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Changes of serum interleukin-6 in healthy pregnant women and establishment of relevant reference intervals
Journal Pre-proofs Changes of serum interleukin-6 in healthy pregnant women and establishment of relevant reference intervals Yaoyang Fu, Lingli Tang, Min Hu, Qinglin Liu, Zhongyuan Xiang PII: DOI: Reference:
S0009-8981(19)32182-5 https://doi.org/10.1016/j.cca.2019.12.013 CCA 15963
To appear in:
Clinica Chimica Acta
Received Date: Revised Date: Accepted Date:
16 September 2019 21 November 2019 16 December 2019
Please cite this article as: Y. Fu, L. Tang, M. Hu, Q. Liu, Z. Xiang, Changes of serum interleukin-6 in healthy pregnant women and establishment of relevant reference intervals, Clinica Chimica Acta (2019), doi: https:// doi.org/10.1016/j.cca.2019.12.013
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Changes of serum interleukin-6 in healthy pregnant women and establishment of relevant reference intervals Yaoyang Fua, Lingli Tanga, Min Hua, Qinglin Liua, Zhongyuan Xianga,1 a Department of Laboratory Medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China 1 Corresponding author at: Department of Laboratory Medicine, The Second Xiangya Hospital, Central South University, #139 Renmin Middle Rd, Changsha, Hunan 410011, China. E-mail address: [email protected] (Z. Xiang).
1
Abstract Objective: To explore the changes of serum interleukin-6 (IL-6) levels in healthy pregnant women and establish reference intervals (RIs). Method: According to the requirements for the RIs study model and the reference population screening criteria in C28-A3 document, Serum IL-6 levels were measured by electrochemiluminescence immunoassay in 480 healthy Chinese women, including 120 pregnant women in each of the first, second and third trimester and 120 non-pregnant women as the negative control. The establishment of RIs for IL-6 were defined using nonparametric percentile. Results: The RIs for serum IL-6 levels in healthy pregnant women is <4.19pg/ml, the RIs for serum IL-6 levels in healthy pregnant women who are in the first trimester is <3.52pg/ml, and the RIs for serum IL-6 levels in healthy pregnant women who are in the second and third trimester is <4.40pg/ml. Conclusions: Serum IL-6 level in healthy pregnant women is higher than the healthy non-pregnant women, and the level of IL-6 who are in the second and third trimester is higher than those in the first. This paper successfully established RIs for serum IL-6 levels in pregnant women, providing a reference for clinical medical staff and laboratory workers. Key words：IL-6, pregnant women, reference intervals.
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1. Introduction As a pleiotropic cytokine, interleukin-6 (IL-6) is usually produced in local tissues and released into the blood circulation in the case of autologous disorders such as endotoxemia, trauma and acute infection. In addition to the known role of IL-6 in combating chronic infections in the cell[1], IL-6 in the circulation is also involved in the innate immune process. Clinically, serum IL-6 is closely related to the severity of inflammation and bacterial infection. In patients with systemic infection, the degree of elevation is more obvious, so it can be used as a sensitive indicator for sepsis. Determination of IL-6 levels in blood is important for assessing disease progression and prognosis. Pregnancy is based on an evolutionary balance of cytokines that undergo changes in cytokine type or concentration at different stages of pregnancy. Specifically, Th2-type anti-inflammatory cytokines predominate in the middle pregnancy, while Th1-type pro-inflammatory cytokines are mainly distributed in the early and late stages of pregnancy and are characterized by inflammatory-like events. As an important indicator of inflammation, the changes of IL-6 are particularly important. Studies have shown[2] that the serum levels of IL-6 in gestational diabetes and gestational hypertension are significantly higher than those in healthy gestational women. The serum level of IL-6 in pregnancy-induced hypertension is associated with pregnancy outcome and is linearly positively correlated with systolic blood pressure. In addition, in a prospective cohort study[3], the study team followed a group of pregnant women with premature rupture of membranes and healthy pregnant women until delivery, and found that the average serum IL-6 level was significantly higher in women with premature rupture of membranes. The fetal outcomes of patients with IL-6≥14pg/mL were poor. Therefore, measurement of maternal serum IL-6 can reflect the adverse effects of the fetal intrauterine environment and the need for necessary interventions. In addition to the above, IL-6 also shows certain indication significance in chorioamnionitis[4], premature delivery[5], intrauterine infection[6], and maternal periodontopathy[7]. However, at present, the domestic clinical laboratory has only the interleukin-6 reference intervals (RIs) for the normal population. As a special group, pregnant women show physiological changes different from the general population. Whether the existing IL-6 RIs is suitable for pregnant women is questionable. Therefore, before using IL-6 levels as indicators of gestational complications, it is necessary to determine the specific RIs for IL-6 in pregnant women[8]. In this study, a standard RIs for maternal serum IL-6 was established according to the Clinical and Laboratory Standards Institute (CLSI) C28-A3 document, providing a useful and reliable clinical reference for physicians.
