Reduced-number-of-regulatory-T-cells-in-maternal-c

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Original Research ajog.org
OBSTETRICS
Reduced number of regulatory T cells in maternal
circulation precede idiopathic spontaneous preterm
labor in a subset of patients
Michal Koucky, MD, PhD; Zdenek Lastuvka, MD, PhD; Helena Koprivova, Mgr; Tereza Cindrova-Davies, MD, PhD;
Jiri Hrdy, RNDr, PhD; Karin Cerna, MD; Pavel Calda, MD, CSc
BACKGROUND: Accumulating evidence suggests that spontaneous CD25þFoxP3þCD45RA, CD4þCD25þFoxP3þHeliosþ, and CD4þ
preterm labor is a syndrome caused by multiple pathological processes.
The breakdown of maternal-fetal tolerance has been proposed as a key
mechanism of idiopathic spontaneous preterm labor, often viewed as a
chronic inflammatory process resulting from the maternal immune
system’s impaired tolerance of the fetus from early pregnancy. Regu-
latory T cells are crucial for maintaining maternal-fetal tolerance. Even a
partial reduction in their levels can disrupt this tolerance, leading to
adverse pregnancy outcomes such as preterm labor. Given the
complexity of the T lymphocyte-mediated immune response, identifying
candidate signaling pathways involved in maternal-fetal tolerance is
challenging. However, current literature highlights the importance of the
functional and developmental markers FoxP3, CD45RA, Helios, and
CD39 due to their immunosuppressive abilities essential for maintaining
pregnancy.
OBJECTIVE: This study aimed to determine whether changes in
numbers of selected regulatory T cell subpopulations in the first trimester
are associated with subsequent spontaneous preterm labor.
STUDY DESIGN: This prospective study enrolled 43 women with early
singleton pregnancies, excluding those with autoimmune diseases, diabetes
mellitus (type 1, type 2), primary hypertension, or who had been treated with
vaginal progesterone prior to sample collection. We analyzed regulatory T cell
subpopulations in maternal circulation using the DURAClone IM T cell kit,
focusing on the following subsets: CD4þCD25þFoxP3þ, CD4þ
Cite this article as: Koucky M, Lastuvka Z, Koprivova H,
et al. Reduced number of regulatory T cells in maternal
circulation precede idiopathic spontaneous preterm labor
in a subset of patients. Am J Obstet Gynecol
2025;232:222.e1-11.
0002-9378
ª 2024 The Author(s). Published by Elsevier Inc. This is an
open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
https://doi.org/10.1016/j.ajog.2024.11.001
222.e1 American Journal of Obstetrics & Gynecology FEBRUARY 2025
CD25þFoxP3þCD39�.
RESULTS: Among the participants, 7 experienced spontaneous preterm
labor between the 23rd and 33rd weeks of gestation, while 36 delivered at
term. The preterm group showed a significant reduction in numbers of all
analyzed regulatory T cell subpopulations: CD4þCD25þFoxP3þ (median
0.0410�10
ˇ
9/L vs median 0.0550�10
ˇ
9/L, P¼.0217), CD4þCD25þ
FoxP3þCD45RA� (median 0.0310�10
ˇ
9/L vs median 0.0420�10
ˇ
9/L,
P¼.0216), CD4þCD25þFoxP3þHeliosþ (median 0.0270�10
ˇ
9/L vs
median 0.0370�10
ˇ
9/L, P¼.0260), CD4þCD25þFoxP3þCD39� (median
0.0300�10
ˇ
9/L vs median 0.0420�10
ˇ
9/L, P¼.0427).
CONCLUSION: Early first trimester alterations in specific regulatory T
cell subpopulations, including diminished levels of CD4þCD25þFoxP3þ,
CD4þCD25þFoxP3þCD45RA�, CD4þCD25þFoxP3þHeliosþ, and
CD4þCD25þFoxP3þCD39�, are associated with idiopathic sponta-
neous preterm labor. These findings suggest that early changes in these
lymphocyte subpopulations may be linked to spontaneous preterm birth.
This highlights the need for further research to understand the mecha-
nisms underlying regulatory T-cell dynamics and their impact on preg-
nancy outcomes.
Key words: anti-fetal rejection, CD4, CD25, CD45, CD39, FoxP3, early
pregnancy, Helios, immunology, immunosuppression, maternal-fetal
tolerance, preterm labor, regulatory T cells
Introduction
Spontaneous preterm labor is a major
concern in obstetrics due to its role as a
leading cause of neonatal morbidity and
mortality, highlighting the complex
interplay between maternal and fetal
health.1 Current knowledge suggests that
it is a syndrome attributable to multiple
pathological processes.2 Increasingly, the
breakdown of the maternal-fetal toler-
ance has been proposed as a mechanism
of idiopathic spontaneous preterm
labor.3
The occurrence of spontaneous pre-
term labor primarily underscores a
chronic inflammatory response facili-
tated by the maternal immune system’s
interaction with the semi-allogeneic
fetus.2,4e7 Similar involvement of regu-
latory T cell (Treg) cells has been shown
in the prevention of autoimmune8 and
graft rejection.9 This highlights the crit-
ical role of immune tolerance mecha-
nisms, particularly the function of Tregs,
in maintaining a successful pregnancy. A
simplified overview diagram of Tregs
development is shown in Figure 1.
