IgGFc-binding protein in pregnancies complicated by spontaneous preterm delivery: a retrospective cohort study

To determine the IgGFc-binding protein (FcgammaBP) concentration in amniotic and cervical fluids in preterm prelabor rupture of membranes (PPROM) and preterm labor with intact membranes (PTL) and to assess the diagnostic indices of FcgammaBP to predict intra-amniotic infection (the presence of both microbial invasion of the amniotic cavity and intra-amniotic inflammation). In this study, we included 170 and 79 women with PPROM and PTL, respectively. Paired cervical and amniotic fluid samples were obtained using a Dacron polyester swab and transabdominal amniocentesis, respectively. The FcgammaBP concentrations in the samples were assessed using an enzyme-linked immunosorbent assay. The presence of intra-amniotic infection was associated with elevated FcgammaBP concentrations in pregnancies with PPROM and PTL [PPROM—presence: 86 ng/mL vs. absence: 13 ng/mL, p < 0.0001, area under receiver operating characteristic curve (AUC) = 0.94; PTL—presence: 140 ng/mL vs. absence: 22 ng/mL, p < 0.0001, AUC = 0.86]. In cervical fluid, the concentrations of FcgammaBP were elevated in the presence of intra-amniotic infection in pregnancies with PPROM only (presence: 345 ng/mL vs. absence: 60 ng/mL, p < 0.0001, AUC = 0.93). FcgammaBP in amniotic fluid might be a marker of intra-amniotic infection in women with both PPROM and PTL However, in cervical fluid, it is only observed in women with PPROM.

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 the amniotic fluid from women with PPROM and PTL are listed in Table 3.
Amniotic fluid FcgammaBP concentrations based on the phenotype of intra-amniotic inflammation. PPROM pregnancies. PPROM pregnancies with intra-amniotic infection and sterile intra-amniot- Table 1. Maternal and clinical characteristics of women with preterm prelabor rupture of membranes based on the presence and absence of intra-amniotic infection. Continuous variables were compared using a nonparametric Mann-Whitney U test. Categorical variables were compared using the Fisher's exact test. Continuous variables are presented as median (IQR) and categorical as number (%). Statistically significant results are marked in bold. CRP: C-reactive protein; IL-6: interleukin-6; IQR: interquartile range; WBC: white blood cells.

Characteristic
The presence of intra-amniotic infection (n = 19) The absence of intra-amniotic infection (n = 151) p-value  Fig. 1a). No differences in amniotic fluid FcgammaBP concentrations were found between the women with colonization and negative amniotic fluid (Table 4). Women with intra-amniotic infection had higher amniotic fluid FcgammaBP than did those without intraamniotic infection (with infection: median IQR 85.6 ng/mL, IQR 37.3-146.0 vs. without infection: median 12.6 ng/mL, IQR 9.1-20.3; Fig. 2a). The amniotic fluid FcgammaBP cutoff value of 60 ng/mL was optimal in the prediction of intra-amniotic infection (area under the ROC curve [AUC] = 0.94; p < 0.0001; Fig. 2b). The diagnostic indices of these cutoff values are in Table 5.
PTL pregnancies. Differences in the concentrations of FcgammaBP were identified among the subgroups of women with intra-amniotic infection, sterile intra-amniotic inflammation, and negative amniotic fluid (infec-  Table 3. The microbial species identified in the amniotic fluid of women with preterm prelabor rupture of membranes and with spontaneous preterm labor with intact membranes.   Fig. 1b) in the crude analysis and after the adjustment for gestational age at sampling (both p-values < 0.0001). Women with intra-amniotic infection had higher amniotic fluid FcgammaBP concentrations than did women with sterile intra-amniotic inflammation and with negative amniotic fluid ( Table 6). Women with sterile intra-amniotic inflammation had higher amniotic fluid FcgammaBP concentrations than those with negative amniotic fluid (  Fig. 3a). The amniotic fluid FcgammaBP cutoff value of 120 ng/mL was found to be optimal in the prediction of intra-amniotic infection (AUC = 0.86; p < 0.0001; Fig. 3b). The diagnostic indices of these cutoff values are in Table 5.  Table 4. IgGFc-binding protein in amniotic fluid from preterm prelabor rupture of membranes: the comparisons among the subgroups of the women with intra-amniotic infection, sterile intra-amniotic inflammation, colonization, and negative amniotic fluid. p-value: a comparison between two subgroups (a nonparametric Mann-Whitney U test); Adj. p-value: a comparison between two subgroups after the adjustment for gestational age at sampling (a Spearman partial correlation). Statistically significant results are marked in bold.