2. Materials and Methods 2.1. Subjects The information of pregnant women who were admitted to the Second Xiangya Hospital of Central South University (Hunan, Changsha) for prenatal check-up or delivery from September 2018 to May 2019 was collected. This study was approved by the ethics committee of the Second Xiangya Hospital of Central South University 3
and all participants have signed the informed consent form. The selection of healthy pregnant and non-pregnant women was based on the exclusion criteria from CLSI C28-A3 guidelines: (1) diagnosed as chronic disease, liver disease or endocrine disease; (2) having a family history of genetic disease; (3) Body mass index (BMI) ≥ 28kg/m2 or ≤ 18.5kg/m2; (4) History of hypertension for more than 3 years (systolic blood pressure ≥140mm Hg or diastolic blood pressure ≥90mm Hg); (5) abnormalities in the heart, liver, lungs and kidneys found by ultrasound or electrocardiogram; (6) undergoing surgery within 4 months before pregnancy or blood transfusion or blood donation within 6 months; (7) taking drugs or abusing antibiotics within 2 weeks; (8) Excessive smoking (more than 20 cigarettes per day) or drinking (30 grams or more per day); (9) excessive labor or exercise; (10) The following laboratory results occurred: triglyceride ≥ 2.26mmol/L, total cholesterol ≥ 6.22mmo/L, fasting blood glucose > 7.0mmol/L, hepatitis B surface antigen, hepatitis C antibody or HIV antibody positive, abnormal urine, red blood cells Count ≥ 5.0 × 1012/L or ≤ 3.0 × 1012/L, hemoglobin ≤ 110g/L, white blood cell count ≥ 12.0 × 109/ L or ≤ 4.0 × 109 / L, platelet count <100 × 109/L. According to the above recruitment conditions, a total of 480 healthy Chinese women, including 120 pregnant women in each of the first, second and third trimester and 120 non-pregnant women as the negative control participated in the study. Non-pregnant women are not in menstruation. The average age of the participants in the experimental groups was 30.9±4.7; 30.5±4.3; 29.8±5.3; 30.3±5.0 for non-pregnant, the first, second and third trimester, respectively. The baseline characteristics of the individuals are presented in Table 1, including body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), glucose (Glu), total cholesterol (TC), triglyceride (TG), hemoglobin (Hb).The blood samples for each trimester are not collected serially from the same pregnant women. 2.2. IL-6 quantification All of the participants were asked to maintain their normal lifestyle and avoid strenuous physical exercise within 3 days of physical examination. The participants fasted overnight for at least 8 h and sat for at least 30 min before specimen collection. The specimens collected in the experiment were free of hemolysis, chyle and jaundice. After the specimen trait is determined, the sample is tested on the machine within 2 hours. The concentration of IL-6 were analyzed on Roche Cabas E602 (Germany) by electrochemiluminescence immunoassay (ECLIA). All of the tests were performed according to standard operating procedures recommended by NCCLS. To evaluate instrument performance, two control concentrations of IL-6 were tested. Quality control results are under control. The precision and accuracy were demonstrated according to the document EP 15-A2 recommended by CLSI. The detection limit of the detection system is 1.5pg/ml. We dilute the low-value control to 2-3pg/ml, dispense and freeze at -20 degrees for intra-assay and inter-assay precision experiments. The intra-assay and inter-assay imprecision are 4.92% and 4.22%, respectively. We collected multiple samples and conducted simultaneous tests with the 4
hospitals that participated in the inter-room quality assessment to verify the accuracy of our results. The bias between the results is within the allowable bias. So, the precision and accuracy is in accordance with the requirements of the current standard documents. 2.3 Statistical analysis All data were analyzed using SPSS 22.0 Statistical Software (SPSS Inc., Chicago, USA). The distribution of the data was detected using Kolmogorov-Smirnov test. Outliers were excluded using Dixon test. For non-normal distribution, Mann-Whitney U test were used to compare variables between two groups and Kruskal-Wallis H test was applied to compare variables among multiple groups. Differences at P<0.05 were considered statistically significant. According to the recommendations of CLSI C28-A3 document, a non-parametric test was used to calculate serum IL-6 RIs. Reference range were expressed between the 0th and 95.0th percentiles, because the upper limit of IL-6 is of clinical significance.
3. Results During the test, the values of IL-6 in 42 non-pregnant women and 6 the first trimester women were lower than the lower limit of detection in the system. Because the reference interval of interleukin 6 is generally only one side, it does not affect the establishment of subsequent reference intervals. In comparing the differences in interleukin-6 between non-pregnant women and pregnant women, we stripped out all such undetected data. If there is still a difference, it indicates that there is indeed a difference between the two. 3.1. IL-6 levels in healthy non-pregnant women and healthy pregnant women. Komlogorov-Smimov test demonstrated serum IL-6 levels were not in normal distribution in both healthy non-pregnant women and healthy pregnant women group (P<0.05). Outliers were removed by Dixon's test. U test showed that serum IL-6 levels were significantly different between healthy non-pregnant women and healthy pregnant women group (P<0.05). Therefore, serum IL-6 RIs for healthy non-pregnant and pregnant women should be established separately (Table 2). 3.2. IL-6 levels in healthy pregnant women in different trimesters According to CLSI C28-A3 guidelines, healthy pregnant women were divided into three groups according to gestational age: first trimester (1-13 week), second trimester (14-27 week), and third trimester (≥28 week) group to calculate RIs. According to Komlogorov-Smimov test, serum IL-6 levels were not in normal distribution in the first and second trimester groups (P<0.05). But for the third trimester group, serum IL-6 is in line with normal distribution (P=0.14). Outliers were removed by Dixon's test. H test confirmed that serum IL-6 levels for healthy women in first, second, and third trimester group were not all significantly different (P>0.05). U tests were thus performed for pairwise comparisons and serum IL-6 levels were significantly different between the first and second trimester group as well as the first and third trimester group (P<0.05). Nevertheless, there was no significant difference 5
between the second and third trimester group (P>0.05). Therefore, healthy pregnant women in the second and third trimester could be combined into one group to calculate serum IL-6 RIs and serum IL-6 RIs for healthy pregnant women in the first trimester should be established separately (Table 3). 3.3 IL-6 RIs for healthy non-pregnant women and healthy pregnant women. IL-6 is only clinically meaningful with reference to the upper limit of the interval. According to CLSI C28-A3 guidelines, the RIs of serum IL-6 for healthy non-pregnant women and healthy pregnant women were calculated (Table 4). Therefore，serum IL-6 RIs is <1.518 for healthy non-pregnant women. For healthy pregnant women, serum IL-6 RIs are <3.52 pg/ml in the first trimester and <4.40 pg/mL for in the second and third trimester.