Tregs, key players in maternal-fetal
tolerance, adapt to the unique immu-
nological environment of pregnancy,
increasing in both systemic circulation
and at the maternal-fetal interface.3,10,11
Adequate Treg numbers and function
play a significant role for numerous
pregnancy complications, such as spon-
taneous abortion, spontaneous preterm
labor, and preeclampsia.12,13 The recruit-
ment and specific response of Tregs to
fetal antigens at the decidua are essential
for sustaining pregnancy, reflecting the
body’s intricate mechanism to foster
maternal-fetal harmony.10,14e16 Tregs
regulate immune system via induction of
apoptosis of target cells or production of
cytokines (especially interleukin 35,
transforming growth factor-beta [TGF-
b], and interleukin 10).17
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https://doi.org/10.1016/j.ajog.2024.11.001
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AJOG at a Glance
Why was this study conducted?
There is a lack of studies examining the relationship between first trimester
regulatory T cell (Treg) subpopulations and the risk of subsequent spontaneous
preterm birth.
Key findings
The study identified a significant reduction in numbers of selected Treg sub-
populations in first trimester peripheral blood samples from women who later
experienced preterm delivery compared to those who delivered at term. These
subpopulations include CD4þCD25þFoxP3þ, CD4þCD25þFoxP3þCD45RA
�, CD4þCD25þFoxP3þHeliosþ, and CD4þCD25þFoxP3þCD39�.
What does this add to what is known?
The first demonstration of a reduction in the number of selected T regulatory cells
in maternal circulation prior to idiopathic spontaneous preterm labor.
ajog.org OBSTETRICS Original Research
Clinical implications arise from the
dynamic immunological shifts observed
during pregnancy. For instance, reduc-
tion of specific Treg counts in the second
and third trimester was associated with
FIGURE 1
Development of T regulatory cells
T regulatory cells originate in the thymus or peripher
become activated. Cytokine stimulation plays an im
clarity, only the CD markers (cluster of differentiatio
mentioned in the text are highlighted. CD4: Helps
CD25: A marker on activated Tregs. CD45: A trans
receptor signaling in both T and B cells. RA: cells th
cells that have encountered specific antigen. IL-2,
regulatory cells.
IL-2, interleukin 2; IL-10, interleukin 10; Treg, regulatory T cell.
higher incidence of spontaneous pre-
term labor.18 Understanding the balance
between different immune cell pop-
ulations, especially the role of Tregs in
promoting tolerance and preventing
al tissue and migrate as naı̈ve T cells, where they
portant role in their differentiation. To maintain
n, membrane receptors) and transcription factors
T cells communicate with other immune cells.
membrane protein involved in regulating antigen
at have not yet encountered specific antigen, RO:
IL-10:Cytokines involved in differentiation of T
FEBRUARY 2025 Ameri
inflammation, is crucial in the context of
pregnancy.
In a clinical setting, monitoring the
immune status of pregnant women,
especially the balance and function of
Tregs, could offer insights into the risk of
spontaneous preterm birth, providing a
basis for early intervention strategies.
This approach necessitates a shift from
purely molecular investigations to clin-
ical applications, focusing on how alter-
ations in immune regulation can be
addressed or utilized to improve preg-
nancy outcomes. As such, advancements
in the field of reproductive immunology
hold significant promise for enhancing
prenatal care and reducing the incidence
of spontaneous preterm births, marking
a pivotal area of interest for specialists in
reproductive medicine and gynecology.
The primary objective of this study
was to explore the association between
spontaneous preterm labor and alter-
ations in the quantity of specific Tregs
subpopulations (CD45þCD3þCD4þ
CD25þFoxP3þ) during the early first
trimester of pregnancy. The secondary
objective was to examine the expression
of markers CD45RA, CD39, and Helios
on Tregs in early gestation among
women who experienced spontaneous
onset of preterm labor, compared to
thosewhodelivered at term. By analyzing
the expression of these markers, we aim
to shed light on the functional charac-
teristics and regulatory mechanisms of
Tregs that may influence the pathophys-
iology of spontaneous preterm labor.
