Method for improving embryo transplantation birth rate of mammals

Abstract

The invention discloses a method for improving embryo transplantation birth rate of mammals, which is characterized in that PBMC (peripheral blood mononuclear cell) is used as a source cell for uterus immune regulation, and the PBMC and an embryo are co-cultured to complete induced transformation of the PBMC. The PBMC subjected to induced transformation and the embryo are transplanted to a receptor uterus together, so that the uterus implantation immune environment is improved, normal implantation of the embryo is helped, and the birth rate of the transplanted embryo is remarkably increased.

Description

Technical Field

[0001] The invention relates to a method for improving the birth rate of mammal embryo transplantation, and belongs to the technical field of animal reproduction. Background Art

[0002] The sperm and egg of mammals combine to form a fertilized egg, which develops into a blastocyst. During the embryo implantation period, the blastocyst hatches from the zona pellucida and invades the receptive endometrium. This process occurs within a limited time called the "implantation window". During this period, the maternal endometrium undergoes drastic changes, collectively referred to as "decidualization". Decidualization is guided not only by maternal factors but also by molecules secreted by the embryo and is highly regulated by multiple factors, which promotes the dialogue between the mother and the fetus at the physiological and molecular levels, thereby initiating the implantation process. Any disorder in the spatiotemporal expression and function of regulatory factors in the embryo-uterus interface may lead to the arrest of embryonic development and death before or after implantation. Therefore, the two-way crosstalk of cells and molecules between the blastocyst and the uterus is a key rate-limiting step for successful pregnancy. Endometrial receptivity requires the carefully coordinated interaction of various types of cells, such as epithelial cells, stromal cells, and immune cells. Embryo implantation is also regulated by several different cellular components, including chemokines, growth factors, and cytokines. Among them, specific types of immune cells and immune molecules play an important role in maternal-fetal communication.

[0003] Assisted reproductive technology plays an important role in the breeding and variety improvement of livestock animals. Despite the continuous progress of assisted reproductive technology, the pregnancy success rate of transplanted embryos is still very low. Studies have shown that embryo implantation is an inflammatory process. It is believed that genetic heterogeneity determines that the embryo will trigger an immune response in the uterus, but little is known about the basic immunological characteristics of the embryo. Maternal-fetal immune interactions at the implantation interface are very important for the successful implantation of the embryo and subsequent normal pregnancy.

[0004] Co-culture of mammalian preimplantation embryos with several somatic cells, such as fallopian tube epithelial cells and endometrial epithelial cells, has been successfully used to simulate the subphysiological environment of embryonic development and improve the embryonic development ability under in vitro culture conditions. However, no studies have revealed the role of embryos in immune cells under physiological conditions, and it is still unclear what the implantation requirements of blastocysts are for the uterine immune environment.

[0005] Peripheral blood mononuclear cells (PBMCs) are a mixed population of cells with a single nucleus in peripheral blood, mainly including monocytes, dendritic cells and lymphocytes (T cells, B cells and NK cells, etc.). PBMCs are distributed to various tissues and organs throughout the body through blood circulation, and participate in immune response and immune regulation. At present, there is no reported method to improve the birth rate of transplanted embryos by improving the immune status of the recipient uterus during the implantation period. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a method for improving the birth rate of mammalian embryo transplantation.

[0007] Technical solution: The method of the present invention for improving the birth rate of mammalian embryo transplantation comprises the following steps:

[0008] (1) Co-incubating peripheral blood mononuclear cells with embryos;

[0009] (2) After co-incubation, the embryo is transplanted into the uterus.

[0010] Furthermore, the mammal is a mouse.

[0011] Furthermore, the method also includes the step of isolating peripheral blood mononuclear cells.

[0012] Furthermore, the specific operation of step (1) is: incubating peripheral blood mononuclear cells and embryos for 24 hours.

[0013] Furthermore, the number of embryos is 10 to 20.

[0014] Furthermore, the number of embryos is 20.

[0015] Furthermore, the incubation conditions were 37°C, 5% CO 2 .

[0016] Furthermore, the total number of immune cells in peripheral blood mononuclear cells after co-incubation decreased significantly, and the IL-6 factor increased significantly.

[0017] Furthermore, the specific operation of step (2) is: transplanting the co-incubated peripheral blood mononuclear cells together with the 8 embryos.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The method of the present invention uses PBMC as the source cell for uterine immune regulation, co-cultures PBMC with embryos, and completes the induced transformation of PBMC. The induced transformed PBMC is transplanted into the recipient uterus together with the embryo, thereby improving the uterine implantation immune environment, helping the embryo to implant normally, and significantly improving the birth rate of the transplanted embryo. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The following is the operation flow of this application method.

