| BackgroundEmbryo implantation is a major limiting step in the process of pregnancy, also the beginning of embryonic development. Embryo implantation is a very complex and sophisticated process of maternal-fetal dialogue. On one hand, embryonic development including disappearation of the zona pellucida of oocyte, syncytiotrophoblast differentiation. On the other hand, under the stimulation of sufficient progesterone in pregnant woman, the uterus has a very short sensitive period called the implantation window, allow embryo implantation which is usually seven days after ovulation. Then blastocyst transfer, positioning, adhesion, and penetrate to the ultimate success of implantation. An exception occurs at any step, may lead to implantation failure. Histological studies on the endometrium showed that angiogenic state is at its peak during implantation period. The function of normal angiogenic microenvironment is essential for embryo implantation, placenta formation and the maintenance of pregnancy.In recent years, macrophage accumulation in the window of implantation in the endometrium and be involved in implantation arouse more and more interest. As an important component of the body’s innate immune cells, decidual macrophages have distinct phenotypes. They cannot be simply categorized as pro-inflammatory M1 or anti-inflammatory M2. Embryo implantation cause inflammation of the decidual microenvironment, raised number of immune cells gather, in which macrophages accounted for about 20%.In repeated implantation failure patients, inducing aseptic inflammation in which macrophage obviously gathered, can promote the embryo implantation, increase the pregnancy rate, but the specific mechanism is unclear. The change of endometrial capillaries environmental during implantation window of mouse is the key to the receptivity of endometrium. The positive correlation exists between angiogenesis and the amount of macrophages in mice endometrium. This observation implies that they may be involved in vascular bed development that is essential for the preparation of endometrial receptivity before implantation in addition to.The process of embryo implantation, to some extent, similar to malignant tumor invasion and growth, and tumor associated macrophages participate in angiogenesis. When the peripheral blood mononuclear cells migrate into the tumor tissue, the tumor microenvironment help for the differentiation of tumor-associated macrophages (TAM). Tumor associated macrophages was found an important role in maintaining the balance of local tumor capillary network in, TAM accelerate the growth of blood vessels is pro-angiogenic growth factors and by upregulating the release and can be achieved. These pro-angiogenic factors are also polarized to M2 or M1/M2 mixed phenotype reflect the tumor microenvironment macrophages. TAM most major pro-angiogenic factor is VEGF-A. In many types of human cancer and the level of VEGF-A TAM Density. Such as breast cancer, the tumor hypoxic avascular region, TAM able to produce VEGF-A. Related studies show that NO TAM on Angiogenesis way adjustment may be mainly through the influence of VEGF synthesis indirectly, that together form NO and VEGF angiogenesis regulating functional protein complexes, NO plays an upstream regulator of VEGF synthesis the role of factors that regulate the function of the local NO concentration gradient factor partial pressure of oxygen, NO donors and other closely related:a small amount of NO under hypoxic environment may mitogen-activated protein kinase (MAPK) by wire or p38 kinase pathway regulated VEGF gene expression, during which there may be hypoxia-inducible protein (HIF)-1 and heme oxygenase (HO)-1 participation; but a lot of NO inhibits the expression of VEGF pathway genes is unknown. TAM in the tumor microenvironment angiogenesis status and tumor target therapy has become a hot issue in basic research. Studies have also found that when pregnant mice embryo implantation was significantly higher decidua Mφ, VEGF, iNOS expression of strength, and there is a positive correlation between microvessel density, then the uterus if macrophages are also involved in embryo implantation Construction decidua local vascular microenvironment, and then participate in embryo implantation? This is the subject of research.So far, endometrial macrophages partial suppression method has not been seen at home and abroad, to study the mechanisms involved in embryo implantation. Systemic knockout mice macrophages presence of ovarian vascular network damage, decreased progesterone levels, estrous cycle disorders, impaired h ypothalamic-pituitary-gonadal axis function, which may be the main cause of the model of embryo implantation failure. Ovarian ovulation and pregnancy hormone secretion important role in doubt, knock systemic Mφ model not only affect ovulation and pregnancy intervention to maintain the most important progestin, and can not explain Mφ itself in the uterine implantation within the critical period the specific role played by local film, so as to avoid the influence of ovarian and systemic Mcp, and we hope that through the establishment of uterine implantation mouse macrophage inhibitory partial model for studies in mice endometrial