Abstract:Objective:To investigate the effect of lymphocyte active immunotherapy on the prevention and treatment of recurrent miscarriage of unknown cause and pregnancy outcome. Methods:900 patients with recurrent miscarriage of unknown cause were randomly divided into treatment group and western medicine control group and Chinese medicine control group, 300 cases in each group. In the treatment group, lymphocyte active immunotherapy was given, and in the western medicine control group, didrogestrel tablets were given 10 mg each time, 3 times a day; in the Chinese medicine control group, fast-acting fetal preservation tablets were given orally, 3 tablets each time, 3 times a day. Results: After treatment, the fetal survival rates at 65 days and 12 weeks of gestation were 88.9% and 84.2% in the treatment group, 58.3% and 46.1% in the western medicine control group, and 51.7% and 40.1% in the traditional Chinese medicine control group, respectively, and the differences between the treatment group and the two control groups were statistically significant (P < 0.05). The miscarriage rates at 65 d and 12 weeks of gestation were 11.1% and 15.8% in the treatment group, 41.7% and 53.9% in the western medicine control group, and 48.2% and 59.9% in the traditional Chinese medicine control group, respectively, and the differences were statistically significant when comparing the treatment group with the two control groups (P < 0.05); the levels of IL-4 and IL-6 increased and the levels of IFN-γ and IL-2 decreased in the treatment group, and the differences were statistically significant when comparing the two control groups (P < 0.05). Conclusion:Lymphocyte active immunotherapy can enhance the immune tolerance of patients with recurrent miscarriage of unknown cause, significantly reduce the miscarriage rate and improve the fetal survival rate by regulating the conversion of Th1-type immune response to Th2-type, which is an effective treatment for recurrent miscarriage of unknown cause. Wei Aiwu, Reproduction Center, The First Affiliated Hospital of Henan College of Traditional Chinese Medicine
Key words: recurrent miscarriage; active immunization of lymphocytes; didrogestrel tablets; fast-acting fetus-protecting spirit tablets; active immunotherapy
The etiology of recurrent miscarriage involves genetic, immune, anatomical, endocrine, infectious, and adverse lifestyle habits, but the cause is still unknown in more than 40%-60% of patients [1]. It is called unexplained recurrent spontaneous abortion (URSA). There are many studies on the treatment of URSA, and most of them show that for a normal pregnancy the mother must develop immune tolerance, and any cause that interferes with immune tolerance can lead to the occurrence of miscarriage, while herbal medicine, progesterone and active immunity can enhance maternal immune tolerance, but the efficacy is uncertain. And there is a great controversy about the involvement of lymphocyte active immunity in the treatment of this disease, and its effectiveness. In this study, we compared the effects of lymphocyte active immunity, didrogestrel and rapid-acting fetal preservation tablets on the miscarriage rate and fetal survival rate of URSA, and preliminarily investigated the mechanism of action of lymphocyte active immunity on URSA.
1. Materials and methods
1.1 General information
The study cases were 900 patients with URSA in the reproductive medicine clinic of the First Affiliated Hospital of Henan College of Traditional Chinese Medicine from August 2011 to September 2012, and all patients were divided into treatment group and western medicine control group and Chinese medicine control group by random number table method using single-blind method, 300 cases in each group. The treatment group was 22-43 (28.35±2.25) years old with 2-8 (2.70±0.40) miscarriages; the western medicine control group was 23-45 (29.30±2.82) years old with 2-8 (2.47±0.45) miscarriages; the traditional Chinese medicine control group was 22-47 (29.19±2.51) years old with 2-7 (2.63± 0.54). The age and number of miscarriages in the three groups were statistically treated (P>0.05) and were comparable.
1.2 Case inclusion criteria
① Recurrent miscarriage, two or more consecutive spontaneous miscarriages in early pregnancy; ② No abnormal findings by the examination of all relevant indexes.
1.3 Case exclusion criteria
(1) abnormal karyotype analysis of both parties; (2) ABO and/or RH blood group incompatibility between husband and wife; (3) abnormal endocrine test of female; (4) organic lesions such as malformation of female reproductive tract; (5) positive closed antibody, anti-endometrial antibody, anti-sperm antibody, anti-cardiolipin antibody, anti-ovarian antibody and anti-nuclear antibody; (6) reproductive tract infection confirmed by TORCH, chlamydia and mycoplasma examination; (7) male partner (7) abnormal semen routine analysis; (8) allergic and hypersensitive to various drugs; (9) history of autoimmune diseases and/or other medical and surgical diseases; (10) combined neurological and psychiatric disorders that prevent cooperation or are unwilling to cooperate.