4. Discussion At present, for pregnant women, partial RIs used in laboratory tests is still the same as that of the normal population, which have not considered the unique physiological and biochemical changes during pregnancy[9]. Establishing a medical RIs for commonly used testing items for pregnant women is conducive to improving the diagnostic level of clinicians for pregnant women, making subsequent examinations and treatments more rational, ensuring greater utilization of medical resources, and so on. This is indeed an urgent problem to be solved in domestic clinical trials. IL-6 is a promising biochemical marker with high sensitivity and specificity, which is an early assessment of infection, and an early warning for some common gestational diseases, such as gestational diabetes, pregnancy hypertension, premature rupture of membranes. IL-6 has an important role in the health of pregnant women and the fetus, so it is necessary to pay attention to changes in serum IL-6 levels during pregnancy. In this study, the reference population was selected in strict accordance with the screening criteria of the CLSI C28-A3 guidelines, and a serum IL-6 RIs was established for a special population of healthy pregnant women. The experiment first compared the serum IL-6 levels between healthy pregnant women and healthy pregnant women, showing a significant difference between the two groups (P<0.001), suggesting that the serum IL-6 RIs for pregnant women should be established separately. Then we analyzed the levels of IL-6 in the three stages of pregnancy, showing significant differences in serum IL-6 levels between the first and the second trimester, the first and the third trimester (P＝0.003). There was no significant difference in serum IL-6 levels between the second trimester and the third trimester (P ＝0.318), suggesting the need to establish a RIs of the first trimester, the second and the third trimester. The RIs of serum IL-6 level in healthy pregnant women is <4.19pg/ml. The RIs of serum IL-6 in the first trimester is <3.52pg/mL, and the RIs of serum IL-6 in the second and the third trimester is <4.40pg/ml. In pregnancy, for the maternal immune system, it is necessary to satisfy two situations at the same time: on the one hand, it is necessary to protect the mother from infection, and on the other hand, to receive a semi-allogene fetus. This process reflects the positive regulation of the inflammatory immune process during pregnancy to ensure 6
specific tolerance to foreign fetal antigens[10]. Pregnancy can be considered as an embryo-induced inflammatory process in the endometrium, and as the pregnancy progresses, the embryo-induced inflammation gradually becomes stronger, which may explain the increase in IL-6 levels in pregnant women. For pregnant women, the pro-inflammatory and anti-inflammatory pathways are involved in the establishment of the receptor endometrium (implantation window) and embryo-endometrial communication. Many of the cytokines produced by embryos are pro-inflammatory, suggesting that implantation is the process by which an embryo induces an inflammatory pathway in the endometrium. The endometrium responds to these embryonic signals by enhancing the expression of receptive genes required for embryo adhesion and invasion during the implantation window. At the time of preparation for implantation, decidualization initially occurs around the spiral arteriole during the secretory phase and occurs remarkably throughout the endometrium if pregnancy occurs. In addition to the differentiation of endometrial stromal cells into decidual cells, decidualization is also involved in triggering inflammatory events such as leukocyte infiltration, modification of extracellular matrices, and increased vascular permeability. Cytokines, particularly IL-6, play an important role in implantation. In vitro studies have shown that their receptors are expressed at the implantation site by several cells. The knockout mouse model has demonstrated that interleukin-11 and leukemia inhibitory factor (LIF) play an important role in implantation[10], while IL-6 affects fertility and implantation efficiency. Leukemia inhibitory factor is a pro-inflammatory cytokine expressed in epithelial and decidual stromal cells and is mediated by several inflammatory mediators such as interleukin-1, tumor necrosis factor (TNF), leptin, insulin-like growth factor, transforming growth factor (TGF-β)[11, 12]. And the recent discovery of prokinetic protein regulation suggests the complexity of regulation of the inflammatory pathway[13]. Thence, it is important to understand the changes of IL-6 during pregnancy in detail. In our study, The changes in IL-6 during pregnancy have been described, and corresponding reference intervals have been established. This will help clinicians to provide some help for the medical diagnosis of pregnant women. In summary, rigorously according to CLSI C28-A3 guidelines, the authoritative document of RIs establishment, we established the RIs of serum IL-6 for healthy pregnant Chinese women. The results of this study will provide a valuable reference for clinical work and laboratory researches.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Acknowledgments: Thanks to Professor Aiguo Tang for the revision and suggestions of this article. The work was supported by the Fundamental Research Funds for the Central Universities of Central South University (2019zzts807).
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References: [1] M. Huang, D. Yang, M. Xiang, J. Wang, Role of interleukin-6 in regulation of immune responses to remodeling after myocardial infarction, Heart Fail Rev 20(1) (2015) 25-38. https://doi.org/10.1007/s10741-014-9431-1 [2] S. Nergiz, O.S. Altinkaya, M. Kucuk, H. Yuksel, S.D. Sezer, I. Kurt Omurlu, A.R. Odabasi, Circulating galanin and IL-6 concentrations in gestational diabetes mellitus, Gynecol Endocrinol 30(3) (2014) 236-40. https://doi.org/10.3109/09513590.2013.871519 [3] S.M.K. Stancu, L.K. Ash, C. Smeding, M.A. Alwan, Predictors of Caesarean Delivery in Preterm Premature Rupture of Membranes, Open Access Maced J Med Sci 7(7) (2019) 1124-1128. https://doi.org/10.3889/oamjms.2019.250 [4] G.E. Osanyin, O. Adegbola, Maternal serum interleukin 6 levels and fetomaternal outcomes in women with preterm premature rupture of membranes in Lagos, South-western Nigeria, J Matern Fetal Neonatal Med 29(21) (2016) 3506-10. https://doi.org/10.3109/14767058.2015.1135123 [5] I. Musilova, C. Andrys, M. Drahosova, O. Soucek, M. Stepan, T. Bestvina, R. Spacek, B. Jacobsson, T. Cobo, M. Kacerovsky, Intraamniotic