Amniotic fluid CD36 in pregnancies complicated by spontaneous preterm delivery: a retrospective cohort study

Abstract Objective The aim of this study was to evaluate CD36 concentrations in amniotic fluid in pregnancies complicated by spontaneous delivery with intact fetal membranes (preterm labor, PTL) and preterm prelabor rupture of membranes (PPROM) with respect to the presence of the intra-amniotic infection. Methods A total of 80 women with PPROM and 71 with PTL were included in the study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid CD36 concentrations were assessed by enzyme-linked immunosorbent assay. Microbial colonization of the amniotic cavity (MIAC) was determined by the cultivation and non-cultivation approach. Intra-amniotic inflammation (IAI) was defined as an amniotic fluid bedside interleukin-6 concentration ≥3000 pg/mL. Intra-amniotic infection was characterized by the presence of both MIAC and IAI. Results Women with PPROM with intra-amniotic infection had higher amniotic fluid CD36 concentrations than women without infection (with infection: median 346 pg/mL, IQR 262–384 vs. without infection: median 242 pg/mL, IQR 199–304; p = .006) A positive correlation between amniotic fluid CD36 concentrations and interleukin-6 concentrations was found (rho = 0.48; p < .0001). In PTL pregnancies, no statistically significant difference was found in the amniotic fluid level of CD36 between intra-amniotic infection, sterile IAI, and negative amniotic fluid. Conclusions The presence of intra-amniotic infection is characterized by higher amniotic fluid CD36 concentrations in pregnancies complicated by PPROM. An amniotic fluid CD36 cutoff value of 252.5 pg/mL was found to be optimal for the prediction of intra-amniotic infection. In PTL pregnancies, no statistically significant change in CD36 concentration was found with respect to the presence of intra-amniotic infection.


Introduction
Preterm birth, that is birth before the 37th week of pregnancy, is a significant problem in contemporary obstetrics [1,2]. Preterm labor can be induced artificially for medical reasons (iatrogenic preterm labor) or triggered spontaneously. Spontaneous preterm births account for approximately two-thirds of all preterm births [1,2]. The etiology of spontaneous preterm births is complex. Such delivery can occur as spontaneous delivery with intact fetal membranes (preterm labor, PTL) or as delivery with preterm prelabor rupture of the membranes (PPROM) [1,2]. These phenotypes are often accompanied by microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation (IAI), and intra-amniotic infection [3][4][5][6]. Intra-amniotic infection is diagnosed when signs of inflammation and MIAC occur simultaneously [7]. If inflammation occurs due to endogenous causes without evidence of an infectious agent, it is called sterile inflammation [3]. Regardless of its origin, IAI is triggered by the release of signals of danger, followed by the migration and action of cells of the immune system. This situation is always accompanied by the release of various cytokines, chemokines, and other substances, mainly into the amniotic fluid; however, a change in the concentration of a number of substances has also been found in other biological materials, such as cervical fluid, umbilical cord blood, or maternal plasma [8][9][10]. Intra-amniotic infection is associated with an increased risk of fetal and neonatal complications [11][12][13][14][15], and its diagnosis is based on the detection of such cytokines and other substances together with the identification of microorganisms. This information is important in clinical decision-making when it is necessary to decide between an active and wait-and-see approach in women at risk of preterm birth [3]. The diagnosis of IAI is based on the determination of interleukin-6 (IL-6), which is currently the most commonly used marker of IAI, and increased concentrations of IL-6 are evident during inflammation in all the above-mentioned biological materials [16][17][18][19][20][21][22].
Cultivation and molecular biology techniques were used to identify microbial agents. Although these are modern and sophisticated procedures, they are timeconsuming. Therefore, efforts of several scientific groups have focused on finding a reliable biomarker whose concentration changes would indicate an ongoing intra-amniotic infection early. Proteomic studies focusing on the detection of IAI and intra-amniotic infection have yielded significant data on changes in the concentrations of many biomarkers. Many molecules have the potential for diagnostic use, and much information has provided more specific insights into the pathophysiology of inflammation-related preterm births [23][24][25][26][27][28][29][30][31]. However, a suitable and reliable marker that could be routinely used to identify intra-amniotic infections has not been found.
This study aimed to provide information about the importance of CD36 in the pathophysiology of preterm birth and intra-amniotic infection. CD36 was first described in the 1970s as "glycoprotein IV" on platelet membranes [32]. The molecule belongs to the scavenger receptor family, and its ligands include thrombospondin-1, oxidized phospholipids, oxidized low-density lipoproteins, fibrillar Ab amyloid peptides, and long-chain fatty acids [33]. The CD36 protein is expressed on many types of cells, especially monocytes and macrophages, platelets, megakaryocytes, myeloid and erythroid precursors, dendritic cells, smooth and skeletal muscle cells, microvascular endothelial cells, adipocytes, and epithelial cells [33][34][35]. Because of its ability to bind the abovementioned substances, the CD36 molecule is important in the process of internalization of apoptotic cells and some bacterial and fungal pathogens, which are associated with the activation of Toll-like receptors 4 and 6; therefore, the action of CD36 is pro-inflammatory [36][37][38]. From our point of view in our study, it is significant that this molecule also participates in the initiation of sterile inflammation, which has been described especially in the pathogenesis of atherosclerotic diseases [34][35][36]. The pro-inflammatory effect of CD36 also lies in its role in the activation of NLRP3 and composition of the inflammasome with subsequent production of the important pro-inflammatory cytokine, IL-1b [39]. Activation of the NLRP3 inflammasome is directly related to the development of IAI and PPROM [40]. According to Deng et al. [41], increased expression of CD36 (together with transforming growth factor b) is also related to increased epithelial-mesenchymal transition. However, according to Menon [42], dysregulation of this process is related to the inappropriate setting of fetal membrane cells, thereby leading to an increased risk of preterm birth. This study aimed to determine the concentration of CD36 in amniotic fluid samples from women with PTL and PPROM in relation to microbial colonization, sterile IAI, and intra-amniotic infection. Our results aimed to provide information on whether the concentration of molecules changes during the abovementioned conditions and whether it can theoretically be used for the diagnosis of intra-amniotic infection.

