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THE EFFECT OF INTRAUTERINE AND OBSTETRIC
FACTORS ON IMMUNE RESPONSES AMONG
ADOLESCENTS BORN BY CAESAREAN SECTION
Hermina Jakupović
Master of Science thesis
Master´s Degree Programme
in Biomedicine
School of Medicine
Faculty of Health Sciences
University of Eastern Finland
21.11.2016
University of Eastern Finland, Faculty of Health Sciences, School of Medicine
Master´s Degree Programme in Biomedicine
Hermina Jakupović: The Effect of Intrauterine and Obstetric Factors on Immune Responses
Among Adolescents Born by Caesarean Section
Master of Science thesis; 34 pages, 19 appendices
Supervisors: Marjut Roponen1 & Leea Keski-Nisula2,3,4
1
The Inhalation Toxicology Laboratory, Department of Environmental and Biological Sciences,
University of Eastern Finland, Kuopio, Finland, 2Department of Environmental Health, National
Institute for Health and Welfare, Kuopio, Finland, 3Department of Obstetrics and Gynecology,
Kuopio University Hospital, Kuopio, Finland, 4Institute of Clinical Medicine, School of Medicine,
University of Eastern Finland, Kuopio, Finland.
21.11.2016
Keywords: birth cohort, Caesarean section, cytokine, immune development, multiplex
immunoassay, obstetric factors
Abstract
Intrauterine and obstetric factors have been suggested to be major determinants in shaping the
developing immune system. However, the long-term effects of these factors have not been studied.
Therefore, the aim of this study was to characterize if factors before and during the parturition serve
as predictors of aberrant immune responses among adolescents born by Caesarean section (CS). To
achieve this objective, peripheral blood mononuclear cells of adolescents were stimulated with
lipopolysaccharide,
combination
of
ionomycin
and
phorbol
myristate
acetate,
polyinosinic:polycytidylic acid and peptidoglycan and the spontaneous and stimulated production of
IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13 and TNF-α cytokines was analysed with Meso Scale
Discovery multiplex immunoassay system (N=79). Data on obstetric factors were collected
prospectively at the time of delivery and health information was assessed from the questionnaires and
clinical data. As main results, advanced cervical dilatation at the time of CS associated with higher
spontaneous and to some extent also with higher stimulated cytokine secretion. In addition, treatment
in the neonatal intensive care unit associated with lower cytokine levels produced in adolescence.
Similar, yet not always significant associations were observed between increased cytokine production
at teenage and factors such as duration of breastfeeding and culturable microbes in amniotic fluid. In
addition, some of the studied factors such as type of CS (elective, emergency, urgent) and neonatal
antibiotic treatment did not associate with altered immune functions. Based on the present findings,
it can be suggested that multiple factors before and during the CS and postnatal period are potent to
modulate the immune responses in adolescence.
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Introduction
The Caesarean deliveries have been increasing dramatically worldwide for several decades, thus
leading to an ongoing debate about the optimal CS rate. A total estimation of the proportion of CS
from all deliveries in the least developed countries is as low as 6 % whereas in developed countries
the number is dramatically higher, as high as 27.2 %, being substantially greater than WHO's
recommended 15 % in developed countries (Betrán et al, 2016; Cavallaro et al, 2016; World Health
Organization, 1985). This suggests that constantly increasing proportion of women is undergoing CS
of elective and prelabour nature without clear medical indications. Therefore, the increasing number
of CSs may lead to the escalating prevalence of their possible contributions to the immune health.
Significant rise in asthma and allergy prevalence has been noted worldwide during the past decades
(Bieber, 2008). However, more specific diagnostic criteria of asthma are probably behind the 30 %
diminish seen in asthma prevalence among children in Finland during the past decade, where it is
currently varying from 7 to 9 % (Harju et al, 2016). Escalating occurrence of atopic diseases and other
immunoglobulin E– mediated (IgE) allergies in both industrialized countries and urban areas suggests
the possible role of the hygiene hypothesis in the background of the related diseases. According to
the hygiene hypothesis, an excessively hygienic environment during the development of the innate
immune system predisposes to atopic diseases, highlighting the importance of the environmental
factors (Bieber, 2008; McLoughlin & Mills, 2011). However, recently adapted microbiota hypothesis
proposes that alterations in the composition of gut microbiota in early life drives the development of
mucosal immune tolerance, resulting in aberrant immune responses (McLoughlin & Mills, 2011).
As the mode of delivery has been suggested to be a major determinant of the composition of
microbiota, it plays a critical role in shaping the developing immune system. A great variation among
microbial exposure that neonate gets from the mother has been observed not only between vaginal
delivery and CS, but also between different section types (Dominguez-Bello et al, 2016; Olszak et al,
2012). Recent epidemiological studies have elucidated that elective CS plays a role in development
of aberrant short-time immune responses in new-borns, and more importantly predisposes to immune
diseases, such as allergies, asthma, different immune deficiencies, celiac disease and type 1 diabetes
(Cho & Norman, 2013; Dominguez-Bello et al, 2016; Penders et al, 2006). In addition, 20 % increase
in the risk of asthma onset has been recorded for children born by the means of elective and
emergency CS in recent meta-analysis (Huang et al, 2015). Therefore, the possible associations
between the section type and neonatal outcomes regarding the immune functions can be postulated.
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Recent scientific advances have suggested that aberrant innate immune- and metabolic maturation
may ensue from hindered gut microbiota, thus contributing to the development of non-communicable
disorders (Rautava et al, 2012). With elective CS, mostly performed before rupture of the fetal
membranes and advanced cervical dilatation, the normal exposure to the vaginal microbiota is
interrupted. Here, much-needed direct microbial contact between vaginal microbial flora and the
neonatal gut is prevented, mostly due to the intact membranes and closed cervix during the operation.
Together they prevent the vaginal microbes from reaching the amniotic fluid (AF) and uterus itself.
This leads to the microbiota that differs from one acquired during the normal delivery, hence resulting
in microbiota enriched in skin and hospital microbiota instead of microbiota in maternal vagina
(Dominguez-Bello et al, 2010). Whereas in non-elective CS, which are generally performed after
onset of labour, rupture of membranes (ROM) and with advanced cervical dilatation, maternal
microbiota easily enters intrauterine cavity even before birth. However, due to the intrauterine
bacterial colonization (and commonly prolonged colonization), intrauterine infections are more
frequent and therefore, may lead to higher need for neonatal antibiotic treatments (Keski-Nisula,
1997; Keski-Nisula et al, 2009b).