inflammation and umbilical cord blood interleukin-6 concentrations in pregnancies complicated by preterm prelabor rupture of membranes, J Matern Fetal Neonatal Med 30(8) (2017) 900-910. https://doi.org/10.1080/14767058.2016.1197900 [6] R. Romero, P. Chaemsaithong, N. Chaiyasit, N. Docheva, Z. Dong, C.J. Kim, Y.M. Kim, J.S. Kim, F. Qureshi, S.M. Jacques, B.H. Yoon, T. Chaiworapongsa, L. Yeo, S.S. Hassan, O. Erez, S.J. Korzeniewski, CXCL10 and IL-6: Markers of two different forms of intra-amniotic inflammation in preterm labor, Am J Reprod Immunol 78(1) (2017). https://doi.org/10.1111/aji.12685 [7] C.C. Otenio, I. Fonseca, M.F. Martins, L.C. Ribeiro, N.M. Assis, A.P. Ferreira, R.A. Ribeiro, Expression of IL-1beta, IL-6, TNF-alpha, and iNOS in pregnant women with periodontal disease, Genet Mol Res 11(4) (2012) 4468-78. https://doi.org/10.4238/2012.September.20.3 [8] Y. Hu, M. Yang, Y. Zhou, Y. Ding, Z. Xiang, L. Yu, Establishment of reference intervals for procalcitonin in healthy pregnant women of Chinese population, Clin Biochem 50(3) (2017) 150-154. https://doi.org/10.1016/j.clinbiochem.2016.10.013 [9] Y. Yang, H. Jiang, A. Tang, Z. Xiang, Changes of serum homocysteine levels during pregnancy and the establishment of reference intervals in pregnant Chinese women, Clin Chim Acta 489 (2019) 1-4. https://doi.org/10.1016/j.cca.2018.11.026 [10] H. Song, H. Lim, Evidence for heterodimeric association of leukemia inhibitory factor (LIF) receptor and gp130 in the mouse uterus for LIF signaling during blastocyst implantation, Reproduction 131(2) (2006) 341-9. https://doi.org/10.1530/rep.1.00956 [11] P.L. Caron, G. Frechette-Frigon, C. Shooner, V. Leblanc, E. Asselin, Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells, Reprod Biol Endocrinol 7 (2009) 80. https://doi.org/10.1186/1477-7827-7-80 [12] C.N. Nagineni, V.K. Kommineni, A. William, J.J. Hooks, B. Detrick, IL-11 expression in retinal and corneal cells is regulated by interferon-gamma, Biochem Biophys Res Commun 391(1) (2010) 287-92. https://doi.org/10.1016/j.bbrc.2009.11.051 [13] H.N. Jabbour, K.J. Sales, R.D. Catalano, J.E. Norman, Inflammatory pathways in 8
female reproductive health and disease, Reproduction 138(6) (2009) 903-19. https://doi.org/10.1530/REP-09-0247
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Highlights Serum IL-6 levels are significantly different between healthy non-pregnant women and healthy pregnant women group. Serum IL-6 levels were significantly different between the first and second trimester group as well as the first and third trimester group, but there was no significant difference between the second and third trimester group. Healthy pregnant women in the second and third trimester could be combined into one group to calculate serum IL-6 RIs and serum IL-6 RIs for healthy pregnant women in the first trimester should be established separately.
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Abstract Objective: To explore the changes of serum interleukin-6 (IL-6) levels in healthy pregnant women and establish reference intervals (RIs). Method: According to the requirements for the RIs study model and the reference population screening criteria in C28-A3 document, Serum IL-6 levels were measured by electrochemiluminescence immunoassay in 480 healthy Chinese women, including 120 pregnant women in each of the first, second and third trimester and 120 non-pregnant women as the negative control. The establishment of RIs for IL-6 were defined using nonparametric percentile. Results: The RIs for serum IL-6 levels in healthy pregnant women is <4.19pg/ml, the RIs for serum IL-6 levels in healthy pregnant women who are in the first trimester is <3.52pg/ml, and the RIs for serum IL-6 levels in healthy pregnant women who are in the second and third trimester is <4.40pg/ml. Conclusions: Serum IL-6 level in healthy pregnant women is higher than the healthy non-pregnant women, and the level of IL-6 who are in the second and third trimester is higher than those in the first. This paper successfully established RIs for serum IL-6 levels in pregnant women, providing a reference for clinical medical staff and laboratory workers. Key words：IL-6, pregnant women, reference intervals.
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Tables Table 1. Baseline characteristic of the participants Pregnant Total
Non-pregnant The first trimester
The second trimester
The third trimester
N
480
120
120
120
120
Age（years）
30.6±4.9
30.9±4.7
30.5±4.3
29.8±5.3
30.3±5.0
BMI（kg/m2）
24.4±2.2
24.3±1.9
24.2±2.6
24.6±2.0
24.7±2.8
SBP（mm Hg） 133.5±15.9
125.7±12.6
134.6±14.5
135.8±17.5
138.2±22.6
DBP（mm Hg） 77.0±11.0
72.6±9.9
73.7±9.5
77.6±9.4
84.2±11.6
Glu（mmol/L） 5.25±0.61
5.19±0.54
5.16±0.60
5.31±0.71
5.34±0.78
TC（mmol/L） 4.71±0.43
4.78±0.36
4.73±0.40
4.68±0.52
4.66±0.59
TG（mmol/L） 1.17±0.60
1.20±0.63
1.18±0.49
1.14±0.55
1.16±0.6
117.4±6.2
120.5±6.6
119.8±5.5
116.9±6.0
112.2±5.9
Hb（g/L）
Values are presented as the mean ± SD unless otherwise indicated.
Table 2. Serum IL-6 levels in healthy non-pregnant women and healthy pregnant women (pg/ml). Standard N
Median
2.5th percentile
97.5th percentile
deviation Non-pregnant
78
1.510
0.026
1.500
1.518
Pregnant a
354
2.318
0.913
1.511
4.550
a
Comparison of serum IL-6: has significant difference from non-pregnant group (p<0.001).
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Table 3. Serum IL-6 levels in healthy pregnant women in different trimesters (pg/ml). N First trimester (1–13 week)
Standard
2.5th
deviation
percentile
Median
97.5th percentile
114
1.975
0.696
1.500
3.875
120
2.335
0.853
1.525
4.343
120
2.644
1.058
1.542
5.687
Second trimester (14–27 week) Third trimester (≥28 week)a, b a
Comparison of serum IL-6: has significant difference from the first trimester group (P<0.05). Comparison of serum IL-6: has no significant difference from the second trimester group (P>0.05). b
Table 4. The RIs of serum IL-6 for healthy non-pregnant women and healthy pregnant women (pg/ml). 95.0th N
RIs percentile
Non-pregnant
120
1.518
<1.518
The first trimester
120
3.520
<3.520
The second and third trimester
240
4.400
<4.400
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Author Contributions: Zhongyuan Xiang contributed to the conception of the study. Yaoyang Fu and Lingli Tang contributed significantly to analysis and manuscript preparation; Yaoyang Fu and Qinglin Liu performed the data analyses and wrote the manuscript; Min Hu helped perform the analysis with constructive discussions.
