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\begin{document}

\title{The periconceptional maternal intake of ultra-processed foods and the
impact on imaging markers of early utero-placental vascular development:
a hospital-based prospective observational cohort study}



\author[1]{Regine Steegers-Theunissen}%
\author[1]{Eline S. DE VOS}%
\author[1]{Annemarie Mulders}%
\author[2]{Anton Koning}%
\author[1]{Hilco S. SMIT}%
\author[1]{Lenie van Rossem}%
\affil[1]{Erasmus MC Afdeling Verloskunde en Gynaecologie}%
\affil[2]{Erasmus MC Afdeling Pathologie}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Objective: To investigate whether periconceptional maternal intake of
ultra-processed foods (UPF) impairs first-trimester utero-placental
vascular development, and whether macronutrients and dietary patterns
substantiate the associations. Design: Prospective observational cohort.
Setting: Academic hospital. Population or Sample: Ongoing pregnancies.
Methods: 93 women completed a food frequency questionnaire from which we
calculated percentage of energy intake from UPF, intake of energy and
macronutrients and adherence to dietary patterns. We performed
sequential three-dimensional power Doppler ultrasounds of the
first-trimester utero-placental vasculature. VOCAL software, Virtual
Reality segmentation and a skeletonization algorithm were applied to
measure placental volume (PV), utero-placental vascular volume (uPVV)
and generate the utero-placental vascular skeleton (uPVS). Absolute
vascular morphology was quantified by assigning a morphologic
characteristic to each voxel in the uPVS (end-, bifurcation-, crossing-
or vessel point) and used to calculate density of vascular branching.
Main Outcome Measures: PV, uPVV, uPVS characteristics and density of
vascular branching. Results: Fully adjusted linear mixed models showed a
10\%/day higher UPF intake was associated with increased first-trimester
density of vascular branching (bifurcation points: \selectlanguage{greek}\emph{β}\selectlanguage{english}=0.465[?]n,
95\%CI=0.148;0.782). Higher carbohydrate intake of 10g/day was
associated with increased trajectories of uPVV (\selectlanguage{greek}β\selectlanguage{english}=0.017,
95\%CI=0.001;0.032) and uPVS (end points (\selectlanguage{greek}β\selectlanguage{english}=0.286, 95\%CI=0.062;0.511),
bifurcation points (\selectlanguage{greek}β\selectlanguage{english}=0.004, 95\%CI=0.003;0.006), vessel points
(\selectlanguage{greek}β\selectlanguage{english}=0.772, 95\%CI=0.137;1.408). The associations were substantiated by
the adherence to the ``Snack'' dietary pattern. Conclusions:
Periconceptional maternal intake of UPF is associated with impaired
first-trimester utero-placental vascular development, whereas the intake
of carbohydrates and strong adherence to a `Snack' dietary pattern, is
positively associated with first-trimester utero-placental vascular
development.%
\end{abstract}%



\sloppy


TITLE PAGE

TITLE: \textbf{The periconceptional maternal intake of ultra-processed
foods and the impact on imaging markers of early utero-placental
vascular development: a hospital-based prospective observational cohort
study}

AUTHORS:

\begin{enumerate}
\tightlist
\item
  Ms. Eline S. DE VOS, MD, Department of Obstetrics and Gynecology,
  Erasmus MC University Medical Center, Rotterdam, The Netherlands
\item
  Dr. Annemarie G.M.G.J. MULDERS, MD, PhD, Department of Obstetrics and
  Gynecology, Erasmus MC University Medical Center, Rotterdam, The
  Netherlands
\item
  Dr. Anton H.J. KONING, PhD, Department of Pathology, Erasmus MC
  University Medical Center, Rotterdam, The Netherlands
\item
  Mr. Hilco S. SMIT, BSc, Department of Obstetrics and Gynecology,
  Erasmus MC University Medical Center, Rotterdam, The Netherlands
\item
  Dr. ir. Lenie van ROSSEM, PhD, Department of Obstetrics and
  Gynecology, Erasmus MC University Medical Center, Rotterdam, The
  Netherlands\selectlanguage{ngerman}\textsuperscript{¶}
\item
  Prof. Dr. Régine P.M. STEEGERS-THEUNISSEN, MD, PhD, Department of
  Obstetrics and Gynecology, Erasmus MC University Medical Center,
  Rotterdam, The Netherlands\textsuperscript{¶}
\end{enumerate}

\textsuperscript{¶} Authors contributed equally.