The microbial colonization of AF and inflammatory reaction, which are recognized by the appearance
of leukocytes in AF without any clinical signs of infection, have previously been shown to occur
during the normal parturition in KEISARI study (Keski-Nisula, 1997, pp. 70-71). When infant is born
by vaginal delivery, the active swallowing of the AF ensures that the gastrointestinal tract gets into
contact with the normal vaginal microbial flora. This happens also in those operations, where the CS
is done hours after the disruption of membranes (Keski-Nisula et al, 1997). If the AF membranes
have already been disrupted while the cervix dilates during the birth, the connection between the
vaginal microbial flora and intrauterine cavity is formed (Keski-Nisula et al, 1997). In cases where
the parturition is prolonged, fetal membranes have ruptured, the number of vaginal digital
examinations is increased or the internal monitoring has been prolonged, the intrauterine microbial
colonization is highly common (Keski-Nisula et al, 2009b). This microbial invasion of gut and
respiratory tract during the parturition and right after it, have been shown to play an essential part of
the development of normal and healthy gut flora during the first months after the birth. In addition,
they have been shown to influence on the immune responses later in life (Kranich et al, 2011;
McLoughlin & Mills, 2011). Therefore, the possible association between cervical dilatation, ROM,
culturable microbes in AF and immune responses can be speculated. In addition, potential impact of
infections or inflammations in amniotic membranes on immune functions can be postulated.
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The role of intrauterine microbial invasion in the evaluation of peripartal intrauterine and maternal
infection, has been previously demonstrated from this same birth cohort group. It was shown, that AF
microbial invasion was as common as 66 to 71 % among women (N=805) who underwent operations
after the onset of delivery and ROM and less common, when operated with still intact membranes
(range, 13-23 %). As reported earlier, no correlation was found between AF microbial colonization
by perinatal infections and potential pathogens among full-term infants delivered by CS with intact
membranes (Keski-Nisula, 1997, p. 65). In addition, no association has been recorded between
allergic sensitization in teenagers and AF quality (Keski-Nisula et al, 2010b).
Noteworthy, specific microbial growth at the time of birth seems to increase the risk of developing
asthma and allergies among offspring (Keski-Nisula et al, 2009b). This initiation is mediated through
still unclear inflammatory mechanisms (Cho & Norman, 2013). Surprisingly, it has been shown that
children who were born by CS have significantly less allergic sensitization at the age of one year
when compared to children born by vaginal delivery. The observed allergic sensitization seems to
associate with the duration of the labour (Keski-Nisula et al, 2010b). This may be explained by the
microbiota hypothesis (McLoughlin & Mills, 2011). Interestingly, it has been suggested that the
rarely occurring transmission of maternal vaginal microbiota seen in elective CS, might not even have
as significant role in asthma development among offspring as it has been thought (Keski-Nisula et al,
2010b). Furthermore, the dogma of sterile fetal life before the onset of labour has been challenged
(Rautava et al, 2012). A distinct composition and activity of unique microbiota harboured by
intrauterine compartment has been revealed. It has been observed, that certain features and specific
bacteria found in the neonatal meconium are similar to those found in placenta and AF, which
supports the hypothesised initiation of fetal gut microbiota already in utero (Collado et al, 2016).
Colonization resistance against pathogens provided by commensal species. is critically involved in
maintaining the systemic immune tolerance. Administration of antibiotics (AB) to the mother during
the pregnancy and parturition as well as to the infant, breast-feeding (BF) and environmental factors,
such as presence of older siblings, have previously been reported to influence the development of
gastrointestinal microbial flora (Kranich et al, 2011; Laursen et al, 2015; McLoughlin & Mills, 2011;
Molloy et al, 2012; Penders et al, 2006). Moreover, neonatal AB exposure has been associated with
increased risk of different diseases, such as asthma later in life. Most neonatal AB use has been shown
to be of empirical nature towards suspected infection, not proven infection, which is precarious
(Schulman et al, 2015). However, the previously reported results discussing the role of ABs are partly
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contradictory (Ly et al, 2011). It seems like bacterial diversity and taxonomic composition found in
the vaginal microbiota of mothers who have received ABs, do not differ from unexposed. This
indicates that the effects of perinatal ABs on the microbiome received by the infant need yet to be
clarified (Dominguez-Bello et al, 2016). The important role in establishment of the gut microbiome
by specific microbes found in breast milk, has been revealed recently (Lodge and Dharmage, 2016).
BF has been shown to protect from early-life infections, obesity and type 2 diabetes (Victora et al,
2016). This protective role of optimal BF period may also be behind the recommendation of exclusive
BF for 6 months after birth by WHO (Lang, 2015). However, the current understanding of the impact
of intrauterine and breast milk microbes as well as ABs in the gut colonization and priming of the
immune development, is relatively scarce and remains to be fully understood. Based on the current,
although partly even contrary data, relationship between BF- and AB treatments and long-term
immune responses can be hypothesized especially among children born by CS.
Moreover, it has been postulated that CS, gynaecological and respiratory infections in pregnancy, low
birth weight as well as prenatal exposure to smoking, may have a profound effect on infants’ immune
system and increase the risk of developing allergic diseases (Harju et al, 2016; Lodge & Dharmage,
2016). Maternal smoking has been proposed to modulate fetal immune development in dosedependent manner through activating or silencing fetal genes, thus affecting fetal growth, increasing
the risk for preterm birth and low birth weight and predisposing offspring to asthma. The prevalence
on maternal smoking in European countries varies from 7 to 25 %, being 16 % in Finland (Harju et
al, 2016). In recent meta-analysis, maternal smoking during prenatal or postnatal period has
associated with significantly increased risk of wheeze and asthma in offspring before the age of 11
years (Burke et al, 2012). Therefore, it can be suggested that the profound effect of maternal smoking
revealed by the previous studies, can be seen affecting the immune responses in adolescence as well.
Noteworthy, maternal cessation of smoking has been shown to have protective effect against asthma
and therefore, should broach a discussion in maternity clinics more thoroughly. This is, especially
since in Finland, the maternal smoking has been at the same level as in the late 1980s (Harju et al,
2016).