Study population
This retrospective cohort study included pregnant women admitted to the Department of Obstetrics and Gynecology at the University Hospital Hradec Kralove in the Czech Republic between March 2019 and November 2021. Inclusion criteria were as follows: (1) singleton pregnancy; (2) maternal age !18 years; (3) gestational age between 24 þ 0 and 36 þ 6 weeks for PPROM and between 22 þ 0 and 34 þ 6 weeks for PTL; (4) PPROM or PTL; and (5) transabdominal amniocentesis at the time of admission to determine IAI. Exclusion criteria were as follows: (1) pregnancy-related and other medical complications, such as fetal growth restriction, gestational or pre-gestational diabetes, gestational or chronic hypertension, and preeclampsia; (2) structural or chromosomal fetal abnormalities; (3) signs of fetal hypoxia; and (4) significant vaginal bleeding. Gestational age was determined using first-trimester fetal biometry.
PPROM was diagnosed by examining the women using a sterile speculum for pooling amniotic fluid in the posterior fornix of the vagina. In cases of clinical uncertainty in diagnosing PPROM, amniotic fluid leakage was confirmed by the presence of insulinlike growth factor-binding proteins (Actim PROM test; Medix Biochemica, Kauniainen, Finland) in the vaginal fluid.
PTL was diagnosed as the presence of regular uterine contractions (at least two contractions every 10 min), along with cervical length (measured using transvaginal ultrasound) shorter than 15 mm or within 15-30 mm with a positive PartoSure test (Parsagen Diagnostics Inc., Boston, MA) [43].
Transabdominal amniocentesis is a part of clinical management of PPROM and PTL and it is offered to all pregnant women with PPROM and PTL weeks to assess the intra-amniotic environment. Women with PPROM were treated with antibiotics. Patients with IAI received first-line treatment with intravenous clarithromycin for seven days. Unless delivery occurred earlier, antibiotic treatment was eventually modified under the condition of MIAC; women without IAI received benzylpenicillin (clindamycin was used in women allergic to penicillin).
Women with PPROM below the gestational age of 35 þ 0 weeks received corticosteroids (betamethasone) to accelerate fetal lung maturation and reduce neonatal mortality and morbidity. Women with PPROM were managed expectantly, except those with intraamniotic infection beyond the gestational age of 28 þ 0 weeks, wherein labor was induced or an elective cesarean section was performed within 72 h of admission.
Women with PTL received a course of corticosteroids (betamethasone) and tocolytic therapy with intravenous atosiban (for gestational age 28 weeks) or nifedipine, which was administered orally, for 48 h. Patients with proven IAI received treatment with intravenous clarithromycin for seven days unless delivery occurred earlier. Antibiotic treatment was eventually modified by the MIAC. Women with PTL who were positive for group B Streptococcus (GBS), as determined from the vaginal-rectal swab, or with an unknown GBS status received intravenous benzylpenicillin (clindamycin, in case of penicillin allergy) during active labor.
All the participants provided informed written consent prior to the collection of amniotic fluid samples. The sample collection for this research was approved by the Institutional Review Board of the University Hospital Hradec Kralove (July 2014; No. 201408 S07P). All the experiments were performed in accordance with the relevant guidelines and regulations. All the participants were Caucasians.

