This retrospective cohort study included 154 patients with pPROM admitted to our institution between January 2012 and April 2022. The following inclusion criteria were applied: 1) singleton pregnancy; 2) gestational age from 20+0 to 33+6 weeks; and 3) AF obtained by transabdominal amniocentesis to evaluate the microbiological status and fetal lung maturity. In cases where repeated amniocentesis procedures were performed (n=12), only the microbiological and biochemical results from the initial procedure were used for analysis to ensure methodological consistency across the entire study population. No patients were excluded from the analysis based on the results of subsequent procedures or persistent clinical symptoms following the intention-to-treat principle. The exclusion criteria were as follows: 1) cerclage placement before amniocentesis and 2) fetal death in utero. Clinical and pregnancy outcome data were extracted from the medical records. The clinical information collected included maternal age, nulliparity, gestational age at amniocentesis, gestational age at delivery, and cervical length. The assessed pregnancy outcomes included birth weight, 1-minute and 5-minute Apgar scores, cord arterial blood pH, and neonatal intensive care unit (NICU) admission. This study adhered to the ethical standards for human experimentation outlined in the Declaration of Helsinki. The Institutional Review Board (IRB) approved the analysis of the clinical records, which included both AF analysis outcomes and supplementary research data (IRB No. B-2211-790-106). As this was a retrospective study based on a review of the medical records of patients who had already completed treatment and were discharged, the requirement for informed consent was waived by the IRB.

pPROM was defined as fluid leakage from the cervix or fluid pooling in the vagina being observed, followed by a nitrazine test for confirmation [23]. Patients with pPROM who were admitted before 34 weeks of gestation underwent transabdominal amniocentesis to assess the microbiological status of AF and/or lung maturity, in line with our institution’s established clinical protocol [24]. Routine assessment involved determining the maternal serum CRP concentrations alongside the white blood cell (WBC) counts. The need for informed consent was waived by the Institutional Review Board of this retrospective study.

AF samples were cultured to detect aerobic and anaerobic bacteria, genital mycoplasmas (Ureaplasma species or Mycoplasma hominis), and fungi. Bacterial culture was performed by placing the AF in BACTEC culture bottles (Becton Dickinson, Franklin Lakes, NJ, USA) containing resin, followed by incubation in a BACTEC FX automated system (Becton Dickinson). Positivity was indicated by an alarm signal. The final culture test results were determined 5 days later. A portion of the AF was transported to a microbiology laboratory, which cultivated genital mycoplasma using Mycoplasma IST-2 (BioMérieux, Marcy l’Etoile, France) for identification. A positive finding of AF was used to define MIAC.

Within a 24-hour period post amniocentesis, maternal blood samples were obtained. A complete blood count, including leukocyte differentials and CRP levels, was performed. NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count.

We analyzed the clinical characteristics and pregnancy outcomes, along with CRP levels and NLR, in relation to the presence of MIAC. Proportional data were analyzed using either the Pearson chi-square test or Fisher’s exact test, while continuous variables were compared using the Mann-Whitney U-test. Statistical significance was defined as a P-value <0.05. To assess diagnostic accuracy, receiver operating characteristic (ROC) curve analysis was conducted to evaluate the specificity and sensitivity of CRP and NLR. The area under the curve (AUC) was calculated and used to obtain the diagnostic cutoff values. Statistical analyses were performed using SPSS software version 27.0 (IBM, Armonk, NY, USA).

The main findings of the present study were as follows: 1) NLR exhibited an independent positive association with MIAC in patients with pPROM; 2) NLR and CRP showed comparable diagnostic performance for detecting MIAC; and 3) patients with high values for both parameters showed the strongest association with MIAC. Nonetheless, it should be kept in mind that 18.8% (12/64) of patients with low NLR and low CRP had MIAC.

Neutrophils are crucial in defending against infections, and it has been proposed that the NLR can reliably indicate inflammation in various disease conditions [19,20,22,25,26]. A meta-analysis examining leukocyte ratios in peripheral blood for infectious diseases found that patients with bacterial infections exhibited markedly higher NLR levels [27]. Another study assessed maternal serum markers in cases of no placental inflammation, histologic chorioamnionitis, and histologic chorioamnionitis with funisitis among patients who underwent preterm delivery due to preterm labor or pPROM. This study underscored the effectiveness of NLR in diagnosing placental inflammatory responses and reported that it achieved the highest AUC value of 0.80 for predicting these responses. With a cutoff value of 6.5, the NLR showed a sensitivity of 71% and a specificity of 78% [28]. Consistent with these findings, our study demonstrated that patients with pPROM with MIAC had significantly higher WBC counts and NLR in maternal blood than those without MIAC.

Maternal CRP levels are one of the most widely used clinical parameters for assessing inflammation [11,26,29]. As maternal CRP secretion increases in response to intra-amniotic inflammation, it has been established as a marker for MIAC [16,30].

Multiple studies have demonstrated that NLR has a diagnostic value for predicting intra-amniotic inflammation that is comparable to or even greater than that of CRP [20,28,31]. In our study, ROC curve analysis revealed that maternal serum NLR and CRP have similar diagnostic effectiveness, reinforcing the potential of NLR as a predictive marker for MIAC on par with CRP. Additionally, when patients were categorized into four subgroups based on high or low levels of NLR and CRP, the highest incidence of MIAC was observed in the subgroup with elevated NLR and CRP levels. Notably, the subgroup with high NLR and low CRP showed a significantly higher rate of MIAC than the subgroup with low NLR and high CRP, indicating that the NLR may offer additional predictive value beyond CRP in diagnosing MIAC.

The strength of this study lies in its meticulous diagnostic approach, as MIAC was confirmed through direct sampling of AF via transabdominal amniocentesis, followed by culturing. This gold standard method significantly reduces diagnostic uncertainty. Maternal blood was obtained within 24 hours of amniocentesis to ensure that the results from both maternal blood and AF accurately reflected the infection status at the same time. Additionally, by directly comparing biomarkers such as NLR and CRP with a definitive MIAC diagnosis, this study delivers reliable and clinically meaningful results that can guide patient management.

One limitation of this study is that it involved patients from a single hospital, which may have restricted the generalizability of the findings. Additionally, the use of AF culture for diagnosing MIAC presents a limitation, potentially underestimating MIAC diagnosis rates, as molecular techniques, such as polymerase chain reaction, were not employed [32]. Our study focused primarily on the diagnostic accuracy of maternal markers for MIAC. We were unable to obtain outcome data for 21 patients who were transferred to other institutions because of imminent delivery, unavailable NICU beds, or patient preferences, as our IRB approval did not extend to data from external institutions. Although this resulted in missing data for gestational latency, we believe that it did not affect our primary findings regarding the independent association between maternal NLR and MIAC.

Numerous markers in maternal blood have been studied as noninvasive methods for the diagnosis of MIAC. Although analyzing maternal blood offers valuable clinical insights, the definitive diagnosis of MIAC still depends on amniocentesis to collect and analyze the AF. The limitation of noninvasive tests lies in the fact that a negative result cannot exclude the presence of MIAC because maternal blood tests do not directly reflect the condition of the amniotic cavity [16,28,33].

Consistent with other studies that utilized maternal CRP levels, our research determined that using an NLR cutoff value of 6.2 and a CRP cutoff level of 0.7 mg/dL, MIAC was diagnosed in 18.8% (12/64) of patients with low NLR and low CRP. These findings suggest that although NLR and CRP levels can be valuable predictors of MIAC, the presence of MIAC cannot be ruled out based on the low levels of both markers.