(2024) 22:295
Moreno et al. BMC Medicine
https://doi.org/10.1186/s12916-024-03495-9

BMC Medicine

RESEARCH

Open AccessFoetal gluten immunogenic peptides
during pregnancy: a new determinanton the coeliac exposome
María de Lourdes Moreno1, María González‑Rovira1, Cristina Martínez‑Pancorbo2, María Martín‑Cameán3,
Ana María Nájar‑Moyano1, Mercedes Romero2, Esther de la Hoz2, Cristina López‑Beltrán2,
Encarnación Mellado1, José Luis Bartha3, Petter Brodin4, Alfonso Rodríguez‑Herrera5,
José Antonio Sainz‑Bueno6,7 and Carolina Sousa1*

Abstract
Background The increasing incidence of coeliac disease is leading to a growing interest in active search for asso‑
ciated factors, even the intrauterine and early life. The exposome approach to disease encompasses a life course
perspective from conception onwards has recently been highlighted. Knowledge of early exposure to gluten immu‑
nogenic peptides (GIP) in utero could challenge the chronology of early prenatal tolerance or inflammation, rather
than after the infant’s solid diet after birth.
Methods We developed an accurate and specific immunoassay to detect GIP in amniotic fluid (AF) and studied theiraccumulates, excretion dynamics and foetal exposure resulting from AF swallowing. One hundred twenty-five preg‑
nant women with different gluten diets and gestational ages were recruited.
Results GIP were detectable in AF from at least the 16th gestational week in gluten-consuming women. Althoughno significant differences in GIP levels were observed during gestation, amniotic GIP late pregnancy was not alteredby maternal fasting, suggesting closed-loop entailing foetal swallowing of GIP-containing AF and subsequent excre‑
tion via the foetal kidneys.
Conclusions The study shows evidence, for the first time, of the foetal exposure to gluten immunogenic peptides
and establishes a positive correlation with maternal gluten intake. The results obtained point to a novel physiologicalconcept as they describe a plausible closed-loop circuit entailing foetal swallowing of GIP contained in AF and its sub‑sequent excretion through the foetal kidneys. The study adds important new information to understanding the coe‑liac exposome.
Keywords Gluten immunogenic peptides, Coeliac disease, Gluten, Exposome*Correspondence:
Carolina Sousa
csoumar@us.es
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Moreno et al. BMC Medicine(2024) 22:295

BackgroundThe notable increase in the incidence of gastrointestinal(GI) pathologies has led to the search for a deeper understanding of the role of the GI immune system. There has
been interest in the ontogeny, early growth and development of the intestine during the prenatal stages becauseof its potential to play a role in gut physiology or pathological challenges in later life [1–3]. Ontogenesis of theimmune system begins as early as 3 weeks after conception and continues after birth and childhood [4]. Studies
based on intrauterine transplantation placed the period
for first immune function development at 20–24 weeks
of gestation [5]. The small intestine is largely mature inutero by the end of the first trimester; absorptive function
is partially detectable at 26 weeks [1]. Adaptive immune
memory in the foetal intestine is particularly abundant in
T cells in mucosal tissues. However, it is known to hosta variety of innate immune cells that exhibit memorylike properties (innate lymphoid cells, tissue-resident
memory T cells, natural killer or gamma/delta T cells)[6–11]. Human immune systems after birth and in particularly in adulthood vary mostly as a consequence ofenvironmental exposures [12]. When most of this environmental imprinting occurs is not clear and likely varies for different types of responses. After birth a dynamic
period begins, triggered by environmental exposures,and largely shaped by microbial interactions with specificmicrobes exerting specific influences and tilting the balance between tolerance and resistance [13, 14].
Amniotic fluid (AF) is a unique liquid that surrounds
the foetus throughout gestation and plays essential rolein foetal development and maturation [15–18]. The AF
provides nutrients and other factors required for foetal
growth, as well as a mechanical cushioning and immunological barrier against antigens [19]. During early development, AF is an extension of the foetal extracellularmatrix. As the placenta and foetal vessels emerge, waterand solutes from the maternal plasma diffuse into the AF.
By 8 weeks of gestation, the urethra is formed; the foetal
kidneys begin producing urine. Shortly thereafter, foetal
swallowing begins. However, these processes do not contribute to AF volume until the second half of pregnancy.
After 25 weeks of gestation, the foetal skin is fully keratinised and can no longer absorb or transfer fluids. Respiration, swallowing and urination are the main routes of
exchange between the foetus and AF to maintain homeostasis and volume [20–22].
The prenatal environments, such as maternal allergens or microbial exposure enhanced by foetal swallowing activity, contribute to both immunity at birthand immune maturation and antigen exposure even in
the foetal GI mucosa [4, 23–26]. Studies conducted on
AF in relation to allergens have been based on the use

