Pre-conceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype

PROBLEM: The aim of the present study was to determine whether pre-conceptional natural killer (NK) cell activity and percentage are predictive of subsequent spontaneous abortion in women with recurrent spontaneous abortion (RSA). METHOD OF STUDY: Pre-conceptional NK cell activity and percentage in peripheral blood of women who had a history of two or more RSA was prospectively assessed. The 51Cr release assay and flow cytometric analysis were performed. A total of 113 RSA women were recruited, and 85 conceived later. RESULTS: Pre-conceptional NK cell activity/percentage values in women whose next pregnancies ended in biochemical pregnancy and spontaneous abortion with normal fetal karyotype (n = 17, median 47%/17.1%), but spontaneous abortion with abnormal karyotype (n = 9, 27%/15.7%), were higher than those in live births (n = 59, 33%/13.1%). High values of pre-conceptional NK cell activity (> 46%; relative risk 3.6, 95%CI 1.6-8.0) and percentage (> 16.4%; 4.9, 1.7-13.8) were found to predict biochemical pregnancy and spontaneous abortion with normal karyotype in the next pregnancy. CONCLUSION: Pre-conceptional NK cell abnormalities were predictive of spontaneous abortion with normal fetal karyotype.     

T辅助细胞、NK细胞与原因不明复发性自然流产关系的研究进展

目前母-胎界面的免疫耐受机制是国内外的研究热点之一,URSA所表现的母-胎界面免疫耐受异常与T辅助细胞及NK细胞有密切的关系。本文首先对T辅助细胞和NK细胞的免疫状态、免疫耐受机制以及它们与URSA之间的关系进行了阐述,最后对目前与二者有关的URSA的主动免疫治疗研究进展作一综述。     

Maternal KIR repertoire is not associated with recurrent spontaneous abortion

BACKGROUND: In view of evidence suggesting an immunological cause of recurrent spontaneous abortions (RSA) and the large number of maternal natural killer (NK) cells present in the pregnant uterus, we investigated the genetic polymorphism of the killer cell immunoglobulin-like receptors (KIR) in women with RSA. METHODS: KIR gene repertoire and KIR2DL4 (a receptor for HLA-G) genotyping were determined by SSP and SSCP respectively, in women experiencing RSA and controls. RESULTS: The KIR repertoire did not differ between RSA patients and controls in terms of: (i) the number of inhibitory receptors; (ii) the number of activating receptors; (iii) the ratio of inhibitory to activating receptors. KIR2DL4, a receptor for HLA-G, has different transmembrane alleles, which produce functionally different phenotypes. The frequency of KIR2DL4 transmembrane genotypes differed significantly between RSA patients and controls (P=0.03). However, although homozygosity for a membrane-bound receptor was more frequent in patients (25%) than controls (10%), other genotypes that would produce the same phenotype were not more frequent in patients than controls. CONCLUSIONS: The data provide little evidence that KIR polymorphism plays a role in predisposition to RSA.     

Decrease in a specific killer cell immunoglobulin-like receptor on peripheral natural killer cells in women with recurrent spontaneous abortion of unexplained etiology

PROBLEM: The aim of this study was to investigate immunophenotypic characteristics of natural killer (NK) cells by assessing specific molecules expressed in women with recurrent spontaneous abortion (RSA) of unexplained etiology. METHOD OF STUDY: Peripheral blood cells were obtained from 20 RSA women and 15 fertile controls. The expression of perforin, CD94, CD161, CD158a, CD158b, and CD244 on CD3- CD56+ NK cells was analyzed by flow cytometry. RESULTS: A significant decrease in CD158a expression was demonstrated in RSA women (mean +/- SD, 22.9 +/- 8.7%) as compared with that in controls (33.6 +/- 15.7%) (P < 0.05). The percentage of NK cells showing dual expression of CD94 and CD161 was relatively higher in RSA women (55.1 +/- 10.2%) than in the controls (47.1 +/- 19.0%), but without statistically significant (P = 0.096). The expression of perforin, CD158b, or CD244 in RSA women did not differ from that in the controls. CONCLUSIONS: A divergence of the specific NK cell repertoire might be related to the etiology of RSA.     