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S0009-8981(19)32182-5 https://doi.org/10.1016/j.cca.2019.12.013 CCA 15963
To appear in:
Clinica Chimica Acta
Received Date: Revised Date: Accepted Date:
16 September 2019 21 November 2019 16 December 2019
Please cite this article as: Y. Fu, L. Tang, M. Hu, Q. Liu, Z. Xiang, Changes of serum interleukin-6 in healthy pregnant women and establishment of relevant reference intervals, Clinica Chimica Acta (2019), doi: https:// doi.org/10.1016/j.cca.2019.12.013
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Changes of serum interleukin-6 in healthy pregnant women and establishment of relevant reference intervals Yaoyang Fua, Lingli Tanga, Min Hua, Qinglin Liua, Zhongyuan Xianga,1 a Department of Laboratory Medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China 1 Corresponding author at: Department of Laboratory Medicine, The Second Xiangya Hospital, Central South University, #139 Renmin Middle Rd, Changsha, Hunan 410011, China. E-mail address: [email protected] (Z. Xiang).
1
Abstract Objective: To explore the changes of serum interleukin-6 (IL-6) levels in healthy pregnant women and establish reference intervals (RIs). Method: According to the requirements for the RIs study model and the reference population screening criteria in C28-A3 document, Serum IL-6 levels were measured by electrochemiluminescence immunoassay in 480 healthy Chinese women, including 120 pregnant women in each of the first, second and third trimester and 120 non-pregnant women as the negative control. The establishment of RIs for IL-6 were defined using nonparametric percentile. Results: The RIs for serum IL-6 levels in healthy pregnant women is <4.19pg/ml, the RIs for serum IL-6 levels in healthy pregnant women who are in the first trimester is <3.52pg/ml, and the RIs for serum IL-6 levels in healthy pregnant women who are in the second and third trimester is <4.40pg/ml. Conclusions: Serum IL-6 level in healthy pregnant women is higher than the healthy non-pregnant women, and the level of IL-6 who are in the second and third trimester is higher than those in the first. This paper successfully established RIs for serum IL-6 levels in pregnant women, providing a reference for clinical medical staff and laboratory workers. Key words：IL-6, pregnant women, reference intervals.
2
1. Introduction As a pleiotropic cytokine, interleukin-6 (IL-6) is usually produced in local tissues and released into the blood circulation in the case of autologous disorders such as endotoxemia, trauma and acute infection. In addition to the known role of IL-6 in combating chronic infections in the cell[1], IL-6 in the circulation is also involved in the innate immune process. Clinically, serum IL-6 is closely related to the severity of inflammation and bacterial infection. In patients with systemic infection, the degree of elevation is more obvious, so it can be used as a sensitive indicator for sepsis. Determination of IL-6 levels in blood is important for assessing disease progression and prognosis. Pregnancy is based on an evolutionary balance of cytokines that undergo changes in cytokine type or concentration at different stages of pregnancy. Specifically, Th2-type anti-inflammatory cytokines predominate in the middle pregnancy, while Th1-type pro-inflammatory cytokines are mainly distributed in the early and late stages of pregnancy and are characterized by inflammatory-like events. As an important indicator of inflammation, the changes of IL-6 are particularly important. Studies have shown[2] that the serum levels of IL-6 in gestational diabetes and gestational hypertension are significantly higher than those in healthy gestational women. The serum level of IL-6 in pregnancy-induced hypertension is associated with pregnancy outcome and is linearly positively correlated with systolic blood pressure. In addition, in a prospective cohort study[3], the study team followed a group of pregnant women with premature rupture of membranes and healthy pregnant women until delivery, and found that the average serum IL-6 level was significantly higher in women with premature rupture of membranes. The fetal outcomes of patients with IL-6≥14pg/mL were poor. Therefore, measurement of maternal serum IL-6 can reflect the adverse effects of the fetal intrauterine environment and the need for necessary interventions. In addition to the above, IL-6 also shows certain indication significance in chorioamnionitis[4], premature delivery[5], intrauterine infection[6], and maternal periodontopathy[7]. However, at present, the domestic clinical laboratory has only the interleukin-6 reference intervals (RIs) for the normal population. As a special group, pregnant women show physiological changes different from the general population. Whether the existing IL-6 RIs is suitable for pregnant women is questionable. Therefore, before using IL-6 levels as indicators of gestational complications, it is necessary to determine the specific RIs for IL-6 in pregnant women[8]. In this study, a standard RIs for maternal serum IL-6 was established according to the Clinical and Laboratory Standards Institute (CLSI) C28-A3 document, providing a useful and reliable clinical reference for physicians.