CORRESPONDING AUTHOR:

Name: Régine P.M. Steegers-Theunissen

Erasmus MC, University Medical Centre

Department of Obstetrics and Gynaecology

PO Box 2040, 3000 CA, Rotterdam, The Netherlands

Phone: +31 10 7038254 / Fax: +31 10 7036815

E-mail: r.steegers@erasmusmc.nl

RUNNING TITLE: Ultra-processed foods and utero-placental vascular
development

ABSTRACT

\emph{Objective:} To investigate whether periconceptional maternal
intake of ultra-processed foods (UPF) impairs first-trimester
utero-placental vascular development, and whether macronutrients and
dietary patterns substantiate the associations.

\emph{Design:} Prospective observational cohort.

\emph{Setting:} Academic hospital.

\emph{Population or Sample:} Ongoing pregnancies.

\emph{Methods:} 93 women completed a food frequency questionnaire from
which we calculated percentage of energy intake from UPF, intake of
energy and macronutrients and adherence to dietary patterns. We
performed sequential three-dimensional power Doppler ultrasounds of the
first-trimester utero-placental vasculature. VOCAL software, Virtual
Reality segmentation and a skeletonization algorithm were applied to
measure placental volume (PV), utero-placental vascular volume (uPVV)
and generate the utero-placental vascular skeleton (uPVS). Absolute
vascular morphology was quantified by assigning a morphologic
characteristic to each voxel in the uPVS (end-, bifurcation-, crossing-
or vessel point) and used to calculate density of vascular branching.

\emph{Main Outcome Measures:} PV, uPVV, uPVS characteristics and density
of vascular branching.

\emph{Results:} Fully adjusted linear mixed models showed a 10\%/day
higher UPF intake was associated with increased first-trimester density
of vascular branching (bifurcation points: \selectlanguage{greek}\emph{β} \selectlanguage{english}=0.465[?]n,
95\%CI=0.148;0.782). Higher carbohydrate intake of 10g/day was
associated with increased trajectories of uPVV (\selectlanguage{greek}β\selectlanguage{english}=0.017,
95\%CI=0.001;0.032) and uPVS (end points (\selectlanguage{greek}β\selectlanguage{english}=0.286, 95\%CI=0.062;0.511),
bifurcation points (\selectlanguage{greek}β\selectlanguage{english}=0.004, 95\%CI=0.003;0.006), vessel points
(\selectlanguage{greek}β\selectlanguage{english}=0.772, 95\%CI=0.137;1.408). The associations were substantiated by
the adherence to the ``Snack'' dietary pattern.

\emph{Conclusions:} Periconceptional maternal intake of UPF is
associated with impaired first-trimester utero-placental vascular
development, whereas the intake of carbohydrates and strong adherence to
a `Snack' dietary pattern, is positively associated with first-trimester
utero-placental vascular development.

\emph{Funding:} This research was funded by the Department of Obstetrics
and Gynaecology of the Erasmus MC, University Medical Centre Rotterdam,
Rotterdam, The Netherlands.

\emph{Keywords} :

\begin{itemize}
\tightlist
\item
  Early pregnancy
\item
  Periconceptional nutritional intake
\item
  Ultra-processed foods
\item
  Placental (vascular) development
\item
  3D power Doppler Ultrasound
\end{itemize}

FUNDING:

This research was funded by the Department of Obstetrics and Gynaecology
of the Erasmus MC, University Medical Centre Rotterdam, Rotterdam, The
Netherlands.

\section*{INTRODUCTION}

{\label{introduction}}

Maternal dietary intake in the periconception period affects fertility,
prenatal development and pregnancy outcome with long-lasting
consequences for offspring health \textsuperscript{1-4}. In the typical
Western diet, the intake of fruits, vegetables and whole grains is
generally below recommended levels and the proportion of sodium, fats
and carbohydrates is relatively high \textsuperscript{5}. With the
global exponential increase of ultra-processed food (UPF) consumption in
the last decade, the nutritional status of women has
worsened\textsuperscript{4, 6, 7}. Therefore, there is an urgent need to
investigate how UPF consumption impacts prenatal development.