Amniotic fluid sampling
Ultrasonography-guided transabdominal amniocentesis was performed and approximately 5 ml of amniotic fluid was aspirated. Amniotic fluid was dispensed into polypropylene tubes and used for the assessment of IL-6, aerobic/anaerobic cultivation, and molecular biological analyses to identify microbial nucleic acids. The remaining amniotic fluid was centrifuged and used for research purposes. The supernatant was aliquoted and stored at À80 C until further analysis.

Assessment of IL-6
The concentrations of IL-6 in the amniotic fluid were assessed using an automated electrochemiluminescence immunoassay method. Interleukin-6 concentrations were measured using the immuno-analyzer Cobas e602, which is a component of the Cobas 8000 platform (Roche Diagnostics, Basel, Switzerland) [44]. The basic measuring range was 1.5-5000 pg/mL, and this could be extended to 50.000 pg/mL with a 10-fold dilution of the sample. The coefficients of variation for the inter-and intra-assay precisions were <10% [44].

Detection of Ureaplasma species, M. hominis and C. trachomatis
We isolated DNA from the amniotic fluid using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Real-time polymerase chain reaction (PCR) was conducted on a Rotor-Gene 6000 instrument (Qiagen, Hilden, Germany) using commercial AmpliSens.
The C. trachomatis/Ureaplasma/M. hominis-FRT kit (Federal State Institution of Science, Central Research Institute of Epidemiology, Moscow, Russia) was used to detect DNA from Ureaplasma species, M. hominis and C. Trachomatis, in the same multiplex PCR tube (multiplex format). We included a PCR run for b-actin, a housekeeping gene that served as the control, to examine the presence of PCR inhibitors.

Detection of other bacteria in amniotic fluid
Bacterial DNA was identified by PCR targeting the 16S rRNA gene with the following primers: 5 0 -CCAGACT CCTACGGGAGG CAG-3 0 (V3 region) and 5 0 -ACATTTCAC AACAC-GAGCTGACGA-3 0 (V6 region) [45,46]. Each reaction contained 3 lL target DNA, 500 nM forward and reverse primers, and Q5 high-fidelity DNA polymerase (NEB, Ipswich, MA, USA) in a total volume of 25 lL. Amplification was carried out on a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The products were visualized on an agarose gel. Positive reactions yielded 950 bp products that were subsequently analyzed by sequencing. The 16S rDNA PCR products were purified and subjected to sequencing with the above-mentioned primers and BigDye Terminator kit v.3.1 (Thermo Fisher Scientific, Waltham, MA, USA). The bacteria were then typed by searching for the obtained sequences using BLAST and SepsiTest BLAST.

Aerobic and anaerobic cultures of amniotic fluid
The amniotic fluid samples were cultured on Columbia agar with sheep blood, Gardnerella vaginalis selective medium, MacConkey agar, Neisseria-selective medium (modified Thayer-Martin medium), Sabouraud agar, and Schaedler anaerobe agar. The samples were cultured for six days and checked daily. Species were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using MALDI Biotyper software (Bruker Daltonics, Bremen, Germany).

Quantification of CD36 in amniotic fluid
The concentrations of CD36 in amniotic fluid samples were assessed using an enzyme-linked immunosorbent assay (ELISA)the Human CD36 Kit (LifeSpan BioSciences, Inc., Seattle, WA, USA), according to the manufacturer's instructions. Amniotic fluid samples were not diluted. The sensitivity of the kit was 156 pg/mL. Absorbance was read at 450 nm using a Multiskan RC ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA).

