Gestational Age, Infection, and Suboptimal Maternal Prepregnancy BMI Independently Associate with Placental Histopathology in a Cohort of Pregnancies without Major Maternal Comorbidities

Abstract

Background: The placenta undergoes morphological and functional adaptations to adverse exposures during pregnancy. The effects ofsuboptimal maternal body mass index (BMI), preterm birth, and infection on placental histopathological phenotypes are not yet well understood, despite the association between these conditions and poor offspring outcomes. We hypothesized that suboptimal maternal prepregnancy BMI and preterm birth (with and without infection) would associate with altered placental maturity and morphometry, and that altered placental maturity would associate with poor birth outcomes. Methods: Clinical data and human placentae were collected from 96 pregnancies where mothers were underweight, normal weight, overweight, or obese, without other major complications. Placental histopathological characteristics were scored by an anatomical pathologist. Associations between maternal BMI, placental pathology (immaturity and hypermaturity), placental morphometry, and infant outcomes were investigated for term and preterm births with and without infection. Results: Fetal capillary volumetric proportion was decreased, whereas the villous stromal volumetric proportion was increased in placentae from preterm pregnancies with chorioamnionitis compared to preterm placentae without chorioamnionitis. At term and preterm, pregnancies with maternal overweight and obesity had a high percentage increase in proportion of immature placentae compared to normal weight. Placental maturity did not associate with infant birth outcomes. We observed placental hypermaturity and altered placental morphometry among preterm pregnancies with chorioamnionitis, suggestive of altered placental development, which may inform about pregnancies susceptible to preterm birth and infection. Conclusions: Our data increase our understanding of how common metabolic exposures and preterm birth, in the absence of other comorbidities or complications, potentially contribute to poor pregnancy outcomes and developmental programming.

Full text

Citation: Duffley, E.; Grynspan, D.;
Scott, H.; Lafrenière, A.; Borba Vieira
de Andrade, C.; Bloise, E.; Connor,
K.L. Gestational Age, Infection, and
Suboptimal Maternal Prepregnancy
BMI Independently Associate with
Placental Histopathology in a Cohort
of Pregnancies without Major
Maternal Comorbidities. J. Clin. Med.
2024,13, 3378. https://doi.org/
10.3390/jcm13123378
Academic Editor: Michal Kovo
Received: 1 April 2024
Revised: 22 May 2024
Accepted: 30 May 2024
Published: 8 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Journal of
Clinical Medicine
Article
Gestational Age, Infection, and Suboptimal Maternal
Prepregnancy BMI Independently Associate with Placental
Histopathology in a Cohort of Pregnancies without Major
Maternal Comorbidities
Eleanor Duffley 1, David Grynspan 2,3, Hailey Scott 1, Anthea Lafrenière 4,5, Cherley Borba Vieira de Andrade 6,
Enrrico Bloise 7,8 and Kristin L. Connor 1, *
1Department of Health Sciences, Carleton University, Ottawa, ON K1S 5B6, Canada;
eleanorduffley@cmail.carleton.ca (E.D.); haileyscott@cmail.carleton.ca (H.S.)
2Children’s Hospital of Eastern Ontario, Department of Pathology, Ottawa, ON K1H 8L1, Canada;
david.grynspan@interiorhealth.ca
3Department of Pathology and Laboratory Medicine, University of British Columbia,
Vancouver, BC V6T 2B5, Canada
4Department of Pathology and Laboratory Medicine, The Ottawa Hospital, The University of Ottawa,
Ottawa, ON K1H 8L6, Canada; anthea.lafreniere@gmail.com
5Department of Pathology and Immunology, Baylor College of Medicine, Texas Children’s Hospital,
Houston, TX 77030, USA
6Histology and Embryology Department, Roberto Alcantara Gomes Institute of Biology, Rio de Janeiro State
University, Rio de Janeiro 20551-030, Brazil; cherleyborba@gmail.com
7Department of Morphology, Federal University of Minas Gerais, Belo Horizonte 31270-901, Brazil;
ebloise@icb.ufmg.br
8Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada
*Correspondence: kristin.connor@carleton.ca; Tel.: +1-613-520-2600 (ext. 4202)
Abstract: Background: The placenta undergoes morphological and functional adaptations to adverse
exposures during pregnancy. The effects ofsuboptimal maternal body mass index (BMI), preterm
birth, and infection on placental histopathological phenotypes are not yet well understood, despite
the association between these conditions and poor offspring outcomes. We hypothesized that
suboptimal maternal prepregnancy BMI and preterm birth (with and without infection) would
associate with altered placental maturity and morphometry, and that altered placental maturity
would associate with poor birth outcomes. Methods: Clinical data and human placentae were
collected from 96 pregnancies where mothers were underweight, normal weight, overweight, or
obese, without other major complications. Placental histopathological characteristics were scored by
an anatomical pathologist. Associations between maternal BMI, placental pathology (immaturity
and hypermaturity), placental morphometry, and infant outcomes were investigated for term and
preterm births with and without infection. Results: Fetal capillary volumetric proportion was
decreased, whereas the villous stromal volumetric proportion was increased in placentae from
preterm pregnancies with chorioamnionitis compared to preterm placentae without chorioamnionitis.
At term and preterm, pregnancies with maternal overweight and obesity had a high percentage
increase in proportion of immature placentae compared to normal weight. Placental maturity
did not associate with infant birth outcomes. We observed placental hypermaturity and altered
placental morphometry among preterm pregnancies with chorioamnionitis, suggestive of altered
placental development, which may inform about pregnancies susceptible to preterm birth and
infection. Conclusions: Our data increase our understanding of how common metabolic exposures
and preterm birth, in the absence of other comorbidities or complications, potentially contribute to
poor pregnancy outcomes and developmental programming.
Keywords: underweight; obesity; placental pathology; placental morphometry
J. Clin. Med. 2024,13, 3378. https://doi.org/10.3390/jcm13123378 https://www.mdpi.com/journal/jcm