1.4 Treatment methods
In the treatment group, lymphocyte active immunotherapy was given as follows: 30 mL of venous blood from the elbow was stored after the husband or third-party infectious diseases were excluded by examination, and lymphocytes were routinely isolated and extracted under aseptic conditions, and the concentration of lymphocytes was adjusted to ( 2-3) × 1010L-1, and the amount of suspension was prepared to be about 1 mL. 1 mL was injected every 7 d. In the western control group, the tablets of dydrogesterone (Solvay Pharmaceuticals B.V., Netherlands) were used. The western drug control group was administered with 10 mg of drospirenone tablets (manufactured by Solvay Pharmaceuticals B.V. Netherlands, approval number: H20090470) orally twice a day. In the Chinese medicine control group, 3 tablets were taken orally 3 times a day. All three groups started treatment from 30 d of menopause when blood β-HCG was measured to indicate pregnancy, and the treatment was continued until 45 d of pregnancy when ectopic pregnancy was detected by ultrasound, and the rest of the cases continued treatment until 12 weeks of pregnancy.
1.5 Observed indicators
(1) Fetal survival rate and miscarriage rate from 65 d to 12 weeks of gestation in the three groups;
(2) Fetal survival rate and miscarriage rate from 12 weeks of gestation in the three groups;
③The levels of Th1 cytokines IFN-γ and IL-2; the levels of Th2 cytokines IL-4 and IL-6; the levels of Th1/Th2 before and after treatment in the three groups (Th1 is
the sum of IFN-γ and IL-2 values, Th2 is the sum of IL-4 and IL-6 values).
Methods: 2 ml of sterile peripheral venous blood was drawn from the subjects before and after treatment, and the serum was separated and the levels of IFN-γ, IL-2, IL-4 and IL-6 in the serum were determined by enzyme-linked immunosorbent double antibody sandwich (ELISA) method, and the assay procedure was carried out strictly according to the kit instructions.
1.7 Statistical methods
SPSS15.0 software was used for statistical analysis, and the measurement data were expressed as mean ± standard deviation, and the comparison between groups before and after treatment was performed by paired samples t-test, and the comparison between the three groups was performed by one-way ANOVA q-test, and the count data were tested with the test level a=0.05, and P < 0.05 was considered statistically significant.
2. Results
There were 0 cases of ectopic pregnancy and 300 cases of intrauterine pregnancy in the treatment group, including 2 cases of poor compliance and 1 case of shedding, and 297 cases were actually involved in the statistical cases. In the western medicine control group, there were 2 cases of ectopic pregnancy and 298 cases of intrauterine pregnancy, including 1 case of poor compliance and 2 cases of shedding, and the actual number of cases involved in the statistics was 295. In the Chinese medicine control group, there were 1 ectopic pregnancy and 299 intrauterine pregnancies, including 3 cases of poor compliance and 2 cases of shedding, and 294 cases were actually involved in the statistics.
2.1 Fetal survival rate and miscarriage rate from pregnancy to 65 d of gestation in the three groups
After treatment, the treatment group could significantly improve the fetal survival rate and reduce the miscarriage rate compared with the western medicine control group and the Chinese medicine control group, and there were significant differences (P<0.05). See Table 1.
Table 1 Pregnancy outcome at 65 d after treatment in the three groups
Group Number of cases 65d fetal survival Number of 65d miscarriage Fetal survival rate Miscarriage rate
Western medicine control group 295 172 123 58.3▲▲▲ 41.7★★
Chinese medicine control group 294 152 142 51.7 48.2
Treatment group 297 264 33 88.9▲ 11.1★★
Note: Compared with the two control groups ▲ P<0.05, ★ P<0.05, compared with the herbal control group ▲▲ P>0.05,★★★ P>0.05,
2.2 Fetal survival rate and miscarriage rate from pregnancy to 12 weeks of gestation in the three groups
After treatment, the treatment group could significantly improve the fetal survival rate and reduce the miscarriage rate compared with the Western medicine control group and the Chinese medicine control group, and there were significant differences (P<0.05). See Table 2.