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of antibody arrays [27]; but to date, no study has been
undertaken using specific monoclonal antibodies todemonstrate the immunogenicity of these allergens.
Within the group of immune-mediated GI pathologies, coeliac disease (CD) is an immune-mediatedchronic small bowel disorder triggered in genetically
predisposed individuals after gluten exposure. CD is asystemic disease characterised by intestinal and extraintestinal symptoms that can present individually orin combination. This pathology involves key immune
factors including human leukocyte antigens (HLA)
DQ2 and DQ8 and anti-tissue transglutaminase (tTG)
antibodies. Following CD diagnosis, patients must follow a strict, life-long gluten-free diet (GFD), the only
treatment currently available; this not only reduces disease symptoms but also allows for healing of the intestinal epithelium and prevents long-term complications[28–30].
Gluten is a complex mixture of prolamins with highproline and glutamine content making them resistant toenzyme-mediated hydrolysis [31]. Many gluten proteins
are not fully degraded in the gastrointestinal tract. Therefore, some gluten peptides can enter the intestinal epithelium, perpetuating intestinal inflammation in the context
of gluten-related pathologies [32]. Thus, most immune
responses against gluten are mediated by a group of gluten epitopes [33]. The α-gliadin 33-mer peptide has been
described as one of the most immunodominant gluten
peptides, harbouring several T cell epitopes [34]. The
anti-α-gliadin 33-mer antibodies A1 and G12 can specifically and sensitively detect excreted gluten immunogenic
peptides (GIP) in stool and urine highlighting the peptides’ resistance to digestion and their role in activating
T cells in patients with CD [35–37]. Lateral flow immunoassay (LFIA) tests in urine provide qualitative data;
however, quantitative results can also be obtained using
LFIA coupled with a lateral flow reader showing a limit ofdetection (LDT) of 2.25 ng GIP/mL in urine with a sensitivity and specificity of 97% and 100%, respectively [37].
In this study, we have overcome technical challenges todevelop for the first time an accurate and specific methodology to detect the presence of GIP in AF from at least
the 16th gestational week using LFIA. Exposure to these
immunogenic peptides can occur before birth. We werealso able to distinguish prenatal levels of gluten peptide
exposure in foetuses of non-CD pregnant women compared to CD pregnant women; we established a positive
correlation with the maternal gluten intake. We foundthat there is a gluten balance in the AF during late pregnancy that is not altered by maternal fasting and consider
plausible that there is a closed-loop of foetal ingestionof GIP containing AF and subsequent excretion via the
foetal kidneys after maternal gluten consumption. This

Moreno et al. BMC Medicine(2024) 22:295

circuit could influence the amount of GIP to which the
foetal immune system is exposed during gestation.

Methods
Patient and public involvement

The patients and/or the public were not involved in thedesign, conduct, reporting or dissemination of this study.
Study design and patients

The present study is a proof-of-concept and subsequent
implementation of basic research on physiological trials that include samples from two cohorts: cohort ofsamples from a biobank and newly recruited pregnant
women. Ten biobank AF samples (five pregnant women
without CD and five pregnant women with confirmedCD) were obtained from the La Paz University Hospital(Spanish IdiPAZ, Madrid, Spain). The newly recruitedcohort included 125 pregnant women (124 non-CD pregnant women and one CD-confirmed pregnant woman)from two hospitals, Virgen de Valme University Hospital(Seville, Spain) and Quirónsalud Sagrado Corazón Hospital (Seville, Spain), between November 2015 and October 2020 (Fig. 1).
This study was conducted in accordance with the principles of the Declaration of Helsinki. The study protocol
was reviewed and approved by the research ethics committee (REC) of each institution involved (ID/Numbers:
Celifluid and PI-2611). Written informed consent was
obtained from all the participants. The participants consented to IdiPAZ for the use of their data and/or samples
for health-related research purposes.
The 10 AF samples collected during pregnancy screening were obtained from the Spanish IdiPAZ biobank.
The maternal age ranged from 18 to 44 years. The entire
biobank cohort had available primary care and specialty data. The inclusion criteria for the biobank cohort
included pregnant women underwent amniocentesis
in the second trimester (15–25 weeks): (1) five non-CD
pregnant women on a gluten-containing diet (GCD) and(2) five CD pregnant women on a gluten-free diet (GFD)
for ≥ 24 months before the time of sampling. Women
with CD were diagnosed by the detection of positiveanti-endomysium and tTG antibodies in the serum, confirmed by a small intestinal biopsy and cleared of digestive symptoms suggestive of CD.
The inclusion criteria for the newly recruited cohortof pregnant women were as follows: (1) second trimester non-CD pregnant women on a GCD who underwent
amniocentesis (16–24 weeks), indicated for clinical reasons and not for the purpose of this study; (2) a cohort ofterm non-CD pregnant women who gave birth with GCD
and were classified according to their mode of delivery(caesarean section or vaginal delivery) and (3) a full-term

