The possible role of interleukin-6 (IL-6) and survivin in the pathogenesis of embryonic damage

Xiu-Rong Wang,Kui-Mei Zhang,Xiao-Ying Meng,Xue-Xue Li,Wen-Ming Cao*

1Changle People's Hospital,Weifang 262400,Shandong,China.

Abstract Objective:To investigate the role of interleukin-6 (IL-6) and survivin in the pathogenesis of embryonic damage and to provide theoretical basis for the diagnosis and treatment of embryonic arrest.Methods:A total of 50 patients with embryo arrest for unknown reasons in People's Hospital of Changle County,Shandong Province were selected as the embryo termination group,and 50 patients with normal pregnancy were selected as the normal pregnancy group.Serum and chorionic tissue of each group were collected,and the serum IL-6 concentration of the subjects in both groups was determined by enzymatic linked immunosorbency assay.The mRNA expression levels of interleukin 6 and survivin in villus were determined by real-time quantitative PCR.The expression and distribution of interleukin 6 and survivin in villi were detected by SP immunohistochemical method.Results:The mRNA and protein levels of survivin in embryo suspension group were significantly lower than those in normal pregnancy group.Conclusion:The normal expression of interleukin-6 and survivin in the early pregnancy villus tissue is very important to maintain the normal development of early embryo.

Key words:Interleukin-6 (IL-6),Survivin,Embryo damage

Background

With the comprehensive opening of the two-child birth policy in China,the smooth birth of healthy babies plays a more important role in maintaining family stability,women's health and fertility.At present,the early embryonic arrest etiology in addition to the known chromosomal abnormalities,endocrine,infection,immune,environmental and other factors,50% of patients with unknown causes,embryo damage seriously affect women's physical and mental health.Studies showed that embryo arrest and the incidence of spontaneous abortion are rising trend,natural abortion caused by embryonic arrest accounted for 10% to 20%of pregnancy [1-2].It has been found that successful pregnancy depends on necessary trophoblast cell apoptosis,and embryo termination may be related to excessive trophoblast cell apoptosis [1-2].

This study by discussing interleukin 6 (IL-6) and survivin unexplained early embryonic damage and at the same time in normal pregnancy abortion voluntary requirements suction palace organization,villi tissue and serum in patients with uterine decidua expression differences in the organization,and to explore its role in the embryonic development stops,aimed at from the perspective of cell and molecular elucidate the pathogenesis of early embryonic damage,provide theoretical basis for the diagnosis and treatment of embryonic damage.

Materials and methods

Data Sources

A total of 50 patients with unexplained embryonic arrest in the department of gynaecology of our hospital from October 2019 to October 2020 were selected as the embryo termination group,and another 50 patients with normal pregnancy during the same period were selected as the normal pregnancy group.The mean age and gestation time of the embryo suspension group were (29.3±4.2) years and (74.1±23.1) days.The mean age and gestation time of normal pregnancy group were (28.13±5.9) years and (72.5±19.6) days.There was no significant difference in age and other general data between the two groups (P＞0.05),indicating comparability.All subjects had no serious infectious diseases,medical diseases,reproductive organ diseases,adverse pregnancy history,and marital chromosomal abnormalities that might affect the study.

Methods

Determination of serum IL-6 concentration

Fastening venous blood (4ml) was collected from both groups of subjects and centrifuged to obtain two serum samples.Serum interleukin-6 concentration was determined by enzyme-linked immunosorbent assay(ELISA),strictly following the instructions of the human interleukin-6 kit.

The m RNA expression l evels o f I L-6 and s urvivin i n villus tissue were determined by real-time quantitativePolymerase Chain Reaction(PCR)

Part of the villus tissue of the embryo suspension group and the normal pregnancy group was taken and rinsed with normal saline.The blood stains on the surface of the villus tissue were absorbed by sterile filter paper and frozen for storage under aseptic conditions.Real-time quantitative fluorescence PCR was used to detect mRNAs of interleukin-6 and survivin in the villous tissues of the two groups.After the mRNAs were reverse transcripted,we performed real-time quantitative PCR using dye method,and actin was set as the internal reference gene.

IL-6 and s urvivin protein expression were detected by SP immunohistochemical method

The villi of the embryo suspension group and normal pregnancy group were fixed,rinsed,dehydrated,transparent,paraffin embedded after paraffin immersion,sliced into 5μm wax pieces,and baked overnight.Paraffin sections were dewaxed into water,repaired by thermal antigen,sealed with 3% hydrogen peroxide for 30min and 10% sheep serum for 30min.IL-6 and survivin were added overnight,respectively.The secondary antibody was incubated,DAB color was developed,dehydrated and transparent,neutral gum was sealed,and photos were taken.Medical image analysis software IPWIN60 was used for analysis.To each section for the cells under a microscope,the image was shown on the monitor,the determination of the area's cell integral optical density(IOD),area (area) the value of the random observation again 10 vision (400),respectively in each section of the protein content was measured as the average of the group,and finally the average for the whole was as relative strength of the protein in the villus tissues expression [3].