Increased natural killer cell cytotoxicity and IL-2 production in recurrent spontaneous abortion

PROBLEM: To determine whether natural killer (NK) cells cytotoxicity in peripheral blood is altered in patients with a history of recurrent spontaneous abortion (RSA); also, if there is any correlation between cytokine production and NK cytotoxicity. METHOD OF STUDY: In this case-control study, 21 patients with RSA within 24 hr of the last abortion (group I), and 32 pregnants with no history of abortion (group II) were surveyed. NK cell cytotoxicity was evaluated by flow cytometry, and IL-2, IL-10, transforming growth factor beta1 were measured in cell culture supernatant by ELISA method. RESULTS: Group I showed higher NK cytotoxicity than group II at all of effector to target (E:T) ratios (P < or = 0.045).The correlation between production of IL-2 and NK cytotoxicity was positively significant (R = 0.350, P = 0.001). Group I had significantly higher levels of IL-2 than group II (P = 0.001). In group II, the production of IL-10 by peripheral blood mononuclear cells was higher than group I (P = 0.002). CONCLUSION: Increased NK cell cytotoxicity and high level of IL-2 may be considered as a risk factor for RSA.     

Proportion of CD56+3+ T cells in decidual and peripheral lymphocytes of normal pregnancy and spontaneous abortion with and without history of recurrent abortion

PROBLEM: The present study investigated the proportion of CD56+3+ T cells in maternal peripheral and decidual lymphocytes in normal pregnancy and spontaneous abortion with and without history of recurrent spontaneous abortion (RSA). METHOD OF STUDY: Maternal peripheral blood and decidua were taken from normal pregnancies and missed abortions with and without RSA. Decidual lymphocytes were prepared from decidual tissue and analyzed by flow cytometry. RESULTS: In normal pregnancy, the percentages of CD56+3+ T cells in decidual lymphocytes did not differ from those in the peripheral blood. However, the proportion of CD56+3+ T cells in decidual CD3+ T cells increased higher than that in the peripheral CD3+ T cells. The percentages of decidual CD56+3+ T cells in missed abortions with and without RSA were lower than those in normal pregnancies. CONCLUSION: CD56+3+ T cells may play a role in the maintenance of pregnancy. The phenomenon, where the proportion of CD56+3+ T cells in decidual lymphocytes decreases, may be due to an immunologic event leading to missed abortion.     

Massive Intravenous Immunoglobulin Treatment in Women with Four or More Recurrent Spontaneous Abortions of Unexplained Etiology: Down-Regulation of NK Cell Activity and Subsets

PROBLEM: The aims of this study were to investigate the efficacy of massive intravenous immunoglobulin (MIVIg) treatment for women with recurrent spontaneous abortion (RSA) of unexplained etiology, and to investigate changes in peripheral natural killer (NK) cell activity and subsets. METHOD OF STUDY: MIVIg treatment was performed in 18 pregnancies from 15 women with 4 or more consecutive RSA of unexplained etiology. NK cell activity and subsets were assessed in 8 of the pregnancies. RESULTS: 14 pregnancies resulted in live births and 4 resulted in abortions with chromosome abnormality. The pre-infusion NK cell activity (mean + SD. 40.9 + 17.0%) at 4.4 +/- 0.5 weeks of gestation (GW) decreased to 15.0 +/- 7.90% at post-infusion status (5.4 +/- 0.5 GW). Pre-infusion percentages of CD56+ CD16- cells (3.5 +/- 2.1%) and CD56+ CD16- cells (16.8 +/- 8.8%) decreased to 3.0 +/- 2.2% and 11.1 +/- 6.9%, respectively, after MIVIg treatment. CONCLUSIONS: MIVIg treatment was effective in all 14 pregnancies from RSA women of unexplained etiology, excluding 4 abortions with chromosome abnormality. Peripheral NK cell activity and subsets were suppressed by MIVIg treatment.     