Page 3 of 14

pregnant woman with GFD who was diagnosed with CD
by the detection of positive anti-endomysium and tTG
antibodies in the serum and confirmed by a small intestinal biopsy. She had consumed a GFD for ≥ 24 months
before the time of inclusion in the study.
The exclusion criteria for all pregnant women included
the presence of known severe medical conditions and theuse of prescription medications and antibiotics 2 months
prior to inclusion in the study.
We recruited AF samples from two different stagesof pregnancy: (1) amniocentesis from weeks 15 to 25 ofpregnancy. AF collection before week 14th may lead tocomplications and miscarriage; (2) at term delivery (after
37 weeks). The newly enrolled pregnant women included
92 at term deliveries, of which 50 had vaginal deliveries(28 eutocic and 22 instrument-assisted) while 42 underwent caesarean sections (32 elective and 10 emergency
C-sections). The amniocentesis samples included 33 prenatal diagnostic amniocentesis samples. The amniocentesis cohort included 14 pregnancies with normal foetuses
and 19 pregnancies with foetal anomalies (Table S1).
Amniotic fluid and urine collection

AF samples were collected in a sterile fashion under careful conditions to avoid blood contamination and storedat − 20 °C until analysis. The collection of AF samples
was conducted as follows: (1) at vaginal delivery, after
cervical opening and amniotomy by lancet; (2) during elective (planned, with no ongoing labour) or acute(labour already started) caesarean delivery, at the time of
caesarean section by aspirating through intact amniotic
membranes using a sterile probe and 20-mL syringe; and(3) during amniocentesis in the second trimester after
abnormal findings on foetal ultrasound or biochemicalmarkers, by inserting a needle through the abdomen into
the utero to remove a sterile 5-mL AF sample. Additionally, all pregnant participants were instructed to collect
50–100 mL of midstream urine samples in a sealed sterile
container. This sample was taken at the time of AF collection and stored at − 20 °C until the time of processing.
Total protein quantitation

Frozen AF samples were analysed for total protein in
duplicate batches using the Pierce BCA Protein Assay
Kit (Thermo Fisher Scientific, Madrid, Spain), as recommended by the manufacturer. Bovine serum albumin,
ranging from 25 to 2 mg/mL, was used as a standard. A
96-microplate well was placed in a shaker for 30 s andincubated at 37 °C for 30 min. The absorbance of eachsample was measured at 562 nm using a plate reader(UVM340; Asys Hitech GmbH, Eugendorf, Austria).

Moreno et al. BMC Medicine(2024) 22:295

Fig. 1 Flowchart of study design and pregnancy outcome. AF, amniotic fluid; GIP, gluten immunogenic peptides

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Gluten peptide concentration and conditioning

The AF samples were centrifuged at 4550 g for 5 min. The
supernatant was mixed with three volumes of ice-coldacetone for 2 h, followed by centrifugation at 11,000 g for
10 min. The supernatant was removed, and the protein
pellets were resuspended in 1% trifluoroacetic acid (TFA)
and concentrated by solid-phase extraction (SPE) withpreconditioned cartridges. For urine samples, 3 mL of a
mixture of 50% urine in TFA was centrifuged for 10 min
at 4550 × g. The resulting supernatant was concentrated
and cleaned using SPE [37].
The SampliQ C18 cartridges (Agilent Technologies,
Wilmington, DE, USA) were prepared according to the
manufacturer’s recommendations. The resulting supernatants from the AF and urine samples were applied tothe cartridge. The target compounds were eluted with 0.5
to 1 mL of phosphate-buffered saline for further use in
the immunochromatographic assays.
Immunochromatographic test for GIP detection