macrophages (macrophages, Mφ) on endometrial receptivity and embryo implantation process.Liposomes are closed vesicles having a synthetic bilayer structure. Membrane fusion and the use of energy characteristics of liposomes as drug carriers into the interior of a cell, in order to achieve the purpose of targeting. Clodronate soluble state can not cross the liposome and cell membrane phospholipid bilayer, when encapsulated in liposomes, you are ready to be macrophage phagocytosis, macrophages destroy lipid esterase phosphine after the body clodronate is released into the cytoplasm, reach a certain concentration can cause apoptosis in macrophages. Some scholars have reported that the local effects of liposomal clodronate (clodronate liposomes, CL) in the testis, ovary experimental research, can effectively eliminate local macrophages. Therefore, this study macrophage-specific scavenger clodronate liposomes, macrophage inhibitory established mouse model; and critical implantation period in mice suppress endometrial local Mφ, watching bed point and non-point in its implantation VEGFA, iNOS expression influence and impact on the embryo implantation.The study of angiogenesis regulatory mechanism endometrial microenvironment, help to further understand the meaning of endometrial receptivity, and to explore new targets for intervention endometrial receptivity adjusted to provide more adequate scientific basis, and to promote implantation dysfunction (repeated in vitro fertilization and embryo transfer failure and habitual abortion), unexplained infertility angiogenic therapy experimental basis, but also to carry out abnormal uterine bleeding, immune contraception, organ transplantation provide new ideas. Mechanism of implantation is important for improving female fertility and infertility and recurrent miscarriages and other related diseases.PurposeEstablish endometrium macrophages suppressed mice model to observe its influence on embryo implantation, and on vascular endothelial growth factor A (VEGFA), iNOS expression during implantation. To observe the different distribution of VEGFA, iNOS at implantation sites and non-implantation sites and further explore its role in embryo implantation.Method30 Estrus mice were taken for the study, determined by the vaginal cells histological changes.Mice were randomly divided into two injection routes of administration, intravenous injection (n=15) and intrauterine injection (n=15). Tail vein group, the mice were randomly divided into three groups, the experimental group (n=5) slow injection from the tail vein wrapped clodronate liposome suspension 0.1ml/10g, the control group (n=5) wrapped PBS liposomes, control group (n=5) was injected with sterile PBS. Intrauterine injection, the mice were randomly divided into experimental group (n=5), the control group (n=5) and control group (n=5), with a micro-syringe, surgery under direct vision from the ovaries to the uterine horn side Palace articular injection wrapped clodronate liposome suspensions, liposomes, and wrapped with PBS sterilized PBS15ul. After injection 48h, using immunohistochemistry, flow cytometry method to detect radio clodronate liposomes suppression efficiency of the uterus F4/80-positive macrophages. Establishing merit-based selection injected mouse endometrium during implantation macrophage inhibitory model within the uterine cavity. Take D3.5 (see the next morning after a vaginal suppository for the female mating DO.5) pregnant rats were randomly divided into three groups, the experimental group and control group and blank group. Experimental group (n=10) using unilateral intrauterine injection wrapped with clodronate liposomes control group (n=10) have PBS liposomes to package unilateral intrauterine injection, the control group (n=10) injected into the unilateral uterine volume autoclaved PBS solution. Each group in the uterine cavity after injection 48H, get D5.5 mouse uterus and ovaries, according to the packet, the number of statistics for each embryo implantation unilateral uterus, staining tissue morphology. Immunohistochemical of the endometrium, ovary Mφ number of changes. Flow cytometry to detect uterine F4/80+CD11b+Mφ relative percentages, selective injection of CL Verify on implantation in the uterus Mφ inhibition. The number of Mφ, VEGFA, iNOS expression in implantation sites and non-implantation sites localized and quantified by immunohistochemistry and to explore the relationship between these indicators.Result1) after intrauterine injection, HE staining of mouse uterine shows complete, clear glandular structures, no inflammatory cell infiltration.2) after 48h of tail vein injection or intrauterine injection, estrus mice with immunohistochemical methods shows that there were statistically significant differences in terms of macrophages numbers among the three groups. Two methods in the degree of inhibition of endometrial macrophages was no significant difference (P=0.788).3) Through intrauterine injection, the number of macrophages in ovary among the experimental group the control group, and the blank group showed no significant difference (P=0.762). With the tail vein injection of liposome, control group and blank group are similar to the intrauterine injection, but