Histological chorioamnionitis and pathological stages on very preterm infant outcomes

Histological chorioamnionitis (HCA) is a condition linked to preterm birth and neonatal infection and its relationship with various pathological stages in extremely preterm neonates, and with their associated short‐ and long‐term consequences, remains a subject of research. This study investigated the connection between different pathological stages of HCA and both short‐term complications and long‐term outcomes in preterm infants born at or before 32 weeks of gestational age.


Introduction
Chorioamnionitis, an ascending infection of the amniotic fluid, placenta or fetus, results from pathogenic microorganisms entering the amniotic cavity through the maternal vagina, and is a prevalent clinical cause of intrauterine infections. 1 Incidence rates are approximately 5% in term infants and range from 30 to 70% in preterm infants. 2 In addition to the major cause of preterm birth, chorioamnionitis has been shown to be associated with short-term complications and long-term severe adverse outcomes, especially perinatal brain injury and neurological sequelae such as cerebral palsy. 3,4Based on the presence or absence of maternal clinical signs of infection, chorioamnionitis is classified into clinical chorioamnionitis and histological chorioamnionitis (HCA).HCA, indicated by polymorphonuclear leucocyte infiltration in maternal placental tissue and membranes, serves as a biomarker for perinatal inflammation, with placental histopathology as the diagnostic gold standard. 5owever, due to its inconspicuous onset and absence of characteristic early clinical signs, prenatal diagnosis of HCA remains challenging and often relies upon postnatal placental examination.Amniocentesis with analysis of microbes or immuno-inflammatory markers in the amniotic fluid has been proposed, but is considered to be too invasive and is not used on a routine basis in most centres. 6Recently, the combination of concentrations of cervical fetal fibronectin, maternal serum C-reactive protein, cervical dilatation and gestational age was suggested to identify patients at risk for intra-amniotic infection and/or inflammation. 7horioamnionitis or intrauterine inflammation is a frequent factor in triggering preterm birth 8 and contributes significantly to perinatal mortality and adverse long-term outcomes. 9,10Advances in neonatal care have led to increased survival rates for very low gestational age and birth weight infants, thus assessing and enhancing long-term neurodevelopmental outcomes has become a critical necessity.Previous clinical investigations have yielded inconsistent findings regarding HCA's association with brain damage and with poor long-term neurological outcomes in preterm infants. 2 One prospective cohort study found no disparities in punctate leucomalacia and brain white matter microstructure between neonates with or without HCA. 11Reports on HCA's influence on long-term neurological outcomes during schoolage years also diverge, with some showing no differences. 12,13However, HCA has been linked to various adverse neonatal complications such as respiratory distress syndrome (RDS), 14 bronchopulmonary dysplasia (BPD), intraventricular haemorrhage (IVH), periventricular leucomalacia (PVL) and sepsis, 15,16 and thus HCA might also be associated with an elevated risk of long-term neurodevelopmental impairments. 17,18However, the association between HCA and neurodevelopmental outcomes and neonatal death is still a matter of debate. 19,20This is potentially influenced by variations in chorioamnionitis definitions, gestational age, follow-up durations, outcome assessments, sample sizes and single-centre versus multicentre recruitment.
The purpose of this study was to analyse the association between HCA and neurological complications at 40 gestational weeks of corrected age and its association with long-term outcomes in preterm infants born before 32 weeks of gestational age followed-up to 18-24 months in a single centre with a large sample size.The impact of different pathological stages of HCA on short-and long-term outcomes was further investigated according to placental pathology.

S T U D Y D E S I G N A N D P A R T I C I P A N T S
A total of 1071 preterm infants with a gestational age ≤ 32 weeks who were born at the Third Affiliated Hospital of Zhengzhou University and admitted to the neonatal intensive care unit between January 2019 and December 2021 were initially screened for eligibility.Infants with chromosomal diseases, genetic metabolic disorders or unconfirmed maternal clinical chorioamnionitis, as well as those with parental refusal, withdrawal from treatment or who were lost to follow-up to 18-24 months, were excluded from the final analysis.Ultimately, a cohort of 481 preterm infants (24-32 weeks gestational age) met the eligibility criteria.This preterm cohort comprised 38 infants who unfortunately did not survive and 443 infants who survived, all of whom had available maternal placental pathology data.Additional followup data were obtained for these surviving infants.The study was granted ethical approval by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University (2021-158-013).