Table 2 Pregnancy outcome at 12 weeks of gestation after treatment in three groups of patients with unexplained recurrent miscarriage
Group Number of cases Number of fetal survival at 12 weeks Number of miscarriages at 12 weeks Fetal survival rate Miscarriage rate
Western medicine control group 295 136 159 46.1▲▲ 53.9★★★
Chinese medicine control group 294 118 176 40.1 59.9
Treatment group 297 250 47 84.2▲ 15.8★★
Note: Compared with the two control groups ▲ P<0.05, ★ P<0.05, compared with the Chinese medicine control group ▲▲ P>0.05, ★★★ P>0.05
2.3 Comparison of Th1, Th2 and Th1/Th2 levels before and after treatment in fetal survivors at 65 days of gestation in each group, see Table 3
The differences in serum IFN-γ, IL-2, IL-4, IL-6 and Th1/Th2 levels before treatment were not statistically significant in the three groups (P>0.05); the serum IFN-γ, IL-2, Th1/Th2 levels decreased significantly and IL-4 and IL-6 levels increased significantly after treatment compared with those before treatment in the three groups (P<0.05); after treatment, the treatment group could significantly increase IL-4 and IL-6 levels compared with the two control groups. The treatment group was able to significantly increase IL-4 and IL-6 levels (P<0.05) and significantly decrease IFN-γ, IL-2, and Th1/Th2 levels (P<0.05) compared with the two control groups.
Table 3 Th1, Th2 and Th1/Th2 levels (pg/ml) before and after treatment in patients with unexplained recurrent miscarriage in three groups with a viable fetus at 65 days of gestation
Group Number of cases IFN-γ IL-2 IL-4 IL-6 Th1/Th2
Western medicine control group 172 Before treatment 19.65±8.89 21.93±6.37 0.62±0.44 0.59±0.35 2.19±0.37
After treatment 10.46±4.59★●10.89±3.62★●1.10±0.37★●1.08±0.39★●1.51±0.44★●
Chinese medicine control group 152 Before treatment 19.88±8.54 22.10±7.15 0.64±0.40 0.52±0.32 2.12±0.34
After treatment 10.21±4.36★ 11.06±3.68★ 0.99±0.50★ 1.04±0.35★ 1.60±0.32★
Treatment group 264 Pre-treatment 18.88±9.37▲ 20.84±7.21▲ 0.59±0.38▲ 0.52±0.36▲ 2.10±0.32▲
After treatment 7.69±3.66★◆6.27±3.78★◆ 1.88±0.41★◆ 1.76±0.40★◆1.25±0.42★◆
Note: Comparison of the three groups before treatment ▲P>0.05, comparison of the three groups after treatment and before treatment ★P<0.05, comparison of the treatment group after treatment and the two control groups ◆P<0.05, comparison of the western control group after treatment and the herbal control group ●P>0.05.
22.4 Comparison of Th1, Th2 and Th1/Th2 levels before and after treatment among the three groups of fetal survivors at 12 weeks of gestation
There was no significant difference in the comparison of serum IFN-γ, IL-2, IL-4, IL-6 and Th1/Th2 levels before treatment in the three groups (P>0.05); serum IFN-γ, IL-2, Th1/Th2 levels decreased significantly and IL-4 and IL-6 levels increased significantly after treatment in each group (P<0.05); after treatment, the treatment group could significantly reduce IFN- γ, IL-2, Th1/Th2 levels and significantly increased IL-4, IL-6 levels after treatment compared with the two control groups (P<0.05). See Table 4.
