- Academic Editor
Background: To investigate fractalkine and MIP-1
Premature rupture of membranes (PROM) is defined as spontaneous rupture of fetal membranes before 37 weeks of pregnancy [1] and complicates 5–7% of all pregnancies [2]. In addition to its spontaneous occurrence, it can also develop as a complication of invasive procedures such as amniocentesis, fetal surgery, cerclage placement. Mid-trimester preterm premature rupture of membranes (PPROM) develops before 28 weeks of gestation and is seen in approximately 0.4–0.7% of all pregnancies, and is associated with high neonatal mortality and severe short- and long-term morbidity [3]. Studies have reported that serum inflammatory marker levels are elevated in PROM [4, 5] and perinatal mortality is associated with gestational week at birth, birth weight, and presence of fetal growth restriction and anhydramnios [6, 7].
It has been shown that the matrix metalloproteinase-9 (MMP-9) concentrations
increase in the membrane rupture mechanism in fetuses with PROM, but
interleukin-1 beta (IL-1
Chemokine ligand 1 (CX3CL1) is the only known representative of the
Fractalkine, together with other cytokines such as chemokine CC motif ligand 7
(CCL7), CCL4 (MIP-1
In recent years, various potential biomarkers such as placental protein 14 in amniotic fluid and high-mobility box-1 have been investigated for use in the clinical diagnosis of PROM [19, 20]. An ideal test should be non-invasive, fast, accurate, cost-effective, easily applicable and easily accessible [21].
Currently, analyzes of amniotic fluid (AF) samples obtained through
amniocentesis are generally considered the gold standard approach to identify
microbial invasion of the amniotic cavity (MIAC)/intraamniotic infection (IAI) in
PROM. In particular, IL-6, IL-8 and matrix metalloproteinase (MMP)-8 and -9 in AF
have been shown to be the strongest markers of intra-amniotic infectious and
inflammatory states in PROM [22]. However, their measurements in current practice
may be of limited use due to their invasiveness (e.g., amniocentesis) and
relatively low sensitivity [23]. Although assessment of maternal blood markers
may serve as a non-invasive, desirable and inexpensive approach for the
anticipated management of women with PROM, these inflammatory biomarkers have not
been adequately studied in plasma samples from women with PPROM in relation to
MIAC/IAI [22]. In our single-center, prospective cohort study, we aimed to
investigate whether fractalkin and MIP-1
This prospective cohort study was carried out on 80 patients who applied to the Firat University, Faculty of Medicine, Gynecology and Obstetrics Clinic within the period of 1 January 2022 to 1 September 2022 after obtaining the approval of the Firat University Clinical Ethics Committee (number 2021-13-46). The patients were divided into 2 groups. We selected 40 patients with the required inclusion criteria during the time period for each group who met the study criteria.
Group 1: 40 healthy pregnant patients without PROM who were pregnant between 37 and 41 weeks and had cesarean section due to elective reasons.
Group 2: 40 pregnant patients who had PROM 24 hours before delivery and
were pregnant between 24 weeks and 36
The patients were questioned in terms of age, parity, drug use, and whether they had a systemic disease.
Pregnant women with preterm birth, pregnant women younger than 24 weeks of gestation, hypertensive pregnant women, acute or chronic infections, pregnant women with preeclampsia or eclampsia, gestational diabetes, and intrauterine growth retardation.
Pregnant women who applied to the clinic with the pre-diagnosis of PROM were
examined with a sterile speculum (REF T201793, TMS Medikal ve plastik ürünler san. dış. tic. ltd., Istanbul, Turkey). About 5 mL of amniotic fluid accumulated in the
lower spoon of the speculum from the cases with active amniotic fluid
presentation was drawn into the injector, put into Eppendorf tubes, and quickly
stored in a deep freezer at –80 °C until the working day. In the
meantime, the diagnosis of PROM was confirmed by performing placental alpha
microglobulin-1 protein test (AmniSure test). At the same time, approximately 5
mL of venous blood was collected from the patients, centrifuged and stored at
–80 °C until the study day. All PROM cases were followed up in the
hospital until the pregnancy was terminated. In the follow-up, fever, heart rate
and blood pressure were monitored. At daily visits, foul-smelling vaginal
discharge was investigated and the presence of fundal tenderness was checked with
fundal examination. Weekly sedimentation, leukocyte, C-reactive protein (CRP) and procalcitonin values
were measured. In all cases under 34 weeks of age, 2 doses of betamethasone
(Celestone Chronodose, Schering-Eczacıbaşı, Lüleburgaz, Turkey), 24
mg in total, were administered intramuscularly at 24-hour intervals for lung
maturation. Ampicillin (Ampisina
Pregnancies that reached 34th gestational week were delivered according to the indication (normal labor induction or cesarean section delivery) and were followed up in the clinic for 2 more days postoperatively. Pregnancies admitted with the diagnosis of PROM between 34–37 weeks of gestation were hospitalized and their pregnancies were terminated according to the indication, and blood and amniotic fluid samples were collected and stored as described above.