P L A C E N T A L H I S T O L O G I C A L A S S E S S M E N T
Upon delivery, fresh placental specimens underwent macroscopic and microscopic analyses using a standardised protocol. 21 Chorioamnionitis and preterm infant outcomes 1025 placental membranes, which was then classified by inflammatory response into maternal inflammatory response (MIR) and fetal inflammatory response (FIR), according to the established criteria. 22In the context of MIR, stage 1 is characterised by the presence of neutrophils in the chorion or subchorionic space, stage 2 refers to neutrophilic infiltration of the chorionic connective tissue and/or amnion or the chorionic plate and stage 3 is necrotising chorioamnionitis with degenerating neutrophils.Any vasculitis in the umbilical cord or vasculitis in the chorionic plate of the placenta was considered to indicate a FIR.FIR stage 1 refers to chorionic vasculitis or umbilical phlebitis, stage 2 is characterised by involvement of the umbilical vein and one or more umbilical arteries and stage 3 is necrotising funisitis (Figure 1).For the purpose of describing the degrees of severity of HCA, we defined MIR/FIR stage 1 as mild HCA and MIR/FIR stages 2-3 as moderatesevere HCA.
Comprehensive perinatal information collection included gestational age, birth weight, sex, intrauterine distress, birth asphyxia and maternal factors such as gestational diabetes mellitus, pregnancy-induced hypertension, placental abruption, premature rupture of membranes, amniotic fluid volume, mode of delivery, antenatal steroid and antibiotic therapy and clinical chorioamnionitis. 23The short-term complications of sepsis, pneumonia, pulmonary haemorrhage, BPD, 24 RDS, necrotising enterocolitis (NEC), 25 IVH, PVL and retinopathy of prematurity (ROP) 26 at 40 weeks of corrected age were documented.
F O L L O W -U P Scheduled follow-ups occurred every 3 months until the infants reached a corrected age of 18-24 months.Blinded examiners proficient in the Bayley Infant Development Scale (2nd edition) assessed the mental development index (MDI) and psychomotor development index (PDI) at 18-24 months.The primary outcome was death or neurological disability.Cerebral palsy was identified by non-progressive central nervous system dysfunction causing abnormal muscle tone and movement control. 27Hearing impairment was defined as the need for external hearing aid intervention 28 and vision impairment was defined by low visual acuity or reduced visual radius. 29PDI < 70 was considered motor development deficiency and further evaluated for rehabilitation to determine the presence of cerebral palsy, and MDI < 70 was considered mental retardation. 30Neonates experiencing cerebral palsy, MDI score < 70, PDI score < 70 or hearing or vision impairment were categorised as having neurological disability. 31

C O H O R T D E M O G R A P H I C A N D C L I N I C A L C H A R A C T E R I S T I C S
Of the initial 1071 infants in the study, 533 lacked placental pathological assessment, including 68 with clinical chorioamnionitis but no confirmed placental pathology, and 15 with genetic, metabolic or chromosomal disorders.This left us with 523 infants for analysis.Among these, 42 infants (8.0%) were lost to follow-up and 38 infants (7.3%) died, leaving 443 infants who survived and were followed-up to 18-24 months of corrected age.Among these, 203 were in the non-HCA group, while 240 were classified as HCA-exposed based on placental pathology.Within the HCA-exposed group, 142 infants (59.2%) had MIR stage 1 and 98 (40.8%) had MIR stages 2-3, while 198 infants (82.5%) had no FIR or FIR stage 1 and 42 (17.5%)had FIR stages 2-3, according to placental pathology staging (Figure 2).
HCA was detected in 240 of the 443 preterm infants (54.2%) in the cohort.Only 18 infants (7.5%) were born to mothers diagnosed with clinical chorioamnionitis.The clinical characteristics of the preterm infants, both with and without HCA, are summarised in Table 1.Infants in the HCA group exhibited a lower likelihood of being born to mothers with pregnancy-induced hypertension (P < 0.001), but had a higher incidence of premature rupture of membranes lasting more than 18 h (P = 0.012), reduced amniotic fluid levels (P < 0.001) and the use of antenatal antibiotic therapy (P = 0.025).Additionally, these infants had an increased incidence of early-onset sepsis (P = 0.015) and BPD (P = 0.002) when compared to infants not experiencing HCA.
Table 2 presents the clinical characteristics of infants categorised into MIR stage 1 and MIR stages 2-3 groups.Similarly, infants in the moderate-severe HCA group showed a reduced likelihood of being born to mothers with pregnancy-induced hypertension (P = 0.007).They also had lower gestational age (P = 0.021) and birth weight (P = 0.012), as well as a higher prevalence of amniotic fluid reduction (P = 0.006) and early-onset sepsis (P = 0.006) compared to infants in the mild HCA group.Table 3 Chorioamnionitis and preterm infant outcomes 1027 shows the clinical features of infants exposed to various FIR stages of HCA.Additionally, this cohort exhibited lower gestational age (P = 0.012) and birth weight (P = 0.013).Moreover, there was a heightened prevalence of sepsis (P = 0.020) and early-onset sepsis (P = 0.032) within the FIR stages 2-3 HCA group.