Materials and methods
Study design
Thisprospective study included a cohortof
43 Caucasian patients between 10þ0 and
11þ6 weeks of singleton pregnancy
recruited between January 2020 and
January 2021. All patients were selected
froma cohort ofwomenwith a history of 2
or more spontaneous abortions (12e21
weeks of gestation). Blood samples were
obtained during routine prenatal care ap-
pointments. Patients with autoimmune
diseases, diabetes mellitus, and primary
hypertension and those treated with
vaginal progesteroneprior toblood sample
collection were excluded from the study.
can Journal of Obstetrics & Gynecology 222.e2
http://www.AJOG.org
TABLE 1
Patient details
Characteristics
Preterm labor cohort,
n (%) (n¼7)
Control cohort,
n (%) (n¼36)
Age (median) 35 (29e43) 34 (32e40)
Cigarette smoking 1 (14.2) 6 (17.1)
Gestational diabetes 0 (0) 2 (5.5)
Gestational hypertension 0 (0) 2 (5.5)
Placenta abruption 0 (0) 0 (0)
Bleeding during pregnancy 1 (14.2) 0 (0)
Urinary tract infection 1 (14.2) 1 (2.8)
In vitro fertilization 1 (14.2) 4 (11.4)
Body mass index (median) 23 (19e25) 26 (20e32)
Previous C-section 1 (14.2) 3 (8.3)
Cerclage in previous pregnancy 0 (0) 0 (0)
Vaginal birth 5 (71.4) 32 (88.9)
C-section
Emergency 2 (28.6) 2 (5.5)
Planned 0 (0) 2 (5.5)
Previous spontaneous preterm labor 1 (14.3) 3 (8.3)
Descriptive characteristics of controls who had term vaginal deliveries (n¼36) and patients with idiopathic spontaneous preterm
labor (n¼7).
For statistical evaluation, a 2-sample t test was used and all the values are nonsignificant.
The P value equals .1483.
Differences were considered not statistically significant.
The mean of group 1 minus group 2 equals 0.23862, and the 95% confidence interval of this difference is from�0.09592 to
0.57317.
Original Research OBSTETRICS ajog.org
Gestational agewas calculatedbasedon the
first trimester ultrasound scan.
Blood sample collection and
processing
Peripheral blood (6 mL in total) was
collected in EDTA tubes (Vacutai-
nerBecton Dickinson, San José, CA) and
analyzed within 5 hours of collection.
The PerFix-nc kit (Beckman Coulter,
Indianapolis, USA) was used for fixation,
lysis and permeabilization, as per the
manufacturer’s recommendations.
Flow cytometry analysis
The DURAClone IM Tregs kit (Beckman
Coulter, Brea, California, USA) was used
to analyze Tregs subpopulations. This kit
contains a set of tubes with dried mono-
clonal antibodies (mAbs) intended for
surface and intracellular labeling, as well
as a set of antibodies intended for setting
panel compensations on aflowcytometer.
The combination of mAbs was used for
surface labeling included: CD45RA-FITC
(clone 2H4LDH11LD
B9(2H4), CD25-PE (clone B1.49.9),
CD39-PC5.5 (clone BA54), CD4-PC7
(clone SFCI12T4D11(T4), CD3-APC
A750 (clone UCHT-1), and CD45-KrO
(clone J33). FoxP3-Alexa Fluor 647
(clone 259D) andHelios-PB (clone 22F6)
mAbs were used for intracellular labeling.
Data were measured using a NAVIOS
flow cytometer (Beckman Coulter, Indi-
anapolis) and analyzed with Kaluza 5.1
software (Beckman Coulter, Indian-
apolis). Lymphocyteswere gated based on
forward (FSC) and side (SSC) scatter. The
CD45 marker was used to purify lym-
phocytes. After removing debris, mono-
cytes, and granulocytes, the CD3þCD4þ
population was gated, from which the
FoxP3þCD25þ population emerged and
was defined as Tregs. Treg populations
were also monitored using Helios tran-
scription factor expression, CD45RA
expression to differentiate the naïve and
effector populations, and CD39 expres-
sion. Values were converted to absolute
numbers (ie, 109/L).
Gating strategy
Regarding the gating strategy to identify
Tregs, lymphocytes were gated on FSC/
SSC, and Tregs were gated as CD45þ
222.e3 American Journal of Obstetrics & Gynecol
CD3þCD4þCD25þFoxP3þ Tregs.
CD45RA, CD39, and Helios-positive and
Helios-negative populations were detec-
ted within this population.
Statistical analysis
The analyses were performed using the R
statistical package, version 4.2.1. The
Shapiro-Wilk test assessed the normality of
variables. Given the nonnormal
distribution of data, nonparametric
statistical methods were employed.
Continuous variables are presented as
medians and interquartile ranges (IQRs),
and categorical variables are shown as ab-
solute and relative counts. The Mann-
Whitney U test was used to compare the
analyzed groups. A P value of �.05 was
deemed statistically significant.
Results
The pregnancy outcomes of 43 women
were tracked from the initial trimester
ogy FEBRUARY 2025
through childbirth (see Tables 1 and 2).
Throughout the study period, we
observed no spontaneous abortions or
fetal deaths. A subset of the cohort,
hereafter designated as the spontaneous
preterm labor cohort, comprised 7 of the
43 participants, who experienced spon-
taneous preterm delivery occurring be-
tween the 23rd and 33rd weeks of
gestation. The remaining 36 participants
successfully carried their pregnancies to
term, delivering after the completion of
the 37th week of gestation, and thus
formed the control cohort.