J. Clin. Med. 2024,13, 3378 2 of 20
1. Introduction
Maternal underweight and obesity are global health burdens and there has been a
substantial increase in the prevalence of these conditions among women of reproductive
age worldwide [
1
,
2
]. Mothers who are underweight or have obesity are at an increased
risk of delivering preterm [
3
], which is associated with neurodevelopmental disorders and
cardiometabolic diseases later in life [
4
–
6
]. The mechanisms that drive the relationship
between suboptimal maternal body mass index (BMI) and adverse offspring outcomes in
preterm and term pregnancies remain poorly understood, in part because cases are often
confounded by multiple comorbidities and adverse perinatal events, making it difficult to
disentangle the effects of specific exposures on fetoplacental development.
The placenta responds to cues in the pregnancy environment through morphological
and functional changes in an effort to maintain proper fetal growth and development [
7
].
For example, delayed maturation of the placenta has been observed in response to in-
creasing maternal BMI [
7
]. This altered placental maturity may result in poor gas and
nutrient exchange at the maternal–fetal interface and, subsequently, suboptimal infant out-
comes [
8
,
9
]. For example, delayed placental maturation, including the persistent thickness
of vasculosyncytial membranes, forfeits optimal gas exchange and has been associated with
placental insufficiency [
10
] and fetal macrosomia [
11
,
12
]. In pregnancies with gestational
diabetes mellitus, placentae from pregnancies with macrosomic babies have been character-
ized by reduced umbilical artery Pulsatility Index compared with controls (non-macrosomic
newborns). Thus, suboptimal maternal metabolic status associates with structural and func-
tional changes in the placenta, influencing infant growth [
13
]. While the effects of maternal
undernutrition on placental maturity are less studied, animal models of undernutrition
have shown evidence of abnormal placental development, including reduced relative
proportion of the junctional zone and lower cross-sectional area of fetal blood spaces [
14
],
which may have functional consequences. The histomorphology of the placenta ultimately
determines placental function, and histological markers of placental maturity and mor-
phometry are thus clinically useful and may reveal mechanisms underlying poor offspring
outcomes in the context of suboptimal maternal BMI. Yet, the limited evidence on the effect
of suboptimal maternal BMI on placental maturity and morphometry stems predominantly
from complicated pregnancies, and the effects of suboptimal maternal BMI alone across
gestational age/infection groups on placental histomorphology remain unclear.
Independent of maternal BMI, morphofunctional changes in the placenta also occur
in response to increasing maternal–fetal exchange demands throughout fetal develop-
ment [
15
]. Compared to term placentae, preterm placentae have distinct gross and mi-
croscopic characteristics, including decreased presence of syncytial knots, thickening of
the syncytiotrophoblast [
16
,
17
], and increased placental vascular lesions and evidence of
malperfusion [
18
], pathologies that have functional consequences for the placenta. For
example, accelerated maturation of the placenta has been observed in preterm pregnancies
and has been interpreted as an attempted compensatory adaption [
19
], yet it associates with
adverse fetoplacental outcomes, including relative placental insufficiency and late onset
intrauterine growth restriction (IUGR) [
20
]. Moreover, chorioamnionitis due to infection of
the fetal membranes results in an inflammatory cascade within the fetal membranes causing
premature rupture ofmembranes (PROM) and preterm birth (PPROM), which can further
contribute to poor fetoplacental outcomes [
21
]. Collectively, placental (mal)adaption in re-
sponse to suboptimal maternal metabolic status, infection, and/or preterm birth may have
negative effects on placental function, and thus, offspring growth and development [
9
].
While previous studies have associated maternal BMI with placental pathology at term,
these studies do not include the full range of suboptimal maternal BMI groups, or consider
preterm pregnancies with or without infection [
22
,
23
]. Others have observed placental
pathological and inflammatory lesions, yet fail to exclude maternal conditions associated
with inflammation and placental pathology (such as chorioamnionitis and chronic maternal
inflammatory conditions) [
7
]. Thus, the effects of suboptimal maternal BMI on placental
pathological and morphometric phenotypes in preterm (with and without chorioamnioni-

J. Clin. Med. 2024,13, 3378 3 of 20
tis) and term pregnancies without obstetric complications or comorbidities have been
poorly quantified. This limits our understanding of how common metabolic exposures
influence placental development at term and preterm and potentially contribute to the
programming of offspring development.
To address this gap, we assessed the effects of suboptimal maternal prepregnancy BMI,
without other major comorbidities, on histopathological indicators of placental maturity
and morphometry in preterm (with and without chorioamnionitis) and term pregnan-
cies. We also investigated the effect of gestational age and infection status on placental
maturity and morphometry inclusive of maternal BMI, as gestational age and infection
independently associate with altered placental histopathology. Lastly, we explored whether
altered placental maturity was associated with suboptimal infant anthropometry and Apgar
scores at birth. We hypothesized that maternal underweight (UW), overweight (OW), and
obesity (OB) would associate with suboptimal placental maturity and morphometry, and
that altered placental maturity would associate with poor infant birth anthropometry and
Apgar scores. By characterizing the placental pathological and morphological phenotypes
of pregnancies complicated by suboptimal maternal metabolic status, preterm birth, and
infection, our work may uncover mechanisms that can explain poor offspring development
in these pregnancies, and placental-specific histological markers that could predict altered
postnatal health trajectories.
2. Materials and Methods
2.1. Study Population
This study was approved by the Mount Sinai Hospital Research Ethics Board (17-0186-
E) and Carleton University Research Ethics Board (106932). Clinical data and placentae
from 96 pregnancies were collected through the Research Centre for Women’s and Infants’
Health (RCWIH) BioBank at Mount Sinai Hospital, Toronto. Inclusion criteria were sin-
gleton pregnancies and live birth with no known fetal anomalies. Exclusion criteria were
gestational diabetes mellitus (GDM), hypertension (including pregnancy induced hyper-
tension), HELLP syndrome, lupus, antiphospholipid antibody syndrome, Crohn’s disease,
ulcerative colitis, colitis, Guillain–Barrésyndrome, sexually transmitted infections, gastritis,
urinary tract infections, smokers, documented recreational drug use during pregnancy,
pelvic inflammatory disease, and
in vitro
fertilization. Women were categorized as having
delivered preterm with chorioamnionitis (PTC, n = 29), preterm without chorioamnionitis
(PT, n = 31), or at term (T, n = 36; there were no term pregnancies with chorioamnionitis),
and were further classified as underweight (UW, n = 21), normal weight (NW, n = 24),
overweight (OW, n = 27), or as having obesity (OB, n = 24). The RCWIH Biobank estab-
lished chorioamnionitis status by identifying suspected cases through signs and symptoms
reported on patient charts (i.e., maternal fever, fetal tachycardia, tenderness, distinct smell
of amniotic fluid during delivery), then confirming these cases with the pathologist and/or
by recently obtained bloodwork. Gestational age was calculated based on the last menstrual
period to the nearest week. Among 60 preterm pregnancies, 12 women were classified
as UW by prepregnancy BMI, 17 as NW, 18 as OW, and 13 as OB, and among 36 term
pregnancies, 9 were classified as UW, 7 NW, 9 OW, and 11 OB.
2.2. Maternal and Infant Characteristic Data Collection
The primary exposure of interest was maternal prepregnancy BMI classified according
to the World Health Organization and American College of Obstetricians and Gynecolo-
gists guidelines [
24
] with one exception; due to a low prevalence of women considered
underweight in the study region, a prepregnancy BMI of <19 kg/m
2
was considered to
be underweight. Maternal underweight, overweight (BMI
≥
25–29.9 kg/m
2
), and obesity
(BMI
≥
30.0 kg/m
2
) groups were compared to normal weight controls (19–24.9 kg/m
2
).
Maternal BMI was extracted directly from participant patient charts, and was considered
as both a continuous and categorical exposure variable. A retrospective medical chart
review was conducted to extract antenatal and birth data. Maternal characteristics have