H C A A N D N E O N A T A L B R A I N I N J U R Y
There was no significant difference in the incidence of IVH between infants exposed to HCA (75 of 240 cases, 31.3%) and those not exposed to HCA (62 of 203 cases, 30.5%).Although infants exposed to moderate-severe HCA (34 of 98, 34.7%) showed a slightly higher IVH rate than those exposed to mild HCA (41 of 142, 28.9%), this difference was not statistically significant (P > 0.05).However, when evaluating severe IVH (grades III-IV), a higher rate of occurrence was observed in the HCA group (29 of 240, 12.1%) compared to the unexposed group (12 of 203, 5.9%) (Table 1).There was a more pronounced difference between the MIR stages 2-3 group (19 of 98, 19.4%) and the MIR stage 1 group (10 of 142, 7.0%) (Table 2).Similarly, an analysis of newborns exposed to varying FIR stages revealed a statistically significant difference (23.8 versus 9.6%, P = 0.010) in the context of severe IVH (Table 3).Additionally, the analysis revealed no significant correlation between HCA and PVL in neonates, regardless of HCA exposure or different stages, albeit with a noticeable increasing trend.
No significant differences were observed in the mortality rate or the incidence of neurological disability at 18-24 months of corrected age between HCAexposed infants and those not exposed (P > 0.05).Similarly, no notable variations were observed in the occurrence of cerebral palsy (P = 0.064), PDI score < 70 (P = 0.174), hearing impairment (P = 0.793) or vision impairment (P = 0.981) between these two groups.However, a univariate analysis revealed a significant difference in MDI scores < 70 between infants with and without HCA  Chorioamnionitis and preterm infant outcomes 1029  Chorioamnionitis and preterm infant outcomes 1031 at 18-24 months of corrected age [odds ratio (OR) = 0.36; 95% confidence interval (CI) = 0.18-0.70;P = 0.003; Table 4].After adjusting for relevant perinatal factors, including pregnancy-induced hypertension, premature rupture of membranes >18 h, amniotic fluid reduction, antenatal antibiotic therapy, sepsis, early-onset sepsis, BPD and severe IVH (grades III-IV), no statistically significant differences were found between the groups, regardless of HCA exposure, at 18-24 months of corrected age.Moreover, even for MDI scores < 70, post-adjustment for confounding factors showed that infants in the HCAexposed group did not exhibit a significant difference when compared to the unexposed HCA group (5.9 versus 15.0%; OR = 2.53; 95% CI = 0.24-5.17;P = 0.061), although a notable trend was observed (Table 5).