UPF, such as soft drinks, instant meals and confectionery, are produced
by various processing techniques, and additives are used to produce
durable, appetizing, ready-to-eat packaged foods \textsuperscript{8}.
UPF differ in composition compared to less processed foods as they are
generally high in energy and contain higher levels of sodium, sugars and
saturated fat \textsuperscript{9, 10}. Foods rich in simple
carbohydrates and saturated fatty acids are typically associated with a
negative impact on overall health and promote
obesity\textsuperscript{11}. Indeed, several studies have shown a
negative effect of high UPF consumption on multiple health domains,
including an increased risk of obesity, cardiovascular disease and
certain type of cancers \textsuperscript{12, 13}.

High consumption of UPF in the periconception period is associated with
impaired embryonic growth, increased gestational weight gain and higher
neonatal body fat \textsuperscript{14, 15}. Yet research on associations
between maternal UPF consumption and obstetric outcomes are scarce and
the underlying mechanisms are not fully understood. As the placenta
forms the crucial link between the mother and the growing conceptus and
is essential for the delivery of oxygen, energy and
nutrients\textsuperscript{16, 17}, we hypothesize high UPF consumption
impairs the utero-placental vascular development with consequences for
pregnancy outcomes and prenatal growth\textsuperscript{18}.

In the present study, our primary aim is to investigate whether
periconceptional maternal consumption of UPF impairs first-trimester
utero-placental vascular development using recently developed imaging
markers \textsuperscript{19, 20}. We further explore whether the
associations can be substantiated by macronutrient intake and the
adherence to specific dietary patterns.

\section*{METHODS}

{\label{methods}}

\subsection*{\texorpdfstring{\emph{2.1 Study
design}}{2.1 Study design}}

{\label{study-design}}

The VIRTUAL Placenta cohort was embedded in the ongoing prospective
Rotterdam Periconception Cohort \textsuperscript{21, 22}. Between
January 2017 and March 2018, women who were at least 18 years old,
carried a singleton pregnancy \textless{}10 weeks gestational age (GA),
and gave written informed consent were recruited from an academic
hospital. Both naturally conceived pregnancies and pregnancies achieved
via in vitro fertilization (IVF) with or without intracytoplasmic sperm
injection (ICSI) were eligible for inclusion. Pregnancies achieved via
oocyte donation and miscarriages were excluded from analyses. At
enrolment, participants filled out a questionnaire on general
characteristics, medical and obstetrical history and lifestyle
behaviours, and a Food Frequency Questionnaire (FFQ).

For all participants, two or more study visits were scheduled in the
first trimester at 7, 9 and 11 weeks GA during which 3D PD transvaginal
ultrasound scans of the whole gestational sac including the placenta and
utero-placental vasculature were obtained using the GE Voluson E8 (GE,
Zipf, Austria). Standardized ultrasound settings were previously
described (quality: max; pulse repetition frequency (PRF): 0.6; wall
motion filter (WMF): low1; compound resolution imaging (CRI): off; power
Doppler (PD) gain: -8.0) \textsuperscript{19}. Ultrasound examinations
were performed according to international guidelines on safe use of
Doppler ultrasound in the first trimester of pregnancy
(ALARA-principle)\textsuperscript{23}.

At the first study visit, height and weight were measured according to
protocol to calculate the body-mass index (BMI). Pregnancy outcomes were
collected through a questionnaire filled out by the participant within 1
month after giving birth and complemented with medical delivery records.

\subsection*{\texorpdfstring{\emph{2.2 Pregnancy
dating}}{2.2 Pregnancy dating}}

{\label{pregnancy-dating}}

For naturally conceived pregnancies in regular cycles (25-35 days), GA
was calculated from the first day of last menstrual period (LMP). In
case of unknown LMP or irregular cycle, GA was calculated from
Crown-Rump-Length (CRL). If the two methods varied \textgreater{}6 days,
the CRL-based GA was assumed the true GA. For fresh IVF/ICSI
pregnancies, GA was calculated from oocyte pick-up day +14 days. In case
of cryopreserved embryo transfer, GA was calculated from transfer date
+19 days.

\subsection*{\texorpdfstring{\emph{2.3 Periconceptional maternal dietary
intake}}{2.3 Periconceptional maternal dietary intake}}

{\label{periconceptional-maternal-dietary-intake}}

We used a standardized semi-quantitative food frequency questionnaire
(FFQ) validated for women in the reproductive age \textsuperscript{24}.
The FFQ consists of 191 food and beverage items and collects detailed
information about dietary intake, the frequency of consumption, portion
size and method of preparation over the previous four weeks. Energy and
nutritional intake of each food item was determined with the Dutch food
composition table by Wageningen University.

