Placental inflammation and pregnancy outcomes: A prospective, observational study in South Asia: The PURPOSe study

To examine inflammatory lesions in placentas of stillbirths, preterm neonatal deaths and term controls in India and Pakistan.


| I N TRODUC T ION
][7][8][9][10][11][12][13][14] It is possible that each type of inflammation has a different relationship to specific adverse pregnancy outcomes.Stillbirth and neonatal mortality are common adverse outcomes potentially related to various types of placental inflammation.Understanding these relationships may provide insight into how placental inflammation impacts pregnancy outcomes in LMIC.
Every year, more than 2.6 million third-trimester stillbirths occur worldwide. 3According to a recent study, the stillbirth rates for India and Pakistan were 25 and 57 per 1000 births, respectively, compared with rates of 2 to 3 per 1000 births in some high-income countries. 4Some of the proposed causes of stillbirth in LMIC include placental infection, fetal infection, placental malfunction, birth defects, umbilical cord complications and maternal conditions such as diabetes and hypertension.The final pathway in LMIC often ends in fetal asphyxia. 2here are also more than 2 million neonatal deaths worldwide each year.According to a recent study, the neonatal mortality rates for India and Pakistan were approximately 20 and 40 per 1000 live births, respectively, compared with rates of 2 to 3 per 1000 live births in some high-income countries.Complications related to preterm birth are often cited as the most common cause of neonatal mortality.
We wanted to better understand the relationship between different types of placental inflammation and pregnancy outcomes.Therefore, in this study we describe placental inflammatory changes in pregnancies with stillbirths, preterm neonatal deaths and term controls from a prospective multicentre study, The Project to Understand and Research Preterm Pregnancy Outcomes and Stillbirths in South Asia (PURPOSe), conducted in India and Pakistan. 15

| M ET HODS
We conducted a multicentre, prospective, observational cohort study, the Project to Understand and Research Preterm Pregnancy Outcomes and Stillbirths in South Asia (PURPOSe), in India and Pakistan from July 2018 to February 2020.The PURPOSe study protocol has been published previously. 15Briefly, participants were recruited in India at three hospitals associated with the Jagadguru Jayadeva Murugarajendra Medical College, Davangere, and in Pakistan, at the Jinnah Postgraduate Medical Centre, and the National Institute of Child Health, Karachi.Women who experienced a fetal death or a preterm birth were screened and, with consent, enrolled in the study.The study screened 5961 pregnant women at delivery across all sites with an aim to recruit 350 women who had a stillbirth and 350 women who experiencing a preterm neonatal death at each site.We also planned to enrol 100 term live birth controls at each site.For this sub-study, we restricted the analytic population to singleton pregnancies that involved either a stillbirth, or a preterm birth with a neonatal death by 28 days, and term controls.The term controls were a convenience sample chosen randomly at all three sites.For the Indian site only, placental evaluations were also performed among women delivering preterm with the infant alive at day 28, and these cases were also included in one of the analyses.For this study, stillbirth was defined as any fetus of 20 weeks or more gestation born with no heartbeat, no respirations and no movement.A neonatal death was defined as the death of a liveborn infant in the first 28 days of life regardless of its gestational age or birthweight.A preterm infant was defined as one born at before 37 weeks of gestation.

| Placental sampling and histology
Our overall objective was to collect and analyse placentas from all consenting women enrolled in the study with a stillbirth or a preterm birth, as well as 100 term controls at each site.The placentas collected for the study were subsequently evaluated using the Amsterdam criteria by trained pathologists at each Conclusions: Chorioamnionitis was the most common type of placental inflammatory lesion regardless of whether the placentas evaluated were from term controls, stillbirths or neonatal deaths.For stillbirths, inflammation in each inflammation category was more common than in the term controls and significantly more so for any inflammation, chorioamnionitis, intervillitis and villitis.For neonatal deaths, compared with the placentas of term controls, all inflammation categories were more common, but only significantly so for chorioamnionitis.Ureaplasma spp.were the most common organisms found in the placentas and were significantly associated with inflammation.