Natural killer (NK) cell receptors' repertoire in couples with recurrent spontaneous abortions

PROBLEM: Natural killer (NK) cell receptors (NKRs) have been suggested to protect trophoblast, but their function at the fetomaternal interface remains unknown. To investigate if the outcome of pregnancy depends on women's NKRs, we studied the NKR repertoire in couples with recurrent spontaneous abortions (RSA). METHODS: Twenty-six childless couples with > or = 2 abortions, characterized by alloimmune abnormalities, and 26 control couples were genotyped for five killer immunoglobulin-like receptors (KIR) and two CD94/NKG receptors, known to have as ligands human leukocyte antigen (HLA) class I molecules with trophoblastic expression: inhibitory 2DL1,2,3 and activating 2DS1,4 KIRs, inhibitory NKG2A and activating NKG2C. Detected repertoires of women and partners were compared between the two groups. RESULTS: Less aborters than controls were found to have all three inhibitory KIRs (30.77% versus 69.23%, P = 0.01), some of them had only one inhibitory KIR (19.23% versus 3.85%, P = 0.08) and most of them were lacking inhibitory KIRs possessed by their husbands (57.69% versus 15.38%, P = 0.001). CONCLUSIONS: Women with alloimmune abortions have a limited inhibiting KIR repertoire and such miscarriages may occur because trophoblastic HLA class I molecules are recognized by decidual NK cells lacking the appropriate inhibitory KIRs.     

Natural killer cell activity and cytokine production after in vitro immunoglobulin treatment of lymphocytes derived from pregnant women with or without risk for spontaneous abortion

OBJECTIVE: To investigate the possible mechanism of action effective in immunoglobulin G (IgG) treatment of recurrent spontaneous abortion (RSA). The effect in vitro of a commercially available intravenous immunoglobulin (IvIg) on the rate of interleukin (IL)-10 and IL-12 positive cells (Th1/Th2 balance) and on natural killer (NK) cell activity in populations of peripheral lymphocytes of healthy pregnant women and women at risk for premature pregnancy termination was studied. Primary habitual aborters as well as women showing clinical symptoms (bleeding or regular uterine contractions) of threatened premature pregnancy termination were included. METHODS: Lymphocytes of 20 pregnant women were tested. Five different batches of an IvIg with reported immunomodulatory potential were used at a concentration of 10 mg/mL. Cytokine profiles of the lymphocytes were determined by immunocytochemistry. For testing of NK cell activity, the 4 hr single cell cytotoxicity assay was used. RESULTS: Incubation with IgG of lymphocytes from recurrent spontaneous aborters concomitantly and significantly decreased the rate of IL-12 positive cells (P < 0.01) and increased the rate of IL-10 positive cells (P < 0.01), whereas such treatment had no significant effect on lymphocytes of pregnant women not at risk of abortion. Dialysis or heat treatment (56 degrees C, 30 min) of the IgG preparations did not modify the effect. Elevated NK cell activity of women at risk for premature pregnancy termination significantly decreased after IgG incubation of cells in all cases, whereas NK cell activity of normal pregnancy lymphocytes was not altered. CONCLUSION: This study suggests that incubation of peripheral lymphocytes from RSA patients with polyclonal polyspecific IgG alters cytokine profiles and NK activity while the same treatment does not affect lymphocytes of healthy pregnant women. These data might add to the understanding of mechanisms of action of IvIg in prevention of recurrent pregnancy loss.     

Th1 and Th2 cytokine profiles in recurrent aborters with successful pregnancy and with subsequent abortions

BACKGROUND: This study compared Th1-Th2 cytokine profiles in a subgroup of recurrent aborters who had an abortion with those in a subgroup of recurrent aborters who had a successful pregnancy. METHODS: Fifty-four women with a history of at least three normal pregnancies, 24 women with a history of recurrent spontaneous abortion (RSA) followed by abortion (RSA-->A) and 39 women with a history of RSA followed by normal pregnancy (RSA-->N) were studied. Blood samples and placentas were obtained at the time of delivery or abortion; peripheral blood mononuclear cells were stimulated separately with phytohaemagglutinin and with autologous placental cells, and the secreted cytokines estimated. RESULTS: Peripheral blood mononuclear cells from the RSA-->N subgroup secreted higher concentrations of Th1-type cytokines as compared with normal pregnant women, indicating a higher Th1 bias in these women. However, women in the RSA-->N subgroup had significantly higher concentrations of Th2 cytokines as compared with women in the RSA-->A subgroup. A comparison of Th1:Th2 cytokine ratios indicated a higher Th2 bias in RSA-->N women as compared with RSA-->A women. CONCLUSIONS: We conclude that abortion-prone women who proceed to have successful pregnancy are more Th2-biased than abortion-prone women who abort, and that recurrent aborters who undergo spontaneous abortion have a stronger Th1 bias than aborters who have normal pregnancy.     