J. Clin. Med. 2024,13, 3378 4 of 20
been previously reported [
25
]. Infant data included gestational age, infant sex, Apgar
scores (at one, five, and ten minutes), and newborn anthropometry (including birthweight,
a secondary outcome). Standardized birthweight by infant sex and gestational age were
calculated based on singleton data reported by Kramer et al. [26].
2.3. Placental Collection and Processing
Placentae were collected immediately after birth by trained staff at the RCWIH BioBank
(Toronto, ON, CA) by sampling a nearly full-thickness tissue core of approximately 1.5 cm
by 1.5 cm by cutting from the maternal surface and excluding the chorionic plate. Placen-
tal samples were obtained from all four placental quadrants, at least 1.5 cm away from
the edge and from the centre of the placental disc, the umbilical cord insertion site, and
areas of thrombosis, infarcts, or other abnormalities. Biopsies were processed for histology.
Formalin-fixed, paraffin-embedded placental biopsies were sectioned (6
µ
m) and stained
with haematoxylin (Gill’s Number 1, Sigma-Aldrich, St. Louis, MO, USA) and eosin (Eosin
Y-Solution, Sigma-Aldrich) and stained with (H&E) according to standard protocols. The
primary outcomes of interest were placental pathologies, specifically microscopic placental
pathologies related to morphometry and placental maturity. Histological chorioamnionitis
was not a pre-defined histological feature evaluated here. Rather, chorioamnionitis sta-
tus was determined by the BioBank from which the samples were obtained. Previously,
chorioamnionitis stage and grade were assessed by a clinical pathologist in fetal membrane
samples matched with this cohort [
25
]. Most preterm pregnancies with chorioamnionitis
had fetal membranes with chorioamnionitis stage and grade 2, and all but one preterm
with chorioamnionitis case had a stage and grade > 0 [25].
2.4. Placental Morphometry
Placental morphometry analysis was undertaken using methodology previously de-
scribed in the literature with specific adaptations, which are outlined below [
27
]. Image
acquisition and analysis were performed on a subset (n = 87) of H&E-stained sections using
an Aperio AT2 microscope (Leica Biosystems, Richmond, IL, USA), coupled with a com-
puter using the software ImageScope x64. The histomorphological analysis was undertaken
by an experienced examiner, blinded to exposure groups, using the Fiji ImageJ (v1.0; Im-
ageJ, Madison, WI, USA). Relative volume estimates of placental histological components
(syncytiotrophoblast, syncytial knots, cytotrophoblasts, villous stroma [connective tissue
and the villous core], and fetal capillaries) were quantified by superimposing placental
histological photomicrographs with a grid of equidistant points (measuring 25
µ
m distance
between two points). Previous studies using morphometric analysis in human placentae
recorded 190–1000 points (grid intercepts) to evaluate the volumetric proportion of each
placental histological component [
27
–
29
]. In this study, we assessed placentae by recording
1500 points overlapping with each of the histological components for the first 39 placentae.
For the remaining 48 placentae, in order to optimize the recordings, we reduced the number
of points recorded to 600 points overlapping with each of the histological components,
while still exceeding the number of points typically assessed [
29
]. We analysed volumetric
proportions of each histological component across BMI or gestational age groups for placen-
tae from 1500- and 600-point morphometric analyses separately. There were no differences
in volumetric proportions of histological components between the placentae assessed using
the 1500-point approach and the 600-point approach when comparing outcomes across
maternal BMI or gestational age groups. Thus, placentae assessed using 1500 and 600 points
were pooled for statistical analyses. The total average area of evaluated histological sections
per placenta was 393,173.22
µ
m
2
, and there were no differences in median area (
µ
m
2
)
assessed across maternal BMI groups (UW: 324,043 [273,407, 587,270]; NW: 505,332 [259,336,
557,864]; OW: 276,020 [257,202, 505,674]; OB: 502,106 [270,320, 532,097]). The volumetric
proportion (VP) of each histological component was calculated as VP = NP
×
100/600 for
placentae for which 600 points were recorded, and as VP = NP
×
100/1500 for placentae

J. Clin. Med. 2024,13, 3378 5 of 20
for which 1500 points were recorded, where NP = number of equivalent points on each
histological component [27,30,31].
2.5. Placental Maturity
Histopathological characteristics were scored on H&E-stained sections by an anatom-
ical pathologist following the Amsterdam criteria [
32
] to assess placental maturity and
chorangiosis relative to gestational age. Hypercapillarisation and characteristics of immatu-
rity (1. villous immaturity and 2. stromal immaturity) and hypermaturity (1. distal villous
hypoplasia and 2. accelerated villous maturation) were scored as either 0 (absent) or 1
(present). Descriptions of all characteristics are included in Table 1. As previous studies
in term cohorts have included accelerated villous maturation as diagnostic criteria [
23
,
33
],
we assessed placental hypermaturity at both preterm and term. No placentae had both
immature and hypermature characteristics.
Table 1. Methodology of placental histopathological assessment.
Histopathological Characteristic Definition Scoring
Immature
1. Villous immaturity [monotonous villi (≥10
villi) with centrally placed capillaries]
2. Stromal immaturity [decreased
vasculosyncytial membranes resembling villi
in early pregnancy present in at least 30% of
full thickness section]
[0 (absent) or 1 (present)]
Hypermature
1. Distal villous hypoplasia [reduced size of
intermediate villi with dispersed terminal villi
and reduced number that appear thin and
elongated, widening of intervillous space;
adjusted for gestational age; involving at least
30% of a full thickness slide]
2.
Accelerated villous maturation [the presence of
term-appearing and/or hypermature villi for
gestational age, not in areas adjacent to
infarction].
[0 (absent) or 1 (present)]
Hypercapillarisation 1.
Chorangiosis defined as >10 terminal villi with
≥10 capillaries. [0 (absent) or 1 (present)]
Placentae with presence of one or more immaturity characteristic but absent of all hypermaturity characteristics
were deemed ‘immature’ for gestational age. Placentae with presence of one or more hypermaturity characteristic
but absent all immaturity characteristics were deemed ‘hypermature’ for gestational age. Placentae absent all
immaturity and hypermaturity characteristics were deemed ‘normal’ for gestational age.
2.6. Statistical Analyses
2.6.1. Univariate Analyses
The primary exposure of interest was maternal prepregnancy BMI, specifically ma-
ternal UW, OW, and OB compared to NW controls. As gestational age and infection
can independently affect outcomes, we also assessed these variables as secondary ex-
posures. The primary outcomes of interest were placental maturity (immature, normal,
hypermature), chorangiosis, and placental morphometry (syncytiotrophoblast, syncytial
knots, cytotrophoblasts, villous stroma, and fetal capillaries). Data were stratified by term
(37–42.2 weeks gestation) and preterm (<37 weeks gestation) to assess the relationships
between maternal prepregnancy BMI and outcome variables in preterm and term preg-
nancies separately. Associations between maternal prepregnancy BMI groups or preterm
with chorioamnionitis/preterm without chorioamnionitis and continuous outcomes (pla-
cental morphometry measures) were tested using one-way ANOVA or Kruskal–Wallis
test with Tukey’s post hoc or Steel–Dwass post hoc. Likelihood Ratio Chi Square tests
were used to evaluate the associations between maternal BMI groups or preterm with
chorioamnionitis/preterm without chorioamnionitis/term group and categorical outcome

J. Clin. Med. 2024,13, 3378 6 of 20
variables (placental maturity outcomes). To explore sex differences in placental maturity
and morphometry in response to both BMI and gestational age/infection, we also strat-
ified data by fetal sex to analyze outcomes in males and females separately. Data were
analysed using JMP statistical software (version 14.2, SAS Institute, Cary, NC, USA). Data
are presented as median (interquartile range; non-parametric data), mean and standard
deviation (parametric data), or frequency (percentage; categorical variables). Statistical
significance was defined as p< 0.05. We also report false discovery rate (FDR) adjusted
p-values (q-values).
To support the objectivity and reproducibility of our histopathological assessments,
we associated our placental pathology data with morphometry histological components
for which we would expect associations with placental pathology. We assessed volumetric
proportion of syncytial knots or fetal vascular endothelium stratified by placental maturity
(immature, normal, and hypermature) or placental hypercapillarisation using Kruskal–Wallis
test with Steel–Dwass post hoc. Data are presented as median (interquartile range; non-
parametric data). Statistical significance was defined as p< 0.05.
2.6.2. Multivariable Analyses
Multivariable regression analyses were conducted to assess the relationship between
maternal prepregnancy BMI (continuous) and placental maturity and morphometry sepa-
rately for preterm and term pregnancies. Covariables were identified a priori. Covariables
of interest were identified a priori and included fetal sex [
34
],maternal GWG [
35
,
36
],
chorioamnionitis [
37
,
38
], and degree of prematurity (gestational age) [
39
]. First, an un-
adjusted model was used to identify the associations between prepregnancy BMI and
placental maturity in preterm and term pregnancies (model A). An adjusted nominal lo-
gistic regression model was then used to determine the associations between maternal
BMI and placental maturity (model B) adjusted for fetal sex (male/female), and GWG
(continuous) for term pregnancies, and also adjusting for chorioamnionitis (yes/no) and
gestational age (continuous) for preterm pregnancies. Odds ratios were derived from the
exponential function of the regression coefficient. Data are presented as odds ratios (OR) (or
adjusted OR [aOR]) and 95% confidence intervals, and pvalue from Likelihood Ratio Chi
Square test. Thirdly, an unadjusted model was used to identify the associations between
prepregnancy BMI and placental morphometry data (model C). An adjusted Standard
Least Squares regression model was used to determine the associations between maternal
BMI and placental morphometry data (model D; adjusted for the same covariates as model
B. Data are presented as
β
(or adjusted
β
[a
β
]) and 95% confidence intervals, and pvalue
from Standard Least Squares regression models.
3. Results
3.1. Maternal BMI Has Limited Effect on Placental Maturity or Morphometry
There was no effect of maternal prepregnancy BMI on placental anthropometry among
preterm or term pregnancies (Table 2). However, based on histopathologic classification by
an anatomic pathologist, we found that, among preterm pregnancies with chorioamnionitis,
placental hypermaturity was more prevalent in NW pregnancies compared to UW and OW
pregnancies, where UW and OW pregnancies had a 150% and 400% decrease in proportion
of hypermature placentae, respectively, compared to NW pregnancies (Figure 1). At
preterm (without chorioamnionitis), placental hypermaturity was not highly prevalent with
suboptimal BMI, but immaturity was more prevalent in OW and OB preterm pregnancies
compared to NW pregnancies, representing a 300% and 200% increase in the proportion
of immature placentae, respectively (Figure 1). At term, placental immaturity was more
prevalent in OW and OB pregnancies, representing a 400% increase in proportion of
placentae that were immature in OW and OB pregnancies, respectively, compared to
NW pregnancies. However, there were no differences in placental maturity (immature,
normal, hypermature) across maternal BMI groups when stratifying by preterm with
chorioamnionitis, preterm without chorioamnionitis, and term pregnancies (Figure 1).