Statistical analysis

SPSS 22.0 statistical software was used,the data were expressed by Mean±SD,and the measurement data were compared by LSD-t test.P<0.05 was considered statistically significant.

Ethics statement

The study has been approved by the Ethics Committee of Changle people's Hospital and put on record in the Ministry of Medicine and the Ministry of Science and Education.

Results

Determination of serum IL-6 concentration

The expression of interleukin-6 in the embryo suspension group was lower than that in the normal pregnancy group,but the difference was not statistically significant (P>0.05),as shown in Table 1.

The mRNA expression levels of IL-6 and survivin in villus determined by real-time quantitative PCR

At the mRNA level,interleukin-6 and survivin were expressed in both the embryo suspension group and the normal pregnancy group,but the positive expression of interleukin-6 (1.35±0.48) and survivin (0.13±0.04) in the embryo suspension group were lower than that in the normal pregnancy group (2.45±1.6,0.88±0.68),and the difference between the two groups was statistically significant (P=0.022,0.0014).

Expression of IL-6 and survivin was detected by SP immunohistochemical method

IL-6 and survivin were positively expressed in villi of the two groups to varying degrees,with tan or yellowish-brown staining.IL-6 was mainly localized in the membrane and cytoplasm of villus tissue,while survivin was in the nucleus and cytoplasm of villus tissue.The expression of both IL-6 and survivin in the embryo termination group was lower than that in the normal early pregnancy group,with statistical significance (P<0.05).See Table 3.

Table 1 Serum expression of IL-6 (Mean±SD)

Table 2 The levels of mRNA expression of IL-6 and Survivin (Mean±SD)

Table 3 Expression of IL-6 and survivin (Mean±SD)

Discussion

First,endocrine disorders,maternal estrogen,progesterone,villous gonadotropin and other factors cannot meet the needs of embryo growth and development,leading to the occurrence of sterility.In addition,luteal insufficiency is also a major factor.Due to insufficient production of progesterone,endometrial dysplasia or endometrial maturation delay for more than 2 days affects egg implantation and development.Second,that is immune factors.Embryos are allotransplantation from the mother,which is a fusion of the parents' genetic genes,different from the mother.Therefore,the immune maladjustment between the mother and the fetus will cause the rejection of the fetus.Common autoimmune diseases include scleroderma and dermatomyositis.Third,uterine abnormalities,intrauterine adhesion,uterine fibroids,thin endometrium and so on can cause sterility [4].Fourth,chromosomal abnormality is the common reason for inducing sterility,accounting for about 55%of the sterility caused by gestational age <8 weeks.The common abnormal karyotype is triploid.Abnormal chromosomal structures include overlap,deletion of chromosomes,balanced translocation,female age over 35 years old,and aging of eggs,which lead to polyploid embryos and abortion [5].Fifth,genital tract infection,rubella virus,cytomegalovirus infection can lead to fetal death in utero.Endocrine changes occur in women during pregnancy,and lower immunity reduces the ability to prevent and control pathogens.Immune deficiency is the direct factor of infection.Viral infections include parvovirus,cytomegalovirus,and hepatitis virus.Seventh,there are many unknown reasons.Through data research,it is found that vascular insufficiency and cell death are also direct factors causing sterility during placenta formation.Abnormal expression of angiogenesis and angiogenic factors in decidua [6].

There are sertoli cell apoptosis in normal physiological activities of pregnancy women in a certain degree [7],which mainly occurred in trophoblast cells.At the same time in the process of embryonic development,the balance between cell apoptosis and proliferation plays an important role in maintaining pregnancy [8],which is conducive to form villous vascular and branch,and to keep related ability.Studies have found that successful pregnancy depends on the necessary trophoblastic cell apoptosis,embryo arrest may be associated with excessive trophoblastic cells apoptosis [9].

The result of this study showed that mRNA and protein levels of survivin in the embryo suspension group were significantly lower than those in the normal pregnancy group,suggesting that the decreased expression of survivin in the cytotrophoblast may affect cell mitosis and hinder cell proliferation and differentiation [10].Trophoblast cells cause a large number of cell apoptosis,block the normal structure of the placenta and endometrial blood vessels,causing the embryo to stop.IL-6 effectively regulates survivin through the STAT transduction pathway [11].The results of this study confirm that low expression of IL-6 in the villous tissue of patients with fetal abortion leads to low expression of survivin,chorionic degeneration and necrosis that affect normal placental function,hinder further development of the embryo,and ultimately cause the embryo to stop developing.In conclusion,the normal expression of IL-6 and survivin in the villi of early pregnancy is important for the maintenance of normal development of early embryos.IL-6 can effectively regulate survivin expression through the STAT transduction pathway.Low expression of IL-6 and survivin may cause the embryo to stop developing.