Lateral flow immunoassays were performed on AF
and urine samples to detect GIP. After SPE, 100 µL of
the blind concentrate was added to a G12 lateral flow
immunochromatographic (LFI) strip for 30 min. If gluten peptides are present, they react with the conjugatedantibodies previously fixed on the LFI strip, thus producing a red line in the resulting window. A control Abantigen reaction was performed to indicate correct test
performance (green line). To quantify the signal, the LFIstrip was introduced into the cassette of an LFI reader(iVYCHECK®Reader, Biomedal S.L., Seville, Spain) andirradiated with light; the reflection was subsequently
measured. The limit of quantification (LOQ) of the technique was established as 6.25 ng GIP/mL. The limit ofdetection (LDT) was 2.25 ng GIP/mL.
Statistical analysis

The sample size required for each experiment was calculated. The minimum expected difference was the LOQ(0.062) and the dropout rate was 0.2, with an alpha risk
of 5%, a beta risk of 20% (80% statistical power) and astandard deviation of 0.101. The calculation was madeusing the GRANMO v7.12 April 2012 (Institut Municipald’Investigació Mèdica, Barcelona, Spain).
All results are expressed as mean standard deviation(SD). Each human specimen was analysed in triplicate.
Positive controls and buffer blanks were used for eachassay. Statistical analyses were performed using GraphPad Prism 6 for Windows (GraphPad Software Inc., San
Diego, CA, USA). An unpaired two-tailed Student’s t-test
was used; statistical significance was set at p < 0.05.
The Mann–Whitney U test was used to compare
quantitative variables in independent groups, and thePage 5 of 14

Wilcoxon signed-rank test was used to compare quantitative variables in dependent groups. SAS version 9.4(Cary, NC, USA) was used for all statistical calculations.

Results
Detection of gluten immunogenic peptides in amniotic
fluid samples from biobank

To date, no studies have demonstrated the presenceof GIP in AF or whether its presence plays a role in the
immune response in the prenatal environment. In thisstudy, we attempted to determine whether maternal GIP
is absorbed from the diet, crosses the placental barrier,accumulates in the amniotic cavity and can be detectedin AF samples.
Based on our previous experience with the detection of
GIP in human samples, we have overcome to optimise amethodology specific and reliable in AF samples (Fig. 2).
Because it is known that the AF to be a dialysate identicalto foetal and maternal plasma, but with a lower protein
concentration, we decided to perform an extraction withacetone to improve GIP detection in samples and applieddifferent methodologies for subsequent peptide resuspension (see ‘Methods’).
Initially, as a proof-of-concept, 10 AF samples werecollected from the biobank of five pregnant women with
CD following a GFD and five non-CD pregnant women
on a GCD (Fig. 3). No visible signals in sticks (no GIP)
were found in the AF of pregnant women with CD following a GFD. However, all LFI strips from pregnant
women without CD under unrestricted gluten conditions were positive (> LDT); three of the five AF samples
could also be quantified ranging from 7.3 to 13.2 ng GIP/
mL AF (> LOQ). These results indicated that the signal
was dependent on gluten intake and revealed the existence of maternal-foetal gluten peptide exchange in the
AF (p = 0.008), which could potentially contain sequencesthat specifically stimulate T cells as the immunoassay
antibody has specific affinity for such sequences.
Concentration of gluten immunogenic peptides
in amniotic fluid and urine samples in different trimestersof gestation

Once the methodology developed for the detection of
GIP in AF samples was proven to work correctly, we
conducted an active new recruitment. AF and urine
samples from women at different periods of gestation and with different dietary habits were collected
to explore various aspects of the physiology of gluten
peptides and foetuses. We collected 130 AF samples
from 124 healthy pregnant women and one pregnant
woman with CD. Of these, 25 AF samples (five vaginal
deliveries, 17 caesarean sections and three amniocenteses) were discarded for blood contamination because

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Fig. 2 Workflow for gluten immunogenic peptides detection in AF samples. AF, amniotic fluid; GIP, gluten immunogenic peptides; IC,
immunochromatographic; LFT, lateral flow test; LFIA, lateral flow immunoassay; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid

Fig. 3 Detection and quantification of GIP content in AF samples from amniocentesis of coeliac and non-coeliac pregnant women according
to the gestational age. Presence of GIP visible and quantifiable in AF (> LOQ); visual presence of GIP not quantifiable in AF (> LDT, 2.25 ng GIP/
mL AF and < LOQ, 6.25 ng GIP/mL AF); absence of GIP in AF (< LDT). Differences in GIP levels in amniotic fluids between the 2 groups were tested
with a Mann–Whitney U test. LDT, the limit of technique detection; LOQ, the limit of quantification; AF, amniotic fluid; GIP, gluten immunogenic
peptides; GFD, gluten-free diet; GCD, gluten containing dietthey could falsify the results of the immunological
techniques for gluten detection. Sixty-nine AF samples(45 vaginal deliveries and 24 caesarean sections) fromterm deliveries (38–40 weeks) and 36 samples fromamniocentesis (16–24 weeks) were analysed. As shownin Fig. 4, GIP was detectable in 87.62% (n = 92) of allconcentrated AF samples (> LDT); however, only 12 ofthese samples could be quantified (> LOQ). We determined that the quantified GIP content ranged from6.25 to 25.5 ng GIP/mL and from 6.25 to 8.05 ng GIP/
mL, in deliveries at second and third trimesters, respectively. No significant differences of GIP concentrations
between mid and late trimester were found (p = 0.05).
We did not detect GIP in the AF sample from pregnant

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Fig. 4 Comparison of GIP content in AF samples of healthy pregnant women on diverse periods of pregnancy. Visual presence of GIP
not quantifiable in AF (> LDT and < QL); presence of GIP visible and quantifiable in AF (> QL); absence of GIP in AF (< LDT). AF from deliveries
in the third trimester (at term) (n = 69) and in the second trimester (amniocentesis) (n = 36). Two mothers with monochorionic diamniotic twins
were included. Coeliac mother is represented by a circle; non-coeliac mothers in second trimester are indicated by rhombuses; non-coeliac mothersin third trimester are indicated by triangles. Differences in GIP levels in amniotic fluids between the 2 groups were tested with a Mann–Whitney U
test. LDT, the limit of technique detection; LOQ, the limit of quantification; GIP, gluten immunogenic peptides; AF, amniotic fluid

woman with CD, which is similar to previous results
obtained with AF samples from the biobank.
To study and compare the concentration of gluten
ingested and subsequently excreted in the urine of pregnant women with respect to the gluten levels in AF, weanalysed GIP in the urine of 83 women (among the 94
recruited) at different gestational ages and compared
the results with those obtained from AF samples. As thiswas a non-interventional study, urine and AF measurements were performed once, and neither the amount of
fluid ingested, nor the feeding pattern of the pregnant
women was modified. As shown in Fig. 5, the spot measurement of GIP excreted in the urine samples was higher
in number of positive quantitative samples (> LOQ) thanAF samples, and significant differences were detectedin urine samples in both gestational stages in relation
to AF samples (p = 0.006). Of all the urine samples, 88%(77/83) were GIP-positive; 36.1% (30/83) could be quantified, ranging from 6.25 to 19.7 ng GIP/mL. In comparison, 78.3% (65/83) of AF samples were positive; ofthese, 13.3% (11/83) were quantified (ranged from 6.25
to 25.5 ng GIP/mL and from 6.25 to 8.05 ng GIP/mL, indeliveries at second and third trimesters, respectively).
The concordance between the punctual measurementof GIP content in the AF and urine samples of pregnant women is showed in Fig. 6. All urine samples fromwomen in whom GIP content in the AF was quantifiable(GIP + +) were positive for GIP. Furthermore, GIP was
detected in the urine in 85.5% of AF samples with positive
visual results, but not quantifiable GIP (GIP +). Samples
negative for GIP in AF corresponded mainly to samples
negative for GIP in urine or positive for visual GIP. In
contrast, we did not detect GIP in the AF sample from
CD woman, in accordance with previous results obtainedwith AF samples from the biobank in the absence of gluten intake. These results demonstrate the specificity ofthe technique and its association with ingested gluten, as
well as the high correlation between the absence of gluten in AF and adherence to a GFD in women with CD.
GIP flow dynamics in foetus and pregnant woman

Previous studies have shown that from week 12 onwards,
the foetus is also involved in the renewal of AF by providing urine, which is the main component in the following
weeks. To determine whether the amniotic cavity could
be a closed-loop circuit in relation to GIP, we selected
pregnant women with strict fasting, such as preoperative fasting in caesarean sections, and subsequently compared GIP excretion in elective and emergency caesarean
sections in healthy pregnant women. We compared GIPlevels in AF and urine samples from 15 women (among
the 94 recruited pregnant women) who underwent elective and emergency caesarean sections. Pregnant women
undergoing elective caesarean sections were subjectedMoreno et al. BMC Medicine(2024) 22:295