Sterile intra-amniotic inflammation Colonization Negative
Intra-amniotic infection x p = 0.11 adj. p = 0.51 www.nature.com/scientificreports/   Table 6. IgGFc-binding protein in amniotic fluid from preterm labor with intact membranes: the comparisons among the subgroups of the women with intra-amniotic infection, sterile intra-amniotic inflammation, and negative amniotic fluid. p-value: a comparison between two subgroups (a nonparametric Mann-Whitney U test); Adj. p-value: a comparison between two subgroups after the adjustment for gestational age at sampling (a Spearman partial correlation). Statistically significant results are marked in bold. www.nature.com/scientificreports/  Fig. 4a) in the crude analysis as well as after the adjustment for gestational age at sampling (both p < 0.0001). Women with intra-amniotic infection had higher cervical fluid FcgammaBP concentrations than women with sterile intra-amniotic inflammation, colonization, and negative amniotic fluid (Table 7). Women with intra-amniotic infection had higher cervical fluid FcgammaBP than those without intra-amniotic infection (with infection: median 345.0 ng/mL, IQR 201.9-480.0 vs. without infection: median 59.6 ng/ mL, IQR 31.9-111.5; Fig. 5a). The cervical fluid FcgammaBP cutoff value of 300 ng/mL was found to be optimal in the prediction of intra-amniotic infection (AUC = 0.93; p < 0.0001; Fig. 5b). The diagnostic indices of these cutoff values are in Table 5. Meaning of the study. FcgammaBP was discovered more than 30 years ago as a specific site for the fragment of crystallizable (Fc) region of the immunoglobulin (Ig) G antibody in the small intestinal and colonic epithelia 65 . This specific site differed from previously recognized receptors in the Fc region of IgG 65 . The specific www.nature.com/scientificreports/ site for the Fc region of IgG was later termed FcgammaBP and identified as a protein primarily localized in the mucosal granules of the small intestinal and colonic epithelia that are secreted into the intestinal lumen 66 . Based on the current knowledge, FcgammaBP is considered to be a protein that provides immunologic protection of the intestinal tissue and facilitates the interaction between the intestinal mucus and potentially harmful stimuli (microorganisms, alarmins) with the ultimate goal of protecting the mucosal surface 62,65,66 . However, its exact biological function has yet to be fully elucidated. The production of FcgammaBP has been described to occur in the intestinal epithelial cells, placenta, and thyroid tissue 62,63 . However, its expression has not been observed in the brain, heart, kidney, liver, lung, and skeletal muscles 62 . Interestingly, the ability to produce FcgammaBP was confirmed only in humans and monkeys, but not in mice, rats, rabbits, dogs, bovines, and porcines 62 .

Concentration of FcgammaBP in cervical fluid based on the phenotype of intra-amniotic in
FcgammaBP has been found in low concentrations in human serum from healthy individuals 64 . However, its serum concentrations were elevated in the presence of autoimmune diseases such as Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, and progressive systemic sclerosis 64 Table 7. IgGFc-binding protein in cervical fluid from preterm prelabor rupture of membranes: the comparisons among the subgroups of the women with intra-amniotic infection, sterile intra-amniotic inflammation, colonization, and negative amniotic fluid. p-value: a comparison between two subgroups (a nonparametric Mann-Whitney U test); adj. p-value: a comparison between two subgroups after the adjustment for gestational age at sampling (a Spearman partial correlation). Statistically significant results are marked in bold.