First, we extracted total daily energy intake (kcal/day) from the FFQ.
Using the Goldberg cut-off, designed for an average population as
described by Black \textsuperscript{25}, participants reporting an
unrealistically low value of energy-intake were excluded from analysis.

Next, we calculated the percentage energy intake (PEI) of each food
item. Then, using the NOVA classification, each food item in the FFQ was
categorized as `unprocessed or minimally processed food', `processed
culinary ingredient', `processed food' or `ultra-processed
food'\textsuperscript{9}. The classification of all items was performed
by three researchers independently. In case of discrepancies, items were
discussed with a nutritional epidemiologist until consensus was reached.
Hereafter, we calculated the percentage of energy intake from
ultra-processed food consumption (PEI-UPF, \%) for each participant.

To assess the intake of macronutrients, we used the FFQ to calculate the
total daily intake of carbohydrates, proteins and fats (g/day). In
addition, we calculated the total daily intake of macronutrient
compounds, for which we distinguished between mono-/disaccharides and
polysaccharides, animal proteins and plant-based proteins, and saturated
fatty acids and unsaturated fatty acids (g/day).

To identify distinct dietary patterns, we first reduced all 191 food
items into 25 food groups based on similarities in origin and nutrient
content, which we adapted from the European Prospective Investigation
into Cancer and Nutrition (EPIC) project \textsuperscript{26}, see Table
S1. All food groups were entered in a principal component analysis (PCA)
to identify dietary patterns (principal components) based on the degree
of reciprocal correlation between specific food groups. We extracted
dietary patterns with eigenvalues \textgreater{}1.0 and used a scree
plot to only select dietary patterns that explain a large proportion of
the variance in the food groups and exclude the residual
components\textsuperscript{27}. We provided a nutritional summary per
dietary pattern. The PCA automatically calculated a factor loading for
each food group, showing the extent to which that specific food group is
correlated with each dietary pattern. Finally, participants received a
factor score representing their adherence to each dietary pattern.

\subsection*{\texorpdfstring{\emph{2.4 Imaging markers of first-trimester
utero-placental vascular
development}}{2.4 Imaging markers of first-trimester utero-placental vascular development}}

{\label{imaging-markers-of-first-trimester-utero-placental-vascular-development}}

Image quality was scored on a four-point scale ranging between zero
(optimal) and three (unusable) based on the presence of artefacts, the
ability to distinguish between myometrium and trophoblastic tissue, and
completeness of the placenta. Images with a quality score of three were
excluded from the analyses.

The placental volume (PV) was measured using VOCAL software according to
the previously published validation study \textsuperscript{28}. In
short, the placental outline and gestational sac contours were
repeatedly traced in rotational steps of 15 degrees to calculate total
pregnancy volume and gestational sac volume respectively. The
gestational sac volume was subtracted from the total pregnancy volume to
calculate PV (cm\textsuperscript{3}) \textsuperscript{28}.

The utero-placental vascular volume (uPVV) was measured using a virtual
reality (VR) desktop system with the V-Scope volume rendering
application. First, the threshold for 8-bit Doppler magnitude data was
set at a value of 100 and PD artefacts were removed with a virtual
eraser. Then, VR segmentation was used to erase the Doppler signal in
the embryo, the umbilical cord and the uterine tissue surrounding the
placenta (Figure S1A-B). The V-Scope application automatically
calculated the volume of all remaining PD voxels to measure the uPVV
(cm\textsuperscript{3}), a volumetric vascular characteristic, as
published previously \textsuperscript{19} (Figure S1C).

The utero-placental vascular skeleton (uPVS) was generated by applying a
skeletonization algorithm to the uPVV segmentations\textsuperscript{20}.
The skeletonization algorithm repeatedly peels off the outermost layer
of voxels from the uPVV, reducing the diameter of the PD signal at each
point in the vascular network until one central voxel remains, thereby
creating a network-like structure representing the vascular morphology
(Figure S1D) \textsuperscript{(18)}. Following the construction of the
network, the skeletonization algorithm classifies each 26-connected
voxel based on the number of neighbouring voxels as endpoint (n) (1
neighbour), bifurcation point (n) (3 neighbours), crossing point (n) (4
neighbours) or as normal vessel point (n) (2 neighbours). Voxels with
\textgreater{}4 neighbours are considered an anomaly and excluded from
analyses. Further, the algorithm measures total network length and
average vascular thickness (mm) (Figure S1E). The 6 uPVS characteristics
represent absolute morphologic development of the first-trimester
utero-placental vasculature. Also, we calculated ratios of the uPVS
end-, bifurcation- and crossing points to the uPVV
(n/cm\textsuperscript{3}) to identify 3 imaging markers to represent the
density of vascular branching in the utero-placental vascular volume.
Women who had no PV, uPVV or uPVS measurement available were excluded
from analysis.