Modulatory effect of intravenous immunoglobulin on Th17/Treg cell balance in women with unexplained recurrent spontaneous abortion

Recurrent spontaneous abortion (RSA) is a growing problem worldwide. In a majority of cases, the cause remains unknown but there is increasing evidence that immunologic factors play an important role. Intravenous immunoglobulin (IVIg) therapy has been proposed to have immune modulatory effects and therefore been applicable for the treatment of patients with RSA. Although its efficacy is still controversial, several recent studies suggest that IVIg treatment may improve pregnancy outcomes. CD4⁺ T cells and their related cytokines play an important role in maternal‐fetal immune regulation, and an imbalance of Th17/Treg cell ratio has been proposed as a cause for RSA. We review the scientific evidence supporting a modulatory effect of IVIg on Th17/Treg cell balance and discuss the potential mechanisms how IVIg might enhance Treg cells function. We propose that correction of Th17/Treg cell dysregulation could be one of the mechanisms that can explain the positive therapeutic effects of IVIg therapy. Consequently, selecting patients with abnormal Th17/Treg cell ratios could increase the success of IVIg therapy.     

Decidual natural killer cells in recurrent spontaneous abortion with normal chromosomal content.

PROBLEM: The maternal local immune responses in unexplained recurrent spontaneous abortion (RSA) are not yet well known. Maternal peripheral and decidual natural killer (NK) cells were evaluated in RSA with normal chromosomal content. METHOD OF STUDY: Maternal peripheral blood, villous trophoblast, and decidua were taken from 15 normal pregnancies and 9 RSA patients with normal chromosomes. The NK cells in decidual lymphocytes were evaluated by flow cytometry using monoclonal antibodies for CD56, CD16, and CD3. RESULTS: The percentages of CD56+ CD16- CD3- cells in decidual lymphocytes in RSA were lower than in normal pregnancies (P < 0.002). The CD56+CD16+/CD56+CD16- cells ratio in RSA was higher than in normal pregnancies (P < 0.02). CONCLUSION: The lower percentages of CD56+CD16-CD3- cells in RSA cases may show an inappropriate accumulation of NK cells in the decidua, and this finding may be a factor involved in RSA.     

Leukocyte activation in the decidua of chromosomally normal and abnormal fetuses from women with recurrent abortion

As part of our continuing programme to investigate immunological causes of unexplained recurrent pregnancy losses, we studied subpopulations of white blood cells and their activation status in decidua of women with a history of recurrent spontaneous abortion (RSA). We differentiated specifically between normal karyotyped male fetuses and abnormal karyotyped fetuses with trisomy 16 because trisomy 16 is not compatible with life and is thus a non-controversial cause of spontaneous miscarriage. Leukocytes were counted in paraffin-embedded decidua after immunohistological staining for CD45 (LCA), CD3, CD56, CD68, CD69 and CD25. Numbers of activated versus non-activated T lymphocytes, NK cells and macrophages were compared in decidua from women with: (i) unexplained RSA who had a normal male karyotype (n = 17) miscarriage; (ii) unexplained RSA who had a trisomy 16 (n = 21) miscarriage; and (iii) normal gestationally age-matched first trimester pregnancies following elective termination procedures (n = 20). Significantly more activated leukocytes were detected in the decidua of women with unexplained RSA who had a normal male karyotype compared to the other groups (P < 0.0001). In addition, numbers of cells comprising the major leukocyte subpopulation, CD56+ NK cells, appeared reduced in the decidua of women with unexplained RSA compared to decidua from women having elective terminations. Increased numbers of activated leukocytes in the decidua of women with a history of unexplained recurrent pregnancy loss who had a normal karyotyped pregnancy provide evidence that cellular immunity may be involved in unexplained recurrent pregnancy loss.     