[0020] Figure 2 Flow cytometry was used to detect the expression of cell subsets in PBMC after treatment.

[0021] Figure 3 The expression of cytokines in PBMCs after treatment was detected by ELISA.

[0022] Figure 4 To treat PBMCs to improve reproductive efficiency after embryo transfer. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0024] Example 1

[0025] (1) Isolation of PBMCs

[0026] 1.5 mL of blood was collected from the eyeballs of three healthy mice (Experimental Animal Center of Xinjiang Medical University, 6 weeks old), and mononuclear cells were isolated using a PBMC isolation kit (Tianjin Haoyang Huake Biotechnology Co., Ltd., LDS1090). After removing the separation solution, 5 mL of washing solution was added, and the cells were centrifuged at 250 g for 10 min, and the supernatant was discarded. After washing three times, the cells were resuspended in 2 mL of RPMI-1640 medium (containing 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin double antibody). The cells were counted using a hemocytometer and diluted to an appropriate concentration.

[0027] (2) PBMC and blastocyst co-culture

[0028] (2.1) Female mice (Experimental Animal Center of Xinjiang Medical University, 6 weeks old) were induced to superovulate using PMSG (pregnant mare serum gonadotropin) and HCG (human chorionic gonadotropin). Blastocysts were collected from the uterus 3.5 days after cohabitation with male mice. The collected blastocysts were cultured at 37°C and 5% CO 2 Cultivate in a saturated air humidity environment for later use.

[0029] (2.2) In a 96-well culture plate, 100 μL of PBMC (2×10 4 The experiment was divided into an experimental group and a control group. The experimental group was inoculated with 10 or 20 blastocysts per well (the commonly used reproductive model ICR mice give birth to 10-20 pups per litter, so 10 and 20 embryos were selected); the control group was not inoculated with blastocysts. The culture plate was kept at 37°C and 5% CO 2 Incubate in saturated air humidity environment for 24h.

[0030] (3) Detection of cell subpopulations in cultured cells by flow cytometry

[0031] After 24 hours of co-culture, the changes in the number of PBMCs and the changes in the proportions of several key immune cells were detected by flow cytometry. Specifically, the 96-well plate was removed from the incubator, the culture medium was aspirated into a 1.5 mL centrifuge tube, 400 μL PBS was added, vortexed and mixed, centrifuged at 1200 rpm for 10 min, and 350 μL of supernatant was aspirated into a new centrifuge tube for standby use (for ELISA detection, short-term storage at 4 ° C, long-term storage at -20 ° C). 0.5 μL CD16 / 32 blocking antibody was added to the remaining liquid in the original centrifuge tube, incubated at room temperature for 15 min, and then 1 μL of antibodies CD45-ER780 (Elabscience), CD3-EV450 (Elabscience), CD19-APC (Elabscience), CD4-PE (Elabscience), CD11b-PC5.5 (Elabscience), CD49b-PC7 (Elabscience), and CD11c-FITC (Elabscience) were added, mixed, and incubated at room temperature and away from light for 15 min. After incubation, add 1 mL of PBS and mix well, centrifuge at 1200 rpm and 4°C for 10 min, discard the supernatant, resuspend the cells with 200 μL of PBS, and detect the changes of leukocyte subsets in PBMC by flow cytometry.

[0032] The results show that Figure 2 ), the total number of immune cells (LEU) decreased after treatment. Among them, the number of T cells, which accounted for the largest proportion, decreased significantly. Compared with the control group, the proportion of T cells in the 20 blastocysts treatment group decreased more significantly than that in the 10 blastocysts treatment group.