\subsection*{\texorpdfstring{\emph{2.5 Statistical
analysis}}{2.5 Statistical analysis}}

{\label{statistical-analysis}}

Baseline characteristics were presented as mean with standard deviation.
If needed, non-volumetric parameters were transformed using a square
root transformation to approximate a normal distribution. For volumetric
parameters and ratios a cubic root and natural log transformation were
used, respectively.

We used linear mixed models to estimate the association between maternal
intake of PEI-UPF, total energy, macronutrients and their compounds and
dietary patterns, and imaging markers of utero-placental vascular
development, assessed with PV, uPVV and uPVS morphologic and density
characteristics. We constructed three different models to explore the
potential effects of confounding: model 1 (adjusted for gestational age
only); model 2 (model 1 additionally adjusted for maternal age, BMI,
parity, conception mode, foetal sex and periconceptional alcohol
consumption, smoking and folic acid supplement use); and model 3 (model
2 additionally adjusted for total energy intake). Possible confounders
were selected based on literature and discussion amongst authors using a
directed acyclic graph.

All analyses were performed using SPSS (version 25.0; SPSS Inc.,
Chicago, IL, USA) and R (version 4.2.2, R Core Team, Vienna, Austria,
2022). P-values \textless{}0.05 were considered statistically
significant.

\section*{RESULTS}

{\label{results}}

\subsection*{\texorpdfstring{\emph{3.1 Study
population}}{3.1 Study population}}

{\label{study-population}}

The flowchart of participant selection is depicted in Figure S2. A total
of 93 women were included in the analyses. Table S2 shows participant
characteristics at baseline and periconceptional maternal dietary intake
of total energy, PEI-UPF, macronutrients and its compounds.

\subsection*{\texorpdfstring{\emph{3.2 Ultra-processed foods and total
energy}}{3.2 Ultra-processed foods and total energy}}

{\label{ultra-processed-foods-and-total-energy}}

First, we investigated associations between periconceptional maternal
intake of UPF and first-trimester imaging markers of utero-placental
vascular development. All models showed higher PEI-UPF is associated
with increased density of vascular branching for bifurcation points and
crossing points in the uPVS but not with absolute morphologic
development, see Table 2. Model 3 shows a statistically significant
association between PEI-UPF and density of bifurcation points
{[}\selectlanguage{greek}β\selectlanguage{english}=0.465, 95\%CI=0.148;0.782, p-value=0.006{]}.

Next, we investigated associations between the periconceptional maternal
intake of total energy (kcal/day) and first-trimester imaging markers of
utero-placental vascular development. Total energy intake was negatively
associated with density of bifurcation points in the uPVS {[}\selectlanguage{greek}β\selectlanguage{english}=-0.053,
95\%CI=-0.101;-0.004, p-value=0.039{]}, see Table S3. We found no
associations with the imaging markers of absolute morphologic
utero-placental vascular development.