Demographically, the average maternal
age at enrollment was 35 years (ranging
from 29e43 years) within the preterm
labor cohort, and 34 years (ranging from
32e40 years) within the control cohort.
No significant discrepancies in maternal
age between the 2 cohorts were observed,
indicating that agewas not a distinguishing
factor in the observed outcomes.
http://www.AJOG.org
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ajog.org OBSTETRICS Original Research
FEBRUARY 2025 Am
eri
In terms of pregnancy-related health
conditions, the control cohort reported
instances of gestational hypertension and
gestational diabetes in 2 participants.
Additionally, urinary tract infections
were documented in one participant
from each cohort, highlighting the
occurrence of common pregnancy-
associated complications irrespective of
the gestation duration.
Delivery modes varied across both
cohorts with a majority, 37 out of 43,
opting for vaginal delivery, while cesar-
ean section was performed in 6 cases.
The prevalence of cigarette smoking, a
known risk factor for various pregnancy
complications, was comparable between
both cohorts, suggesting that it did not
significantly influence the incidence of
preterm labor in our study population.
In the spontaneous preterm labor
cohort the birth weights of the newborns
ranged from 620 g to 1860 g, including
both male (3) and female (4) infants.
Funisitis was absent in all cases. Chronic
chorioamnionitis was identified in 3
cases, 2 of which also presented with
villitis. These cases were linked to earlier
gestational ages (23e25 weeks). Impor-
tantly, no other placental inflammatory-
immune conditions, such as chronic
deciduitis, chronic intervillositis, or
acute chorioamnionitis, were observed,
consistent with the criteria outlined in
the Amsterdam Placental Workshop
Group Consensus Statement.19
None of the patients experienced pre-
term premature rupture of membranes.
All newborns were admitted to the
neonatal intensive care unit. One infant
required patent ductus arteriosus ligation,
and another underwent retinopathy of
prematurity intervention, both in-
terventions involved the baby born at
23 weeks of gestation. Necrotizing entero-
colitis was not observed in any of the
newborns, though one infant, born at
24 weeks, was diagnosed with grade 3/4
intraventricular hemorrhage (see Table.2).
Absolute numbers of
CD4DCD25DFoxP3D Tregs and
pregnancy outcomes
Tregs were identified and characterized
based on the co-expression of CD4, CD25,
and FoxP3 markers, thereby delineating
can Journal of Obstetrics & Gynecology 222.e4
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Original Research OBSTETRICS ajog.org
the CD4þCD25þFoxP3þ population as
the focal point of our analysis. The
expression level of FoxP3 is intrinsically
linked to the suppressive function and
identity of Tregs. Thus, analyzing numbers
of CD4þCD25þFoxp3þ cells allow for a
comprehensive evaluation of the total
Tregs population, providing insights into
their quantitative presence within the
gestational milieu. Our findings, as illus-
trated in Figure 2, revealed a statistically
significant reduction in numbers of
CD4þCD25þFoxp3þ cells among the
Tregs population in the preterm labor
cohort compared to the control cohort
(median 0.0410�10
ˇ
9/L, IQR 0.0380e
0.0465�10
ˇ
9/L vsmedian 0.0550�10
ˇ
9/L,
IQR 0.0445e0.0645�10
ˇ
9/L, P¼.0217).
Different immunophenotypes of
Tregs and pregnancy outcomes
Treg subpopulations were selected based
on the expression of specific markers
FIGURE 2
Absolute numbers of CD4DCD25DFox
term labor cohorts
Data are represented as individual data points with
N, sample size; NA’s, missing data; SD, standard deviation; Treg, re
222.e5 American Journal of Obstetrics & Gynecol
that reflect their functional and devel-
opmental states: CD4þCD25þFoxP3þ
CD45RA�, CD4þCD25þFoxP3þ
Heliosþ, and CD4þCD25þFoxP3þ
CD39�.
Our findings revealed a pronounced
diminution in the effector Tregs
(CD45RA�) population within the early
gestational period in the preterm labor
cohort of women, as illustrated in
Figure 3 (median 0.0310�10
ˇ
9/L, IQR
0.0300e0.0365�10
ˇ
9/L vs median
0.0420�10
ˇ
9/L, IQR 0.0355e0.0535�
10
ˇ
9/L, P¼.0216). This particular sub-
population is critical for understanding
the adaptive immune responses during
pregnancy, as effector Tregs are known
for their immunosuppressive functions,
which are essential for fetal-maternal
tolerance.
Moreover, the analysis identified a
significantly lower frequency of the Tregs
Heliosþ population (CD4þCD25þ
P3D T regulatory cells in preterm vs
medians. Mann-Whitney U test: P¼.0217.
gulatory T cell.
ogy FEBRUARY 2025
FoxP3þHeliosþ) in the spontaneous
preterm birth group, as referenced in
Figure 4 (median 0.0270�10
ˇ
9/L, IQR
0.0265e0.0320�10
ˇ
9/L vs median
0.0370�10
ˇ
9/L, IQR 0.0315e0.0495�
10
ˇ
9/L, P¼.0260). Helios, a transcription
factor, is often associated with the Tregs’
suppressive abilities and their thymic
origin, suggesting that alterations in this
subpopulation could reflect disruptions in
the regulatory landscape essential for
pregnancy maintenance.