[0033] (4) ELISA detection of cytokines

[0034] The same method as in (3) was used to collect 100 μL of the culture supernatant and to detect the levels of TNF-α, IL-6, and IL-10 in the culture supernatant using ELISA kits (Elabscience, E-EL-M3063, E-EL-M0044, E-EL-M0046). The ELISA experimental method was operated according to the instructions of the kit: standard wells, blank wells and sample wells were set respectively (three replicate wells in each group, 100 μL of culture supernatant in each well), and the ELISA plate was coated and incubated at 37°C for 90 min; after the incubation, the liquid in the wells was shaken off, patted dry, 100 μL of biotinylated antibody working solution was added to each well, coated, and incubated at 37°C for 1 h; the liquid in the wells was shaken off, patted dry, 200 μL of washing solution was added to each well and soaked for 1 min and then patted dry, after repeated washing three times, 100 μL of enzyme conjugate working solution was added to each well, coated, and incubated at 37°C for 30 min; after drying, the plate was washed 5 times, 90 μL of substrate solution was added to each well, coated, and incubated at 37°C in the dark for 15 min (the time can be shortened or extended as appropriate according to the degree of color development, but not more than 30 min); 50 μL of stop solution was added to each well, and the OD value of each well at a wavelength of 450 nm was measured using an ELISA reader. The standard curve was drawn according to the concentration of the standard wells and the corresponding OD values ​​(TNF-α: y = 48.667x 2 +64.4x+2.7966, R 2 =0.9985;IL-6:y=104.43x 2 +344.83x-35.107, R 2 =0.9995;IL-10:y=506.85x 2 +9.94x+14.67, R 2 =0.9979), and then the cytokine concentration of each well was calculated according to the OD value of the sample well.

[0035] The results show that Figure 3 ), IL-6 increased significantly in the 20 embryos treated group, and decreased significantly after TNF-α treatment.

[0036] Flow cytometry and ELISA data showed that embryo-treated PBMCs could regulate the differentiation of cell subsets and induce immune cell function changes to express differential cytokines. In particular, the 20-embryo treatment group had a significant effect, so the 20-embryo treatment group was selected for subsequent experiments.

[0037] (3) Processed PBMC and blastocysts co-transplanted into recipient mouse uterus

[0038] The above PBMCs co-cultured with 20 blastocysts (treated PBMCs (P20), 5000-8000 cells) or PBMCs not co-cultured with blastocysts (untreated PBMCs (PBMCs)) were surgically transplanted into one side of the uterus (15 recipients) of synchronous estrus recipient mice (Experimental Animal Center of Xinjiang Medical University, 8 weeks old); the control group (Control) was transplanted with only 8 blastocysts. Two days after transplantation, the 5 recipient mice in each group were euthanized and dissected to detect uterine pregnancy, and the embryo implantation rate was calculated; 10 days after transplantation, the 5 recipient mice were euthanized and dissected to detect uterine pregnancy, and the embryo reabsorption rate was calculated. The 5 recipient mice began to give birth to mice 16 days after transplantation, and the mouse birth rate was calculated.

[0039] The results show that Figure 4 ), treated PBMC (P20) for embryo transplantation, the birth rate reached 77.3%, significantly higher than the 43.5% of the untreated PBMC group and the 38.4% of the embryo transplantation control group; further analysis found that the treatment of PBMC increased the embryo implantation rate and significantly reduced the embryo reabsorption rate in the middle and late pregnancy. Compared with the control group (34.5%), the embryo resorption rate of the treated PBMC (P20) group was only 7.7%. The results of the study showed that the treatment induced the functional transformation of PBMC, promoted the implantation of transplanted embryos, reduced the abnormal development of the pregnant fetus, and ultimately significantly improved the terminal development rate of the fetus.

Claims

1. A method for improving the birth rate of mammalian embryo transplantation, characterized in that: The method comprises the following steps: (1) Co-incubating peripheral blood mononuclear cells with embryos; (2) After co-incubation, the embryo is transplanted into the uterus.

2. The method for improving the birth rate of mammalian embryo transplantation according to claim 1, characterized in that: The mammal is mouse.

3. The method for improving the birth rate of mammalian embryo transplantation according to claim 1, characterized in that: The method further comprises the step of isolating peripheral blood mononuclear cells.

4. The method for improving the birth rate of mammalian embryo transplantation according to claim 1, characterized in that: The specific operation of step (1) is: incubating peripheral blood mononuclear cells and embryos for 24 hours.

5. The method for improving the birth rate of mammalian embryo transplantation according to claim 4, characterized in that: The number of embryos is 10 to 20.

6. The method for improving the birth rate of mammalian embryo transplantation according to claim 4, characterized in that: The number of embryos is 20.

7. The method for improving the birth rate of mammalian embryo transplantation according to claim 4, characterized in that: The incubation conditions were 37°C and 5% CO2.

8. The method for improving the birth rate of mammalian embryo transplantation according to claim 1, characterized in that: After co-incubation, the total number of immune cells in peripheral blood mononuclear cells decreased significantly, and the IL-6 factor increased significantly.

9. The method for improving the birth rate of mammalian embryo transplantation according to claim 1, characterized in that: The specific operation of step (2) is: transplanting the co-incubated peripheral blood mononuclear cells together with 8 embryos.

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