\subsection*{\texorpdfstring{\emph{3.3
Macronutrients}}{3.3 Macronutrients}}

{\label{macronutrients}}

We investigated the intake of separate macronutrients and observed
positive associations between the intake of carbohydrates (g/day) and
first-trimester development of uPVV {[}\selectlanguage{greek}β\selectlanguage{english}=0.017, 95\%CI=0.001;0.032{]}
and imaging markers of absolute morphologic development: uPVS end points
{[}\selectlanguage{greek}β\selectlanguage{english}=0.286, 95\%CI=0.062;0.511{]}, bifurcation points {[}\selectlanguage{greek}β\selectlanguage{english}=0.004,
95\%CI=0.003;0.006{]}, vessel points {[}\selectlanguage{greek}β\selectlanguage{english}=0.772, 95\%CI=0.137;1.408{]}
and total length {[}\selectlanguage{greek}β\selectlanguage{english}=0.700, 95\%CI=0.106;1.295{]}, all
p-values\textless{}0.05, see Table 3 (only model 3 is shown). No
associations were found between carbohydrate intake and imaging markers
representing density of vascular branching. Further, we observed
positive associations between the periconceptional maternal intake of
mono-/disaccharides and first-trimester development of uPVV {[}\selectlanguage{greek}β\selectlanguage{english}=0.021,
95\%CI=0.003;0.039{]} and imaging markers of absolute morphologic
development: uPVS end points {[}\selectlanguage{greek}β\selectlanguage{english}=0.336, 95\%CI=0.072;0.603{]},
bifurcation points {[}\selectlanguage{greek}β\selectlanguage{english}=0.380, 95\%CI=0.057;0.703{]}, crossing points
{[}\selectlanguage{greek}β\selectlanguage{english}=0.257, 95\%CI=0.011;0.502{]}, vessel points {[}\selectlanguage{greek}β\selectlanguage{english}=0.916,
95\%CI=0.170;1.662{]} and total length {[}\selectlanguage{greek}β\selectlanguage{english}=0.856,
95\%CI=0.158;1.554{]}, all p-values\textless{}0.05, but not for the
intake of polysaccharides, see Table 3. We found no associations between
carbohydrate intake and imaging markers representing density of vascular
branching.

These analyses were repeated for the total intake of fats and proteins
and their respective compounds, see Table S4 (only model 3 shown). We
observed no associations between the periconceptional maternal intake of
fats, proteins and their compounds and first-trimester imaging markers
of utero-placental vascular development in any of our models.

\subsection*{\texorpdfstring{\emph{3.4 Dietary
patterns}}{3.4 Dietary patterns}}

{\label{dietary-patterns}}

Using the PCA analysis we identified three distinct dietary patterns.
The first dietary pattern is associated with higher intake of fresh and
processed meats, cereal products, potatoes, eggs, cakes and sauces, and
is therefore referred to as a Western dietary pattern, which explains
16.2\% of the variance. The second dietary pattern is associated with
higher intake of vegetables, fruits, fish and shellfish, nuts and seeds,
vegetable oils and soy- and other plant-based meat-/dairy substitutes.
Accordingly, this dietary pattern is referred to as a Mediterranean
dietary pattern, which explains 11.7\% of the variance. The third
dietary pattern is associated with higher intake of sugar and
confectionary, savoury snacks, milk and dairy products, soft drinks,
fruit/vegetable juices and butter. We refer to this last dietary pattern
as a Snack dietary pattern, which explains 8.4\% of the variance. A
nutritional overview of the dietary patterns is depicted in Table S5.

We investigated associations between periconceptional maternal adherence
to the three dietary patterns and imaging markers of utero-placental
vascular development. There are no associations between adherence to the
Western or Mediterranean dietary patterns and imaging markers of
utero-placental vascular development in all three models, see Table 4.
Model 2 shows (borderline-) statistically significant positive
associations between adherence to the Snack dietary pattern and
first-trimester development of uPVV and imaging markers of absolute
morphologic development: uPVS end points, bifurcation points, crossing
points, vessel points and total length. Model 3 shows similar effect
estimates with wide confidence intervals and no statistically
significant associations, see Table 4.

\section*{DISCUSSION}

{\label{discussion}}

\subsection*{\texorpdfstring{\emph{4.1 Main
findings}}{4.1 Main findings}}

{\label{main-findings}}

This study shows a 10\%/day higher maternal UPF intake is positively
associated with first-trimester density of vascular branching
(bifurcation points: \selectlanguage{greek}\emph{β} \selectlanguage{english}= 0.465 [?]n, \emph{p} = 0.006). The intake
of carbohydrates, in particular of mono-and di-saccharides, is
associated with increased absolute morphologic development of the
first-trimester utero-placental vasculature, but not with density of
vascular branching. We established positive associations between
adherence to a Snack dietary pattern and absolute morphologic
development of the utero-placental vasculature in model 2, but no clear
associations in model 3. A graphic summary of the results is depicted in
Figure 4.

\subsection*{\texorpdfstring{\emph{4.2 Strengths and
Limitations}}{4.2 Strengths and Limitations}}

{\label{strengths-and-limitations}}

Strengths of our study are the use of validated FFQ's to study
periconceptional nutritional intake, application of the validated
Goldberg cut-off to exclude cases with unreliable nutritional intake and
the use of EPIC project-based food groups to categorize food items for
the identification of the dietary patterns \textsuperscript{24-26}.