Sterile intra-amniotic inflammation
Colonization Negative  36,37 . The observation from this study, where we found FcgammaBP as a constituent of amniotic fluid from pregnancies with PPROM and PTL, is in line with the abovementioned findings. Previously, concentrations of FcgammaBP in amniotic fluid have been shown to be higher in women with PPROM with microbial invasion of the amniotic cavity and acute histological chorioamnionitis than in those without these complications 36 . Interestingly, no differences in the amniotic fluid concentration of FcgammaBP between the presence and absence of the abovementioned complications were identified in women with PTL, where amniotic fluid was obtained from the forewaters at the end of the first stage of labor 37 .
In this study, we found elevated amniotic fluid concentrations of FcgammaBP in the presence of both phenotypes of intra-amniotic inflammation. Interestingly, in women with PTL, the concentrations of FcgammaBP in amniotic fluid were higher in the presence of intra-amniotic infection than in the presence of sterile intraamniotic inflammation. Collectively, the results from this study clearly show that both infectious and noninfectious stimuli might trigger the production of FcgammaBP.
In this study, the concentrations of FcgammaBP were measured in paired amniotic and cervical fluid samples obtained from women with both phenotypes of spontaneous preterm delivery. Interestingly, the FcgammaBP concentrations were higher in the cervical fluid samples than in the amniotic fluid samples, despite the fact that cervical fluid samples obtained with a swab were diluted in 1.5 mL of the buffer. These observations suggest that epithelial cells and/or immune cells in the endocervical canal are able to produce FcgammaBP. This finding supports the key role of the cervix during pregnancy, which is its immunologic protection against the ascension of microorganisms from the vagina and the cervix toward the upper genital tract [67][68][69][70] .
It is obvious that the protein composition of a cervical fluid sample from pregnancies with PPROM may be substantially affected by amniotic fluid that has leaked from the amniotic cavity. Therefore, the cervical fluid samples of PPROM pregnancies may reflect such a situation in both the intra-amniotic and cervical compartments. This is a possible explanation as to why women with intra-amniotic infection had higher cervical fluid FcgammaBP concentrations than those without intra-amniotic infection in PPROM but not in PTL pregnancies. However, a weak positive correlation between amniotic and cervical fluid FcgammaBP protein concentrations was also found in PTL.
This study suggests that FcgammaBP might be a new biomarker for intra-amniotic infection in both phenotypes of spontaneous preterm delivery. This finding is clinically very relevant since confirmation of intra-amniotic infection represents a challenge for clinicians. The necessity to rule in or rule out the presence of microorganisms in amniotic fluid makes the diagnosis of intra-amniotic infection time-consuming and more expensive when the techniques used to identify either non-culturable or difficult-to-culture microorganisms are employed. Therefore, from a clinical point of view, there is an urgent need to discover a single marker of intra-amniotic infection that has reliable sensitivity and specificity. In this study, FcgammaBP in amniotic fluid was identified as a potential Comparing diagnostic indices among amniotic fluid FcgammaBP, MMP-8, and IL-6 to identify intra-amniotic infection in pregnancies with PTL, amniotic fluid FcgammaBP has the highest likelihood ratio [FcgammaBP (cut-off value of 120 ng/mL) 7.8, MMP-8 (cut-off value of 10 ng/mL) 3.3, IL-6 (cut-off value of 745 pg/mL) 2.6, and IL-6 (cut-off value of 1000 pg/mL) 3.0] 21 . In addition, in women with PPROM, FcgammaBP in cervical fluid was also revealed as a potential marker of intra-amniotic infection. Particularly, cervical fluid FcgammaBP can be a clinically relevant marker given the non-invasive nature of cervical fluid sampling. Moreover, its diagnostic indices are better than previously published diagnostic indices of IL-6 in cervical fluid (AUC = 0.78, the positive likelihood ratio of 4.8) in PPROM pregnancies 71 . In addition, cervical fluid sampling can be safely repeated during the latency period of pregnancy complicated by PPROM. Therefore, FcgammaBP assessment in cervical fluid might be used to monitor the development of secondary intra-amniotic infection during the latency interval in women with PPROM.
Strengths and limitations of the study. The strength of this study is the relatively large cohort of paired samples of amniotic and cervical fluid. Second, the fluid samples were collected from well-defined phenotypes of spontaneous preterm delivery (PPROM and PTL). Finally, the thorough assessment of microbial invasion of the amniotic cavity, by a combination of culture and non-culture methods, provided an opportunity to precisely distinguish the subsets of women with intra-amniotic infection and sterile intra-amniotic inflammation.
This study also has some limitations that are worth mentioning. For example, there was a small number of women with intra-amniotic infection (n = 19 and n = 12). To confirm whether the concentration of FcgammaBP in amniotic fluid and cervical fluid is a reliable marker of intra-amniotic infection, the results need to be replicated in independent cohorts. Next, despite the FcgammaBP expression in the placenta that was described 62 , the questions of which part of the placenta is a source of FcgammaBP and whether fetal membranes produce Fcgam-maBP still remain unanswered. A body of evidence has shown that intestinal epithelial cells produce FcgammaBP (97, 102, 103), but no data are available on whether amniotic epithelial cells can produce FcgammaBP. Given the importance of the amniotic epithelium as a barrier against the ascension of microorganisms into the amniotic cavity [72][73][74] , some similarities between intestinal and amniotic epithelial cells might be identifiable such as, (1) to serve as mechanical barriers 72-76 ; (2) to have spatially expressed toll-like receptors 77,78 ; and (3) to indicate that the expression of toll-like receptors changes when inflammation is present [77][78][79] . Therefore, we hypothesize that the amniotic epithelium might be involved in FcgammaBP production.