Clinical definitions
Microbial invasion of the amniotic cavity was determined based on positive PCR analysis of Ureaplasma species, M. hominis, C. Trachomatis, or a combination of these species or positivity for the 16S rRNA gene, aerobic/anaerobic cultivation of the amniotic fluid, or a combination of these parameters. Intra-amniotic inflammation was defined as amniotic fluid IL-6 concentration !3000 pg/mL [44]. Intra-amniotic infection was defined as both microbial invasion of the amniotic cavity and IAI. Sterile IAI was defined as the presence of IAI without concomitant MIAC. Colonization was defined as MIAC without IAI. Negative amniotic fluid was defined as MIAC and IAI.

Statistical analyses
Women's demographic and clinical characteristics were compared using the nonparametric Mann-Whitney U test for continuous variables and are presented as median values (interquartile range [IQR]).
Categorical variables were compared using Fisher's exact test and are presented as number (%). Data normality was tested using the Anderson-Darling test. Because the CD36 concentrations in the amniotic fluid were not normally distributed, nonparametric Kruskal-Wallis and Mann-Whitney U tests were performed for statistical analyses, and the results were presented as median values (IQR). Receiver operating characteristic (ROC) curves were constructed to assess the predictive value of amniotic fluid CD36 for IAI. Cutoff values were determined based on the highest positive likelihood ratio. Differences were considered statistically significant at p < .05. All p values were obtained using two-tailed tests, and all statistical analyses were performed using GraphPad Prism version 8.4.3. for Windows OS (GraphPad Software, San Diego, CA, USA).

Results
A total of 80 women with PPROM and 71 with PTL were included in the study. The presence of intraamniotic infection, sterile IAI, colonization, and negative amniotic fluid was observed in 20% (16/80), 9% (7/80), 15% (12/80), and 56% (45/80) of the women with PPROM, respectively. Intra-amniotic infection, sterile IAI, and negative amniotic fluid were found in 10% (7/71), 30% (21/71), and 60% (44/71) of women with PTL, respectively. None of the women with PTL had colonization. The demographics of all the women in this study and clinical data of the women with PPROM and PTL, based on the presence and absence of intra-amniotic infection, are shown in Tables 1 and 2, respectively. The microorganisms identified in amniotic fluid from women with PPROM and PTL are listed in Table 3.
No differences in amniotic fluid CD36 concentrations were found between women with colonization and negative amniotic fluid. Women with intra-amniotic infection had higher amniotic fluid CD36 concentrations than those without intra-amniotic infection (with infection: median 346 pg/mL, IQR 262-384 vs. without infection: median 242 pg/mL, IQR 199-304; p ¼ .006; Figure 2(a)). No differences in amniotic fluid CD36 concentrations were observed between women with sterile IAI and those with infection.

PTL pregnancies
In PTL pregnancies, no statistically significant difference was found in the amniotic fluid level of CD36 between intra-amniotic infection, sterile IAI, and negative   Figure 1(b)). No differences in amniotic fluid CD36 concentrations were found between women with and without intra-amniotic infection. No correlation was found between amniotic fluid CD36 and IL-6 concentration (rho ¼ 0.04; p ¼ .72). Table 3. Microbial species identified in the amniotic fluid of women with preterm prelabor rupture of membranes and spontaneous preterm labor with intact membranes.
Preterm prelabor rupture of membranes Preterm labor with intact membranes Ureaplasma spp.

Discussion
Principal findings of the study The main findings of this study are as follows: (i) the CD36 molecule is present in a soluble form in the amniotic fluid of women with preterm birth; (ii) in women with PPROM, amniotic fluid CD36 levels are elevated during inflammatory conditions, regardless of their origin (infectious, sterile); (iii) CD36 levels do not increase in the case of colonization without signs of an inflammatory response; (iv) comparison of CD36 levels in women with intra-amniotic infection and sterile inflammation showed no statistically significant differences; (v) in women with PPROM, a correlation was found between CD36 and IL-6 levels; (vi) a value of 252.5 pg/mL of the CD36 molecule in amniotic fluid appears to be optimal for the detection of intraamniotic infection in women with PPROM; and (vii) in women with PTL, no significant differences were found in the concentration of CD36 in the amniotic fluid during intra-amniotic infection, sterile inflammation, and negative findings.