the experimental group of ovarian macrophages indeed decreased, the difference was statistically significant (P <0.01). Ovarian macrophages significantly inhibited in tail vein injection compared to intrauterine (P<0.05).4) flow cytometry to detect F4/80+macrophages in mice by tail vein injection, intrauterine injection after 48h. F4/80 macrophages in experimental group were significantly decreased, compared to the control group and the blank group, the difference was statistically significant.5) at embryo implantation stage, immunohistochemical shows the left side of the experimental group injected CL endometrial Mφ number is 22.50 ± 8.15 was significantly lower than PL side(83.60 ± 12.15),as well as the left side of the control group and blank group (P<0.05), the right of the three groups had no significant difference. Non-implantation sites alike. Macrophages at IS were higher than that at nonIS, macrophages have a tendency to gather at IS.6) 48h after intrauterine injection of CL, with single-cell suspensions flow cytometry to detect the relative proportions of F4/80+CD11b+Mφ. the left uterine of the experimental group, decreased significantly, inhibition was 74%, compared to uterus tissue of mice the experimental group were injected with PL side, and the control group each side of the blank group.7) after injection of CL in the experimental group, Mφ number in ovaries has no significant difference compared to the control group and blank group.8) When the macrophages were suppressed in uterus during implantation, trypan blue staining showed significant reduction in the number of embryo implantation, embryo implantation is partially suppressed. The left side of the experimental group after injection of CL embryo implantation average number is 2.20 ± 1.81, obviously lower than the right side of the experimental group were injected with PL, the left side of the blank group and the left side of control group, the average bed point respectively: 5.10 ± 1.91,5.00 ± 2.21,5.80 ± 2.25 (P<0.05), no significant difference between the three groups on the right.9) from the uterus of pregnant mice with IS section histology observed in the experimental group were injected with CL side of the bed only embryonic point, although embryonic structures visible, but not closed uterine decidua range of the experimental group compared with PL narrow side. PL mice with bed side point epithelial edema, interstitial cells of decidua obviously, uterine closure.10) in the IS, with the suppression of uterus Mφ, VEGFA expression in endometrial of experimental group injected with CL was significantly lower than the right side of the injection PL (P<0.05), non-implantation site expression in the experimental group injected CL side VEGFA decreased, but the difference was not statistically significant (P=0.069), VEGFA expression in the experimental group on both sides of the bed points were higher than the point of beds The difference was statistically significant (P<0.05).11) iNOS distribution in the uterus of the experimental group,,there is no significant difference between uterus injected with CL and PL(P=0.552) the number of iNOS positive cells in the endometrium. No significant difference (p=0.711) expression in non-implantation sites between CL and PL side. iNOS positive cells in IS higher than that in non-IS in Experimental group PL side of the uteru (P<0.05). The CL side is not (P=0.118).Conclusion1) Strict adherence to aseptic intrauterine injection does not affect the integrity of the uterine tissue, as intrauterine route of administration.2) the use of specific macrophage scavenger clodronate liposomes, in two ways:by intravenous injection and intrauterine injection can effectively inhibit uterine mouse macrophages, and intrauterine injection in surgery operation does not affect the integrity of the uterus, by intrauterine injection can effectively avoid the impact of the ovaries, and the drug can be administered in one side of each other on both sides of the uterus so that the control for precise endometrial macrophage function It provides a good animal model, at home and abroad for the first time.3) partial inhibition of uterine macrophages leads to embryo implantation rate decreased implantation site Decidualization incomplete uterine cavity can not be closed, prove endometrium Mφ necessary for the embryo implantation.4) When inhibiting implantation of endometrial Mφ after implantation point VEGFA expression consistent with reduced inhibition of macrophages, suggesting Mφ possibly by regulating the expression of VEGFA, and participate in the construction of local decidual angiogenesis microenvironment, thereby influence endometrial receptivity and embryo implantation.5) uterine iNOS expression does not reduce with uterine inhibition of macrophage, and the expression may be regulated by other ways not only by macrophages.6) Mφ, VEGFA, iNOS expression in the implantation point higher than that of the implantation point, the difference is further illustrated Mφ, VEGFA, iNOS is not only associated with the implantation of the embryo, may also be involved in the selection of implantation point, the specific mechanisms needed further research. |
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