The prospective observational study design allowed us to collect a
comprehensive set of patient characteristics, which we used as
covariates in advanced statistical models to minimize the effect of
confounding bias. Although we cannot exclude the possibility of residual
bias from any unknown factors, the direction of the effect estimates is
similar in all three models. Therefore we consider it unlikely our
findings result from residual confounding.

We used a unique longitudinal data collection of power Doppler
ultrasounds in the first trimester of pregnancy to perform validated
uPVV measurements (ICC above 0.80 and relative differences of less than
20\%) \textsuperscript{19} and subsequently generate the uPVS, our
imaging markers of utero-placental vascular
development\textsuperscript{20}.

Recruitment from an academic hospital might affect the generalizability
of our results. Although our study population contains a relatively high
number of IVF-ICSI pregnancies and participants have a higher risk of
developing pregnancy complications \textsuperscript{21}, we believe it
is unlikely the direction of the associations will be different in the
general population.

We performed multiple statistical analysis, which raises some concern
about multiple testing. However, previously uPVV and uPVS
characteristics were highly correlated and are inversely correlated with
the density of vascular branching\textsuperscript{20}. Therefore, the
positive correlations between the effect estimates of uPVV and uPVS
characteristics and the inverse correlation with density of vascular
branching found in the present study can be interpreted as internal
validation.

\subsection*{\texorpdfstring{\emph{4.3 Interpretation (in light of other
evidence)}}{4.3 Interpretation (in light of other evidence)}}

{\label{interpretation-in-light-of-other-evidence}}

\subsubsection*{\texorpdfstring{\emph{4.3.1 Ultra-processed
foods}}{4.3.1 Ultra-processed foods}}

{\label{ultra-processed-foods}}

So far, no studies have investigated associations between the
consumption of UPF and placental development. High UPF intake is
associated with increased total energy intake, an overall unhealthy
dietary pattern and an increased risk of obesity \textsuperscript{15,
29-31}. UPF intake is negatively associated with embryonic growth and
most studies on the effect of UPF-rich diets on birthweight suggests
high UPF intake is associated with lower birth
weight\textsuperscript{14, 32-35}. In the present study, UPF is
positively associated with first-trimester density of vascular
branching, which has previously been associated with decreased embryonic
and foetal growth and lower birth weight percentiles\textsuperscript{36,
37}. Associations with UPF persist in model 3, which implies our
findings are not solely mediated by higher energy content in UPF. We
propose a high UPF exposure induces oxidative stress resulting in
aberrant first-trimester vascular development and a compensatory
increased density of vascular branching. This way the first-trimester
utero-placental vascular development might act as a mediator in the
relation between periconceptional maternal intake and pregnancy
outcomes. This mechanism is substantiated by previous research on
associations between UPF intake, oxidative stress, first-trimester
placental development and prenatal growth\textsuperscript{21, 22,
38-42}.

\subsubsection*{\texorpdfstring{\emph{4.3.2
Carbohydrates}}{4.3.2 Carbohydrates}}

{\label{carbohydrates}}

In our study, periconceptional maternal carbohydrate intake, more
specifically of mono-and disaccharides, is positively associated with
volumetric and absolute morphologic utero-placental vascular development
in the first trimester. Carbohydrates seem to affect a different aspect
of the utero-placental vascular development than UPF, which likely
results from the high heterogeneity of the type and amounts of
carbohydrates in UPF-rich foods.

During the first trimester, the placental microenvironment is
hypoxic\textsuperscript{43}. Rather than on oxidative phosphorylation,
the placenta relies on glycolysis for energy production, supported by
the rich supply of glucose from the endometrial glands
\textsuperscript{44}. With increased carbohydrate intake maternal serum
glucose concentration rises and subsequently the expression of glucose
transporter 1 (GLUT1) increases. GLUT1 is an important cargo molecule of
extracellular vesicles that augment decidualization, stimulate
angiogenesis, and modulate trophoblast differentiation in the
endometrial stroma\textsuperscript{45}. The physiological interplay
between serum glucose and placental development is substantiated by
previous research indicating an insufficient glucose metabolism during
gestation results in placental aberrations and foetal growth restriction
(FGR)\textsuperscript{46-50}.