Previously, it has been adduced that season of birth increases the risk of asthma and wheezing in
offspring. Variations in cytokine responses and asthma risk observed between seasons of birth may
result from different exposures to sunlight and pollen during gestation. This may drive the maternal,
and further, prime infant’s cytokine environment towards Th2-polarized responses, thus alterng the
promotion of regulatory T cell (Treg) expansion by different vitamin D levels. Lately, lower cytokine
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levels have been observed in umbilical cord blood samples particularly among children born during
spring or winter when compared to children born during autumn (Keski-Nisula et al, 2010a).
Therefore, it can be postulated that this season dependent association can be observed also in
adolescence.
Recently, gender has also been noted as a potential factor affecting the immune development (KeskiNisula et al, 2009a; Keski-Nisula et al, 2010a; Markle et al, 2013). Sex hormone levels and microbiota
have been shown to exert potent effects on autoimmune disease development in genetically
susceptible individuals with a higher incidence of autoimmunity in female gender (Markle et al,
2013). In addition, it has been demonstrated that those adolescents whose mothers either had preeclampsia or placental abruption at the time of delivery, have more atopy and more severe form of
atopy as measured by the incidence of skin prick tests positive for 5 or more allergens. This increased
risk has been observed particularly in male adolescents earlier (Keski-Nisula et al, 2009a). Therefore,
gender-related differences in long-term immune responses can be speculated. Apart from the limited
data, also widely used prostaglandin induction has been shown to increase the risk of early-onset
persistent asthma among children (Keski-Nisula et al, 2010a). In contrary to the possible
predisposition to asthma, no allergic sensitization has been associated with the induction of the birth
process (Keski-Nisula et al, 2010b). Since the induction of labour is relatively common procedure
among deliveries, it is of great importance to study its possible long-term effects on immune
responses. The possible impact of induction can be speculated based on previous, although
incongruent data.
Regardless of the relatively limited information about neonatal outcomes after neonatal intensive care
unit (NICU) treatment, a wide variation among findings, such as candidiasis, central line associated
bloodstream infection (CLABSI) and sepsis have been documented. These observations have
concerned especially infants with low birth weight (Aliaga et al, 2014; Schulman et al, 2015). Since
the need for NICU admission has concerned especially low birth weight infants, the factors
influencing neonatal development as well as possible preterm deliveries may be of explanatory role
to the need for NICU admission. Noteworthy, it has been observed that CS and preterm delivery are
more common among primiparous and advanced aged mothers and their children seem to be affected
more frequently by low birthweight and NICU admission. The significantly higher rates of perinatal
death and fetal growth retardation have been documented among primiparous women (Kalayci et al,
2016). In addition, a relationship has been found between high birth weight (> 4000 g) and increased
risk for obesity from childhood to early adulthood, which again is linked to multiple diseases (Yu et
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al, 2011). Therefore, since there is a lack of previous studies concerning the possible long-term
impacts on the immune responses regarding these factors, the need for more relevant comparative
studies can be pinpointed. Furthermore, a puzzling question about the optimal conditions for the
immune development during pregnancy and infancy and their fine interplay can be raised.
A significant body of evidence suggests, that immune responses tend to develop mostly during the
first months after the delivery, which is the period when the predisposition to microbes and allergens
is highly increased. The fetus normally encounters microbes that colonize the maternal birth canal
and genital tract during the normal vaginal birth. The uterine cavity is usually free of microbes before
the onset of parturition and rupture of membranes, which protect the fetus against infections and
hinder microbial ascent during delivery (Bieber, 2008; Keski-Nisula et al, 2009b; Romero &
Korzeniewski, 2013). However, outside the uterus a newborn confronts the immunological challenges
and life in an antigen-rich environment. It has been shown, that the events during and before the
labour are potent to modify the neonatal immune system in addition to the birth process itself, which
launches the secretion of proinflammatory cytokines and acute phase proteins (Malamitsi-Puchner et
al, 2005; Marchini et al, 2000; Romero & Korzeniewski, 2013).
Mode of delivery has been proposed to influence immune development by several pathways. First,
by altering epigenetic regulation of gene expression. Secondly, by perturbing bacterial colonization
of gastrointestinal tract and finally, through different levels of adaptive stress caused by parturition
(Cho & Norman, 2013). Chorion as well as decidual and trophoblast tissues produce interleukin (IL)
10 cytokine during gestation and up to term pregnancy, also decidual mononuclear cells secrete IL10 and interferon gamma (IFN-γ) spontaneously. This secretion is occurring in addition to many T
helper (Th) 1 and 2 cytokines, especially after Toll-like receptor (TLR)-stimulation. Mode of delivery
however, may also have an impact on immune function, as seen especially among IL-10 and IFN-γ
responses previously (Keski-Nisula et al, 2010a).
The individual immune responses develop by the fine interplay between immune system and
environmental factors. Innate immune system tends to elicit rapid, non-specific responses against
different microbes, reported as being independent of T or B lymphocytes and attributable to activated
macrophages. On the other hand, adaptive immunity responds more sophistically against distinct
antigens in more targeted manner, characterised by T and B lymphocytes. T cells primarily provide
cell mediated immunity, whereas B cells and their antibodies mainly give rise to humoral immunity
(Sykes et al, 2012). Recently however, the discovery of the capability of innate cells such as
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granulocytes, natural killer (NK) cells and monocytes to display adaptive characteristics by building
cross-protection to secondary infections independently from B and T cells has been proposed. This
novel concept for which the term trained immunity has been introduced, has changed the dogma of
innate immunity of being non-specific. Moreover, the cells of the innate immunity not only are
responsible for providing the early defence against infections, but in addition trigger and drive the
immune system responses to be capable to respond to infections efficaciously (Quintin et al, 2014).
Interestingly, infants born by the means of prelabour form of CS have been found to harbour less
leukocytes, monocytes, NK cells and neutrophils in cord blood when compared to infants born by the
means of vaginal birth (Cho & Norman, 2013).
Recently, the gut microbiota has been recognized for its role in systemic immune regulation and
development. Therefore, alterations in the balance of gut microbiota may lead to dysbiosis, which has
been proposed to be the reason behind the increased prevalence of autoimmune and allergic diseases
(Kranich et al, 2011; McLoughlin & Mills, 2011). Bacterial colonization of the gut tends to complete
in around a week after labour, but bacterial numbers and species remain fluctuating remarkably during
the first few months (Palmer et al, 2007). At first infants tend to reflect immunological tolerance
educated by the mother by preferential induction of Treg lymphocytes but later, most of these
microbes to which newborn is initially exposed, become replaced resulting in distinctive microbiotas
harboured by individual adults. Indeed, also the individual genetics of the host may itself affect the
microbial colonization, which on the contrary may be altered via epigenetic regulation of gene
expression (Cho & Norman, 2013; Costello et al, 2009; Kranich et al, 2011; Ley et al, 2008; Mold et
al, 2008; Palmer et al, 2007).
