criteria: Related factors affecting the endometrium and immunity such as intrauterine adhesions, intrauterine infections, endometrial polyps, submucous myomas of the uterus, uterine malformations (unicornuate uterus, septate uterus, etc.), recurrent miscarriages, and repeated implantation failures.

[0023] This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Committee of Zhongshan Hospital, Fudan University (approval number: B2022-462). All patients undergoing frozen embryo transfer received hormone replacement therapy (HRT) cycles and at least one high-quality blastocyst was transferred. All patients were collected in accordance with the informed consent of the registered study at Zhongshan Hospital, Fudan University. Clinical information was statistically analyzed anonymously, and relevant clinical data were retrospectively analyzed. Biochemical pregnancy was defined as a human chorionic gonadotropin (hCG) value >5 miu / ml, and clinical pregnancy was defined as the observation of a gestational sac by B-ultrasound on the 28th day after frozen embryo transfer. Based on http: / / riskcalc.org:3838 / samplesize, at an efficiency of 90%, a two-sided α = 0.05 significance level, and an assumed dropout rate of 20%, considering a 1:1 ratio between the pregnancy group and the non-pregnancy group, the total sample size required was 12 cases, with 6 cases in the pregnancy group and 6 cases in the non-pregnancy group.

[0024] 1.2 Detection of peripheral blood immune cells: 1-2 ml of peripheral blood was drawn from patients on the morning of the blastocyst transfer day into EDTA anticoagulated blood. CD68 (Y1 / 82A; BD Biosciences, San Jose, CA, USA) was used to label total mononuclear macrophages in peripheral blood, CD86 was used to label M1-like macrophages in peripheral blood, CD163 and CD206 were used to identify M2-like macrophages in peripheral blood, CD19 was used to label B cells, CD3 was used to label T cells, CD4 was used to label Th cells, CD8 was used to label T cells, and CD56+CD16+ was used to label NK cells. The cells were fixed with 4% paraformaldehyde and permeabilized with FACS permeabilization solution (BD PharMingen, San Diego, CA, USA). PBMCs were gated by FSC×SSC. Flow cytometry analysis was performed on a FACS Aria II flow cytometer (BD Biosciences), and data analysis was performed using FlowJo 7.6.1 (Tree Star, San Carlos, CA, USA) software.

[0025] 1.3 Detection of sex hormones and cytokines: A Roche Cobas 6000 (Roche, Switzerland) machine was used to detect hormone concentrations, including β-human chorionic gonadotropin (β-hCG), estrogen, progesterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). The assay was an enzyme-linked immunosorbent assay, and the detectable concentration range of total β-hCG was 0.2 IU / mL to 10,000 IU / ml. Serum concentrations of interleukin (IL)-1β, IL-2a, IL-6, IL-8, and tumor necrosis factor (TNF)-α were measured using an IMMULITE 1000 immunoassay (Siemens Healthcare Diagnostics GmbH, Berlin, Germany), which uses chemiluminescent immunoassay technology.

[0026] 1.4 Statistical analysis: Statistical analysis was performed using IBM SPSS v.23 (IBM Corporation Inc.) statistical software. Before using parametric statistics, continuous variables were tested for normal distribution and presented as mean ± standard deviation (SD). Therefore, the t-test or Mann–Whitney U test was selected to evaluate statistically significant differences. The t-test was used for clinical characteristics. The chi-square test was used to analyze categorical variables, including implantation rate, biochemical pregnancy rate, clinical pregnancy rate, multiple pregnancy rate, miscarriage rate, and live birth rate. The non-parametric Wilcoxon signed-rank test was used to compare differences between groups. The correlation between cytokines and M2-like macrophages was analyzed by Pearson correlation coefficient. A P < 0.05 was set as the statistical significance. The time-dependent receiver operating characteristic (ROC) curve was analyzed using the "survivalROC" package in R.

[0027] 2. Results:

[0028] 2.1 Clinical characteristics of the pregnant and non-pregnant groups

[0029] This study retrospectively analyzed 50 patients who underwent FET for the first time, of whom 27 were pregnant and 23 were non-pregnant. The clinical characteristics are shown in Table 1. There were no statistically significant differences in age, endometrial factors (including serum estrogen, progesterone, LH value, endometrial thickness on the day of secretory transformation, etc.), and the number of good-quality embryos between pregnant and non-pregnant patients (Table 1).