Additionally, the study observed a
substantial reduction in the CD39�
Tregs subpopulation (CD4þCD25þ
FoxP3þCD39�) among those who
delivered preterm, as depicted in
Figure 5 (median 0.0300�10
ˇ
9/L, IQR
0.0275e0.0345�10
ˇ
9/L vs median
0.0420�10
ˇ
9/L, IQR 0.0290e0.0545�
10
ˇ
9/L, P¼.0427). Given CD39’s role in
modulating extracellular adenosine
triphosphate levels and promoting an
antiinflammatory milieu, its decreased
presence among Tregs in early pregnancy
could signify an impaired ability to
manage inflammatory responses,
potentially contributing to the patho-
physiology of spontaneous preterm
labor.
Comment
Principal findings
The study identified a significant reduction
in numbers of selected Treg sub-
populations in first trimester peripheral
blood samples from women who later
experienced spontaneous preterm delivery
between the 23rd and 33rd week of preg-
nancy. These subpopulations include:
CD4þCD25þFoxP3þ, CD4þCD25þ
FoxP3þCD45RA�, CD4þCD25þ
FoxP3þHeliosþ, and CD4þCD25þ
FoxP3þCD39� (Figure 6).
Results in the context of what is
known
Previous studies have evaluated changes
in the number or function of Tregs in
advanced stages of pregnancy.20e23
When early development of the
placenta in the first trimester is
disturbed, disorders, such as preterm
labor or preeclampsia, might manifest
later in pregnancy.24 Regarding pre-
eclampsia, Garcia et al21 in a prospective
http://www.AJOG.org
FIGURE 3
Absolute numbers of CD4DCD25DFoxP3DCD45RAL T regulatory cells in
preterm vs term labor cohorts
Data are represented as individual data points with medians. Mann-Whitney U test: P¼.0216.
N, sample size; NA’s, missing data; SD, standard deviation; Treg, regulatory T cell.
ajog.org OBSTETRICS Original Research
study of maternal blood samples
collected between 5 and 16 weeks of
pregnancy, showed a statisticallysig-
nificant reduction in Tregs in patients
who later developed preeclampsia.
Similar results have previously been
described for other pregnancy compli-
cations, such as spontaneous abortion.
In spontaneous abortion, the percent-
age of the circulating Tregs population
is significantly lower than that in
normal pregnancy.25 Similarly, Ehren-
traut et al26 observed an elevation of
Tregs during early normal pregnancy,
which was not present in spontaneous
abortion.
T regulatory lymphocytes are
considered key players in the patho-
genesis of numerous pregnancy com-
plications, such as preeclampsia,
spontaneous preterm labor, and
recurrent spontaneous abortion.13 A
significant reduction in Tregs levels and
their function in the peripheral blood
and the decidua of pregnant and
nonpregnant women with recurrent
spontaneous abortion has been
demonstrated.27 Insufficient expan-
sion of fetal antigen-specific Tregs in
the decidua has been reported to play a
role in the pathophysiology of pre-
eclampsia.28,29 In addition, several
groups, including those in the current
study, have observed changes in Treg
subpopulations and activity in women
with spontaneous preterm
birth.3,18,20,30
Most studies have investigated Tregs at
the time of preterm labor diagnosis,
whereas our study examines them
prior to the occurrence of this event.
Gomez-Lopez et al3 assessed Tregs
FEBRUARY 2025 Ameri
subpopulations directly in the decidua
and found a decrease in the number of
Tregs at the maternal-fetal interface in a
subgroup of women with spontaneous
pretermbirth,whichwas accompaniedby
a simultaneous increase in the number of
T helper 17 cells. Mechanisms by which
depletion of Tregs induces adverse peri-
natal outcomes include tissue-specific
immune responses, systemic maternal
inflammatory responses, and dysregula-
tion of cellular processes in the placenta,
all in the absence of intraamniotic
inflammation.3 The manifestations of
maternal-fetal rejection have been already
described by Romero et al and Kim et al.
One can only speculate whether down-
regulation of Treg cells may promote
chronic maternal-fetal rejection.
We confirmed the relationship be-
tween reduced Tregs levels
(CD4þCD25þFoxp3þ) and the inci-
dence of preterm labor.18 Xiong et al31
monitored dynamics of Tregs levels
(CD4þCD25þFoxp3þ) throughout
pregnancy and found significant
reduction in patients who subse-
quently had delivered. Although
Steinborn et al30 and Schober et al20
found no significant differences in the
percentage of Tregs between term and
preterm births, both authors noted a
significant reduction in the suppressive
capacity of Tregs in women who
delivered preterm compared to term
controls, suggesting involvement of
Tregs in mediating parturition (See
Table 3).