Discussion
Chorioamnionitis and intrauterine infection present significant challenges in the fields of obstetrics and neonatology, and are linked to spontaneous preterm births and complications in very preterm neonates.However, inconsistent research findings have made it difficult to predict neonatal outcomes following HCA exposure.In our relatively large prospective cohort study, we observed a higher incidence of severe IVH (grades III-IV) among neonates exposed to HCA compared to those not exposed.Additionally, HCAexposed preterm infants exhibited a greater likelihood of early-onset sepsis and BPD during their hospital stay.Furthermore, the severity of HCA was associated  Chorioamnionitis and preterm infant outcomes 1033 with adverse outcomes, particularly an increased risk of cerebral palsy and cognitive deficits.When studying adverse outcomes in preterm infants, both mortality and neurological disorders are important considerations. 32We considered both these factors while investigating the link between HCA and adverse outcomes in this group.In our analysis, both prenatal factors and postnatal factors showed statistical significance in univariate analyses.These factors could potentially confound the relationship between  HCA and neonatal outcomes.Recognising that sociodemographic and perinatal risks can influence outcomes, we conducted multivariate logistic regression to control for these variables.Ultimately, exposure to HCA in neonates did not significantly correlate with mortality or adverse outcomes at 18-24 months corrected age.This suggests that while HCA might not notably impact preterm infant prognosis, perinatal factors might have a more prominent role in determining long-term outcomes, in line with the existing literature. 33,34rior studies have focused primarily on the impact of HCA in newborns, overlooking the potential effect of HCA severity in preterm infants.The quantification system for grade of placental inflammation indicates the intensity of the inflammatory response in a specific area, while the stage indicates the degree of anatomical neutrophil infiltration, reflecting the progression of the disease. 35,36In our study, we used the placental pathology stage to assess HCA severity, because it is considered more relevant than grade for evaluating the severity of placental inflammation and is deemed more consistent. 37,38We categorised HCA into mild and moderate-severe groups according to different MIR and FIR stages, a method that could potentially enhance our understanding of the connection between HCA and newborn outcomes.
In our study, the moderate-severe HCA group showed a trend towards lower gestational age and birth weight compared to the mild HCA group.Chorioamnionitis triggers preterm labour, necessitating prompt iatrogenic delivery to mitigate maternal and fetal risks. 39,40This suggests that profound intrauterine infection might contribute to fetal growth restriction during pregnancy.Interestingly, we found no significant differences in PVL, BPD, RDS, NEC, pulmonary haemorrhage, pneumonia or ROP between the mild and moderate-severe HCA groups.However, the moderate-severe HCA group had a higher susceptibility to early-onset sepsis, aligning with prior research. 41Also, severe IVH (grades III-IV) was more prevalent in the moderate-severe HCA group. 42o adjust for potential influences from prenatal and postnatal factors, especially gestational age and birth weight, on preterm infants' long-term outcomes, we performed multivariate regression analysis.This showed that the moderate-severe HCA group did not have significantly greater mortality or disability at 18-24 months corrected age.However, HCA has been linked to a higher incidence of adverse longterm outcomes, 43 cerebral palsy 43,44 and MDI scores < 70 compared to the mild HCA group. 45This emphasises that advanced HCA stages might predict a poorer long-term prognosis.Clinicians should closely consider HCA stage, recognising the potential complications and unfavourable long-term outlooks in preterm infants with moderate to severe placental inflammation.This study has several limitations.One constraint is the assessment time-frame, which is limited to the period up to 18-24 months corrected age.This leaves room for the possibility of associations between HCA and subtler neuropsychological deficits that may emerge later.][48] Therefore, extending the follow-up period might provide a more comprehensive evaluation in future research.Additionally, the absence of non-histological biomarkers, such as placental cytokines or microorganisms, hampers the assessment of the severity of histological chorioamnionitis using laboratory indicators. 49Lastly, this study focuses solely upon very preterm infants who inherently face an increased risk of neurodevelopmental disorders regardless of HCA exposure.As a result, these findings might not be directly applicable to all gestational age infants.
In summary, our study concludes that moderatesevere HCA increases early-onset sepsis and severe IVH in preterm infant ≤ 32 gestational weeks.Furthermore, this subgroup was associated with unfavourable long-term neurological prognosis at 18-24 months corrected age compared with mild HCA.This highlights the potential for a more challenging clinical trajectory in instances characterised by severe stages of HCA.
Dedicated specialists performed the processing, involving fixation in 10% formaldehyde, embedding in paraffin, sectioning and staining with haematoxylin and eosin.Pathological assessment by senior pathologists in the Department of Pathology determined the presence of HCA based on the presence of an inflammatory response in the Ó 2024 The Authors.Histopathology published by John Wiley & Sons Ltd., Histopathology, 84, 1024-1037.

Figure 1 .
Figure 1.Pathological stages of maternal and fetal inflammatory response.The left two columns depict the stages of the maternal inflammatory response (MIR).'Normal' denotes chorioamniotic membranes free from neutrophil infiltration.In stage 1, neutrophils are confined to the chorion layer.Stage 2 shows neutrophils penetrating the amniotic connective tissue.Stage 3 is distinguished by severe inflammation and the necrosis of amnion epithelial cells.The two right-hand columns illustrate the stages of the fetal inflammatory response (FIR).'Normal' signifies healthy umbilical cord vessels.Stage 1 is characterised by inflammation in the umbilical vein.Stage 2 presents as inflammation in the umbilical artery.The final stage is marked by a distinct arrangement of degenerated neutrophils encircling the vessels in a concentric, perivascular pattern.