K E Y W O R D S
low-to middle-income countries, neonatal deaths, pathogens, placental inflammation, pregnancy outcomes, stillbirths, Ureaplasma site. 16Research staff collected placentas of study participants in a fresh state following parental consent.Placentas were then transported for evaluation to the departments of pathology at each of the study sites.Trained technicians, supervised by a study pathologist, examined the placental surfaces for gross abnormalities and then sliced the placenta at 1-2 cm intervals on the maternal side without cutting all the way through and the samples were processed for histological evaluation.Tissue samples for microscopic examination were also taken from the umbilical cord (two samples 5 cm from both ends), normal appearing parts of the placental disc (two samples) and any lesions such as infarcts or hematomas.
Tissue specimens were fixed in 10% neutral buffered formalin for 24 hours, processed, and 4-to 5-μm-thick sections were taken and stained with haematoxylin & eosin as per standard procedures.Additional special stains such as the periodic acid-Schiff and immunohistochemical markers such as Leukocyte Common Antigen (LCA), CD3 and CD20 were used when required.At all three sites, an experienced histopathologist either performed or oversaw the gross and microscopic examinations of all placenta samples.
All placental tissues were evaluated based on the criteria for placental lesions as described in the Amsterdam Placental Workshop Group Consensus Statement. 16Placental findings were classified by the type of inflammation noted on the examinations.These categories included chorioamnionitis in the membranes, funisitis in the umbilical cord, villitis in the placental body, and intervillitis in the placental body.Placentas could have more than one type of lesion noted.

| Molecular pathogen detection
All sample preparation and molecular procedures were conducted by the Indian and Pakistani teams on-site.The placenta samples were initially processed using proteinase K and homogenised using beads and the Precellys 24 homogeniser (Bertin Technologies).Total nucleic acid was extracted from the homogenised samples using the EZ1 Virus Mini kit (Qiagen) on the EZ1 Advanced XL automated extractor (Qiagen).Polymerase chain reaction (PCR) was performed for molecular detection using customised assays that were developed and validated by the US Centers for Disease Control and Prevention (CDC) and loaded onto TaqMan Array Cards (TACs, ThermoFisher). 15Two array cards were designed; one to specifically detect pathogens in the lungs and the other to detect pathogens from all other biological samples.For the first card, a total of 43 pathogens were targeted which included 19 bacteria, 21 viruses, 1 fungus and 2 bacterial toxins.For the second card, the TAC was designed to detect 54 pathogens which included 33 bacteria, 16 viruses, 2 fungi and 3 parasites (Table S1).In each well, primers and probes for one up to three target pathogens were included.For some, genus-specific primers and probes were used, i.e., Ureaplasma species.
Both TACs contained an assay control (internal positive control) to identify the presence of PCR inhibitors and a sample extraction control (human RNA polymerase) to confirm the presence of sufficient total nucleic acid.Additionally, two loading lanes were designated for a no template control to identify any exogenous contaminants and a positive template control to validate the proper amplification for each custom assay.All newly prepared lots of TACs were validated by the CDC and shipped to the sites.
The no template control, the positive template control and the sample total nucleic acids were loaded on the appropriate TAC and the card was run on the QuantStudio™ 7 Flex Real-time PCR instrument (Qiagen).All PCR data were analysed using a customised QuantStudio™ Real-Time PCR software, version 1.2.Amplification at any cycle threshold (Ct) was considered positive if the amplification curve and fluorescence level were deemed appropriate.However, for some select target pathogen assays (e.g., Escherichia coli, Chikungunya virus, Dengue virus and Klebsiella pneumoniae), specific Ct value cutoffs were designated to indicate a positive or indeterminate result.For the pathogens with multiple target assays (e.g., toxigenic versus non-toxigenic Corynebacterium diphtheriae), an algorithm was used to determine the final outcome based on the combined results of each assay.In the data presented, we count the pathogen as present if it was found in the placental body, the membranes or the umbilical cord.

| Statistical analyses
Data were reviewed and cleaned at each site.Data analyses were performed centrally using SAS 9.4 (SAS Institute).The analyses are presented overall and stratified by site and include descriptive statistics to assess the distribution of pathogens across conditions.Proportions were calculated for stillbirths, neonatal deaths and term controls in which a particular pathogen was found in placental tissue.Significance was defined as a p value less than 0.05.