\subsubsection*{\texorpdfstring{\emph{4.3.3 Fats and
Proteins}}{4.3.3 Fats and Proteins}}

{\label{fats-and-proteins}}

Multiple studies suggest fatty acids to play a regulatory role in the
angiogenesis in tumours and in various organs, including the
placenta\textsuperscript{51-55}. The contradictory pro- and
antiangiogenic effects of individual PUFAs might explain why we did not
find any associations between the periconceptional intake of fatty acids
and utero-placental vascular development \textsuperscript{51, 52}.

Not much is known about dietary intake of proteins and placental
development. Recent studies suggest low-protein diet is associated with
placental morphologic disruption and foetal growth restriction and found
maternal protein intake positively associated with prenatal
growth\textsuperscript{56, 57}. Our findings suggest it is unlikely
associations between protein intake and prenatal growth are mediated by
utero-placental vascular development.

\subsubsection*{\texorpdfstring{\emph{4.3.5 Dietary
patterns}}{4.3.5 Dietary patterns}}

{\label{dietary-patterns-1}}

The Snack dietary pattern in this study is characterized by excessive
consumption of food items with high carbohydrate content. This might
explain why we found a (borderline-) statistically significant positive
associations between adherence to the high snack pattern and
first-trimester density of vascular branching in model 2 similar to the
associations with carbohydrates. However, the snack dietary pattern also
includes food items with high energetic value but relatively low
carbohydrate content, like butter. The heterogeneity in the Snack
dietary pattern and the small sample size might explain why the positive
associations in model 3 were not statistically significant\emph{.}

\subsection*{\texorpdfstring{\emph{4.4
Implications}}{4.4 Implications}}

{\label{implications}}

The intake of UPF seems to impair first-trimester utero-placental
vascular development. These findings are not substantiated by
associations with related macronutrients and dietary patterns as
increased maternal intake of carbohydrates, within healthy quantities,
seems to promote first-trimester utero-placental vascular development.
Associations between UPF intake and placental vascular development could
reflect the effects of `ultra-processing' techniques in the food
industry, but likely result from a coinciding lack of micronutrients in
these food items.\textsuperscript{58, 59} UPF-rich diets are associated
with deficiencies in folate and vitamin B12, important micronutrients
involved in the 1-carbon metabolism. Derangements in the 1-carbon
metabolism are associated with hyperhomocysteinemia and oxidative
stress, which causes aberrant development of the utero-placental
vasculature, associated with placenta-related
complications.\textsuperscript{60}

\section*{CONCLUSION}

{\label{conclusion}}

This study demonstrates associations between periconceptional maternal
intake of UPF, carbohydrates and utero-placental vascular development in
the first trimester. Interestingly, the periconceptional maternal intake
of UPF and carbohydrates and the adherence to the Snack dietary pattern
are associated with different aspects of first-trimester development of
the utero-placental vasculature. Associations between UPF intake and
placental vascular development likely result from a coinciding lack of
nutrients in these food items, which induces oxidative stress and causes
aberrant development of the utero-placental vasculature. This study's
findings substantiate the need of healthy diets and nutritional and
lifestyle coaching in periconception and obstetric care. Future research
should focus on the impact of periconceptional dietary interventions on
first-trimester utero-placental vascular development.

ACKNOWLEDGEMENTS

We thank dr. Sten Willemsen for his advisory involvement in the
statistical analysis.

DISCLOSURE OF INTERESTS:

None.

CONTRIBUTION TO AUTHORSHIP:

E.d.V., A.M., A.K., H.S., R.S.T and L.v.R. were involved with
conceptualization and the study design. E.d.V. performed statistical
analysis and wrote the first draft of the manuscript. A.M., A.K.,
R.S.T., and L.v.R. were responsible for reviewing and editing of the
manuscript. L.v.R. and R.S.T. had primary responsibility for final
content. All authors have read and approved the final manuscript.

DETAILS OF ETHICS APPROVAL:

This study was conducted in accordance with the ethical principles for
medical research set out in the Declaration of Helsinki and was approved
by the Institutional Review Board of the Erasmus Medical Centre on 2
June 2015 (MEC 2015-494).

FUNDING:

This research was funded by the Department of Obstetrics and Gynaecology
of the Erasmus MC, University Medical Centre Rotterdam, Rotterdam, The
Netherlands.

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