The results of our work may be
related to the findings of the above-
mentioned studies,3,18,31 with abnormal
numbers of selected populations of
Tregs found as early as in the first
trimester. Women’s immune systems
are constantly exposed to paternal and
fetal antigens before conception
through exposure to sperm and during
pregnancy through exposure to fetal
cells.32 This leads to the formation of
memory T cells specific for paternal and
fetal antigens. Memory T lymphocytes
generally have the ability to remember
antigens they have previously encoun-
tered and thus mount a faster, more
can Journal of Obstetrics & Gynecology 222.e6
http://www.AJOG.org
FIGURE 4
Absolute numbers of CD4DCD25DFoxP3DHeliosD T regulatory cells in
preterm vs term labor cohorts
Data are represented as individual data points with medians. Mann-Whitney U test: P¼.0260.
N, sample size; NA’s, missing data; SD, standard deviation; Treg, regulatory T cell.
Original Research OBSTETRICS ajog.org
pronounced, and more targeted im-
mune response when reencountering
the antigen. Knowledge of memory
T cells in pregnancy has shown that
these cells can play a beneficial role in
maternal-fetal tolerance.33 Memory/
effector Treg fractions, CD4þCD25þ
FoxP3þCD45RA� (TregCD45RA�),
help maintain anergy to fetal antigens.34
Rowe et al14 developed a mouse model
in which they showed an increase in the
number of antigen-specific T regulatory
memory cells during a first pregnancy,
which persisted in lower numbers after
delivery. They further observed a signif-
icantly faster increase in the number in
an ensuing pregnancy compared to a first
pregnancy. In our cohort of pregnant
women who gave birth prematurely,
representation of the TregCD45RA�
population was lower than in the group
222.e7 American Journal of Obstetrics & Gynecol
of women who gave birth at term, which
supports the irreplaceable role of the
memory/effector Treg populations in the
setting of maternal-fetal tolerance. Pre-
vious studies investigating the role of
memory Tregs in pregnancy complica-
tions show that memory Tregs may be
beneficial for reproductive success in
subsequent pregnancies in mice; how-
ever, there are conflicting data from
studies in humans. The fact that some
studies found higher levels of memory
Tregs in pregnancy complications, such
as preeclampsia and gestational dia-
betes,35,36 and others found that lower
levels before embryo transfer in vitro
fertilization/intracytoplasmic sperm in-
jection treatment are associated with
pregnancy success,37 might indicate
specific roles ofmemoryTregs depending
on the stage of pregnancy.
ogy FEBRUARY 2025
There are very few studies that explore
the role of Heliosþ Tregs in pregnancy.
Our research group detected relatively
stable levels of this subpopulation during
uncomplicated pregnancy.38 Inada
et al39 demonstrated that a reduction in
Heliosþ Tregs in the decidua is associ-
ated with spontaneous abortion. Our
research demonstrates decreased levels
of the Treg Heliosþ population in first
trimestermaternal blood in patients who
subsequently had a spontaneous preterm
delivery. These results suggest that
adequate representation of this Treg
population may play a role in the
maintenance of pregnancy. The CD39
signature is an increasingly appreciated
regulatory marker expressed not only
among Treg populations but also on
other T cells (CD8 Tregs, B regulatory
cells, and gd T cells).40,41 Among other
properties, this inhibits production of
interleukin 6 while simultaneously pro-
moting that of TGF-b. Antigenic stim-
ulation of T cells in the presence of TGF-
b leads to FoxP3 expression. The fre-
quency of CD39 expression on Tregs is
stable over time, but not all Tregs display
this feature. Furthermore, the frequency
of the CD39� Treg subgroup changes
depending on age and ongoing immune
processes, suggesting that this marker
might play an important diagnostic role
in the future. We found a significant
reduction in CD39� Tregs in women
who gave birth prematurely. Samudra
et al42 revealed CD39 and CD73 activity
to be protective in a mouse model of
antiphospholipid antibody-induced
spontaneous abortion. Rissiek et al43
showed that CD39þ Tregs suppressed
T-cell proliferation and inflammatory
cytokine production more effectively
than CD39� Tregs, while Yousefzadeh
et al44 reported reduced expression of
CD39 in the peripheral blood and
decidua of women with preeclampsia.
Similarly, the work of Sato et al45 sug-
gests that CD39 downregulation is a
useful marker of chronic intervillositis of
unknown etiology and associated with
poor pregnancy outcomes, leading to
various pathological conditions, partic-
ularly fetal death and fetal growth
http://www.AJOG.org
FIGURE 5
Absolute numbers of CD4DCD25DFoxP3DCD39L T regulatory cells in
preterm vs term labor cohorts
Data are represented as individual data points with medians. Mann-Whitney U test: P¼.0427.
N, sample size; NA’s, missing data; SD, standard deviation; Treg, regulatory T cell.
ajog.org OBSTETRICS Original Research
restriction. McRare et al46 showed in a
mouse model that expression of CD39
plays a protective role against the devel-
opment of preeclampsia.