Immunology: Outcome of pregnancy in women with recurrent spontaneous abortion following immunotherapy with allogeneic lymphocytes

The efficacy of immunotherapy in the prevention of habitualabortion remains controversial. It has been suggested that thebenefits are predominantly due to psychological factors. Wehave evaluated the success of pregnancy outcome following immunotherapywith allogeneic lymphocytes, in relation to the subsequent developmentof anti-paternal cytotoxic antibodies (APCA). It was observedthat in women who developed an APCA titre of 1:16, live birthsoccurred in 16 out of 21 cases (76%), while only two out ofseven (28%) women who failed to achieve an APCA titre of 1:16had successful pregnancies (P <0.05). In eight women whohad an APCA titre of 1: 16 on initial screening, and were, therefore,excluded from the trial, successful pregnancy outcome was notedin 62.5% of cases. Although these results are based on a smallsample and on an open, non-randomized trial, they show thatthe efficacy of immunotherapy is related to immune responseto allogeneic lymphocytes, and is not simply a placebo effect.Measurement of APCA titre could serve as a marker for immunopotentiation.     

Lack of soluble TNF-receptors in women with recurrent spontaneous abortion and possibility for its correction

PROBLEM: Tumor necrosis factor (TNF) and soluble TNF receptors (sTNF-Rs) system related with Th1 and Th2 and activity of NF-kappaB/IkappaB regulatory system. This study was designed to compare sTNF-R1 and sTNF-R2 production (shedding) and levels of late activated CD8+ T-lymphocytes in non-pregnant (n = 30) and pregnant (n = 20) normal women and non-pregnant (n = 20) and pregnant (n = 30) RSA women. Effects of progesterone (natural structure) injections in RSA women were studied. METHODS OF STUDY: Levels of sTNF-R1, sTNF-R2, TNF in peripheral blood serum were detected by enzyme-linked immunosorbent assay. Lymphocyte subsets were estimated by multicolor flow cytometry. NK cell cytotoxic activity of peripheral blood lymphocytes (PBL) in whole blood against K562 targets was determined using Europium-release cytotoxicity assay. Mitogen-induced proliferative response of PBL to PHA-P, Con A and PWM were determined by standard 3H-thymidine incorporation assay. RESULTS: Levels of soluble TNF-R1 and TNF-R2 in normal pregnancy were elevated when compared with non-pregnant normal women and pregnant RSA women. Levels of late activated CD8+ T-lymphocytes in normal pregnancy were decreased but no changes were detected in RSA women. After progesterone therapy (i.m. injections of 2.5% oil solution) in RSA women elevation of sTNF-R1 and sTNF-R2 to normal pregnancy ranges was observed. No changes in levels of late activated CD8+ T-lymphocytes after progesterone treatment were detected. CONCLUSIONS: Elevation of levels of sTNF-R1, sTNF-R2 and decrease of late activated cytotoxic T-lymphocytes are pronounce markers of normal human pregnancy. In RSA women there are no elevation of sTNF-R1 and sTNF-R2 levels during pregnancy. This deficiency may be restored by progesterone treatment.     

Endocrine abnormalities during the follicular phase in women with recurrent spontaneous abortion.

The frequency of endocrine abnormalities during the follicular phase in non-pregnant women with a history of recurrent abortion was investigated in a case-control study. A total of 42 consecutive women with recurrent spontaneous abortion (three or more consecutive abortions, mean +/- SD: 3.9 +/- 1.1 range 3-8) with no parental chromosome rearrangement or uterine abnormality were studied during the early follicular phase under standardized conditions. Serum concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, androstenedione, testosterone, dehydroepiandro-stenedione, 17-OH-progesterone, oestradiol, progesterone and thyroid stimulating hormone (TSH) were measured by commercially available radioimmunoassays. Controls were 42 nulligravid females with tubal or male factor infertility without miscarriage. Mean (SD) concentrations of prolactin and androstenedione were 14.2 +/- 6.7 ng/ml versus 10.5 +/- 3.5 ng/ml (95% CI 0.8-6.1) and 2.3 +/- 0.9 ng/ml versus 1.7 +/- 0.6 ng/ml (95% CI 0.2-0.9) in the study and control groups respectively. The other endocrine parameters were comparable in both groups. Obesity [BMI weight (kg)/height (m2) > or = 25] was more prevalent (23 versus 5 women, P = 0.0001) in the study than the control group. Recurrent spontaneous abortion is associated with abnormalities in prolactin and androgen secretion during the follicular phase, suggesting an endocrine aetiology in this disorder. Reduction of body weight and correction of hyperprolactinaemia and of hyperandrogenism may reduce the rate of miscarriage in a subsequent pregnancy in these women.     