2. Materials and Methods 2.1. Subjects The information of pregnant women who were admitted to the Second Xiangya Hospital of Central South University (Hunan, Changsha) for prenatal check-up or delivery from September 2018 to May 2019 was collected. This study was approved by the ethics committee of the Second Xiangya Hospital of Central South University 3
and all participants have signed the informed consent form. The selection of healthy pregnant and non-pregnant women was based on the exclusion criteria from CLSI C28-A3 guidelines: (1) diagnosed as chronic disease, liver disease or endocrine disease; (2) having a family history of genetic disease; (3) Body mass index (BMI) ≥ 28kg/m2 or ≤ 18.5kg/m2; (4) History of hypertension for more than 3 years (systolic blood pressure ≥140mm Hg or diastolic blood pressure ≥90mm Hg); (5) abnormalities in the heart, liver, lungs and kidneys found by ultrasound or electrocardiogram; (6) undergoing surgery within 4 months before pregnancy or blood transfusion or blood donation within 6 months; (7) taking drugs or abusing antibiotics within 2 weeks; (8) Excessive smoking (more than 20 cigarettes per day) or drinking (30 grams or more per day); (9) excessive labor or exercise; (10) The following laboratory results occurred: triglyceride ≥ 2.26mmol/L, total cholesterol ≥ 6.22mmo/L, fasting blood glucose > 7.0mmol/L, hepatitis B surface antigen, hepatitis C antibody or HIV antibody positive, abnormal urine, red blood cells Count ≥ 5.0 × 1012/L or ≤ 3.0 × 1012/L, hemoglobin ≤ 110g/L, white blood cell count ≥ 12.0 × 109/ L or ≤ 4.0 × 109 / L, platelet count <100 × 109/L. According to the above recruitment conditions, a total of 480 healthy Chinese women, including 120 pregnant women in each of the first, second and third trimester and 120 non-pregnant women as the negative control participated in the study. Non-pregnant women are not in menstruation. The average age of the participants in the experimental groups was 30.9±4.7; 30.5±4.3; 29.8±5.3; 30.3±5.0 for non-pregnant, the first, second and third trimester, respectively. The baseline characteristics of the individuals are presented in Table 1, including body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), glucose (Glu), total cholesterol (TC), triglyceride (TG), hemoglobin (Hb).The blood samples for each trimester are not collected serially from the same pregnant women. 2.2. IL-6 quantification All of the participants were asked to maintain their normal lifestyle and avoid strenuous physical exercise within 3 days of physical examination. The participants fasted overnight for at least 8 h and sat for at least 30 min before specimen collection. The specimens collected in the experiment were free of hemolysis, chyle and jaundice. After the specimen trait is determined, the sample is tested on the machine within 2 hours. The concentration of IL-6 were analyzed on Roche Cabas E602 (Germany) by electrochemiluminescence immunoassay (ECLIA). All of the tests were performed according to standard operating procedures recommended by NCCLS. To evaluate instrument performance, two control concentrations of IL-6 were tested. Quality control results are under control. The precision and accuracy were demonstrated according to the document EP 15-A2 recommended by CLSI. The detection limit of the detection system is 1.5pg/ml. We dilute the low-value control to 2-3pg/ml, dispense and freeze at -20 degrees for intra-assay and inter-assay precision experiments. The intra-assay and inter-assay imprecision are 4.92% and 4.22%, respectively. We collected multiple samples and conducted simultaneous tests with the 4
hospitals that participated in the inter-room quality assessment to verify the accuracy of our results. The bias between the results is within the allowable bias. So, the precision and accuracy is in accordance with the requirements of the current standard documents. 2.3 Statistical analysis All data were analyzed using SPSS 22.0 Statistical Software (SPSS Inc., Chicago, USA). The distribution of the data was detected using Kolmogorov-Smirnov test. Outliers were excluded using Dixon test. For non-normal distribution, Mann-Whitney U test were used to compare variables between two groups and Kruskal-Wallis H test was applied to compare variables among multiple groups. Differences at P<0.05 were considered statistically significant. According to the recommendations of CLSI C28-A3 document, a non-parametric test was used to calculate serum IL-6 RIs. Reference range were expressed between the 0th and 95.0th percentiles, because the upper limit of IL-6 is of clinical significance.
3. Results During the test, the values of IL-6 in 42 non-pregnant women and 6 the first trimester women were lower than the lower limit of detection in the system. Because the reference interval of interleukin 6 is generally only one side, it does not affect the establishment of subsequent reference intervals. In comparing the differences in interleukin-6 between non-pregnant women and pregnant women, we stripped out all such undetected data. If there is still a difference, it indicates that there is indeed a difference between the two. 3.1. IL-6 levels in healthy non-pregnant women and healthy pregnant women. Komlogorov-Smimov test demonstrated serum IL-6 levels were not in normal distribution in both healthy non-pregnant women and healthy pregnant women group (P<0.05). Outliers were removed by Dixon's test. U test showed that serum IL-6 levels were significantly different between healthy non-pregnant women and healthy pregnant women group (P<0.05). Therefore, serum IL-6 RIs for healthy non-pregnant and pregnant women should be established separately (Table 2). 3.2. IL-6 levels in healthy pregnant women in different trimesters According to CLSI C28-A3 guidelines, healthy pregnant women were divided into three groups according to gestational age: first trimester (1-13 week), second trimester (14-27 week), and third trimester (≥28 week) group to calculate RIs. According to Komlogorov-Smimov test, serum IL-6 levels were not in normal distribution in the first and second trimester groups (P<0.05). But for the third trimester group, serum IL-6 is in line with normal distribution (P=0.14). Outliers were removed by Dixon's test. H test confirmed that serum IL-6 levels for healthy women in first, second, and third trimester group were not all significantly different (P>0.05). U tests were thus performed for pairwise comparisons and serum IL-6 levels were significantly different between the first and second trimester group as well as the first and third trimester group (P<0.05). Nevertheless, there was no significant difference 5
between the second and third trimester group (P>0.05). Therefore, healthy pregnant women in the second and third trimester could be combined into one group to calculate serum IL-6 RIs and serum IL-6 RIs for healthy pregnant women in the first trimester should be established separately (Table 3). 3.3 IL-6 RIs for healthy non-pregnant women and healthy pregnant women. IL-6 is only clinically meaningful with reference to the upper limit of the interval. According to CLSI C28-A3 guidelines, the RIs of serum IL-6 for healthy non-pregnant women and healthy pregnant women were calculated (Table 4). Therefore，serum IL-6 RIs is <1.518 for healthy non-pregnant women. For healthy pregnant women, serum IL-6 RIs are <3.52 pg/ml in the first trimester and <4.40 pg/mL for in the second and third trimester.