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Fig. 5 Amniotic and urinary gluten peptide content and gestational age. Visual presence of GIP not quantifiable (> LDT and < LOQ, scatter plot
in white area); absence of GIP (< LDT, scatter plot in grey area) and presence of GIP visible and quantifiable (> LOQ, box and whiskers). Differences
between groups were tested with a Wilcoxon signed-rank test in dependent groups and Mann–Whitney U test in independent groups. LDT,the limit of technique detection; LOQ, the limit of quantification; GIP, gluten immunogenic peptides; AF, amniotic fluid

to a strict fasting of at least 9 h; pregnant women who
underwent emergency caesarean section did not undergo
any consumption restriction. As shown in Fig. 7, rapid
and nearly complete urinary elimination of the remaining dietary gluten was observed in pregnant women
after fasting as compared with those who did not fast(p < 0.05). All urine samples from emergency caesarean
sections without fasting were positive (> LDT < LOQ),
unlike the 77.8% negative (< LDT) urine samples fromelective caesarean sections of pregnant women subjected
to fasting. In contrast, GIP content in AF in both situations was similar, unlike that in urine. These preliminary
data suggest that fasting is not sufficient to affect gluten
content in AF samples; therefore, amniotic GIP could berecycled through the foetal kidneys (Fig. 8).
Assessment of protein profiles and GIP in normal
and pathology‑affected amniotic fluids

Patients with Down syndrome have a sixfold increased
risk of developing CD as compared with the general
population. High levels of alpha-feto-protein in AF hasproved to be of great value for the prenatal diagnosis ofin abnormal foetal development. To address this question, we studied the profiles of proteins, including GIP of
the 94 AF samples from pregnant women; we selected 26
diagnostic amniocenteses that were classified according
to the underlying condition. The total protein content forsuspected foetuses with Down syndrome was analysedand compared with that of other pathologies and healthyfoetal counterparts to test whether Down syndrome correlates with an alteration in the membrane permeabilityof proteins, including gluten. In accordance with previous studies, we found a statistically significant increase
in total protein values in foetuses with abnormalities,
including Down syndrome, beyond 16–22 weeks with anarrow scatter around the mean (p < 0.05) (Fig. 9). In contrast, of the seven AF samples classified with suspicion of
Down syndrome, all presented GIP levels similar to thoseof their normal and foetal abnormality counterparts;
therefore, we did not observe significant differences in
the influence of membrane permeability in relation togluten peptides in the studied cases (p = 0.879). GIP content was not correlated with the total protein level in any
sample. Among healthy foetuses, a slight increase in total
protein content was observed in the AF from the second
trimester with respect to term delivery.

Discussion
To date, clinical trials have been based on the postnatal stage, attempting to demonstrate the doses of gluten
and the timing of its introduction. However, studies do
not agree on the most suitable age to introduce it withminimal risk [38]. Nevertheless, it is unknown whether
there are immunogenic fragments of these proteins

Moreno et al. BMC Medicine(2024) 22:295

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Fig. 6 Concordance between the punctual measurement of GIP content in the AF and urine samples of pregnant women. According to the QLof technique, individuals with a higher or equal GIP value than LOQ were considered positive (GIP + +) for the presence of GIP, while thosewith lower GIP content but higher than LDT were considered positive not quantifiable (GIP +) and those with lower GIP content than LDT wereconsidered negative (GIP −). LDT, the limit of technique detection; LOQ, the limit of quantification; GIP, gluten immunogenic peptides

in AF to which the foetal immune system is exposed
before birth. In this study, we describe a specific and reliable approach for detecting and monitoring GIP in AF
using G12 IC strips (LFIA). The recovery of measurable
amounts of gluten peptides in the AF samples of womenindicated foetal exposure to immunogenic fragments ofgluten related to maternal ingestion, emphasising thatamniotic GIP is absorbed through foetal swallowing during the maturation of the foetal gut (Fig. 8). Therefore,
after ingesting gluten-containing food, these proteins are
partially digested by GI enzymes into immunogenic peptides that pass from the pregnant woman’s bloodstream
through the placenta and into the AF, foetal respiratory
tract and gut by suction, thereby constituting the firstpossible route of sensitisation or tolerance. In this study
too, we have been able to distinguish prenatal levels ofgluten peptide exposure for the foetuses of non-CD pregnant women with regard to CD pregnant women andestablished a positive correlation with maternal gluten
intake.
Up to the 20th week of pregnancy, there is a significant maternal contribution to the formation of AF. After
25 weeks of gestation, the foetal skin is fully keratinisedand can no longer absorb or transfer fluids easily. Consequently, respiration, swallowing and urination are theprimary means of exchange between the foetus and AF tomaintain homeostasis and volume [20–22]. To study andcompare potential differences in GIP content in healthy