| R E SU LTS
Of 5961 pregnancies screened, 4656 were eligible and enrolled in the overall PURPOSe study.Of these, 4055 were singletons and of those, 3065 had an outcome of interest and a placenta available.The enrolled participants with placentas available for this study, were organised into four groups, stillbirths (n = 814), preterm live births who died within 28 days of life (n = 618), term live birth controls (n = 201) and, only from India, preterm live births who were alive at day 28 (n = 1432).The number of placental samples available for the PCR analysis is also displayed (Figure 1).
Maternal characteristics of the study participants are presented in Table 1.Most of the women at both sites were between 20 and 30 years old.Indian women were of lower gravidity, had higher education and received more antenatal care compared with women from Pakistan.The reported rates of maternal morbidities such as anaemia and antepartum haemorrhage were higher at the Pakistan sites compared with the Indian site.Women with hypertensive disease represented about one-third of the women who delivered either a stillbirth or a preterm infant with a neonatal death at all three sites.Women in the term control group were generally more educated, had higher rates of antenatal care and had lower rates of adverse maternal conditions.
We first evaluated the difference in rates of the types of inflammation between the Indian liveborn infants who died and those who lived.Inflammation of any kind (26.2% versus 16.6%, p = 0.0002), chorioamnionitis (25.8% versus 16.3%, p = 0.0002) and funisitis (4.1% versus 1.5%, p = 0.005) were all more common in the infants who died.Villitis and intervillitis occurred rarely in these placentas and the differences between the rates in those preterm infants who lived and who died were not significant (data not shown).
The results of the histological examinations for inflammation of placental tissue from stillbirths, neonatal deaths and term controls in both sites combined are shown in Table 3.In the placentas of the 201 term controls, 18.9% had any inflammation, 16.9% had chorioamnionitis, 5.5% had funisitis, 0.5% had intervillitis and none had villitis.Overall, for stillbirths, any inflammation was observed in 30.2%; chorioamnionitis in 26.9%, funisitis in 5.7%, intervillitis in 6.0% and villitis in 2.2%.For stillbirths, inflammation in each category was significantly more common than in the term controls for any inflammation, chorioamnionitis, intervillitis and villitis, but not funisitis.
For the neonatal deaths, any inflammation was present in the placentas of 24.9%, chorioamnionitis in 23.3%, funisitis in 8.1%, intervillitis in 1.9% and villitis in 0.5%.Compared with the placentas of term controls, all inflammation categories were more common in neonatal deaths, but the results only approached significance for any inflammation (24.9% versus 18.9%, p = 0.08) and chorioamnionitis (23.3% versus 16.9%, p = 0.05).
We also examined placental inflammation in stillbirths and neonatal deaths by birthweight and gestational age (Table 4).Among stillbirths, the rates of any inflammation, chorioamnionitis, intervillitis and villitis were similar across the birthweight groups.However, funisitis was more common in the placentas of stillbirths weighing ≥2500 g (13.8%) compared with 1.0% for fetuses weighing <1000 g and 4.8% for fetuses weighing 1000-2499 g (p < 0.0001) A similar pattern was seen for funisitis in the stillbirths delivered at term compared with those delivered preterm (p < 0.0001).For the preterm neonatal deaths, there were no significant differences in any of the inflammatory category distributions by birthweight or gestational age.We next compared placental inflammation status to the number of different pathogens present for the term controls, neonatal deaths and stillbirths (Table 5).For each outcome, the absence of placental inflammation was more commonly associated with the absence of pathogens whereas any inflammation was commonly associated with three or more pathogens.Villitis and intervillitis were rarely seen in the term control and neonatal death placentas but were more commonly seen in the stillbirth placentas, and when present were more commonly associated with three or more pathogens.Funisitis was most commonly associated with three or more pathogens placentas of each of the outcomes.In the placentas of both neonatal deaths and stillbirths, chorioamnionitis was more commonly found when three or more pathogens were present.
Finally, in Table 6, we describe the specific pathogens present in the placentas of stillbirths, neonatal deaths and term controls.While more than 40 distinct pathogens were identified in one or more placentas, only a few, including Ureaplasma species (U. urealyticum and/or U. parvum), Streptococcus agalactiae, E. coli, Staphylococcus aureus, cytomegalovirus, K. pneumoniae and Enterococcus faecium were present in >3% of the specimens with any type of inflammation.Only Ureaplasma spp.were present in more than 8% of the specimens associated with any condition and were generally present in 20%-30%.Overall, for any condition, Ureaplasma spp.were by far the most common pathogens identified and were always more common in those placentas with inflammation than those without inflammation.In neonatal deaths, E. coli, Staphylococcus aureus and cytomegalovirus were more commonly found in the placentas of those with inflammation than those without inflammation.In stillbirth placentas, with or without inflammation, E. coli and Staphylococcus aureus were more commonly found than in the term controls, but the prevalence was not very different between those with and without inflammation.