[0030] Table 1: Comparison of clinical characteristics of patients in the pregnant and non-pregnant groups in the first frozen embryo transfer

[0031]

[0032]

[0033] 2.2 Comparison of peripheral blood immune cells and cytokines between the pregnant and non-pregnant groups

[0034] The percentage of CD163+ M2-like monocytes in pregnant women was higher than that in non-pregnant women (68.78±5.02% vs 52.41±6.27%, P = 0.01, Table 2). In contrast, there were no changes in the immune cells of CD19+ B cells, CD3+ T cells, CD4+ Th cells, CD8+ Ts cells, CD4+ / CD8+ ratio, CD56+ NK cells, CD68+ monocytes, CD86+ M1-like monocytes, and D206+ M2-like monocytes in the peripheral blood of the two groups of patients (P>0.05, Table 2). In addition, there were no significant changes in the cytokine expression levels of TNF-a, IFN-r, IL-1β, IL-2, IL-6, and IL-8 in the peripheral blood of the two groups of patients (P>0.05, Table 2).

[0035] Table 2: Comparison of peripheral blood immune cells and immune factors

[0036]

[0037]

[0038] 2.3. Correlation analysis of peripheral blood CD163+ M2-like monocytes and other immune factors

[0039] As shown in Table 3, CD163+ M2-like monocytes were significantly positively correlated with Th cells (r = 0.525, P = 0.01), and CD163+ M2-like monocytes were positively correlated with the CD4+ Th / CD8+ Ts ratio (r = 0.445, P = 0.02). The correlation between CD163+ M2-like monocytes and TNF-a was almost negative (r = -0.380, P = 0.055). There was no significant correlation between CD163+ M2-like monocytes and T cells, B cells, NK cells, TNF-a, IL-1β, IL-2r, IL-6, and IL-8.

[0040] Table 3: Correlation analysis of CD163+ M2-like macrophages and other immune cells / factors

[0041] Immune factor r value P value CD19+B cell -0.140 0.50 CD3+T cell 0.227 0.27 CD4+Th cell 0.525 0.01 CD8+Ts cell -0.261 0.20 CD4+ / CD8+ ratio 0.445 0.02 CD56+NK cell -0.144 0.48 TNF-α -0.380 0.06 IL-1β -0.28 0.16 IL-2r -0.361 0.07 IL-6 -0.198 0.33 IL-8 -0.278 0.17

[0042] 2.4 CD163+ M2-like monocytes in the prognostic model of the first pregnancy in FET women

[0043] Among the patients with the first FET pregnancy, the abortion rate was 14.8%, and the continuous pregnancy rate and live birth rate were 63.0% and 63.0% respectively (Table 1). It is worth noting that the percentage of peripheral CD163+ M2-like monocytes in the continuous pregnancy subgroup was higher than that in the non-pregnant subgroup (P = 0.029, Figure 1A). In contrast, there were no significant differences in the percentage of peripheral CD163+ M2-like monocytes between the non-pregnant subgroup and the biochemical pregnancy subgroup, and between the non-pregnant subgroup and the miscarriage subgroup ( Figure 1 A). The ROC curve of the first clinical pregnancy of FE patients was established based on multivariate logistic regression, with a critical value of 40.6 and a nomogram AUC of 0.671( Figure 1 B). According to this critical value, the newly recruited FET patients were divided into a CD163+ high-expression subgroup and a CD163+ low-expression subgroup. The pregnancy rate of women in the high-expression group of CD163+ M2-like monocytes was significantly higher than that of women in the low-expression group of CD163+ M2-like monocytes (66.7% vs 16.7%, P = 0.01, Figure 1 C).

[0044] 3. Discussion

[0045] The experiments of the present invention confirmed that during the implantation window period of the first FET patients, the expression of CD163+ M2 monocytes in peripheral blood of the pregnancy group was higher than that of the non-pregnant group, and the expression of M2-like monocytes in the first FET women was positively correlated with CD4+ Th cells. According to the critical value of 40.6, the pregnancy rate of patients with a higher number of peripheral blood CD163+ M2-like monocytes was higher than that of patients with a lower number of CD163+ M2-like monocytes. These results provide evidence for the peripheral blood CD163+ M2 monocyte value to predict the clinical pregnancy outcome of embryo transfer in the first FET patients, which may help patients make clinical decisions in the first embryo transfer cycle. The importance of peripheral blood CD163+ M2-like monocytes in predicting the first embryo implantation and continued pregnancy in FET patients suggests that it may be a biomarker for the FET cycle.

[0046] This specific embodiment is only an interpretation of the present application and does not limit the present application. Those skilled in the art can make modifications to this embodiment without creative contributions according to their needs after reading this specification, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A kit for predicting the clinical pregnancy outcome of the first frozen embryo transfer, characterized in that, The kit includes immune cells and immune factors in blood; The immune cells include CD19+ B cells, CD3+ T cells, CD4+ Th cells, CD8+ Ts cells, CD4+ / CD8+ ratio, CD56+ NK cells, CD68+ monocytes, CD86+ M1-like monocytes, D206+ M2-like monocytes, CD163+ M2-like monocytes; The immune factors include IL-1β, IL-2r, IL-6, IL-8, and TNF-α.

2. The kit according to claim 1, wherein The immune cells are selected from CD163+ M2-like monocytes.

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