Figure 2 .
Figure 2. Flowchart of the study population for exclusion and inclusion in both the HCA and non-HCA groups.

Table 1 .
Demographic and clinical characteristics of newborns with and without HCA The data are displayed as n (%) or mean [standard deviation (SD)] unless specified otherwise.The P-value was computed using an independent two-sample t-test or the v 2 test to compare the groups with and without HCA.A P-value of less than 0.05 was regarded as indicating statistical significance.IVH, intraventricular haemorrhage; NEC, necrotising enterocolitis; ROP, retinopathy of prematurity.Ó 2024 The Authors.Histopathology published by John Wiley & Sons Ltd., Histopathology, 84, 1024-1037.

Table 2 .
Demographic and clinical characteristics of newborns exposed to different MIR stages of HCA The data are expressed as n (%) or mean [standard deviation (SD)], unless specified otherwise.The P-value was computed using an independent two-sample t-test or the v 2 test to compare MIR stage 1 and MIR stages 2-3 groups.A P-value < 0.05 was considered a statistically significant difference.MIR, maternal inflammatory responses.Ó 2024 The Authors.Histopathology published by John Wiley & Sons Ltd., Histopathology, 84, 1024-1037.

Table 3 .
Demographic and clinical characteristics of newborns exposed to different FIR stages of HCA The data are expressed as n (%) or mean [standard deviation (SD)] unless specified otherwise.The P-value was computed using an independent two-sample t-test or the v 2 test to compare FIR no/stage 1 and FIR stages 2-3 groups.A P < 0.05 was considered a statistically significant difference.FIR, fetal inflammatory responses.Ó 2024 The Authors.Histopathology published by John Wiley & Sons Ltd., Histopathology, 84, 1024-1037.

Table 4 .
Univariate analysis for poor outcomes with or without HCA at 18-24 months of corrected ageThe data are expressed as n/N (%) unless specified otherwise.Disability was characterised by surviving infants experiencing one or more of the following complications: cerebral palsy, MDI < 70, PDI <70, hearing impairment, and vision impairment.A P-value of less than 0.05 was considered to indicate statistical significance.
CI, confidence interval; MDI, mental developmental index; PDI, psychomotor developmental index.a One infant with multiple disabilities contributed to multiple counts.

Table 5 .
Multivariate analysis for poor outcomes with or without HCA at 18-24 months corrected age P-values were calculated to assess the significance of adjusting for confounding factors, including pregnancy-induced hypertension, premature rupture of membrane > 18 h, amniotic fluid reduction, antenatal antibiotics, sepsis, early-onset sepsis, bronchopulmonary dysplasia and IVH grades III or IV.
CI, confidence interval; MDI, mental developmental index; PDI, psychomotor developmental index.a One infant with more than one disability counted multiple times.

Table 7 .
Univariate analysis for poor outcomes with different FIR stages of HCA at 18-24 months corrected age Data are presented as n/N (%) unless otherwise indicated.Disability was defined as surviving neonates experiencing any of the following complications: cerebral palsy, MDI < 70, PDI < 70, hearing impairment, or vision impairment.A significance level of P < 0.05 was deemed statistically significant.CI: confidence interval; MDI: Mental Developmental Index; PDI: Psychomotor Developmental Index; FIR: fetal inflammatory responses.
aOne infant with more than one disability counted multiple times.

Table 8 .
Multivariate analysis for poor outcomes with different MIR stages of HCA at 18-24 months corrected age a One infant with more than one disability counted multiple times.

Table 9 .
Multivariate analysis for poor outcomes with different FIR stages of HCA at 18-24 months corrected ageThe significance of FIR stage adjusting for confounding factors such as gestational age, birth weight, sepsis, early-onset sepsis, IVH and IVH grades III or IV were assessed using P-values.
CI: confidence interval; MDI: Mental Developmental Index; PDI: Psychomotor Developmental Index; FIR: fetal inflammatory responses.a One infant with more than one disability counted multiple times.