Commensal species are capable to actively interact with the innate immune system and modulate the
adaptive one by activating distinct tolerogenic dendritic cells, thus driving Treg and Th1 cell
differentiation (Kranich et al, 2011; McLoughlin & Mills, 2011). This fine interplay enables the
microbiota to suppress the unwanted inflammation by actively inducing immune tolerance and
thereby, affect the susceptibility to disease. Moreover, early exposure to commensals can also repress
cells involved in the induction of inflammatory responses, which have long-term consequences for
the host capacity to develop inflammatory diseases (Olszak et al, 2012). Recently, the differences
within capabilities of commensal bacteria to trigger immune control mechanisms, such as regulatory
or effector responses, have been discovered (Molloy et al, 2012). Furthermore, the microbiota is a
key player in driving Th1 cell activation, which is important in correcting Th2-type skewed immune
system of newborns during the delivery. It has been postulated, that reduced suppressive activity of
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Treg cells may also maintain the Th2-polarized immunity in atopic subjects (Kranich et al, 2011;
McLoughlin & Mills, 2011). It seems that the diminished microbial burden leads to reduced TLRstimulation, which results in weaker Th1 responses and finally, polarizes the immune system towards
the Th2-biased one (Bauer et al, 2007; Horner, 2006).
As stated above, neonatal intestinal bacterial colonization primes the immune system and changes the
fine balance between Th1, Th2 and Th17 effector CD4+ cells. However, this deviant intestinal
colonization of infants born by the means of CS may prolong postnatal immunological immaturity
and prevent appropriate immunological priming, which leads to predisposition to increased risk for
immune diseases later in life (Cho & Norman, 2013; Kranich et al, 2011; McLoughlin & Mills, 2011).
These alterations, such as delayed microbial colonization with beneficial microbial load, might
interfere with the development of the immune system if the proper immunosuppressive Treg cells are
absent, thus resulting in Th1/Th2 imbalance (van Nimwegen et al, 2011). However, it is important to
distinct development of allergy and immune system from each other regardless of their close
relationship. Noteworthy, the priming of allergies tends to occur at an early stage, probably already
in utero during the normal immunologic maturation process due to the neonatal antigen specific
reactivity at birth (Jones et al, 2000; Kranich et al, 2011; Warner et al, 2000). Children with atopic
heredity have been shown to result in delayed maturation of cytokine responses, such as Th1 cytokine
IFN-γ secretion. This is possibly due to the lack of attenuation of Th2 responses as observed among
children, who become atopic later in childhood. This delayed immune development could also explain
the phenomena, where some atopic children become non-atopic later in childhood (Jones et al, 2000;
Kranich et al, 2011; Prescott et al, 1998).
The common conception about deficient and fully immature neonatal immune system has been
proved wrong. The fetal immune system has been shown to be capable of recognizing antigen-like
stimuli as early as during the second trimester of pregnancy (Holt & Jones, 2000; Kranich et al, 2011;
McLoughlin & Mills, 2013). The fine interplay between genes controlling the immune development,
microbial and other potential maternal exposure factors during pregnancy, such as diet and maternal
stress, may contribute to the development of the immune functions (Dunstan et al, 2003; Pfefferle et
al, 2010; Prescott et al, 2007; Wright et al, 2010). Different signals promote differentiation into
particular Th cell subsets provided by the distinct pattern of cytokines, which are elaborated upon
triggering innate immune sensors, such as TLRs, leading to regulation of an immune system that
tends to develop in an age-dependent manner (Chaudhry et al, 2011). All 10 TLRs expressed in
human, play a critical role in detection and recognition of pathogens and differences in signalling
10
between adult and infant immune systems. These differences may lead to increased infection
susceptibility. Moreover, TLRs can recognize bacterial, such as LPS (TLR4), parasitic or viral, such
as POLYI:C (TLR3) products (Guenca et al, 2013). These different products can be used as
stimulants, which are commonly used to study the dissimilar cytokine profiles produced by different
cells. Furthermore, the potent effects of the TLR signalling pathways have been highlighted in
protection against allergic diseases (McLoughlin & Mills, 2011).
The function of the immune system can be investigated by measuring cytokine production. Cytokines
are peptides produced by variety of cell types such as endothelial cells, monocytes, fibroblasts and
lymphocytes. Cytokines are used to modulate the microenvironment at the site and for the
communication between inflammatory and immune cells, which control both local and systemic
immune responses, healing, inflammation and response to injuries (Savinko, 2013; Whicher & Evans,
1990). Cytokines can be classified according to several different characteristics. One classification
system is to categorize their functions into pro-inflammatory, non-inflammatory and regulatory
cytokines, where different Th cell subsets secrete certain cytokine patterns predominantly. Th1/Th2
polarization is widely used despite of the fact they are not representing two distinct CD4+ T cell
subsets in reality. In this division, Th1 cells predominantly secrete IFN-γ, tumour necrosis factor α
(TNF-α) and IL-2 cytokines, whereas Th2 cells tend to secrete cytokines that intensify the secretion
of IgE antibodies from B-cells, such as IL-4, IL-5, IL-9 and IL-13. The most important cytokine
secreted by Th2-cells is IL-4, which is the cause for the observed Th2-biased immune system in
allergic individuals and which in addition, slows down the development of Th1 cells and causes the
switch of isotype of B-cells to IgE. IL-5 is important regulator of function and development of
eosinophils, while IL-13 tends to activate the same signalling paths as IL-4. These are also two
characteristics of an allergic immune response. In addition, Treg cell associated cytokines, such as
IL-10 and IL-2, either suppress or regulate immune responses (Bieber, 2008; McLoughlin & Mills,
2011; Savinko, 2013). Pro-inflammatory cytokines, such as IL-1, IL-6, IL-8, TNF-α are associated
with sepsis and pathogenesis of inflammation (Martin et al, 2001; Whicher & Evans, 1990). However,
as mentioned above the secretion of cytokines from T-cells is not as precise as it is usually presented.