Absence of expression of the CD39
trait on Tregs, associated with reduced
frequency of Treg subpopulationin the
group of women who deliver preterm,
might promote inflammatory processes
that underlie premature birth. These
changes are already apparent in very
early stages of pregnancy and can be
detected noninvasively in maternal
blood.
Research and clinical implications
The significant reduction in specific Treg
subpopulations (eg, CD4þCD25þ
FoxP3þ, CD4þCD25þFoxP3þ
CD45RA�, CD4þCD25þFoxP3þ
Heliosþ, and CD4þCD25þFoxP3þ
CD39�) in women who deliver preterm
indicates that early immunological
profiling could serve as a predictive
marker for a subset of patients with
idiopathic spontaneous preterm birth.
This could guide interventions aimed at
reducing immune responses to support
themaintenance of pregnancy. Given the
study’s limitations in sample size and
scope, these preliminary findings can be
the basis for larger multicenter studies
that could validate and expand upon the
observed associations. This could facili-
tate the development of robust clinical
protocols and interventions aimed at
reducing preterm birth rates.
Strengths and limitations
This is the first study evaluating the
relationship between first trimester
Tregs and subsequent idiopathic spon-
taneous preterm birth in human preg-
nancy. One of the principal constraints
FEBRUARY 2025 Ameri
stems from the relatively small sample
size of 43 participants, which, although
yielding statistically significant findings,
may limit the generalizability of the re-
sults to a broader population. This fac-
tor is particularly relevant given the
complex, multifactorial nature of
spontaneous preterm labor, suggesting
that larger studies are needed to validate
these preliminary findings and to
explore the potential influence of other
confounding factors not accounted for
in this study. Additionally, the study’s
focus on a specific set of Treg sub-
populations, based on the expression of
markers such as CD45RA, Helios, and
CD39, while instrumental in advancing
our understanding of Treg-associated
mechanisms, does not encompass the
full spectrum of Treg diversity and
functionality. Future research could
benefit from incorporating a wider
array of markers to capture a more
comprehensive picture of Treg hetero-
geneity and its implications for preg-
nancy outcomes. Another limitation is
the observational nature of the study,
which, although effective for identifying
associations between Treg dynamics
and preterm labor, does not establish
causality. Experimental studies,
possibly involving animal models or
in vitro systems, are necessary to eluci-
date the causal mechanisms by which
alterations in Treg populations
contribute to the pathogenesis of idio-
pathic spontaneous preterm labor.
Furthermore, the study’s reliance on
peripheral blood samples as the pri-
mary source for Treg analysis, while
practical and minimally invasive, may
not fully reflect the immunological
status at the maternal-fetal interface.
Future studies could explore more
direct methods of assessing immune cell
populations within the decidua or am-
niotic fluid to provide a more localized
understanding of immunoregulatory
processes during pregnancy.
Conclusions
This study identified a significant reduc-
tion in several Treg subpopulations,
can Journal of Obstetrics & Gynecology 222.e8
http://www.AJOG.org
FIGURE 6
Summary of study results
In pregnancies with premature delivery, early changes in selected lymphocyte subpopulations (CD4þCD25þFoxP3þ, CD4þCD25þFoxP3þCD45RA�,
CD4þCD25þFoxP3þHeliosþ, and CD4þCD25þFoxP3þCD39�) are already present at the end of the first trimester.
Original Research OBSTETRICS ajog.org
including CD4þCD25þFoxP3þ, CD4þ
CD25þFoxP3þCD45RA�, CD4þ
CD25þFoxP3þHeliosþ, and CD4þ
CD25þFoxP3þCD39�, in first trimester
TABLE 3
A review of studies evaluating the role
controls
Authors
Study
Point
Gomez-Lopez et al III. trim
Decidu
Koucký et al III. trim
Mater
Xiong et al II. and
Mater
Steinborn et al II. and
Mater
Schober et al II. and
Mater
Max. suppressor activity (%): Percentage of the Treg cells that c
PTL, spontaneous preterm labor; Treg, regulatory T cell.
222.e9 American Journal of Obstetrics & Gynecol
peripheral blood samples from women
who later experienced preterm delivery
compared to those who delivered at term.
Results suggest that in pregnancies
of Treg in pregnant women with spontane
groups
of interest
ester PTL vs term controls
a
ester PTL vs term controls
nal blood
III. trimester PTL vs term controls
nal blood
III. trimester PTL vs term controls
nal blood
III. trimester PTL vs term controls
nal blood
an suppress the proliferation of highly activated CD4þ responder.
ogy FEBRUARY 2025
resulting in idiopathic spontaneous pre-
term labor, early changes in selected
lymphocyte subpopulations are already
present at the end of the first trimester.
ous preterm birth compared to term
Statistically significant main results
Decreased numbers of Tregs
Decreased suppressor activity of Tregs
Decreased numbers of Tregs
-
Decreased numbers of Tregs
-
-
Decreased suppressor activity of Tregs
-
Decreased suppressor activity of Tregs
http://www.AJOG.org
GLOSSARY
Flow cytometry and gating strategy. A technique used to count and classify different types of cells in a sample. It helps in:
� Excluding dead cells: Using a special dye to mark and remove dead cells.