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Fig. 7 Influence of fasting food withdrawal in elective caesarean section in comparison with emergency mode. LDT, the limit of techniquedetection; LOQ, the limit of quantification; GIP, gluten immunogenic peptides; AF, amniotic fluid

Fig. 8 Maternal and foetal immune system exposure to gluten immunogenic peptides and immunological detection. GIP, gluten immunogenic
peptidesMoreno et al. BMC Medicine(2024) 22:295

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Fig. 9 Quantitative comparison of normal versus Down syndrome protein and gluten content in AF samples. Black points represent values of total
protein content each sample. Grey bars show the GIP content. Visual presence of GIP not quantifiable (> LDT and < LOQ, scatter plot in white area);absence of GIP (< LDT, scatter plot in grey area) and presence of GIP visible and quantifiable (> LOQ, box and whiskers). LDT, the limit of techniquedetection; LOQ, the limit of quantification; GIP, gluten immunogenic peptides; AF, amniotic fluid

pregnant women, we recruited AF samples to compare
the GIP content of healthy pregnant women at various stages of pregnancy, specifically in the second (15–25 weeks) and third trimesters (37–40 weeks). Despite
the major maternal contribution to AF formation until
week 20 [39], no significant differences in GIP levels wereobserved at any stage (Fig. 4). Gluten levels in AF samples at the end of gestation were not significantly different from those in the second trimester. Inter-individual
diversity (weight, age, etc.) and differences in feeding
patterns, such as type of gluten, daily amount of liquid
intake and accompanying diet, may have a considerable
impact on the gluten absorbed in the circulation and thenpassed into the AF.
Previous studies have shown that pregnancy involves
important physiological changes, such as increased solute permeability [39]. We conducted a comparative studyof GIP levels in spot AF and urine samples in a newly
recruited group of pregnant women in the second andthird trimesters of gestation. Despite the lower concentration of GIP in the amniotic cavity than in urine samples (Fig. 5), a strong correlation was observed between
both samples (Fig. 6). This agrees with previous studies
arguing for a 73% match between AF and maternal urine
peptidome [40, 41]. Regarding urinary GIP excretion inpregnant women, we found lower levels than those previously reported in healthy non-pregnant individuals [37,42, 43]. Therefore, these findings agree with studies arguing that increased permeability of intestinal epithelial
cells during gestation leads to increased absorption andreduced excretion of solutes in the urine [44, 45]. Interindividual differences in urinary GIP measurements
could be due to factors such as body size, physical exercise, environmental conditions and fluid, salt and protein
intakes, as previously reported [43, 46].
From week 12 onwards, the foetus is also involved in
the renewal of AF by releasing urine. We selected a situation of strict fasting in pregnant women, such as thepreoperative fasting period in caesarean sections, andcompared it to the GIP excretion in elective and emergency caesarean sections in healthy pregnant women(Fig. 7). We found that there was a gluten balance in
the AF in late pregnancy; it was not greatly alteredby maternal fasting which is consistent with previous
studies reporting that in late pregnancy, the main contributor to AF elimination is foetal swallowing [19].
The individuals with Down syndrome have a higher
predisposition to autoimmune diseases [2, 47], including CD [48]. The upregulation of a panel of proteins andtheir potential as a biomarker for prenatal diagnosis
and as revealing of pathogenesis of Down syndrome has
also been demonstrated [49]. Therefore, the concentration of total proteins, including GIP, was studied in AF
samples from both normal foetuses and those affectedby Down syndrome, with the aim of quantifying andcomparing them in both groups. Despite a statisticallysignificant increase in total protein values during amniocentesis of foetuses with abnormalities, no significant
differences were found in the level of GIP as comparedto other foetal abnormalities or healthy foetuses withinMoreno et al. BMC Medicine(2024) 22:295

the same gestational period. Therefore, concurrent CD
in individuals with Down syndrome could be justified
by the HLA genetic risk, as described previously [2, 47].
A limitation of this study is the enrolment of only asingle pregnant woman with CD in the prospective
analysis. This constraint arises from the relatively low
prevalence of CD in the population, coupled with thestringent volume and quality requirements of the AF
samples necessary for the developed methodology.
Therefore, future studies involving a larger cohort ofpregnant women with CD are necessary to validate andextend these findings.