Recently, it has been noted that T-cells are capable to modify the cytokine pool they secrete by either
synthesizing new cytokines in a way, which does not effect on previous cytokine secretion or by
simply redifferentiating into different Th cell subtype, such as from Th17 cells into Th1 cells
(Savinko, 2013).
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Several differences have been found in blood biomarkers following CS compared to vaginal birth.
Neverthless, the effect of parturition and different birth-related factors on the blood biomarkers, such
as whole blood, peripheral blood mononuclear cells (PBMC) and cord blood cytokine production is
still relatively unknown. In labours, where infant has been born by the means of prelabour CS, the
total leukocyte as well as their subpopulation counts have been lower in cord blood. The activity of
leukocytes and their proinflammatory cytokine release of IL-4, IL-1β, IL-6 and TNF-α seem to be
lower, thus hindering overall immune cell functions. However, not all results are consistent with lack
of observed differences in TNF-α concentrations of new-borns born by CS compared to vaginal
delivery (Cho & Norman, 2013).
As stated previously, newborns tend to express Th2-polarized immune responses, which may be
resulting from the maternal Th2-balanced cytokine environment needed for successful pregnancy
(Kranich et al, 2011; Prescott et al, 1998; Sykes et al, 2012). Successful pregnancy requires a degree
of immunosuppression, whilst on the other hand, needs to maintain immune function to fight off
infection caused by the fetus (Kranich et al, 2011; Sykes et al, 2012). One mechanism is the proposed
switch from the Th1 cytokine profile towards the Th2 profile. If the maternal proinflammatory (TNFα) and Th1 (IFN-γ) cytokine responses increase, they may lead to pre-term delivery or even abortion
(Daher et al, 2004; Sykes et al, 2012; Vitoratos et al, 2006). The foetomaternal interface is therefore
surrounded by high concentration of Th2 cytokines during the pregnancy. These cytokines involved
in the pregnancy maintenance may lead to prevention of the ability of Th1 cells to damage immune
responses. Th1/Th2 cytokine levels have been found to be lower in blood from children with Th2 bias
at birth. In addition, the role of maternal inflammatory parameters such as IL-10 and TNF-α in
pregnancy, influence the corresponding cytokines blood levels in children at the age of one. Also high
levels of maternal IgE during pregnancy have been reported to correspond with ones measured from
children at birth and at the age of one (Herberth et al, 2011). This indicates that antenatal period might
be involved in priming the immune development among offspring, thus highlighting the importance
of understanding their immunomodulatory role.
The aim of this Master's thesis was to elucidate the postulated long-term effects of CS and its role as
a predictor of compromised immune functions and to investigate if and how different intrauterine
infections and obstetric factors affect immune functions in adolescence. To reach these objectives, it
was essential to investigate whether obstetric factors and different clinical variables determine the
production of unstimulated and TLR-induced production of cytokines among adolescents born by CS
at Kuopio University Hospital during 1990-1992. Secreted cytokine levels were analysed from
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PBMC supernatants by multiplex immunoassay system. To address our hypothesis, the advanced
cervical dilatation at the time of CS and NICU treatment associated with aberrant immune functions
in adolescence as main results. Additional impact on long-term immune functions concerned factors
such as maternal smoking, culturable pathogens in AF, breastfeeding, gender and season of birth.
These findings may reflect the importance of fetal and neonatal regulation on long-term immune
functions especially since there is an absence of relevant comparative studies in the subject.
Therefore, these compelling results represent novel ground for immunology research and strongly
argue in favour of their scientific and clinical relevance. The present study was undertaken as a part
of a larger national Caesarean delivery birth cohort study (KEISARI study).
Materials and Methods
Study Characteristics
The initial study population consisted of 805 consecutive mother-child pairs from the national
Caesarean delivery birth cohort study (KEISARI study) (Fig.1). Children were born by CS at Kuopio
University Hospital during 1990-92. The ethical approval was obtained from The Research Ethics
Committee, Hospital District of Northern Savonia and written informed consent was obtained from
parents. All needed information was gathered by data collection form. To ensure the anonymity and
protection of personal data, the national legal requirements regarding confidentiality were followed.
Infants were born after singleton gestations, where mean gestational age at the time of delivery was
39 weeks (range, 37-43 weeks) and mean maternal age was 30 (range, 18-42). Microbe samples from
the AF or amniotic cavity were taken during CS prospectively from 792 (98 %) women and results
were available for 702 (87 %) women. In addition, all other essential health information, such as
duration of the birth and pregnancy, possible AB treatments administered to mother, CRP-levels,
possible fever, cervical dilation at the time of the section, cause and quality of the CS, the quality of
the AF, time elapsed from rupture of membranes (ROM) to the birth, possible need of NICU or AB
treatments to infants and their infections and infection suspicions were collected during and after the
parturition respectively (Keski-Nisula et al, 2009b).
When children were 15 to 17 years the postal questionnaire based on International Study of Asthma
and Allergies in Childhood’s standardized questionnaire (Pekkanen et al, 1997) was sent to mothers
(N=749, 93 %) of which the final questionnaire data was available for 545 (68 %) children. Later on
2006, children living in the area of the Hospital District of Northern Savonia (N=688, 86 %) were
invited to clinical examination. In total 382 (48 %) children participated the examination including
13
skin prick tests towards 10 most common allergens (timothy, mugwort, birch, cat, dog, cow, horse,
warehouse- and house dust mite and cockroach). A blood sample was taken to measure the allergic
and hormonal state of the child by screening different parameters, such as estradiol-, free estrogen-,
progesterone-, testosterone- and IgE -levels. In addition, parents filled in questionnaires concerning
children’s health, medication, parental smoking etc. (Keski-Nisula et al, 2009b). Another blood
sample was collected from 111 (14 %) children for PBMC isolation and immunological tests.
In this Master’s thesis, the associations between the intrauterine and obstetric factors and immune
responses among teenaged offspring were studied. This objective was achieved by analysing
cytokines released from cryopreserved human white blood cells in response to various stimuli.
Finally, samples from 79 adolescents were selected among subjects with sufficient number of PBMCs
and full term birth (≥37 weeks). Table 1. shows the characteristics of the subjects included in the
present study. Informed consent was obtained from all subjects.