Pregnant women who applied to the clinic between 37–41 weeks of gestation and
met the exclusion criteria listed above and would have elective cesarean section
constituted the control group. Moreover, 5 mL of blood and about 5 mL of sterile
amniotic fluid were collected from these pregnant women during cesarean section,
and then stored at –80 °C until the study day. TNF-
Biochemical measurements were made using human ELISA kits in serum and amniotic fluid in accordance with the relevant kit procedures. Obtained absorbances were read spectrophotometrically at 450 nm in an EPOCH 2 (Bio Tek Instrument, Inc, Winoosky, VT, USA) microplate reader and the results were obtained.
TNF-
IL-1
IFN-
MIP-1
Fractalkine (CX3CL1) (Sun red Biotechnology Company, Shanghai, China; catalog no = 201-12-2102; kit measuring range = 0.2–30 ng/mL; kit sensitivity = 0.102 ng/mL).
SPSS v.21.0 (IBM Corporation, Armonk, NY, USA) package program was used for
statistical analysis of the data. In data analysis, the distribution of
continuous variables was determined by Shapiro-Wilk normality tests. Data that
did not fit the normal distribution were expressed as median (IQR: interquartile
range) values, while qualitative data were expressed as percentage values. Two
independent groups were compared with the Mann-Whitney U test. p
Age was similar between the groups. Gravida, parity, gestational week, birth weight and umbilical cord pH values in the control group compared to the PROM group was significantly higher (Table 1).
Parameters | Group 1 (Control) | Group 2 (PROM) | p values | ||
(n = 40) | (n = 40) | ||||
Median (min–max) | 25%–75% percentile | Median (min–max) | 25%–75% percentile | ||
Age (years) | 31 (24–41) | 27.25–34.75 | 29 (20–41) | 26–36 | 0.434 |
Gestational week | 38 (37–41) | 37.25–38 | 34 (24–36) | 32–35 | |
Gravida | 4 (1–10) | 2–5.75 | 2 (1–9) | 1–3.75 | 0.005* |
Parity | 2 (0–6) | 1–3.75 | 1 (0–4) | 0–2 | 0.010* |
Birth weight (g) | 3000 (2400–3960) | 2900–3337 | 2275 (620–3140) | 1600–2625 | |
pH value | 7.36 (7.31– 7.44) | 7.33–7.37 | 7.33 (7.22–7.48) | 7.30–7.37 | 0.032* |
* = Compared with Group 1. PROM, premature rupture of membranes.