J. Clin. Med. 2024,13, 3378 7 of 20
Among preterm pregnancies, when considering BMI as a continuous variable, odds of
placental immaturity ([model A: OR = 1.06 (
−
0.03, 0.16), p= 0.28]; [model B: aOR = 1.09
(
−
0.03, 0.21), p= 0.21]) and odds of placental hypermaturity ([model A: OR = 0.97 (
−
0.15,
0.07), p= 0.56]; [model B: aOR = 1.01 (
−
0.11, 0.13), p= 0.83]) did not change with each one
unit increase in maternal BMI. Odds of placental immaturity also did not change with each
one unit increase in maternal BMI among term pregnancies ([model A: OR = 1.05, (0.95,
1.16), p= 0.27]; [model B: aOR = 1.07 (0.95, 1.19), p= 0.24]). When data were further stratified
by fetal sex, there were no differences in placental maturity across maternal BMI groups at
preterm and term in male or female placentae (Supplementary Tables S1 and S2). At term,
hypercapillarisation was more prevalent in OW (5 [62.5]) and OB (3 [37.5]) pregnancies,
compared to NW (0 [0.0]) and UW (1 [12.5]) pregnancies (p= 0.01). Hypercapillarisation
was not present in preterm pregnancies or preterm pregnancies with chorioamnionitis.
Table 2. Infant characteristics by prepregnancy BMI in preterm and term pregnancies.
Prepregnancy BMI
UW NW OW OB pValue
Infant characteristics
Preterm pregnancies (n = 12) (n = 17) (n = 18) (n = 13)
Sex (n [%])
0.47
Female 5 (41.7) 5 (29.4) 10 (55.6) 5 (38.5)
Male 7 (58.3) 12 (70.59) 8 (44.4) 8 (61.5)
Z-birthweight −0.34 ±0.94 A−0.02 ±0.71 AB 0.20 ±0.65 AB 0.59 ±1.09 B0.05
Birthweight (g) 1735 (1010, 2118) 1820 (1005, 2176) 1700 (960, 2685) 1820 (1510, 2955) 0.53
Placental weight (g) 410 (350, 490) 400 (330, 530) 448 (323, 690) 480 (388, 670) 0.21
Birthweight (g): placental
weight (g) 4.39 ±1.19 3.96 ±1.23 3.73 ±1.19 3.90 ±1.00 0.52
Apgar score
1 min 8 (6.25, 8) 8 (4.5, 9) 8 (5, 9) 8.5 (3.75, 9) 0.93
5 min 8.5 (8, 9) 9 (8, 9) 9 (8, 9) 9 (8, 9) 0.87
10 min a8 (8, 8) 8 (2.75, 8.75) 7 (7, 9) 9 (5, 9) 0.90
Gestational age (weeks) 32.1 (29, 34.2) 31.3 (27, 34.7) 31 (27.5, 34.8) 33.3 (29.6, 35.2) 0.81
Term pregnancies (n = 9) (n = 8) (n = 8) (n = 11)
Sex (n [%])
0.05
Female 3 (33.3) 2 (37.5) 6 (66.7) 9 (81.8)
Male 6 (66.7) 5 (62.5) 3 (33.3) 2 (18.2)
Z-birthweight −0.31 (−0.78, 0.11) A0.02 (−0.49, 0.75) A0.14 (−0.27, 0.66) A0.62 (0.09, 2.28) A0.04
Birthweight (g) 3280 (3075, 3480) 3500 (3230, 3820) 3630 (3160, 3780) 3550 (3310, 3940) 0.13
Placental weight (g) 500 (465, 620) 700 (563, 743) 610 (550, 780) 618 (570, 1000) 0.09
Birthweight (g): placental
weight (g) 6.11 ±0.87 5.45 ±1.00 5.46 ±0.74 5.15 ±1.35 0.25
Apgar score (n [%])
1 min 9 (8, 9) 9 (9, 9) 9 (8.5, 9) 9 (6.75, 9) 0.47
5 min 9 (9, 9) 9 (9, 9) 9 (9, 9) 9 (8.75, 9) 0.37
10 min a- - 9 9 (9, 9) 1.00
Gestational age (weeks) 39.3 ±0.52 39.3 ±1.05 39.8 ±1.30 38.7 ±0.88 0.12
Data are means
±
SD (ANOVA; normal distribution/equal variance) or median (IQR; Kruskal Wallis/Wilcoxon
test for non-parametric data with Steel-Dwass post hoc). Data are n (%) (Likelihood Ratio Chi Square test) for
categorical variables. Post hoc differences between groups are denoted by different letters. p< 0.05. Placental
weights reported are from fresh, untrimmed placentae.
a
10 min Apgar scores were not available for 9 (75%)
preterm UW, 13 (76.5%) preterm NW, 15 (83.3%) preterm OW, and 10 (76.9%) preterm OB pregnancies. No 10 min
Apgar scores were obtained for term UW or term NW groups, and scores were not available for 7 (87.5%) term
OW and 9 (81.8%) term OB pregnancies.