| DISCUS SION
This multicentre, prospective, observational cohort study describes inflammatory changes in placental tissues for a large group of women from south Asia who had a stillbirth or neonatal death and compares the results with inflammatory changes seen in placentas of term controls.The results were generally similar between the placentas from India and Pakistan.The most common inflammatory conditions were chorioamnionitis, followed by funisitis, intervillitis and villitis.Chorioamnionitis accounted for most of the observed inflammatory changes.Overall, the inflammatory reactions, except for funisitis, were seen more frequently in the placentas of stillbirths and preterm neonatal deaths than in the placentas of term controls.Funisitis was more commonly found in the placentas of the stillbirths with greater birthweight and later gestational age.The increased frequency of inflammatory placental lesions in stillbirth placentas suggests that placental inflammation may play a role in stillbirth aetiology.
The relationship between chorioamnionitis and funisitis and adverse pregnancy outcomes has been reported previously. 17We are, however, not aware of reports of funisitis becoming more common among larger stillborn fetuses.Inflammation of the placental body and especially intervillitis, as compared to chorioamnionitis and funisitis, has rarely  been reported to be a risk factor for stillbirth, but based on our results, it requires further study.We are impressed that using the Amsterdam criteria to categorise placental lesions provides us with more clarity in understanding the relationship between placental findings and pregnancy outcomes.However, compared with our evaluation of malperfusion lesions, where the relationship of placental malperfusion to stillbirths and neonatal deaths was clear, the message from the inflammatory lesions is less so.The major observation is that compared with controls, the placentas from stillbirths generally have more inflammation than placentas from controls or placentas from neonatal deaths, but the increase is relatively small.Further studies to define the aetiology of this inflammation are needed.
One of the major additions to this study is the use of multiplex PCR to identify the pathogens associated with placental inflammation.Without question, Ureaplasma spp.were the most common pathogens found in the placenta, and were more commonly found in association with any inflammation and with chorioamnionitis.Interestingly in placentas of stillbirths, neonatal deaths and in term controls, the finding of three or more pathogens was also associated with the presence of any inflammation.We did not find any specific pathogen other than Ureaplasma spp. that was associated with any specific type of inflammation.We   Note: Because the placental body with cord and membranes were evaluated separately, we often had two placental specimens for each case.For this presentation, if an organism was identified in any placental specimen it is noted in the table.
should also emphasise that we report associations that do not necessarily imply that causation was involved.This study had its strengths and weaknesses.Its strengths include the large sample size and evaluation of both stillbirths and neonatal deaths compared with a term control group.Standardised formats were used to collect, store and analyse the study data at a central location.That similar results were found in India and Pakistan by independent observers adds strength to the study.Weaknesses include that these results are from a small number of institutions in both countries and may not be representative of placental findings in other institutions in those countries, in Asia, or in other LMIC.

| CONCLUSION
In summary, our study in India and Pakistan, described placental inflammation based on the Amsterdam categorisation (chorioamnionitis, funisitis, intervillitis and villitis) in a large prospective cohort of stillbirths, preterm neonatal deaths and term live births.To our knowledge this is the largest such description from the region and emphasised the relationship between various categories of inflammation and pregnancy outcomes.

AU T HOR C ON T R I BU T ION S
IA, NKG and RLG conceived the idea.IA and NKG wrote the first draft.RLG critically read and provided his expert opinion in finalizing the manuscript.KH, JK and EMM analyzed the data.ZU and VK performed histological examination and IA, NKG, SH and JK oversaw the PCR analyses.SS, SST, MSS, GG, and SSG oversaw study sites and data quality.RLG and EMM provided oversight of overall PURPOSe study.All authors read and approved the final manuscript.

AC K NO W L E D GE M E N T S
We acknowledge the people who worked tirelessly for the study.Most importantly, we thank all parents who consented to participate despite their huge loss in their lives, many of them were first time parents.

F U N DI NG I N FOR M AT ION
This study was funded by a grant from the Bill & Melinda Gates Foundation.

DATA AVA I L A BI L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

E T H IC S A PPROVA L
The study protocol was approved by respective ethical review committees of participating institutions from India and Pakistan and the United States.All procedures were conducted as per hospital protocol following receipt of written informed consent from parents.

F I G U R E 1
Study enrollment.T A B L E 1 Maternal characteristics overall and by site.

T A B L E 5
Inflammation status and the number of different pathogens found in the placentas.

T A B L E 6
Pathogens present in the placentas associated with inflammation in neonatal deaths, stillbirths and term controls.
T A B L E 2a Fetal growth restriction is defined as birthweight less than the INTERGROWTH-21ST 10th centile weight, which are not available for gestational age <24 weeks or ≥43 weeks and fetuses missing sex or birthweight.T A B L E 3 Fetal and neonatal outcomes by placental inflammation and study site.
Birthweight, gestational age and fetal growth restriction by fetal and neonatal outcome.
T A B L E 4