Objective
The purpose of this study was to evaluate circulating and intracellular levels of Th1 and Th2 cytokines in women with threatened miscarriage (TM) and subsequent outcome.
Study Design
Plasma levels of tumor necrosis factor (TNF)-receptors 1 and 2, TNFα, interferon gamma (IFNγ), and interleukins (IL) -6 and -10 were measured by flow cytometric bead assays in 80 women with TM: 53 women with normal outcome and 27 women who miscarried. Fluorescent antibody labeling was also performed on whole blood in a subgroup of 27 women of TM: 16 women with normal outcome and 11 women who miscarried.
Results
Monocyte expression of TNFα and circulating levels of TNFα, IFNγ, IL-10, IL-6, and TNF-R1 were significantly lower, whereas circulating levels of TNFα/IL-10, IFNγ/IL-10, and TNFα/IL-6 ratios were significantly higher, in women with TM who subsequently miscarried, compared with the women with normal outcome.
Conclusion
An increased Th1 type of immune response, which was similar to that observed in preterm delivery, was found in TM cases that were complicated by a subsequent miscarriage.
Threatened miscarriage (TM) is defined as a history of vaginal bleeding in early pregnancy in the presence of a closed cervix and with ultrasonic evidence of an intrauterine gestational sac and a fetal heartbeat. TM is the most common complication of ongoing pregnancy, which occurs in approximately 30% of pregnant women, and is associated with a 10-14% risk of full miscarriage. Various management protocols have been developed that included conservative management and hormonal therapy that were based on progesterone and human chorionic gonadotropin (hCG). Overall, TM can generate great anxiety in patients, especially because bleeding in early pregnancy can lead to pregnancy loss and other obstetric complications.
The pathophysiology of TM is linked to bleeding from uteroplacental vessels at the margin of the placenta with blood accumulating between the chorionic membrane and uterine wall. The formation of a subchorionic hematoma may disrupt the placental bed. If the hematoma expands to the rest of the placental mass, it will induce complete miscarriage within a week of the first symptoms. If bleeding is limited or happens close to the internal cervical os and is evacuated, the pregnancy may continue; however, this can lead to a chronic inflammatory reaction within the decidua and membranes. Within this context, TM is associated with a higher incidence of preterm labor, prelabor rupture of membranes, placental abruption, fetal growth restriction, and low birthweight.
A strong association exists between maternal T helper-1 (Th1)-type immunity and pregnancy loss, whereas a shift towards Th2-type cytokine response has been observed in successful pregnancy. Cytokines are known to play an important role in implantation. An imbalance in cytokine production occurs in early pregnancy loss. Cytokine receptors such as tumor necrosis factor-receptor 1 (TNF-R1) have also been associated with miscarriage in their role as apoptosis-mediators through proinflammatory cytokines such as tumor necrosis factor alpha (TNFα).
Several factors in the maternal history (such as age and smoking, biomarkers that include serum hormones levels, and the presence of a retroplacental hematoma on ultrasound scanning) have been investigated as possible predictors of pregnancy outcome in women with TM. It has been shown that after TM, maternal serum inhibin A in combination with other serum markers, such as hCG, are altered in women who subsequently have a first-trimester miscarriage. The biomarkers that give the best predictive values are a combination of serum progesterone and βhCG, with a sensitivity of 88.1% and specificity of 84.3%. However, no biomarker of inflammation in the peripheral maternal blood has been validated sufficiently to be of any clinical use in counseling women with TM regarding their risks of subsequent miscarriage and other complications or for providing advice about the benefit of specific treatment to reduce these risks. Recent data have indicated that treatment that corrects Th1/Th2 imbalance, such as dydrogesterone, could help prevent TM from resulting in a full miscarriage.
The objective of this study was to evaluate the changes in circulating levels and intracellular expression of Th1 and Th2 cytokines in patients with TM and to investigate whether these cytokines are altered in patients who subsequently miscarry. TNFα and its receptors TNF-R1 and TNF-R2 and interferon gamma (IFNγ) were measured to evaluate the Th1 cytokine response, whereas interleukins (IL)-6 and -10 were measured to evaluate Th2 cytokine response. IL-6 was classified as a Th2 cytokine because of its role in early pregnancy. The role of these cytokines in predicting miscarriage was also investigated.