� Identifying cell types: Using size (Forward Scatter, FSC) and granularity (Side Scatter, SSC) to find specific cell types.
� Staining for T cells: Applying markers to identify T cells with specific proteins like CD39, FoxP3, and Helios.
DURAClone IM Tregs Kit. A tool used for analyzing different types of T regulatory cells (Tregs) using flow cytometry. It includes special
antibodies for labeling cell surfaces and inside the cells.
Interleukin 10 (IL-10). A protein that helps control inflammation and keep the immune system balanced. It is made by various immune cells
and helps prevent overactive immune responses and autoimmune diseases.
Interleukin 35 (IL-35). A protein produced mainly by regulatory T cells (Tregs) and regulatory B cells. It helps suppress the immune system
and maintain tolerance to prevent autoimmune responses.
Immunophenotypes. The specific markers on immune cells that help identify and differentiate various types of Tregs.
Maternal-fetal tolerance. The immune system’s ability to accept the fetus, which is partly foreign to the mother’s body, without rejecting it.
Monoclonal antibodies (mAbs). Lab-made antibodies that are identical and target specific cell markers. In this study, examples include
CD45RA-FITC, CD25-PE, and CD39-PC5.5.
T regulatory cells (Tregs). A type of T cell that helps regulate the immune system and maintain tolerance to the body’s own tissues.Treg
subpopulations. Different types of Tregs distinguished by specific surface markers and proteins. Each subpopulation plays a unique role in
regulating the immune system and maintaining tolerance, which is essential for supporting a healthy pregnancy. Key markers include the
following:
� CD3: Helps activate T cells.
� CD4: Helps T cells communicate with other immune cells.
� CD25: A marker on activated T cells and Tregs.
� CD39: An enzyme that helps create an antiinflammatory environment.
� CD45: A protein involved in regulating T and B cell signaling. RA: cells that have not yet encountered specific antigen, RO: cells that have
encountered specific antigen.
Memory Treg. Regulatory T cells that have previously encountered antigen and are thus primed to respond more quickly and effectively
upon re-exposure. These cells, often characterized by the absence of CD45RA and the presence of markers like CD45RO, retain “memory”
of past immune interactions, which allows them to exert a more rapid and robust suppressive function compared to naı̈ve Tregs.
FoxP3 and Helios. Proteins crucial for the function and stability of Tregs are as follows:
� FoxP3: Essential for Tregs to suppress unwanted immune responses and maintain pregnancy.
� Helios: Supports Tregs in maintaining a tolerant immune environment, which helpsprotect the fetus.
Transforming growth factor-beta (TGF-b). A protein that regulates many cellular functions, including immune responses and cell growth.
It is important for maintaining immune tolerance and the function of Tregs.
ajog.org OBSTETRICS Original Research
Data availability
The datasets used and analyzed during
the current study are available from the
first author on reasonable request. n
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Author and article information
From the Department of Gynecology, Obstetrics and
Neonatology, General University Hospital in Prague and
First Faculty of Medicine, Charles University, Prague,
Czech Republic (Koucky, Lastuvka and Calda); Institute of
Medical Biochemistry and Laboratory Diagnostics, Gen-
eral University Hospital in Prague and First Faculty of
Medicine, Charles University, Prague, Czech Republic
(Lastuvka); Department of Physiology, Development and
Neuroscience, University of Cambridge, Cambridge,
United Kingdom (Cindrova-Davies); Institute of Immu-
nology and Microbiology, General University Hospital in
Prague and First Faculty of Medicine, Charles University,
Prague, Czech Republic (Hrdy); and Department of
Immunology, GENNET, Prague, Czech Republic (Cerna).
Received June 21, 2024; revised Nov. 3, 2024;
accepted Nov. 4, 2024.
The authors report no conflict of interest.
Z.L. is supported by MH CZ e DRO-VFN00064165.
This research was approved by the Ethics Committee
of the First Faculty of Medicine, Charles University, Pra-
gue, Czech Republic (No 481/19, S-IV). The study was
conducted in accordance with the principles of the
Declaration of Helsinki. Informed consent was obtained
from all participants.
Corresponding author: Pavel Calda, MD. Pavel.
Calda@vfn.cz
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mailto:Pavel.Calda@vfn.cz
mailto:Pavel.Calda@vfn.cz
http://www.AJOG.org
	Reduced number of regulatory T cells in maternal circulation precede idiopathic spontaneous preterm labor in a subset of pa ...
	Introduction
	Materials and methods
	Study design
	Blood sample collection and processing
	Flow cytometry analysis
	Gating strategy
	Statistical analysis
	Results
	Absolute numbers of CD4+CD25+FoxP3+ Tregs and pregnancy outcomes
	Different immunophenotypes of Tregs and pregnancy outcomes
	Comment
	Principal findings
	Results in the context of what is known
	Research and clinical implications
	Strengths and limitations
	Conclusions
	Data availability
	References
	Glossary