Current and novel markers for intraamniotic infection after preterm prelabor rupture of membranes

Background
Intraamniotic infection (IAI) is one of the main complications associated with preterm prelabor rupture of membranes (PPROM), affecting up to 60% of such pregnancies. Neonates born following IAI are at high risk of early-onset neonatal sepsis (EONS), acquiring the infection in utero. Preterm neonates are particularly susceptible to multi-organ dysfunction secondary to sepsis, which is associated with high mortality rates. Antibiotic treatment for the mother is insufficient in cases of IAI; evacuation of the pregnancy is necessary to resolve the infection and reduce secondary morbidity and mortality for both the mother and the fetus/neonate. Vigilant monitoring of PPROM pregnancies for signs of IAI is crucial to identify those affected and at high risk of EONS, to initiate timely delivery and thereby reducing adverse outcomes. However, misdiagnosis can lead to unnecessary preterm deliveries. Accurate diagnostic tools for IAI are essential, yet no easily available noninvasive method exists for confirming IAI antenatally and current practices lack reliable markers, possibly resulting in both over- and underdiagnosis of IAI. There is an urgent need to enhance the diagnostic accuracy of IAI and identify those at increased risk of EONS.

The aim of this thesis was to investigate current and novel antenatal markers of IAI following PPROM, with the goal of improving diagnostic accuracy.

Material and methods
The three studies included in this thesis were historical cohort studies derived from a single original study cohort, sharing common inclusion criteria: PPROM in singleton pregnancies, with delivery from 24+0 to 33+6 gestational weeks in Stockholm County, Sweden, from 2012 to 2019. While the studies share a common cohort, they each had slightly different exclusion criteria tailored to their specific research objectives. Data were extracted from existing medical records and cardiotocography traces.

Study I and II examined the association between exposure to IAI and the outcome of changes in short-term variation of fetal heart rate, a potential novel marker of IAI. For the exposure, IAI, we used the diagnosis of EONS in the neonate as a proxy, due to the absence of a reliable method to confirm IAI antenatally and given the strong association between IAI and EONS. Cardiotocography traces were analyzed using a computerized algorithm to assess short-term variation (the primary outcome) and baseline frequency (a secondary outcome). We compared values in the last cardiotocography trace before the start of labor between the groups using linear regression and examined the difference in change over time prior to start of labor using locally estimated scatterplot smoothing curves and linear mixed models.

Study III examined the association between antenatal characteristics and EONS to elucidate their role as markers of IAI. Logistic regression was employed, with EONS as the dependent variable. A risk-indicating model was developed based on complete case analysis, examining the proportion of cases within pairs of risk factors, and utilizing relative excess risk due to interaction tables. The model's performance was evaluated using diagnostic performance metrics.

For all three studies, adjustments were made using meticulously constructed directed acyclic graphs to identify variables as confounders or mediators.

Results
Short-term variation declined significantly more steeply in those exposed to IAI compared to those not exposed, beginning approximately 20 hours before the start of labor. This resulted in a 21.5% (95% confidence interval [CI], -32.6 to -8.7) or 1.41 msec (95% CI, -2.52 to -0.31) lower mean short- term variation in the last cardiotocography trace before start of labor in the IAI-exposed group compared to the non-exposed group. Baseline frequency increased more in the IAI-exposed group than the non-exposed group. The decline in short-term variation was only partially explained by this observed rise in baseline frequency, and it exhibited a larger relative difference and began earlier than the rise in baseline frequency.

A negative correlation was found between short-term variation and EONS, with a 37% increase in the risk of EONS for each 1 msec decrease in mean STV. A short-term variation below 4 msec was associated with a 2.62-fold increase in the risk for EONS. Baseline frequency was positively correlated with the risk of EONS, and a baseline frequency above 160 bpm was associated with a 3.75-fold increased risk of EONS. Maternal diabetes, maternal temperature >38℃, and positive urinary or vaginal/cervical culture were significantly associated with an increased risk of EONS, with odds ratios of 4.37 (95% CI, 1.41 to 13.56), 6.42 (95% CI, 2.94 to 14.02) and 2.62 (95% CI, 1.14 to 6.03), respectively.

A risk-indicating model for EONS was developed which demonstrated an AUC of 0.7348 and surpassed the diagnostic performance of the clinicians' suspicion of IAI. The model indicates an elevated risk of EONS in pregnancies with positive urinary and/or vaginal/cervical culture in conjunction with at least one of the following markers: maternal temperature ≥38℃, maternal diabetes, fetal baseline frequency ≥160 bpm, or fetal short-term variation ≤4 msec. Maternal white blood cell (WBC) count was only marginally associated with EONS, and when categorizing the WBC count at 15,000 cells/mm3 the association was no longer significant. No association was identified with maternal C-reactive protein levels, maternal pulse, or maternal symptoms.

Conclusion
Our findings suggest that short-term variation of fetal heart rate may be a promising novel marker for IAI, warranting further investigation and development. Maternal diabetes also emerged as a novel marker. Among the current antenatal markers for IAI, maternal temperature, positive vaginal and/or cervical cultures, and fetal tachycardia are the most important to consider. A risk-indicating model for EONS, incorporating significant markers for EONS, outperformed clinicians' suspicion of IAI.

List of scientific papers

I. Short-term variation of the fetal heart rate as a marker of intraamniotic infection in pregnancies with preterm prelabor rupture of membranes: a historical cohort study. Birgisdottir BT, Hulthén Varli I, Saltvedt S, Lu K, Abtahi F, Ådén U, Holzmann M. J Matern Fetal Neonatal Med. 2024 Dec;37(1):2345855. https://doi.org/10.1080/14767058.2024.2345855

II. Changes in short-term variation of antenatal cardiotocography to identify intraamniotic infection: a historical cohort study. Birgisdottir BT, Andersson T, Hulthén Varli I, Saltvedt S, Lu K, Abtahi F, Ådén U, Holzmann M. J Matern Fetal Neonatal Med. 2025 Dec;38(1):2434059. https://doi.org/10.1080/14767058.2024.2434059

III. Risk factors and risk-indicating model for early-onset neonatal sepsis after preterm prelabor rupture of membranes: a historical cohort study. Birgisdottir BT, Andersson T, Hulthén Varli I, Saltvedt S, Abtahi F, Ådén U, Holzmann M. [Manuscript]