Conclusion
The concentrations of FcgammaBP observed in amniotic and cervical fluid were elevated in women with intraamniotic infection. Thus, after replication in an independent cohort, FcgammaBP in amniotic fluid might be a potential marker of intra-amniotic infection in pregnancies with PPROM and PTL. Moreover, FcgammaBP in cervical fluid could be a marker of intra-amniotic infection in pregnancies with PPROM.

Methods
This retrospective cohort study included pregnant women who were admitted to the Department of Obstetrics and Gynecology at the University Hospital Hradec Kralove in the Czech Republic between March 2017 and May 2020. The inclusion criteria were the following: (1) singleton pregnancy, (2) maternal age ≥ 18 years, (3) gestational age between 22 + 0 and 36 + 6 weeks, (4) PPROM or PTL, and (5) the performance of transabdominal amniocentesis at the time of admission to determine intra-amniotic inflammation. In contrast, the exclusion criteria were the following: (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.
The gestational age was determined via the use of first-trimester fetal biometry. PPROM was diagnosed by examining the women, using a sterile speculum, for pooling of amniotic fluid in the posterior fornix of the vagina. In the case of clinical uncertainty in diagnosing PPROM, amniotic fluid leakage was confirmed by the presence of insulin-like 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 the 15-30 mm range with a positive PartoSure test (Parsagen Diagnostics Inc., Boston, MA) 80 .
Women with PPROM were treated with antibiotics. Those with intra-amniotic inflammation received firstline treatment with intravenous clarithromycin for seven days. Unless delivery occurred earlier, the antibiotic treatment was eventually modified under the condition of microbial invasion of the amniotic cavity; the women without intra-amniotic inflammation received benzylpenicillin (clindamycin was used in women allergic to penicillin). Women with PPROM below the gestational age of 35 + 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 intra-amniotic infection beyond the gestational age 28 + 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 either intravenous atosiban (for gestational age ≤ 28 weeks) or with nifedipine, which was administered orally, for 48 h. Patients with proven intra-amniotic inflammation received treatment with intravenous clarithromycin for seven days, unless delivery occurred earlier. www.nature.com/scientificreports/ microbial invasion of the amniotic cavity. Women with PTL that 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 an active labor. All participants in this study provided informed written consent prior to the collection of amniotic and cervical fluid samples. Sample collection for this research was approved by the Institutional Review Board of the University Hospital Hradec Kralove (July 2014; No. 201408 S07P). All experiments were performed in accordance with relevant guidelines and regulations. All participants were Caucasian.
Cervical and amniotic fluid sampling. Paired cervical fluid and amniotic fluid samples were collected at the time of admission from all women included in this study, prior to the administration of antibiotics, tocolytics, and/or corticosteroids. Each cervical fluid sample was obtained by placing a Dacron polyester swab in the cervical canal for 20 s to achieve saturation. Once collected, the polyester swab was inserted into a polypropylene tube containing 1.5 mL of phosphate-buffered saline; the tube was then shaken for 20 min. Upon removal of the polyester swab, the tube was centrifuged at 300×g for 15 min at room temperature. The supernatant was divided into aliquots and stored at − 80 °C until further analysis.