Table 4 Th1, Th2 and Th1/Th2 levels (pg/ml) before and after treatment in the three groups of fetal survivors at 12 weeks of gestation
Group Number of cases IFN-γ IL-2 IL-4 IL-6 Th1/Th2
Western medicine control group 136 Before treatment 20.73±7.03 19.93±6.73 0.58±0.38 0.54±0.33 2.11±0.26
After treatment 9.58±3.49★● 9.19±3.76★● 12±0.36★● 1.33±0.40★● 1.36±0.39★●
Chinese medicine control group 118 Before treatment 19.18±7.45 18.49±7.35 0.65±0.29 0.60±0.38 2.20±0.35
After treatment 12.12±3.62★ 10.06±3.75★ 1.68±0.32★ 1.14±0.30★ 1.41±0.40★
Treatment group 250 Before treatment 19.20±6.70▲ 18.47±7.61▲ 0.67±0.31▲ 0.58±0.32▲ 2.03±0.40▲
After treatment 7.23±3.25★ ◆ 6.62±3.57★ ◆ 2.04±0.32★ ◆ 1.84±0.29★ ◆ 1.14±0.27★
Note: The three groups were compared before treatment ▲ P>0.05, before and after treatment in each group ★ P<0.05, after treatment the treatment group was compared with the two control groups ◆ P<0.05, after treatment the western control group was compared with the Chinese medicine control group ● P>0.05
3. Discussion
The etiology of URSA is extremely complex, and the unknown cause is not without a cause, but its pathogenesis is still unclear. Recent studies have shown that abnormal maternal-fetal immune regulation may be the main cause of URSA. Pregnancy is a semi-hybrid transfer process, and the embryo is able to obtain “immune escape” and grow further in the mother mainly due to maternal immune tolerance. The formation of this tolerance state involves humoral, cellular, immunogenetic and uterine immune protection. The various immune factors form a network through organic coordination to achieve a balance in the maternal-fetal immune relationship, thus maintaining the pregnancy. If any factor interferes with this immune balance, the embryo will be immunologically attacked and aborted.
It has been found that in cellular immunity, Th1 cells mainly secrete IFN-γ, IL-2 and TNF-, which promote macrophage activation, strong delayed hypersensitivity and promote cytotoxic effects, and are associated with inflammation and tissue damage; Th2 cells mainly secrete IL-4, IL-6 and IL-10, which promote eosinophilia and mast cell differentiation, promote antibody formation, suppress immune inflammation and reduce excessive damage. There is a mutual suppression between Th1 and Th2 immune response, which regulates the normal immune balance. The normal pregnancy is characterized by a specific Th2 phenomenon, when Th1 cytokines are overexpressed and Th2 is suppressed, leading to the occurrence of miscarriage [2].
Western medicine believes that progestins can mediate immunity and are involved in the process of immune tolerance. Natural progestin preparations are often used clinically for the treatment of URSA, and a certain pregnancy success rate can be achieved [3]. Studies in Chinese medicine have concluded that the disease is mostly due to injury to the punching point then kidney deficiency and weakness of the tethered cells as the root of the disease, and treatment is mostly based on fixing the kidneys and calming the fetus, showing that kidney tonic herbs can promote and increase antibody synthesis and have a certain effect on both cellular and humoral immunity [4]. Studies have also confirmed that kidney tonifying herbs have a good protective effect against DNA damage induced by the chemotherapeutic agent cyclophosphamide, thus reducing the occurrence of teratogenesis [5]. Although the above treatments have achieved some efficacy, many still end in miscarriage. Recently, lymphocyte active immunotherapy has been used to treat URSA with a high pregnancy rate [6, 7]. However, due to the inconsistency and irregularity of individualized treatment protocols, the timing of active immunization, and the dose of lymphocytes per immunization, the efficacy of lymphocyte active immunotherapy varies, and there are adverse effects such as local allergy, septicemia, and mild systemic fever, so many scholars are controversial about the involvement of lymphocyte active immunization in the treatment of this disease and question its effectiveness.
In this study, we compared the effects of lymphocyte active immunity, didrogestrel and rapid-acting fetal preservation tablets on the abortion rate and fetal survival rate of URSA, and initially investigated the mechanism of lymphocyte active immunity on URSA. The study showed that lymphocyte active immunotherapy significantly increased the fetal survival rate and decreased the miscarriage rate of URSA at 65 days of gestation and 12 weeks of gestation, probably by decreasing the levels of IFN-γ and IL-2 water and increasing the levels of IL-4 and IL-6, and regulating the switch from Th1 to Th2 immune response, thus facilitating the success of pregnancy, and the immunization using the lymphocyte dose in this study did not No adverse effects such as local allergy, septicemia and mild systemic fever were found in one case. It is evident that active immunotherapy with lymphocytes is an effective treatment for recurrent miscarriage of unknown origin.
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