J. Clin. Med. 2024,13, 3378 8 of 20
Term
UW
BV
IV
OB
BV
IV
NW
BV
SV
SK
OW
BV
SV
IV
d
200 μm200 μm
200 μm200 μm
TV
e
UW
BV
IV
NW
BV
VSM
SK
MIV
OW
BV
IV
OB
BV
IV
200 μm200 μm
200 μm200 μm
Preterm
ca b
Hype rma ture
Im ma ture
Normal
Propo rti on (%) of placentae with pathology
amon g term pregnancies
UW NW OW OB
62%
37%
14%
62%
UW NW OW OB
22%
50%
12%
11%
38%
Propo rti on (%) of placentae with pathology
amon g preterm pregnancies
UW NW OW OB
Propo rti on (%) of placentae with pathology
amon g preterm + cho rio pregnancies
33%
63%
30% 25%
17% 10%
Hype rma ture
Im ma ture
Normal
Hype rma ture
Im ma ture
Normal
Figure 1. (a). Proportion of placentae with immature, normal, and hypermature placentae across BMI
and gestational age groups among preterm pregnancies with chorioamnionitis. Placental hyperma-
turity was more prevalent in normal-weight pregnancies, representing a 150% and 400% increase
in proportion of placentae that were hypermature in NW pregnancies, compared to UW and OW
pregnancies, respectively. (b). Proportion of placentae with immature, normal, and hypermature pla-
centae across BMI and gestational age groups among preterm pregnancies without chorioamnionitis.
Placental immaturity was more prevalent in OW and OB pregnancies, representing a 300% and 200%
increase in proportion of placentae that were immature in OW and OB, respectively, compared to
NW pregnancies. (c). Proportion of placentae with immature, normal, and hypermature placentae
across BMI and gestational age groups among term pregnancies. At term, placental immaturity was
more prevalent in OW and OB pregnancies, representing a 400% increase in proportion of placentae
that were immature in both OW and OB, compared to NW pregnancies. No hypermaturity was
observed in term placentae. (d). Representative images from H&E-stained placentae from UW,
NW, OW and OB preterm pregnancies. UW, OW, OB = immature pathology. BV = Blood vessel,
IV = immature
villus, TV (large arrowhead) = terminal villus, VSM = Vasculo-syncytial membrane,
SK (small arrowhead) = Syncytial knot, SV = Stem villus. 20
×
Magnification. Scale bar = 200
µ
m.
(e). Representative images from H&E-stained placentae from UW, NW, OW and OB term pregnan-
cies. UW, OW,
OB = immature
pathology. BV = Blood vessel, IV = immature villus, MIV = mature
intermediate villus, VSM = Vasculo-syncytial membrane, SK (large arrowhead) = Syncytial knot. 20
×
Magnification. Scale bar = 200 µm.

J. Clin. Med. 2024,13, 3378 9 of 20
Among preterm and term pregnancies, villous stroma comprised the greatest area of
quantified tissue, followed by fetal capillaries, syncytiotrophoblast, and syncytial knots,
and there were no differences in volumetric proportion of these histologic features across
maternal BMI groups (Figure 2). Placental morphometry also did not differ with increasing
maternal BMI (Table 3). When data were stratified by fetal sex, there was no association
between maternal BMI and volumetric proportion of any histological components at
preterm and term in male or female placentae (Supplementary Tables S1 and S2).
Preterm
Villous stroma Syncytiotrophoblast Syncytial knotsFetal capillaries
a
UW NW OW OB UW NW OW OB UW NW OW OBUW NW OW OB
Volumetric proportion of placental
histological components (%)
UW NW OW OB
Villous strom a Syncytiotrophoblast Syncytial knots
b
Volumetric proportion of placental
his tolog ical components (%)
NW OW OBUWUW NW OW OB
Term
UW NW OW OB
Fetal capillaries
Figure 2. Volumetric proportion of placental histological components (%), including, from left to
right, villous stroma, fetal capillaries, syncytiotrophoblast, and syncytial knots across maternal BMI
groups among (a). preterm and (b). term pregnancies. Data are quantile box plots with a horizontal
line representing the mean across the whole cohort.

J. Clin. Med. 2024,13, 3378 10 of 20
Table 3. Associations between prepregnancy BMI and placental morphometry volumetric proportions
in preterm and term pregnancies.
Model C Model D
Volumetric Proportion of
Placental Histological
Components (%)
β(95% CI) p-Value aβ(95% CI) pValue
Preterm (n = 56)
Syncytiotrophoblast −0.01 (−0.15, 0.12) 0.80 −0.05 (−0.19, 0.08) 0.42
Cytotrophoblast 0.001 (−0.006, 0.008) 0.74 −0.002 (−0.008, 0.004) 0.53
Villous stroma −0.19 (−0.48, 0.09) 0.18 −0.22 (−0.53, 0.08) 0.16
Fetal Capillaries 0.21 (−0.11, 0.54) 0.19 0.28 (−0.05, 0.63) 0.09
Syncytial knots −0.008 (−0.04, 0.02) 0.60 −0.01 (−0.04, 0.02) 0.52
Term (n = 31)
Syncytiotrophoblast −0.11 (−0.29, 0.06) 0.18 −0.08 (−0.31, 0.1) 0.42
Cytotrophoblast −0.006 (−0.02, 0.009) 0.42 −0.002 (−0.02, 0.02) 0.82
Villous stroma 0.03 (−0.31, 0.38) 0.81 −0.07 (−0.52, 0.38) 0.73
Fetal Capillaries 0.06 (−0.32, 0.45) 0.74 0.14 (−0.37, 0.66) 0.58
Syncytial knots −0.005 (−0.03, 0.02) 0.74 −0.002 (−0.04, 0.03) 0.63
Data are
β
and 95% CI and pvalue from Standard Least Squares models. p< 0.05. For preterm and term, model
C is unadjusted. Model D adjusted for fetal sex, maternal gestational weight gain, chorioamnionitis status, and
gestational age. For term, model D includes fetal sex and maternal gestational weight gain.
3.2. Maternal BMI Has Limited Effect on Birth Outcomes
At birth, standardized birthweight increased with increasing maternal BMI among
preterm and term infants (Table 2); although, among term infants there were no differences
in infant birthweight z-scores (BWZ) between BMI groups on post hoc analysis. There were
no differences across maternal BMI groups for infant Apgar scores at 1 or 5 min among
preterm or term infants (Table 2).
3.3. Gestational Age and Infection Status Associate with Altered Placental Maturity and Morphometry
We found that placental weight (p< 0.0001) and birthweight-to-placental weight ratio
(p< 0.0001), sometimes used as a proxy measure for placental efficiency (as well as placen-
tal developmental stage), were decreased in preterm pregnancies with chorioamnionitis
compared to preterm without chorioamnionitis and term pregnancies, inclusive of BMI
(Table 4). As expected, gestational age was also lower in preterm with chorioamnionitis
compared to preterm pregnancies without chorioamnionitis and term pregnancies (Table 4).
Additionally, inclusive of BMI, the greatest proportion of placental hypermaturity was
observed in preterm pregnancies with chorioamnionitis [immature = 1 (3.7), normal = 15
(55.6), hypermature = 11 (40.7)] compared to preterm [immature = 8 (27.6), normal = 18
(62.1)], hypermature = 3 (10.3)] and term [immature = 14 (45.2), normal = 17 (54.8), hyper-
mature = 0 (0.00)] pregnancies (p< 0.0001). In further exploring this association, we found
that, while preterm pregnancies with chorioamnionitis had increased odds of accelerated
villous maturation (AVM), but not distal villous hypoplasia (DVH), compared to preterm
pregnancies without chorioamnionitis (p= 0.01), the significance of this difference was
not retained after adjusted analyses (Supplementary Table S5). Prevalence of immaturity,
normal maturity, and hypermaturity did not differ across gestational age/infection groups
when stratified by fetal sex (Supplementary Table S3).