Figure 1. Flow chart of previously done sample and data collection and methods used in the present study. Obstetric,
clinical and intrauterine data were obtained during and after parturition. In 2006, postal questionnaire data and PBMC
samples (N=111) from adolescents during clinical examination were gathered. In the present thesis, PBMC samples were
further stimulated and analysed (N=79) to study the associations between intrauterine and obstetric factors and longterm immune responses among adolescents born by Caesarean delivery at Kuopio University Hospital (KUH).
14
Table 1. Characteristics of obstetric factors and clinical variables among 79 study parturients and
teenagers undergoing Caesarean delivery.
OBSTETRIC FACTORS AND CLINICAL VARIABLES
MATERNAL AGE, YEARS
GESTATIONAL AGE AT OPERATION, WEEKS
PRIMIPAROUS
SECTION TYPE AT THE CAESAREAN DELIVERY
ELECTIVE
EMERGENCY
URGENT
INDUCTION OF LABOUR
RUPTURED AMNIOTIC MEMBRANES BEFORE OPERATION
CERVICAL DILATATION AT OPERATION
0-1 CM
2-5 CM
≥ 6 CM
TRANSPARENT AMNIOTIC FLUID AT LABOUR
CHANGES/INFLAMMATION IN HISTOLOGICAL SAMPLES OF THE AMNIOTIC MEMBRANES
BACTERIAL CULTURE OF THE AMNIOTIC FLUID
NEGATIVE
APATHOGENIC
PATHOGENIC
NEONATAL INTENSIVE CARE UNIT TREATMENT
NEONATAL ANTIBIOTIC TREATMENT
GENDER OF THE CHILD: GIRL
NEONATAL WEIGHT
> 4000 G
3200-4000 G
< 3200 G
BREASTFEEDING FOR
0-3 MONTHS
4-9 MONTHS
≥ 10 MONTHS
NEONATAL BMI
> 23
20-23
< 20
MATERNAL SMOKING DURING PREGNANCY
ANTIBIOTIC TREATMENT AT PREGNANCY
SEASON OF BIRTH
AUTUMN
WINTER
SPRING
SUMMER
DOCTOR DIAGNOSED ASTHMA IN ADOLESCENCE
DOCTOR DIAGNOSED ATOPIC RASH IN ADOLESCENCE
30.0
(18-42)
39
39
(37-43)
(51 %)
28
18
33
17
49
(35 %)
(23 %)
(42 %)
(23 %)
(62 %)
27
29
23
57
19
(34 %)
(37 %)
(29 %)
(75 %)
(26 %)
39
13
8
22
13
32
(65 %)
(22 %)
(13 %)
(28 %)
(15 %)
(41 %)
20
36
23
(25 %)
(46 %)
(29 %)
22
31
20
(30 %)
(43 %)
(27 %)
18
32
24
11
28
(24 %)
(43 %)
(33 %)
(14 %)
(38 %)
17
16
26
20
14
21
(22 %)
(20 %)
(33 %)
(25 %)
(18 %)
(27 %)
Data are presented as means (range) or number (%).
15
Cell Culture
Since the condition of PBMC samples (N=111) of adolescents was the main critical point of success,
the samples were evaluated and thawing and freezing techniques were tested in a pilot study. Used
thawing method has been previously proved to be appropriate and restore the cells well (Kääriö et al,
2016a; Kääriö et al, 2016b). Samples were thawed by slow thawing method and suspended to 10 %
human AB - medium (RPMI 1640 +1 % glutamine + 10 % human AB serum + 1 %
antibiotic/antimycotic) (Innovative Research, Novi, USA) and washed with medium (RPMI 1640 +
1% L-glutamine + 1% antibiotic/antimycotic) (Invitrogen, Grand Island, USA). Determination of the
numbers and viability of PBMCs (mean, 91.2 %, ±SD 5.3 %) was carried out by trypan blue exclusion
method. Finally, samples were centrifuged for 10 min in RT (1300 rpm) and resuspended in 1x106
cells/ml in 10 % human AB - medium.
Stimulation
The PBMC samples were stimulated as duplicates with lipopolysaccharide (LPS, TLR4) (0.1 µg/ml),
combination of ionomycin (IO) (1.0 µg/ml) and phorbol myristate acetate (PMA) (5.0 ng/ml) (nonspecific stimulation), olyinosinic:polycytidylic acid (POLYI:C, TLR3) (50.0 µg/ml) and
peptidoglycan (PPG, TLR2) (10.0 µg/ml) (all stimuli from Sigma Aldrich Company, St. Louis, USA)
on 96-well plates in incubator (37° C, 5 % CO2), for 5 and 24 hours. After the stimulation, plates
were centrifuged (1800 rpm, 10 min), 220 µl of supernatant was transferred from each sample to deep
well plates and stored at -80° C for further analysis.
Meso Scale Discovery Multiplex Immunoassay
Meso Scale Discovery MULTI-SPOT Assay System Proinflammatory panel 1 (human) V-PLEX Kit
(together with Meso Scale Discovery (MSD) Sector Imager™ 2400A and Discovery Workbench®
3.0.18 software (MSD, Rockville, MD, USA) was used in the analysis as described earlier (Huttunen
et al, 2014). Samples from 79 subjects (5h time point) were analysed according the manufacturer´s
instructions by using the reagents provided with the kit. The limits of detection were: for IFN-γ, 13.212300 pg/ml; IL-1β, 1.8-5040 pg/ml; IL-2, 1.2-13900 pg/ml; IL-4, 0.47-2100 pg/ml; IL-6, 1.84-7580
pg/ml; CXCL8, 1.22-5070 pg/ml; IL-10, 1-3230 pg/ml; IL-12p70, 2.36-4100 pg/ml; IL-13, 8.64-5070
pg/ml and TNFα, 2.32-3160 pg/ml. Non-detectable values were set to the lower or upper detection
limits. Unstimulated and TLR-stimulated IL-4 and IFN-γ and unstimulated, TLR- and PMAIOstimulated IL-12p70, CXCL8 were excluded from the analyses due to high prevalence of values
below or above the limits of detection.
16
Data Analysis
Power calculations were performed and outliers detected and excluded. Main outcomes were the
concentrations of unstimulated and stimulated cytokines. Cytokine variables were log-transformed to
fulfill assumptions of normal distributions in parametric testing. Associations between cytokines and
grouping variables (e.g. the mode of Caesarean delivery, induction of labour, existence of intrauterine
microbial growth, neonatal intensive care) were analysed using T-test or linear regression and
reported as geometric mean ratio (GMR) ± 95 % confidence interval (CI), respectively. Possible
confounding factors, such as maternal and paternal allergic diseases and education were tested, but
GMRs with adjustments were not reported as the change in the estimated effects was less than 10 %
and because they had no impact on the main cytokine results. Statistical significance was set at p <
0.05 and statistical trends were defined by 0.10 < p < 0.05. Softwares SPSS Statistics (Version 23.0.