Meaning of the study
Identification of intra-amniotic infection remains a challenge for obstetricians. Early identification of intraamniotic infection helps to appropriately set up further care for women and tighten their observation. . From these findings, we can conclude that intra-amniotic infection threatens newborns with a higher risk of health complications, owing to lower weight and gestational age. A diagnostic approach that includes a combination of identifying microbial presence and inflammation is also useful in distinguishing between a condition in which the presence of microorganisms is detectable, but an inflammatory response has not yet been triggered (colonization), and an ongoing intra-amniotic infection. However, microbiological analysis is time-and labor-intensive. Therefore, attention is focused on the determination of various biomarkers that can accurately and quickly identify ongoing infections and enable appropriate interventions. Currently, IL-6 is considered the most reliable marker of IAI, although there has been an effort to find a more accurate and suitable biomarker that would indicate ongoing intra-amniotic infection and herald the risk of preterm birth. In this study, such an effort was focused on the CD36 molecule, whose relationship to the prediction of intra-amniotic infection has not yet been investigated. The results of this study revealed that the level of CD36 in patients with PPROM increases with the level of IL-6, and that elevated levels of this molecule reliably distinguish between ongoing intra-amniotic infection and simple microbial colonization or a negative finding. However, these differences were not observed when evaluating samples from women with PTL. Thus, it seems that the CD36 molecule is not a universal biomarker for the detection of infection, regardless of the type of preterm birth, as our group found, for example with the IgGFcBP [31], where elevated levels of the investigated protein were detected in the amniotic fluid of women with PPROM and PTL. However, what leads to this phenomenon can only be speculated. The small number of amniotic fluid samples from women with intra-amniotic infection in PTL (n ¼ 7) may be to blame because the level of free CD36 in the amniotic fluid of women with PTL and infection is generally higher than that in the amniotic fluid of women without infection. Moreover, if we observe a phenomenon in the amniotic fluid of women with PPROM, it would be logical to observe the same phenomenon in the amniotic fluid of women with PTL, especially since we know that IL-6 levels are much higher in women with PTL; it can be assumed that the intensity of inflammation will be higher. Thus, the explanation is that the CD36 molecule could have been released, especially during the rupture of the amniotic membranes, but this is not supported by the fact that the median CD36 levels are very similar in the case of intra-amniotic infection in PPROM and PTL. To verify this finding, it would be appropriate to perform another analysis with a larger cohort of women.

Strengths and limitations of the study
Similar to any previous study, this study has strengths and limitations. The strength of this study lies in its precise microbiological findings. The combination of modern aerobic and anaerobic techniques and cultivation and non-cultivation techniques has enabled precise microbiological findings. These findings, together with the detection of inflammation using IL-6 levels, enabling the precise identification of patients according to the type of inflammation, even with only bacterial colonization without ongoing inflammation. The strict distinction between preterm birth phenotypes (PPROM and PTL) and the separate assessment of amniotic fluid findings is also a strength of this study. A limitation of the obtained results is the small cohort of women, especially the low representation of women with intra-amniotic infection and sterile IAI. We also consider the fact that we do not know the exact source of CD36 molecule to be a limitation of this study. Due to the wide distribution of the protein on leukocytes and non-leukocyte-origin cells, it is impossible to determine the concrete mechanisms involved in the changes in CD36 concentration. It is also necessary to say that the concentration of soluble CD36 was measured. Thus, cellular expression of CD36 can have different dynamics than that observed in the case of free CD36.

Conclusion
CD36 levels in amniotic fluid are elevated in women with PPROM complicated by intra-amniotic infection and sterile IAI. A level of 252.5 pg/mL was defined as the appropriate level for the detection of IAI. In women with PTL, no statistically significant differences were found in CD36 levels in the amniotic fluid with regard to the presence of infection or inflammation. Thus, CD36 does not appear to be a suitable universal biomarker of intra-amniotic infection in women with preterm labor, regardless of phenotype.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The data that support the findings of this study are available from the corresponding author, [Ondrej Soucek], upon reasonable request.