Ultrasonography-guided transabdominal amniocentesis was performed after cervical fluid sampling. Approximately 2-3 mL of amniotic fluid was aspirated, and the amniotic fluid was immediately divided among polypropylene tubes. The samples of amniotic fluid were used for (i) the assessment of amniotic fluid interleukin (IL)-6; (ii) polymerase chain reaction (PCR) analysis of Ureaplasma species, Mycoplasma hominis, and Chlamydia trachomatis; (iii) sequencing of the 16S rRNA gene; and (iv) aerobic and anaerobic cultivation.
Detection of Ureaplasma species, M. hominis, and C. trachomatis. DNA was isolated from amniotic fluid using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Real-time PCR was conducted on a Rotor-Gene 6000 instrument (Qiagen) using the commercial AmpliSens C. trachomatis/Ureaplasma/M. hominis-FRT kit (Federal State Institution of Science, Central Research Institute of Epidemiology, Moscow, Russia) to detect the DNA from Ureaplasma species, M. hominis, and C. trachomatis in the same PCR tube (multiplex format). We included a PCR run for beta-actin, a housekeeping gene that served as the control, to examine the presence of polymerase chain reaction inhibitors.
Detection of other bacteria in amniotic fluid. Bacterial DNA was identified by PCR targeting the 16S rRNA gene with the following primers: 5′-CCA GAC TCC TAC GGG AGG CAG-3′ (V3 region) and 5′-ACA TTT CAC AAC AC-GAG CTG ACGA-3′ (V6 region) 81,82 . Each reaction contained 3 μL of target DNA, 500 nM forward and reverse primers, and Q5 High-Fidelity DNA polymerase (NEB, Ipswich, MA, USA) in a total volume of 25 μL. 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 the BigDye Terminator kit v.3.1 (Thermo Fisher Scientific, Waltham, MA, USA). The bacteria were then typed via searches 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's blood, Gardnerella vaginalis selective medium, MacConkey agar, a Neisseria-selective medium (modified Thayer-Martin medium), Sabouraud agar, or Schaedler anaerobe agar. The plates were cultured for 6 days and checked daily. The species were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using MALDI Biotyper software (Bruker Daltonics, Bremen, Germany).

Clinical definitions.
Microbial invasion of the amniotic cavity was determined based on a 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 concentrations ≥ 3000 pg/mL 22 . Intra-amniotic infection was defined by both microbial invasion of the amniotic cavity and intra-amniotic inflammation. Sterile intra-amniotic inflammation was defined as the presence of intra-amniotic inflammation without the concomitant microbial invasion of the amniotic cavity. Colonization was defined as the microbial invasion of the amniotic cavity without intra-amniotic inflammation. Negative amniotic fluid was defined as the absence of microbial invasion of the amniotic cavity and intra-amniotic inflammation. www.nature.com/scientificreports/ Statistical analyses. The 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 a number (%). The normality of the data was tested using the Anderson-Darling test. Because the FcgammaBP concentrations in the amniotic fluid were not normally distributed, the nonparametric Kruskal-Wallis and Mann-Whitney U tests were performed for statistical analyses, and the results are presented as a median value (IQR). Spearman's partial correlation analysis was performed to adjust the results for gestational age at sampling. Spearman's correlation was used to assess the relationship between the concentrations of amniotic fluid FcgammaBP and cervical fluid FcgammaBP and gestational age at sampling. Receiver operating characteristic (ROC) curves were constructed to assess the predictive value of amniotic fluid and cervical fluid FcgammaBP for the presence of intra-amniotic infection. Cutoff values were determined based on the highest positive likelihood ratio. Differences were considered significant at p < 0.05. All p values were obtained using two-tailed tests, and all statistical analyses were performed using GraphPad Prism, version 8.