J. Clin. Med. 2024,13, 3378 11 of 20
Table 4. Infant characteristics in preterm pregnancies with chorioamnionitis, preterm pregnancies
without chorioamnionitis, and term pregnancies.
Gestational Age and Infection Status
Preterm Chorio
(n = 29)
Preterm
(n = 31)
Term
(n = 36) pValue
Infant characteristics
Sex (n [%])
0.37
Female 13 (44.8) 12 (38.7) 20 (55.6)
Male 16 (55.7) 19 (61.3) 16 (44.4)
Z-birthweight 0.02 (−0.34, 0.33) 0.3 (−0.71, 0.89) 0.14 (−0.49, 0.74) 0.85
Birthweight (g) 1130 (950, 1700) A2340 (1810, 2710) B3485 (3230, 3672) C<0.0001
Placental weight (g) 368 (303, 463) A500 (400, 650) B600 (543, 743) C<0.0001
Birthweight (g): placental weight (g)
3.43 ±1.04 A4.43 ±1.05 B5.54 ±1.05 C<0.0001
Apgar score
1 min 8 (4, 8) A8.5 (6, 9) AB 9 (8, 9) B0.0003
5 min 8 (6, 9) A9 (8, 9) A9 (9, 9) B0.0001
10 min a7 (5, 8) A9 (8, 9) B9 (9, 9) B0.0048
Gestational age (weeks) 28.1 (26.6, 31.4) A34.7 (31.9, 35.6) B39.1 (38.6, 39.7) C<0.0001
Data are means
±
SD (ANOVA; normal distribution/equal variance with Tukey post hoc) or median (IQR;
Kruskal-Wallis/Wilcoxon test for non-parametric data with Steel-Dwass post hoc). Data are n (%) (Likelihood
Ratio Chi Square test) for categorical variables. Post hoc differences between groups are denoted by different
letters. p< 0.05.
a
10 min Apgar scores were not available for 22 (75.9%) preterm chorio, 25 (80.6%) preterm, and
33 (91.7%) term pregnancies.
Fetal capillary volumetric proportion was decreased (p= 0.05, q = 0.13, Table 5) and
villous stromal volumetric proportion was increased (p= 0.02, q = 0.1, Table 5) in preterm
pregnancies with chorioamnionitis compared to preterm pregnancies without chorioam-
nionitis. Although, there were no differences in fetal capillary volumetric proportion on
post hoc analysis, and there were no differences in fetal capillary volumetric proportion
or villous stromal volumetric proportion following FDR adjustment. Infection status at
preterm had no effect on syncytial knots, syncytiotrophoblast, or cytotrophoblast volumet-
ric proportions (Table 5). When data were stratified by fetal sex, there were no differences
in volumetric proportion of histologic features in male or female placentae in preterm preg-
nancies with chorioamnionitis compared to preterm pregnancies without chorioamnionitis
(Supplementary Table S4).
Table 5. Effect of gestational age at birth and infection status on volumetric proportion of placental
histological components.
Volumetric Proportion of
Placental Histological
Components (%)
Gestational Age at Birth
Preterm with
Chorioamnionitis
(n = 27)
Preterm
(n = 29)
Term
(n = 31) †pValue †q Value
Syncytiotrophoblast 20 (19, 22) 21 (19, 23.2) 20 (16.5, 22.5) 0.72 0.9
Cytotrophoblast 0 (0, 0) 0 (0, 0) 0 (0, 0) 0.97 0.97
Villous stroma 58 (54, 63) 55 (48, 58.6) 55 (50, 57.5) 0.02 0.1
Fetal capillaries 20 (17, 26) 22 (18, 29.5) 28 (20.5, 30.5) 0.05 0.13
Syncytial knots 1 (0, 2) 1 (0,1) 0 (0, 1) 0.15 0.25
Data are volumetric proportions of placental histological components. Data are presented as median (IQR).
†
Com-
parisons are calculated only for PTC vs. PT given the impact of gestational age on development/differentiation of
histological components (Kruskal–Wallis test for non-parametric data) p< 0.05.

J. Clin. Med. 2024,13, 3378 12 of 20
3.4. Preterm Pregnancies with Chorioamnionitis Associate with Decreased Infant Apgar Scores
Inclusive of maternal BMI, preterm pregnancies with chorioamnionitis had the lowest
median infant birthweight, followed by preterm pregnancies without infection and term
pregnancies (p< 0.0001, Table 4). However, there were no differences in infant BWZ between
preterm, preterm with chorioamnionitis, and term pregnancies (Table 4). Apgar scores
at one minute (p= 0.0003) were decreased in preterm pregnancies with chorioamnionitis,
compared to scores in term infants, and Apgar scores at 5 min were also decreased in
both preterm pregnancies with and without chorioamnionitis compared to term infants
(
p= 0.0003,
Table 4). Preterm pregnancies with chorioamnionitis also had the lowest
median gestational age at birth, followed by preterm pregnancies without infection and
term pregnancies (p< 0.0001, Table 4). Placental maturity did not associate with infant
BWZ or Apgar scores at 1 and 5 min (Figure 3) among preterm and term pregnancies.
Birthweight (Z-score)
a
b
Birthweight (Z-score)
Immature Normal
Birthweight (Z-score)
Hypermature
Immature
Normal
Immature
Normal
Apgar score (/10)
5 minute1 minute
Apgar score (/10)
1 minute 5 minuteImmature Normal Hypermature
Preterm
Term
Figure 3. Associations between placental maturity (immature, normal, and hypermature) and
birthweight z-scores or Apgar scores at 1 and 5 min among (a) preterm and (b) term pregnancies.
Data are quantile box plots.
3.5. Placental Maturity and Chorangiosis Associate with Placental Morphometry
Inclusive of BMI and gestational age, syncytial knot volumetric proportion was in-
creased (immature 0 [0, 1]; normal 1 [0, 1], hypermature 1 [0, 2]; p= 0.04) and fetal capillary
volumetric proportion was decreased (immature 29 [25, 34]; normal 21 [18, 28]; hyper-
mature 21 [17, 30]; p= 0.003) with advancing maturity; significant differences were only
observed between hypermature placentae compared to immature placentae on post hoc
analysis (Figure 4). Inclusive of maternal BMI and gestational age, fetal capillary volumetric
proportion was increased in placentae with hypercapillarisation compared to placentae
without hypercapillarisation (absent 21 [18, 28.5]; present 29.5 [28.3, 35], p= 0.003, Figure 4).

J. Clin. Med. 2024,13, 3378 13 of 20
Immature Normal Hypermature Immature Normal Hypermature
a
Absent
Hypercapillarisation
Present
ab c
aab c
a
b
Vol umetric proportion of fe ta l
capillaries (%)
Volumetric proportion of fetal
capillaries (%)
Vol umetric proportion of
syncytial knots (%)
Figure 4. Volumetric proportion of syncytial knots or fetal vascular endothelium stratified by placental
maturity (immature, normal, and hypermature) or placental hypercapillarisation. Data are quantile
box plots with a horizontal line representing the mean across the whole cohort. Differences between
groups are denoted by different lowercase letters.
4. Discussion
We examined the effect of maternal prepregnancy BMI, without other major comor-
bidities, on placental maturity and morphometry to quantify how suboptimal maternal
metabolic states influence placental phenotypes and to better understand the mechanisms
that may contribute to poor pregnancy outcomes and fetal (mal)development in these
pregnancies. Reassuringly, we found no major differences in placental maturity or mor-
phometry across maternal prepregnancy BMI groups among preterm or term pregnancies
and placental maturity did not associate with infant birthweight or Apgar scores at birth.
There were limited associations between maternal BMI and infant birth outcomes. We
did observe an influence of gestational age and infection on placental phenotypes, where
the greatest proportion of hypermature placentae were from preterm pregnancies with
chorioamnionitis, compared to placentae from preterm pregnancies without infection and
term pregnancies. Accordingly, preterm pregnancies with chorioamnionitis were associ-
ated with decreased placental weight and efficiency and decreased infant Apgar scores,
suggesting that infection in the context of preterm birth may have negative implications for
placental development and infant outcomes.