Armonk, NY: IBM Corp.) and GraphPad Prism 5 (Inc. San Diego, CA, USA) were used.
Results
Prelabour form of Caesarean section and NICU treatment may lead to impaired cytokine responses
later in life
Advanced cervical dilatation (6 cm or more indicating the active phase of the labour) at the time of
CS associated with higher spontaneous cytokine secretion at teenage (Fig. 2). All studied cytokines
except for regulatory cytokine IL-10 were statistically significantly increased as compared to
teenagers who were delivered before the onset of labour (cervical dilatation 0-1 cm). Similar but not
always statistically significant associations were seen between cervical dilatation and stimulated
cytokine secretion. In addition, NICU treatment was associated with the lower spontaneous and TLRinduced cytokine secretion in adolescence (Fig. 3).
17
Figure 2. Association between cervical dilatation at operation and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood mononuclear cells
(PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D) olyinosinic:polycytidylic
acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated cytokine values relative to
unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in 0-1 cm (N=27) compared with 2-5 cm
(N=29) and ≥ 6 cm (N=23) dilatations. Reference line: Cervical dilatation from 0 to 1 cm. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10;
**p < 0.05.
18
Figure 3. Association between neonatal intensive care unit (NICU) treatment and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in no (N=57)
compared with yes (N=22). Reference line: No NICU admission. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
19
Associations between other birth-related factors and cytokines
Surprisingly, the section type did not associate with spontaneous nor stimulated cytokine production
in statistically significant manner (Appendix Figure A1). However, teenagers born by the means of
induced labour expressed decreased spontaneous proinflammatory cytokine levels, where
proinflammatory cytokine IL-6 showed statistically significant and others trendwise associations
(Appendix Figure A2).
Trendwise associations were seen between ruptured membranes (ROM) and spontaneously increased
proinflammatory IL-1β and LPS-stimulated increased production of TNF-α cytokines (Appendix
Figure A3). On the other hand, histologically normal amniotic membranes were associated with the
increased IL-6 cytokine secretion after LPS-stimulation and increased IL-13 levels after PPGstimulation, when compared to histological changes and inflammation in amniotic membranes
(Appendix Figure A4). In addition, no associations were observed between cytokine secretion at
teenage and the quality of the AF at the labour (Appendix Figure A5). However, the presence of
culturable microbes in AF seemed to increase cytokine secretion at teenage when compared to
negative bacterial cultures (Appendix Figure A6). Though the only statistically significant increase
was observed between IL-13 secretion after PPG-stimulation and presence of culturable pathogens in
AF.
Associations between neonatal antibiotic treatment, breastfeeding and cytokines
Surprisingly, neonatal AB treatment was not associated with the altered function of the immune
system later in life in our study (Appendix Figure A7). BF for 4 to 9 months on the other hand seemed
to increase stimulated cytokine secretion at teenage in contrast to the decreased secretion observed
among BF for longer than 10 months when compared to BF of none to 3 months (Appendix Figure
A8).
Relationship between other studied obstetric factors and cytokines
Maternal smoking during pregnancy associated with altered immune responses in adolescence
(Appendix Figure A9). Unstimulated and TLR-stimulated decrease of secreted cytokines was
observed among adolescents of non-smoking mothers, but only proinflammatory TNF-α cytokine
levels were statistically significant. Surprisingly, maternal AB treatment did not associate in
statistically significant manner with the production of cytokines at adolescence (Appendix Figure
A10).
20
Adolescents whose neonatal weight was less than 4000 g appeared to have increased spontaneous
and POLYI:C-stimulated regulatory IL-2 cytokine secretion when compared to neonatal weight of
more than 4000 g (Appendix Figure A11). Nevertheless, body mass index (BMI) seemed not to be
associated with altered cytokine production at teenage (Appendix Figure A12).
First childbirth seemed to lead to increased cytokine secretion among teenagers in general when
compared to children whose mothers have had previous labours, but the associations were not
statistically significant (Appendix Figure A13). Season of birth appeared to increase the secretion of
spontaneous proinflammatory cytokines and decrease the overall production of stimulated cytokines
in adolescence when compared to autumn (Appendix Figure A14). However, only adolescents born
during winter were shown to express decreased levels of IL-2, IFN-γ and IL-4 cytokines after
PMAIO-stimulation, yet only IL-4 levels were associated with the season of the birth in a statistically
significant manner. In addition, decreased cytokine production was observed in females when
compared to males. Female gender seemed to associate especially with decreases proinflammatory
cytokine secretion among teenagers (Appendix Figure A15). However, no statistically significant
associations were observed systematically among cytokines mentioned above.
Doctor diagnosed atopy and asthma associated with aberrant cytokine production at adolescence
Doctor diagnosed asthma at teenage associated with decreased production of proinflammatory
cytokines following TLR-stimulation (Appendix Figure A16). In addition, a relationship between
asthma and PMAIO-stimulated elevated levels of IL-2, IL-4 and IFN-γ was observed. In contrary to
asthma, doctor diagnosed atopic rash associated with increased production of cytokines following
TLR-stimulation (Appendix Figure A17).
Discussion
In recent years, a lot of effort has been put on research involved in factors affecting immune
maturation. In the present study, the aim was to identify the obstetric and intrauterine factors
orchestrating the possible mediation of the long-term effects of CS on development of the immune
system. To achieve this objective, the possible relationships between the early-life factors and
cytokine productions among 79 adolescents (age range, 15-17 years) from the birth cohort study
KEISARI were studied. To address our primary hypothesis, present findings postulate that the
development of immune system is influenced by CS and multiple obstetric and intrauterine factors as
discussed previously. This influence is mediated by altering the functions of sensitive immature
21
immune system and its development before and after the onset of birth. As observed herein, obstetric
factors, such as NICU admission and advanced cervical dilatation during CS (indicating active phase
of labour), are potent to modulate the development of immune system. There are no previous
longitudinal comparative studies between intrauterine and obstetric factors and long-term immune
functions from adolescents born by the means of CS. Moreover, there is a lack of literature that
discusses the relationship between CS or obstetric factors and altered immune function later in life.