Conclusions
We have suggested that there is a plausible closedloop of foetal ingestion of GIP-containing AF andits subsequent excretion via the foetal kidneys aftermaternal gluten consumption. This circuit can influence the amount of GIP to which the foetal immune
system is exposed during gestation. This study could
be the basis for further research focusing on whether
the foetal exposure pathway of gluten peptides directs
the immune system towards protection or susceptibility in the child, depending on its genetic disposition. In
particular, it will be important to understand whether
there are gluten-reactive cells in the foetus and whether
such cells differ in numbers and regulation in children
with prenatal GIP exposure. Knowledge of early exposure to GIP in utero could lead to primary prevention
strategies based on optimal nutritional therapies inpregnancy to prevent future diseases and could be a
new focus for studies focusing on the timing of introduction and safe amount of gluten.
Abbreviations
AF	Amniotic fluid
CD	Coeliac disease
CS	Caesarean section
GCD	Gluten-containing diet
GFD	Gluten-free dietGI	Gastrointestinal
GIP	Gluten immunogenic peptides
HLA	Human leukocyte antigens
IC	Immunochromatographic
IVF	In vitro fecundation
LDT	Limit of detection
LFIA	Lateral flow immunoassay
LFI	Lateral flow immunochromatographic
LOQ	Limit of quantification
PBS	Phosphate-buffered salineREC	Research ethics committee
SD	Standard deviationSPE	Solid-phase extraction
TFA	Trifluoroacetic acid

Page 12 of 14

tTG	Anti-tissue transglutaminase
VD	Vaginal delivery
W	WeekY	Year

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12916-​024-​03495-9.
Additional file 1: Table S1 Clinical characteristics of pregnant women andnew-born undergoing amniocentesis test (n = 33). CS, caesarean section;
IVF, in vitro fecundation; VD, vaginal delivery; w, week; y, year
Acknowledgements
The authors are grateful to Dr. Jacobo Díaz Portillo for their assistance with thestatistical analysis in this study and also thanks to the midwife Francisca Mar‑tínez Fornieles for her collaboration in the collection of amniotic fluid samples
from Valme Hospital. They are grateful to the generous pregnant volunteers
who enrolled in the study.
Authors’ contributions
Study concept and design: MLM, AR-H and CS; acquisition of data: MLM,
MGR and AN-M; data analysis and interpretation: MLM and CS; techni‑
cal and essential material support: CM-P, MM-C, MR, EH, CL-B, JLB and
JAS-B; manuscript drafting: MLM, AR-H and CS; critical revision of themanuscript: EM, PB, AR-H and CS. All authors read and approved the final
manuscript.
Funding
This work was supported by grant from I + D + I FEDER Andalucía 2014–2020(US-15332) and Federación de Asociaciones de Celíacos de España (FACE)(SUBN/2019/005). María González-Rovira received a fellowship from the Uni‑
versity of Seville, Spain (PIF del VI Plan Propio de Investigación y Transferencia
II.2A).
Availability of data and materials
No datasets were generated or analysed during the current study.

Declarations
Ethics approval and consent to participate
This study was conducted in accordance with the principles of the Declarationof Helsinki. The study protocol was reviewed and approved by the research
ethics committee (REC) of each institution involved (ID/Numbers: Celifluid andPI-2611). Written informed consent was obtained from all the participants.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Microbiology and Parasitology, Faculty of Pharmacy, Univer‑sity of Seville, Seville, Spain. 2 Obstetric and Gynecology Department, Sagrado
Corazón Hospital, Seville, Spain. 3 Obstetric and Gynecology Department, High
Risk Pregnancy Unit, Autoimmune Diseases and Pregnancy Clinic, University
Hospital La Paz, Madrid, Spain. 4 Department of Women’s and Children’s Health,
Karolinska Institutet, Uppsala, Sweden. 5 St. Luke’s General Hospital, UCD
School of Medicine, University College Dublin, Kilkenny &, Kilkenny, Ireland.
6
Department of Obstetrics and Gynecology, Valme University Hospital, Seville,
Spain. 7 Department of Obstetrics and Gynecology, University of Seville, Seville,
Spain.

Moreno et al. BMC Medicine(2024) 22:295

Page 13 of 14

Received: 3 March 2024 Accepted: 20 June 2024

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