Group 1 = Control group; Group 2 = PROM (premature rupture of membranes) group. Values are presented as median (min–max) and 25%–75% percentile,
p
Serum TNF-
While amniotic fluid TNF-
Parameters | Group 1 (Control) | Group 2 (PROM) | p values | ||
(n = 40) | (n = 40) | ||||
Median (min–max) | 25%–75% percentile | Median (min–max) | 25%–75% percentile | ||
TNF- |
113.6 (7.50–18511) | 55.5050–171.7 | 108 (7.5–970.55) | 53–148.66 | 0.840 |
IL-1 |
1180.51 (75–634543) | 632–3003 | 909 (29.27–9824) | 332–1531.4 | 0.138 |
IFN- |
106.28 (1.89–308.91) | 80.3925–177.7 | 107.9 (56.7–629) | 71.4–186.5 | 0.744 |
MIP-1 |
25.54 (1.24–25532) | 17.3–55.7 | 25 (13.68–51013) | 22.6–33.7 | 0.400 |
Fractalkine (CX3CL1), ng/mL | 0.29 (0.25–37) | 0.25–8.27 | 2.88 (0.24–94.5) | 2.12–4.96 | 0.227 |
Group 1 = Control group; Group 2 = PROM (premature rupture of membranes) group;
TNF-
Parameters | Group 1 (Control) | Group 2 (PROM) | p values | ||
(n = 40) | (n = 40) | ||||
Median (min–max) | 25%–75% percentile | Median (min–max) | 25%–75% percentile | ||
TNF- |
83.74 (19.77–9239) | 38.8250–96.9 | 54.16 (7.5–159088) | 12.21–154.6475 | 0.893 |
IL-1 |
625.58 (32.99–1301.2) | 296–1160 | 1558.79 (75–2813,58) | 895–2262 | |
IFN- |
22.9 (15.99–68.12) | 21.33–26.72 | 79.915 (16.07–192.1) | 44.4–129.33 | |
MIP-1 |
11.43 (5.73–23.92) | 6.215–15.21 | 23.125 (1.47–57.7) | 10.335–38.68 | |
Fractalkine (CX3CL1), ng/mL | 0.25 (0.25–2.99) | 0.25–1.81 | 3.84 (0.25–14.63) | 1.4925–7.155 |
Group 1 = Control group; Group 2 = PROM (premature rupture of membranes) group;
TNF-
As a result of our study, we showed that there was a significant decrease in
birth weight of PROM cases because their pregnancies were terminated earlier. In
addition, although the umbilical artery pH value at birth was within clinically
normal limits, it was significantly lower in the PROM group. We detected a
significant increase in the levels of proinflammatory cytokines Fractalkine and
MIP-1
In our study, we found that gravida and parity were significantly lower in our PROM group. The fact that the gestational week and birth weights were also lower in the PROM group is due to the fact that we delivered our PROM cases around the 34th week.
Chemokines [24] act as chemo-attractants that modulate the migration of
leukocytes and the immune response [25]. The major roles of membrane-bound CX3CL1
are by activating target cells, promoting leukocyte attachment and adhesion. In
humans, fractalkine is chemotactic for T cells and monocytes [26]. Many stimuli
that can affect cell homeostasis potentially induce fractalkine secretion [27].
Fractalkine also exerts its effects through the fractalkine receptor (CX3CR1)
[10]. It has been shown that maternal plasma cytokines (IL-1
Generally, chemokines are involved in immune system homeostasis, while inducible chemokines are primarily involved in inflammatory processes. Chemokines are divided into four subfamilies according to the number and spacing of the first two cysteine residues [29]. These four subfamilies are called C, CC, CXC, and CX3C. Here C represents a cysteine and X represents an amino acid residue. It has been shown that the level of fractalkine increases in the first trimester of pregnancy and in term pregnancy complicated by preeclampsia [30, 31, 32]. We also included cases with PROM starting from the 3rd trimester in our study. In addition, we excluded complicated pregnancies such as diabetes, intrauterine growth retardation, hypertension and preeclampsia. Thus, we aimed to prevent the inflammatory cytokine and fractalkine levels in the blood and amniotic fluid from being affected by any other complicated condition. In our control group, we included the cases without complicated pregnancy and at 37 and 41 weeks of gestation, because we thought that fractalkine and other inflammatory cytokines may play a role in the pathophysiology of preterm birth. It could be considered that the control group should be selected from patients with cord prolapse or those who underwent amniocentesis. We did not choose these groups as the control group, because cord prolapse results in PROM and membrane damage. A similar situation may occur in amniocentesis and amniocentesis is usually performed before the third trimester. For these reasons, and also because the third trimester amniocentesis and cord prolapse are very few, we selected our control group from cesarean section cases between 37–41 weeks.
Fractalkine regulates adhesion and migration in fetal-maternal interaction at
different stages of human pregnancy. However, some pregnancy pathologies such as
chorioamnionitis [33] and severe early-onset preeclampsia [34] have been
suggested to be associated with increased placental fractalkine expression.
Hannan et al. [35] reported that serum fractalkine levels were higher in
patients who had abortion in the first trimester, while it was undetectable in
patients who had normal delivery. Li et al. [36] showed that fractalkine
and fractalkine receptors were significantly upregulated in the uterus of
IFN-
Chemokine CC motif ligand 4 (CCL4) (MIP-1) has been shown to be involved in
implantation [37]. However, high serum MIP-1
Based on the study, it is accepted that normal pregnancy is actually a
controlled inflammatory state and many pregnancy-related complications are
associated with an exaggerated local or systemic inflammatory response [39].