Materials and Methods
Subjects and samples
A group of 80 women with first-trimester TM were recruited prospectively from the Early Pregnancy Unit at University College London Hospital after clinical diagnosis of TM (ie, ≤12 weeks 6 days of gestation as calculated from the first day of their last menstrual period) with vaginal bleeding and after an ultrasound confirmation of a singleton viable pregnancy. Exclusion criteria included patients with a history of recurrent miscarriage, presence of twin pregnancy, hydatiform mole or a congenital uterine anomaly, presence of large leiomyomata that was distorting the uterine cavity, known history of cervical incompetence, thrombophilia, or any medical condition that needed chronic drug therapy.
After written informed consent, 10 mL of venous blood were collected by sterile venipuncture. One milliliter of blood was analyzed as part of a prospective study that involved flow cytometric analysis of fluorescent antibody-labeled whole blood. This analysis had to be carried out on fresh whole blood, without prior knowledge of the outcome of the index pregnancy. The remaining 9 mL of blood were centrifuged within 2 hours of collection, and the plasma supernatant was stored at −20°C until it was assayed. Measurement of cytokines/receptors in plasma was done with knowledge of the outcome of the index pregnancy (ie, as a retrospective nested case control study). A normal pregnancy outcome was defined as a singleton live birth at term and of normal weight (≥3 kg). A miscarriage was defined as pregnancy ending spontaneously at <24 weeks of gestation. The study was approved by University College London Hospital Committee on the Ethics of Human Research.
Bioassays
Multiplex bead assays
TNF-receptors 1 and 2, TNFα, interferon gamma (IFNγ), IL-6, and IL-10 were assayed in maternal plasma using Cytometric Bead Array Human Soluble Protein Flex Sets (BD Biosciences, San Jose, CA). The scope of this cytometric bead array was to allow for multiplexed analysis of different cytokines and receptors on smaller quantities of plasma, when compared with the use of conventional enzyme-linked immunosorbent assay. The experiment was carried out according to the manufacturer’s instructions and as previously published. Acquisition of the sample data was performed with a bioanalyzer flow cytometer (BD FACSArray; BD Biosciences). The data were presented in graphic and tabular formats with the FCAP Array software (BD Biosciences). The limit of detection for each assay and the intraassay and interassay coefficients of variation are shown in Table 1 .
| Cytokine/receptor | Assay limit of detection, pg/mL | Intraassay coefficient of variation, % | Interassay coefficient of variation, % |
|---|---|---|---|
| Tumor necrosis factor–α | 0.7 | 10.2 | 5.0 |
| Interferon γ | 1.8 | 10.8 | 5.0 |
| Interleukin-10 | 0.13 | 6.4 | 11.0 |
| Interleukin-6 | 1.6 | 9.4 | 8.0 |
| Tumor necrosis factor–receptor 1 | 5.2 | 2.6 | 10.1 |
| Tumor necrosis factor–receptor 2 | 1.4 | 7.1 | 5.6 |
Flow cytometric analysis of fluorescent antibody-labeled whole blood
Initial whole blood validation experiments showed that 40 ng/mL of lipopolysaccharide (40 LPS) and an incubation period of 12 hours gave the highest increment above basal level (0 LPS) in terms of cytokine expression by the activated monocytes, while retaining a high monocyte population. Dual antibody labeling was carried out with specific antibodies that were conjugated to spectrally distinct fluorochromes to enable easy discrimination. All samples were labeled with mouse anti-human antibody (AbD Serotec, Oxford, UK) that was specific for monocytes (CD 14). Different aliquots were also labeled with antihuman antibodies that were specific to the cytokine/receptor of interest ( Table 2 ).
| Patient code | Monocyte label | Cytokine/receptor label | Lipopolysaccharide, ng/mL |
|---|---|---|---|
| a | CD-14: APC | TNFα receptor I: PE | — |
| b | CD-14: APC | TNFα receptor II: PE | — |
| c | CD-14: APC | IgG1: PE | — |
| d | CD-14: APC | IgG2: PE | — |
| e | CD-14: APC | TNFα: FITC | 40 |
| f | CD-14: APC | TNFα: FITC | — |
| g | CD-14: PE | IFNγ: APC | 40 |
| h | CD-14: PE | IFNγ: APC | — |
| i | CD-14: PE | IL-10: PE | 40 |
| j | CD-14: PE | IL-10: PE | — |
| k | CD-14: PE | IL-6: biotin + streptavidin PE | 40 |
| l | CD-14: PE | IL-6: biotin + streptavidin PE | — |
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