4. Discussion At present, for pregnant women, partial RIs used in laboratory tests is still the same as that of the normal population, which have not considered the unique physiological and biochemical changes during pregnancy[9]. Establishing a medical RIs for commonly used testing items for pregnant women is conducive to improving the diagnostic level of clinicians for pregnant women, making subsequent examinations and treatments more rational, ensuring greater utilization of medical resources, and so on. This is indeed an urgent problem to be solved in domestic clinical trials. IL-6 is a promising biochemical marker with high sensitivity and specificity, which is an early assessment of infection, and an early warning for some common gestational diseases, such as gestational diabetes, pregnancy hypertension, premature rupture of membranes. IL-6 has an important role in the health of pregnant women and the fetus, so it is necessary to pay attention to changes in serum IL-6 levels during pregnancy. In this study, the reference population was selected in strict accordance with the screening criteria of the CLSI C28-A3 guidelines, and a serum IL-6 RIs was established for a special population of healthy pregnant women. The experiment first compared the serum IL-6 levels between healthy pregnant women and healthy pregnant women, showing a significant difference between the two groups (P<0.001), suggesting that the serum IL-6 RIs for pregnant women should be established separately. Then we analyzed the levels of IL-6 in the three stages of pregnancy, showing significant differences in serum IL-6 levels between the first and the second trimester, the first and the third trimester (P＝0.003). There was no significant difference in serum IL-6 levels between the second trimester and the third trimester (P ＝0.318), suggesting the need to establish a RIs of the first trimester, the second and the third trimester. The RIs of serum IL-6 level in healthy pregnant women is <4.19pg/ml. The RIs of serum IL-6 in the first trimester is <3.52pg/mL, and the RIs of serum IL-6 in the second and the third trimester is <4.40pg/ml. In pregnancy, for the maternal immune system, it is necessary to satisfy two situations at the same time: on the one hand, it is necessary to protect the mother from infection, and on the other hand, to receive a semi-allogene fetus. This process reflects the positive regulation of the inflammatory immune process during pregnancy to ensure 6
specific tolerance to foreign fetal antigens[10]. Pregnancy can be considered as an embryo-induced inflammatory process in the endometrium, and as the pregnancy progresses, the embryo-induced inflammation gradually becomes stronger, which may explain the increase in IL-6 levels in pregnant women. For pregnant women, the pro-inflammatory and anti-inflammatory pathways are involved in the establishment of the receptor endometrium (implantation window) and embryo-endometrial communication. Many of the cytokines produced by embryos are pro-inflammatory, suggesting that implantation is the process by which an embryo induces an inflammatory pathway in the endometrium. The endometrium responds to these embryonic signals by enhancing the expression of receptive genes required for embryo adhesion and invasion during the implantation window. At the time of preparation for implantation, decidualization initially occurs around the spiral arteriole during the secretory phase and occurs remarkably throughout the endometrium if pregnancy occurs. In addition to the differentiation of endometrial stromal cells into decidual cells, decidualization is also involved in triggering inflammatory events such as leukocyte infiltration, modification of extracellular matrices, and increased vascular permeability. Cytokines, particularly IL-6, play an important role in implantation. In vitro studies have shown that their receptors are expressed at the implantation site by several cells. The knockout mouse model has demonstrated that interleukin-11 and leukemia inhibitory factor (LIF) play an important role in implantation[10], while IL-6 affects fertility and implantation efficiency. Leukemia inhibitory factor is a pro-inflammatory cytokine expressed in epithelial and decidual stromal cells and is mediated by several inflammatory mediators such as interleukin-1, tumor necrosis factor (TNF), leptin, insulin-like growth factor, transforming growth factor (TGF-β)[11, 12]. And the recent discovery of prokinetic protein regulation suggests the complexity of regulation of the inflammatory pathway[13]. Thence, it is important to understand the changes of IL-6 during pregnancy in detail. In our study, The changes in IL-6 during pregnancy have been described, and corresponding reference intervals have been established. This will help clinicians to provide some help for the medical diagnosis of pregnant women. In summary, rigorously according to CLSI C28-A3 guidelines, the authoritative document of RIs establishment, we established the RIs of serum IL-6 for healthy pregnant Chinese women. The results of this study will provide a valuable reference for clinical work and laboratory researches.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Acknowledgments: Thanks to Professor Aiguo Tang for the revision and suggestions of this article. The work was supported by the Fundamental Research Funds for the Central Universities of Central South University (2019zzts807).
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References: [1] M. Huang, D. Yang, M. Xiang, J. Wang, Role of interleukin-6 in regulation of immune responses to remodeling after myocardial infarction, Heart Fail Rev 20(1) (2015) 25-38. https://doi.org/10.1007/s10741-014-9431-1 [2] S. Nergiz, O.S. Altinkaya, M. Kucuk, H. Yuksel, S.D. Sezer, I. Kurt Omurlu, A.R. Odabasi, Circulating galanin and IL-6 concentrations in gestational diabetes mellitus, Gynecol Endocrinol 30(3) (2014) 236-40. https://doi.org/10.3109/09513590.2013.871519 [3] S.M.K. Stancu, L.K. Ash, C. Smeding, M.A. Alwan, Predictors of Caesarean Delivery in Preterm Premature Rupture of Membranes, Open Access Maced J Med Sci 7(7) (2019) 1124-1128. https://doi.org/10.3889/oamjms.2019.250 [4] G.E. Osanyin, O. Adegbola, Maternal serum interleukin 6 levels and fetomaternal outcomes in women with preterm premature rupture of membranes in Lagos, South-western Nigeria, J Matern Fetal Neonatal Med 29(21) (2016) 3506-10. https://doi.org/10.3109/14767058.2015.1135123 [5] I. Musilova, C. Andrys, M. Drahosova, O. Soucek, M. Stepan, T. Bestvina, R. Spacek, B. Jacobsson, T. Cobo, M. Kacerovsky, Intraamniotic