Therefore, it has been reported that a successful pregnancy depends on the
balance between anti-inflammatory and pro-inflammatory cytokines [40].
MIP-1
Human amniotic epithelial cells (HAEC) are released into the amniotic fluid by
various cytokines such as chemokines and fractalkine, and the chemokine profile
of HAEC during pregnancy can be altered by many factors. Up-regulation of this
chemokine expression by inflammatory stimuli has been considered, since
fractalkine provides regulation of lymphocyte accumulation at sites of
inflammation [41, 42]. However, inflammation is often accompanied by significant
local decreases in oxygen availability [43] and hypoxia has been shown to
significantly inhibit fractalkine production [13, 44]. This is a confusing
situation. Because both inflammation and hypoxia cause a local increase of
TNF-
Chorioamnionitis is inflammation of the amniotic and chorionic membranes and is associated with significant maternal and perinatal adverse outcomes [48]. Clinical chorioamnionitis is characterized by maternal fever, leukocytosis, tachycardia, uterine tenderness and premature rupture of membranes (PROM). Subclinical/histological chorioamnionitis, which is more common than clinical chorioamnionitis, is asymptomatic and is characterized by inflammation of the chorion, amnion, and placenta [49]. The frequency of histologic chorioamnionitis is higher than clinical chorioamnionitis with positive bacterial cultures. Since the incidence of histological chorioamnionitis in PROM is up to 50%, pathological evaluation of the placenta is required for a definitive diagnosis [50]. The role of fractalkine in premature rupture of membranes (PROM) and preterm labor is still under investigation [51]. None of our cases had clinical chorioamnionitis during the study. Therefore, we could not evaluate the proinflammatory and inflammatory cytokine levels in PROM and chorioamnionitis and make a comment.
One study showed that none of the proteins measured in maternal plasma were associated with IAI. In addition, cytokine/chemokine levels in plasma were reported to be weakly correlated with those in amniotic fluid (AF) in this study. These findings also indicate that microbes or microbe-derived cytokines/chemokines in the amniotic cavity are poorly reflected in plasma. Therefore, it is very difficult to identify new non-invasive biomarkers for IAI and/or to perform MIAC using maternal blood samples, especially in women with preterm labor (PTL) [52]. Evaluation of protein levels using maternal blood samples has been shown in one study to have limited clinical value for the noninvasive identification of PROM pregnancies complicated by MIAC/IAI [22]. As a result of our study, we showed that cytokine/chemokine levels were significantly increased in amniotic fluid rather than maternal serum in PROM cases. This shows that amniotic fluid is more valuable in determining PROM cases.
Limitations of our study: (1) Limited number of cases, our parameters could not be studied to compare the 1st, 2nd and 3rd trimester PROM cases of the study. (2) Our study was not grouped according to PROM durations. (3) The absence of a clinical chorioamnionitis group in our study is another limitation. Since chorioamnionitis did not develop clinically in any of our cases during the study, we could not form the chorioamnionitis group. (4) Another limitation of ours is the inability to examine the placenta histologically and examine the presence of inflammation, and the parameters we studied were not evaluated both biochemically and immunohistochemically.
The strengths of our study are that it is a prospective cohort study, and fractalkine levels in maternal serum and amniotic fluid have been studied for the first time. With further studies, the potential of fractalkin levels to be an early biomarker has been emphasized in the early diagnosis of PROM and the determination of cases at risk of developing PROM.
In conclusion, fractalkine and MIP-1
All data generated or analyzed during this study are included in this published article.
ŞP and RA designed the study. ŞP, BÇ, MKA, MDC performed the research, collected data. SCO analyzed the data, helped and advised on writing manuscript. ŞP and RA wrote manuscript. Nİ gave the results of study. MY analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
This study was carried out at Fırat University, Faculty of Medicine, Gynecology and Obstetrics Clinic after obtaining the approval of the Fırat University Clinical Ethics Committee number 2021-13-46. All patient’s informed consent is obtained.
Not applicable.
This research received no external funding.
The authors declare no conflict of interest. Süleyman Cemil Oğlak is serving as one of the Guest editors of this journal. We declare that Süleyman Cemil Oğlak had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Michael H. Dahan.
Publisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.