J. Clin. Med. 2024,13, 3378 14 of 20
Our data showed limited evidence for an effect of low or high prepregnancy BMI on
placental maturity and morphometry among preterm or term pregnancies. Among both
term and preterm pregnancies, we found that placental immaturity was more prevalent
in OW and OB pregnancies, representing a high percentage increase in the proportion
of placentae that were immature in both OW and OB pregnancies, compared to NW
pregnancies, which may suggest the emergence of underlying pathology. We observed
hypercapillarisation only at term, where it was more prevalent in term OW and OB preg-
nancies compared to NW and UW pregnancies. In our study, placentae from pregnancies of
mothers who were overweight or had obesity displayed a phenotype similar to placentae
from pregnancies complicated by type 1 diabetes, which are generally larger than normal,
immature, and hypercapillarised [
12
]. It is well established that maternal obesity promotes
a pro-inflammatory environment within gestational tissues, including elevated levels of cir-
culating interleukin IL-6 during pregnancy and higher levels of placental pro-inflammatory
cytokines [
40
], and associates with poor infant outcomes. Further, a recent study observed
that placentae from pregnancies with GDM and increased prepregnancy BMI or GWG
had increased expression of neoangiogenesis and inflammatory markers, such as vascular
endothelial growth factor (VEGF) and CD31 [
35
]. Diet-induced maternal obesity also asso-
ciates with increased levels of the proinflammatory cytokines tumor necrosis factor TNF-
α
and IL-8 in sheep placentae [
40
]. TNF-
α
can inhibit placental trophoblast motility and mi-
gration, indicating its potential to impact placental development [
41
]. Thus, poor maternal
metabolic health is permissive of a pro-inflammatory environment that may adversely
affect normal placental maturity and structure. However, in contrast to studies showing
increased proportion of macroscopic and microscopic placental pathologies with increasing
maternal BMI [
7
,
42
], our data show limited evidence for altered placental histopathology
in pregnancies with suboptimal maternal BMI. Differences in our findings could be ex-
plained by our study design. We intentionally excluded pregnancies with comorbidities
and complications that are associated with obesity and underweight and have known
effects on placental development and function [
12
,
43
] so that we could more accurately
gain insight into the effects of suboptimal BMI alone on placental pathology and mor-
phometry. Hypertension and GDM are highly prevalent in mothers who are overweight or
have obesity [
44
] and could be driving the placental histopathological changes previously
reported in pregnancies complicated by suboptimal maternal BMI. Given that an estimated
30% of women with overweight or obesity have no other comorbidities [
45
,
46
], our limited
histomorphological findings in placentae from otherwise uncomplicated pregnancies may
be reassuring.
Gestational age and infection may also alter placental pathology and morphometry.
Indeed, our placental morphometric analyses show a modest decrease in fetal capillary
volumetric proportion in preterm pregnancies with chorioamnionitis compared to preterm
pregnancies without chorioamnionitis. Likely due to this global reduction in vascularity,
we also observed increased villous stromal volumetric proportion in preterm pregnan-
cies with chorioamnionitis compared to preterm pregnancies without chorioamnionitis.
Endothelial cell proliferation and elongation is critical for placental vascular remodeling
throughout pregnancy [
47
], and placental endothelial cell dysfunction can contribute to the
development of disorders such as placental insufficiency and pre-eclampsia [
48
]. Thus, de-
creased fetal capillary volume fraction in preterm pregnancies with chorioamnionitis may
suggest inadequate placental vasculature and decreased placental blood flow throughout
gestation, and possible associations between gestational age and infection with placental
morphometry need to be explored in other and larger cohorts to determine if they can be
replicated elsewhere.
In what may have been an attempted compensatory adaptation to decreased fetal
capillaries, placental blood flow, and subsequent placental hypoxia [
49
], preterm pregnan-
cies with chorioamnionitis also showed a greater proportion of placental hypermaturity
compared to preterm and term pregnancies. While we observed placental hypermaturity in
both preterm pregnancies with and without chorioamnionitis, the observation of greater hy-

J. Clin. Med. 2024,13, 3378 15 of 20
permaturity in PTC compared to PT is unlike previous studies [
49
]. In further exploring this
association, we found that preterm with chorioamnionitis pregnancies had increased odds
of AVM, but not DVH, compared to preterm pregnancies without chorioamnionitis, though
there were no differences in odds of AVM or DVH in preterm pregnancies with chorioam-
nionitis compared to preterm pregnancies without chorioamnionitis after adjusting for fetal
sex, maternal GWG, and gestational age. Because our exclusion criteria precluded most
cases with pre-placental major maternal comorbidities and maternal conditions associated
with placental underperfusion, we were left with a cohort that was likely all “spontaneous”;
that is, etiologies that included threatened preterm labor, chorioamnionitis, preterm prema-
ture rupture of the membranes, and cervical incompetence. However, there may be some
indicated preterm births in our cohort, which may be represented to a greater degree at
later gestational ages and may thus influence the placental outcomes we measured here
or could explain the lack of chorioamnionitis we see in these later ages. Our results may
raise the possibility that those with histological chorioamnionitis may be distinct from the
rest of the spontaneous (non-iatrogenic) preterm cluster by having, perhaps, long-standing
adaptation via accelerated maturation to (occult) placental insufficiency, perhaps due to
a global reduction in fetal vasculature (proposed mechanism depicted in Supplementary
Figure 2). This is in line with previous findings of placental molecular changes in cases of
chorioamnionitis. Indeed, others have shown that placental fetal capillaries are stressed by
chorioamnionitis, independent of gestational age; angiogenic factors were decreased, and
factors linked to microvessel maturation were increased in placentae from pregnancies with
chorioamnionitis compared to gestational age-matched controls without placental inflam-
mation [
38
]. Thus, our work may support the hypothesis that chorioamnionitis impairs
fetal capillary angiogenesis, and as a result, may lead to placental hypermaturation as an
attempted compensation. However, as PTC pregnancies delivered at earlier gestational age
compared to the PT and T groups, differences in developmental stage (related to gestational
age) and possible secondary villous edema, which was not assessed here but has been
associated with chorioamnionitis, may also play a role. While we separated term and
preterm pregnancies, our findings remain to be fully explored in future, larger cohorts to
disentangle the effects of gestational age and chorioamnionitis on placental histopathology.
To corroborate our placental pathology data, we compared immature, normal, and
hypermature placentae and found that syncytial knot volumetric proportion was increased
and fetal capillary volumetric proportion was decreased in hypermature placentae com-
pared to immature placentae. Increased syncytial knots in hypermature placentae is to
be expected, as this is a hallmark of placental maturation. However, the decline in fetal
vascular volume fraction with maturity has not yet been studied. Previous studies using
stereology assessed fetal vessels, such as Mayhew and colleagues who used fetal vascular
length and surface area calculations [
50
]. One potential explanation is that along with
the maturational changes in villous shape there is a maturational change in villous stro-
mal and vascular design. The central vessels—in stem villi—become more muscularised
and distributive, and the terminal villi develop less central vascularisation and acquire
vasculosyncytial membranes. Our finding that this results in a global reduction in vol-
ume fraction is novel, and merits further investigation. We also found that fetal capillary
volumetric proportion was increased in placentae with hypercapillarisation compared to
placentae without hypercapillarisation. Given that our morphometric analysis of fetal
capillary volumetric proportion quantified all points falling on villous capillaries, our
finding of greater fetal capillary volumetric proportion in placentae with increased number
of capillaries (chorangiosis) thus corroborates this pathology assessment. Taken together,
these data support the validity of our findings and represent an additional approach for
corroborating histological assessment.
Optimal placental function is necessary for the delivery of nutrients, oxygen, and hor-
mones to the developing fetus [
51
].While we observed no differences in placental maturity
in preterm birth without infection compared to term pregnancies, we found decreased
placental weight and efficiency in preterm pregnancies without infection compared to term