妊娠早期母胎界面NK细胞研究进展

蜕膜作为一种妊娠时特化的子宫内膜,在促进胚胎生长和维持免疫耐受方面起到至关重要作用。研究表明,蜕膜组织中富集以自然杀伤NK细胞和巨噬细胞为主的大量免疫细胞。在正常妊娠时,各种免疫细胞或非免疫细胞互相调节使母胎界面整体处于免疫平衡状态,而一旦该免疫平衡被打破则可能导致各种妊娠疾病,例如复发性自然流产、子痫前期和胚胎宫内生长受限等。本综述重点总结了妊娠前3个月蜕膜NK细胞的来源、亚群、生理功能和病理作用。     

HLA-E polymorphism in patients with recurrent spontaneous abortion

Abstract: The aim of this study was to compare the frequencies of five HLA-E alleles in 82 women with recurrent spontaneous abortions with that of 150 random Danish controls. PCR sequence-specific oligonucleotide typing was carried out to detect polymorphism in exons 2 and 3 of the HLA-E gene. In informative samples sequencing of these two exons was also undertaken to confirm the presence of the HLA-E*01031 allele. HLA-E*0101, HLA- E*01032 and HLA-E*01031 were detected with frequencies of 56.7%, 33.6% and 9.6% in controls and 58,5%, 32.9% and 8.5% in patients with recurrent abortion, respectively. No HLA-E*0102 and E*0104 alleles could be detected. Since the HLA-E allele distribution was similar in women with recurrent spontaneous abortion and controls, it is suggested that maternal HLA-E polymorphism per se does not play any role in the pathogenesis of this disorder of pregnancy.     

Association of the maternal 14-bp insertion polymorphism in the HLA-G gene in women with recurrent spontaneous abortions

Human leukocyte antigen (HLA)-G has been postulated as an important immunotolerant molecule in maintaining fetal-maternal relationship. Recent reports indicated that the 14-bp deletion/insertion polymorphism in exon 8 of HLA-G gene influences HLA-G mRNA stability and isoform splicing patterns, thus modulating the levels of HLA-G expression. This might play an immunomodulatory role of HLA-G during implantation and pregnancy. In the present study, 109 unrelated fertile control women and 79 women who had experienced recurrent spontaneous abortion (RSA) were genotyped for the 14-bp insertion/deletion polymorphism. No significant difference was observed in the distribution of 14-bp insertion/deletion genotype between controls and the RSA group. However, a greater number of 14-bp insertion alleles exist in the RSA group than in the controls.     

复发性自然流产患者绒毛及蜕膜组织中白细胞介素8的表达

目的:研究复发性自然流产患者绒毛及蜕膜组织中白细胞介素8(interleukin-8,IL-8)的表达。方法:应用免疫组化法及图像分析技术,对30例复发性自然流产患者绒毛、蜕膜组织中IL-8的蛋白表达进行定位及半定量分析;H- E染色后光镜下观察绒毛及蜕膜组织的形态学变化。结果:在绒毛组织中,IL-8蛋白定位于绒毛上皮细胞的细胞质内,且病例组的表达明显高于对照组;在蜕膜组织中,病例组蜕膜细胞的胞质内可见IL-8蛋白表达,且高于对照组;H- E染色可见病例组绒毛组织的滋养层变薄,细胞变性甚至坏死、嗜酸性增强,绒毛中轴纤维化程度增强;蜕膜组织中蜕膜细胞失去细胞间连接,部分蜕膜细胞解体、核消失。结论:病例组绒毛及蜕膜组织中IL-8蛋白表达的增强与复发性自然流产有关,IL-8可能参与复发性自然流产的病理过程。