inflammation and umbilical cord blood interleukin-6 concentrations in pregnancies complicated by preterm prelabor rupture of membranes, J Matern Fetal Neonatal Med 30(8) (2017) 900-910. https://doi.org/10.1080/14767058.2016.1197900 [6] R. Romero, P. Chaemsaithong, N. Chaiyasit, N. Docheva, Z. Dong, C.J. Kim, Y.M. Kim, J.S. Kim, F. Qureshi, S.M. Jacques, B.H. Yoon, T. Chaiworapongsa, L. Yeo, S.S. Hassan, O. Erez, S.J. Korzeniewski, CXCL10 and IL-6: Markers of two different forms of intra-amniotic inflammation in preterm labor, Am J Reprod Immunol 78(1) (2017). https://doi.org/10.1111/aji.12685 [7] C.C. Otenio, I. Fonseca, M.F. Martins, L.C. Ribeiro, N.M. Assis, A.P. Ferreira, R.A. Ribeiro, Expression of IL-1beta, IL-6, TNF-alpha, and iNOS in pregnant women with periodontal disease, Genet Mol Res 11(4) (2012) 4468-78. https://doi.org/10.4238/2012.September.20.3 [8] Y. Hu, M. Yang, Y. Zhou, Y. Ding, Z. Xiang, L. Yu, Establishment of reference intervals for procalcitonin in healthy pregnant women of Chinese population, Clin Biochem 50(3) (2017) 150-154. https://doi.org/10.1016/j.clinbiochem.2016.10.013 [9] Y. Yang, H. Jiang, A. Tang, Z. Xiang, Changes of serum homocysteine levels during pregnancy and the establishment of reference intervals in pregnant Chinese women, Clin Chim Acta 489 (2019) 1-4. https://doi.org/10.1016/j.cca.2018.11.026 [10] H. Song, H. Lim, Evidence for heterodimeric association of leukemia inhibitory factor (LIF) receptor and gp130 in the mouse uterus for LIF signaling during blastocyst implantation, Reproduction 131(2) (2006) 341-9. https://doi.org/10.1530/rep.1.00956 [11] P.L. Caron, G. Frechette-Frigon, C. Shooner, V. Leblanc, E. Asselin, Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells, Reprod Biol Endocrinol 7 (2009) 80. https://doi.org/10.1186/1477-7827-7-80 [12] C.N. Nagineni, V.K. Kommineni, A. William, J.J. Hooks, B. Detrick, IL-11 expression in retinal and corneal cells is regulated by interferon-gamma, Biochem Biophys Res Commun 391(1) (2010) 287-92. https://doi.org/10.1016/j.bbrc.2009.11.051 [13] H.N. Jabbour, K.J. Sales, R.D. Catalano, J.E. Norman, Inflammatory pathways in 8
female reproductive health and disease, Reproduction 138(6) (2009) 903-19. https://doi.org/10.1530/REP-09-0247
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Highlights Serum IL-6 levels are significantly different between healthy non-pregnant women and healthy pregnant women group. Serum IL-6 levels were significantly different between the first and second trimester group as well as the first and third trimester group, but there was no significant difference between the second and third trimester group. Healthy pregnant women in the second and third trimester could be combined into one group to calculate serum IL-6 RIs and serum IL-6 RIs for healthy pregnant women in the first trimester should be established separately.
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Abstract Objective: To explore the changes of serum interleukin-6 (IL-6) levels in healthy pregnant women and establish reference intervals (RIs). Method: According to the requirements for the RIs study model and the reference population screening criteria in C28-A3 document, Serum IL-6 levels were measured by electrochemiluminescence immunoassay in 480 healthy Chinese women, including 120 pregnant women in each of the first, second and third trimester and 120 non-pregnant women as the negative control. The establishment of RIs for IL-6 were defined using nonparametric percentile. Results: The RIs for serum IL-6 levels in healthy pregnant women is <4.19pg/ml, the RIs for serum IL-6 levels in healthy pregnant women who are in the first trimester is <3.52pg/ml, and the RIs for serum IL-6 levels in healthy pregnant women who are in the second and third trimester is <4.40pg/ml. Conclusions: Serum IL-6 level in healthy pregnant women is higher than the healthy non-pregnant women, and the level of IL-6 who are in the second and third trimester is higher than those in the first. This paper successfully established RIs for serum IL-6 levels in pregnant women, providing a reference for clinical medical staff and laboratory workers. Key words：IL-6, pregnant women, reference intervals.
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Tables Table 1. Baseline characteristic of the participants Pregnant Total
Non-pregnant The first trimester
The second trimester
The third trimester
N
480
120
120
120
120
Age（years）
30.6±4.9
30.9±4.7
30.5±4.3
29.8±5.3
30.3±5.0
BMI（kg/m2）
24.4±2.2
24.3±1.9
24.2±2.6
24.6±2.0
24.7±2.8
SBP（mm Hg） 133.5±15.9
125.7±12.6
134.6±14.5
135.8±17.5
138.2±22.6
DBP（mm Hg） 77.0±11.0
72.6±9.9
73.7±9.5
77.6±9.4
84.2±11.6
Glu（mmol/L） 5.25±0.61
5.19±0.54
5.16±0.60
5.31±0.71
5.34±0.78
TC（mmol/L） 4.71±0.43
4.78±0.36
4.73±0.40
4.68±0.52
4.66±0.59
TG（mmol/L） 1.17±0.60
1.20±0.63
1.18±0.49
1.14±0.55
1.16±0.6
117.4±6.2
120.5±6.6
119.8±5.5
116.9±6.0
112.2±5.9
Hb（g/L）
Values are presented as the mean ± SD unless otherwise indicated.
Table 2. Serum IL-6 levels in healthy non-pregnant women and healthy pregnant women (pg/ml). Standard N
Median
2.5th percentile
97.5th percentile
deviation Non-pregnant
78
1.510
0.026
1.500
1.518
Pregnant a
354
2.318
0.913
1.511
4.550
a
Comparison of serum IL-6: has significant difference from non-pregnant group (p<0.001).
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Table 3. Serum IL-6 levels in healthy pregnant women in different trimesters (pg/ml). N First trimester (1–13 week)
Standard
2.5th
deviation
percentile
Median
97.5th percentile
114
1.975
0.696
1.500
3.875
120
2.335
0.853
1.525
4.343
120
2.644
1.058
1.542
5.687
Second trimester (14–27 week) Third trimester (≥28 week)a, b a
Comparison of serum IL-6: has significant difference from the first trimester group (P<0.05). Comparison of serum IL-6: has no significant difference from the second trimester group (P>0.05). b
Table 4. The RIs of serum IL-6 for healthy non-pregnant women and healthy pregnant women (pg/ml). 95.0th N
RIs percentile
Non-pregnant
120
1.518
<1.518
The first trimester
120
3.520
<3.520
The second and third trimester
240
4.400
<4.400
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Author Contributions: Zhongyuan Xiang contributed to the conception of the study. Yaoyang Fu and Lingli Tang contributed significantly to analysis and manuscript preparation; Yaoyang Fu and Qinglin Liu performed the data analyses and wrote the manuscript; Min Hu helped perform the analysis with constructive discussions.
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