J. Clin. Med. 2024,13, 3378 16 of 20
pregnancies. Others have suggested that placental insufficiency, including various placental
pathologies such as placental hypermaturity and reduced placental weight [
52
], is one
etiology of idiopathic preterm birth which may arise from oxidative stress due to abnormal
spiral artery remodeling and subsequent suboptimal uteroplacental blood flow [
52
,
53
]. The
histological markers of placental immaturity and hypermaturity are also indicative of a
placenta that may be structurally ill-suited to meet fetal demands [
11
,
12
,
20
]. However, in
our cohort, placental maturity did not associate with infant outcomes at birth in preterm
or term pregnancies. This is surprising, as others have supported the prognostic value
of placental histology, including demonstrating associations between placental maturity
and infant outcomes [
49
,
54
]. Whereas previous studies linking placental maturity and
infant birth outcomes were from complicated pregnancies, our cohort purposefully lacked
major maternal comorbidities apart from suboptimal maternal BMI. This suggests that
altered placental maturity may only predict infant outcomes in complex pregnancies with
specific comorbidities. Altered placental pathology has also been associated with long-term
adverse offspring outcomes. For example, others have demonstrated associations of villous
edema, maternal vascular malperfusion, and funisitis in preterm-born pregnancies with
suboptimal offspring neurodevelopmental outcomes at school age [
55
,
56
]. Also, conditions
that alter placental pathology associate with long-term adverse maternal phenotypes [57].
For example, hypertensive disorders of pregnancy associate with defective spiral artery
remodeling and later maternal cardiovascular disease [
58
,
59
]. Long-term follow up of
mother–infant dyads with placental pathology is required to determine whether there
are long-term adverse offspring and maternal outcomes in pregnancies without major
complications or maternal comorbidities. A key strength of our study is the exclusion of
pregnancies with major comorbidities and conditions that may associate with pathological
placental findings, including GDM, hypertension, pre-eclampsia, pro-inflammatory condi-
tions, and
in vitro
fertilization. Here, we assessed placental morphometry and maturity
among preterm (with and without chorioamnionitis) and term pregnancies to better un-
derstand the influence of the full range of suboptimal maternal BMI at these gestational
periods on placental development. To our knowledge, only one other study has evaluated
placental histopathology in pregnancies with obesity without complications or comorbidi-
ties, and this study reported only moderate associations between increasing maternal BMI
and accelerated villous maturation and chronic villitis among term pregnancies [
23
]. In
contrast, a study by Bar et al. investigating high prepregnancy BMI with maternal condi-
tions including pre-eclampsia and GDM, but not hypertension or other pro-inflammatory
conditions [
22
], showed increased maternal inflammatory lesions among pregnancies com-
plicated by obesity compared to normal-weight pregnancies; these findings were consistent
when comparing mothers with and without complications [
22
]. However, this cohort did
not assess underweight or preterm pregnancies, and as such, did not capture the full scope
of metabolic states or gestational age effects as we did. Another larger study by Huang
et al. that included a cohort of women with pregnancy complications observed increased
placental pathology with increasing maternal BMI, a finding that was also observed in a
subset of women without obstetric complications; however, preterm and term pregnancies
were not examined separately [
7
]. Thus, there are conflicting findings and discrepancies
in cohort selection among the few studies investigating the effects of suboptimal mater-
nal BMI on placental pathology. While future studies are required to confirm the effects
of suboptimal maternal BMI alone on placental pathology, our cohort helps to address
these gaps in knowledge on the impact of maternal prepregnancy BMI, preterm birth,
and infection, in the absence of other major maternal comorbidities, on placental maturity
and morphometry.
5. Conclusions
Our data show that gestational age and infection associate with altered placental matu-
rity and morphometry, and, at term, placental immaturity and hypercapillarisation are more
likely in pregnancies with high maternal prepregnancy BMI, despite suboptimal BMI (in

J. Clin. Med. 2024,13, 3378 17 of 20
the absence of other comorbidities) having few other effects on placental histopathologies.
Limited changes in micro/macroscopic placental pathology do not preclude functional
changes in placentae from pregnancies complicated by suboptimal maternal BMI. Our
results add to the incomplete evidence on the effects of suboptimal maternal BMI, gesta-
tional age, and infection on placental maturity and morphometry in pregnancies without
major comorbidities, and are a step forward in understanding the mechanisms that may
contribute to poor offspring outcomes in pregnancies complicated by suboptimal maternal
BMI and preterm birth (with and without infection).
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/jcm13123378/s1, Supplementary Figure S1: Cohort breakdown;
Supplementary Figure S2: Proposed mechanism of placental villous hypermaturity in preterm preg-
nancies with chorioamnionitis. Decreased fetal vascular endothelium and increased villous stroma,
and subsequently inadequate placental vasculature and blood flow, in preterm pregnancies with
chorioamnionitis may prompt compensatory placental hypermaturation. Syncytiotrophoblast shed-
ding via increased number of syncytial knots and subsequent thinning of the syncytiotrophoblast
layer may be one mechanism leading to placental villous hypermaturity and attempted improved
exchange in preterm pregnancies with chorioamnionitis; Supplementary Table S1: Effect of maternal
BMI group on volumetric proportions of placental histological components and placental maturity
stratified by fetal sex in preterm pregnancies; Supplementary Table S2. Effect of maternal BMI group
on placental maturity, hypercapillarisation and volumetric proportions of placental histological com-
ponents stratified by fetal sex in term pregnancies; Supplementary Table S3. Effect of gestational age
and infection inclusive of maternal BMI on placental maturity stratified by fetal sex; Supplementary
Table S4. Placental morphometry volumetric proportions amongst preterm with chorioamnionitis
and preterm without chorioamnionitis pregnancies stratified by fetal sex; Supplementary Table S5.
Odds of placental pathology for preterm with chorioamnionitis pregnancies in comparison to preterm
pregnancies without chorioamnionitis.
Author Contributions: Conceptualisation, K.L.C., D.G., E.B. and E.D.; Methodology, E.D., K.L.C.,
D.G., E.B., C.B.V.d.A., A.L. and H.S.; Formal analysis and visualisations, E.D., D.G., C.B.V.d.A. and
H.S.; Manuscript writing, E.D. and K.L.C.; Manuscript editing, review, and final approval, E.D.,
K.L.C., D.G., E.B., C.B.V.d.A., A.L. and H.S. All authors have read and agreed to the published version
of the manuscript.
Funding: This research is funded by the Faculty of Science, Carleton University. E.D. was supported
by a Natural Sciences and Engineering Research Council Undergraduate Student Research Award.
K.L.C. is supported by grants from the Canadian Institutes of Health Research, Natural Sciences and
Engineering Research Council of Canada, the Molly Towell Perinatal Research Foundation (New
Investigator), and Carleton University Office of Research. E.B. is funded by Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Braziland Fundação de Amparo àPesquisa do
Estado de Minas Gerais (FAPEMIG), Brazil.
Institutional Review Board Statement: This study was performed in accordance with the Declaration
of Helsinki and was approved by the Mount Sinai Hospital Research Ethics Board (17-0186-E, 2017)
and the Carleton University Research Ethics Board (106932, 2017).
Informed Consent Statement: Informed consent was obtained from all individual participants
included in the study, and all methods were performed in accordance with the Declaration
of Helsinki
.
Data Availability Statement: Data are available from the authors upon reasonable request and with
permission of the Research Centre for Women’s and Infants’ Health BioBank where accessibility
restrictions may apply due to the terms contained within the biobank’s material transfer agreements.
Requests for data from this study can be made to Kristin Connor.
Acknowledgments: The authors thank the staff at the Research Centre for Women’s and Infants’
Health BioBank, the Lunenfeld–Tanenbaum Research Institute, and Mount Sinai Hospital for their
assistance and support, and the pregnant people who donated their placenta for research.
Conflicts of Interest: The authors have no conflicts of interest to declare.

J. Clin. Med. 2024,13, 3378 18 of 20
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