Therefore, this scarcity regarding relevant data stood out as something that should be addressed more
carefully in future studies.
The preceding speculations regarding the capability of birth process itself and microbial exposure
around and during the onset of birth in modifying neonatal immune system, may be of explanatory
nature to these findings. Since it is demonstrated that prolonged and advanced cervical dilatation
present the maternal microbes with the opportunity to descent into uterine cavity, they are capable of
affecting the initial immune priming (Keski-Nisula et al, 2009b). Advanced cervical dilatation at the
time of CS indicates that the birth process has already been induced by yet unknown mechanisms.
This means that different changes, such as hormonal and microbial, have already initiated the
immunological priming, which occurs in vaginal delivery naturally. In the present data, this was seen
as the capability of immune system to recognize different environmental stimulants in more advanced
manner, thus leading to higher cytokine secretions observed. Hence, it can be speculated that the birth
process itself, especially in advanced phase, serves as an essential initiator for normal immune
development.
Regardless of the relatively limited information about neonatal outcomes after NICU treatment, it is
presented as potential factor affecting neonatal outcomes (Aliaga et al, 2014; Schulman et al, 2015).
However, no studies regarding the relationship between the NICU admission and immune functions
have been conducted, hence introducing us with the challenges in ensuring a normal initial immune
priming during crucial period, especially since the present compelling data postulated a strong
association between NICU treatment and immune responses at adolescence. In contrast to cervical
dilatation, this was seen as the weaker cytokine production observed after NICU admission, possibly
causing the aberrant immune reactions to environmental stimulants. It can be speculated that the
isolation during NICU treatment may prolong the postnatal immunological immaturity and prevent
the appropriate immunological priming by diminishing the important environmental contact,
especially the one obtained from mothers during nursing, which may even lead to suboptimal BF.
Although greater insight is needed, it is tempting to speculate that environmental effect can be partly
22
explained by modulation of the microbiome composition at skin and mucosal surfaces. This
postulation is also supported by the hygiene and microbiota hypotheses discussed previously.
The present findings demonstrated, that distinct obstetric and intrauterine factors along with certain
clinical variables were associated with the function of immune system later in adolescence. The most
prominent findings suggested that NICU treatment and advanced cervical dilatation are potent to
drive long-term immune functions as hypothesised. These compelling data about the
immunomodulatory potent harboured by NICU treatment and birth process itself (as seen with
advanced cervical dilatation) may thus reflect the importance of fetal regulation before, during and
after parturition by genetic, maternal and other environmental factors in development of complex
network of immune functions, seen as cytokine production. Noteworthy, since there are no previous
data from the topic, the present findings have important implications in enhancing understanding of
their possible long-term impacts on immune functions. Therefore, these compelling data need to be
further confirmed in future studies.
The possible correlations between studied factors should be considered when interpreting present
results. That is, the advanced cervical dilatation by the time of CS may indicate that the clinical
decision about the need of conducting the emergency CS has been inevitable. Furthermore, this may
have led to NICU admission and potentially to the AB-treatment. Noteworthy, NICU admission and
advanced cervical dilatation predicted cytokine secretion in adolescence also independently (data not
shown). This postulates their role not only in an early-life immunomodulation, but also potential longterm effects up to early adulthood.
The relationship seen between CS and diseases in children and adults are argued to be of direct effect
(Cho and Norman, 2013). However, since CS is mostly considered as a contributor to preterm
delivery, which can cause adherent immune functions and diseases by itself, it should be noted that
increased birth rates of preterm nature have in most cases been recorded to occur among CS,
especially in the late preterm period. However, in present birth cohort the parturients underwent full
term birth where gestational age during the CS was ≥ 37 weeks, which may explain the lacking
association between section type and immune functions in teenage, unlike it was initially
hypothesized. Apart from the limited data, the observed spontaneously decreased proinflammatory
cytokine levels after the induction of the labour were incongruent with the previous findings, where
prostaglandin induction increased the risk of early-onset persistent asthma but did not associate with
allergic sensitization among children (Keski-Nisula et al, 2010a; 2010b). In addition, cord blood
cytokine levels IFN-γ, unlike IL-5 and IL-10, are previously associated with the mode of delivery and
23
inversely associated with induction of the labour (Keski-Nisula et al, 2010b). However, these data
cannot be reflected with the present data as such. These observations could possibly indicate that
unlike it has been previously reported, mode of delivery is not associated with altered immune
function in adolescence among teenagers born by the means of full term, elective CS. All in all, this
speculated gap in the knowledge regarding the possible differences between immune functions among
adolescents born by the means of full term and pre-term CSs represents itself as a novel challenge for
studies yet to come.
As it was postulated, ROM and culturable microbes in AF associated with the long-term immune
responses among offspring born by CS. In the present study, trendwise associations between ROM
and cytokine secretion was partly incongruent with the previous findings, where no association was
found between the type of fetal ROM and neonatal outcomes. As discussed, no correlation is recorded
previously between the AF microbial colonization and potential pathogens among full-term infants
with intact membranes (Keski-Nisula, 1997, p. 65). These results are discrepant with present findings,
where the presence of culturable microbes, especially pathogens in AF, seemed to increase cytokine
secretion at teenage when compared to negative bacterial cultures. This may be explained by the
previously suggested difference between elective and non-elective CS. However, present study is in
line with the previously noted lacking association between AF quality and immune functions later in
life (Keski-Nisula et al, 2010b). On the other hand, changes and inflammation in the amniotic
membranes were associated with the increased proinflammatory IL-6 cytokine secretion after LPSstimulation and increased IL-13 levels after PPG-stimulation. This supports the finding that certain
features and specific bacteria found in the meconium are similar to those found in placenta and AF,
thus supporting the previously hypothesised initiation of fetal intestine and further immune priming
already in utero (Collado et al, 2016).
Interestingly unlike it was postulated, no association was recorded between neonatal nor maternal AB
treatment and long-term functions of immune system in offspring according to present findings.
These results are not in line with some of the previous results, indicating that intestinal microbiota
perturbation caused by AB exposure in the perinatal and postnatal period is able to modulate the
host’s immune system (Kranich et al, 2011; Laursen et al, 2015; McLoughlin & Mills, 2011; Molloy
et al, 2012; Penders et al, 2006). However, as mentioned earlier also contrary data regarding AB
treatments is available that supports present findings (Ly et al, 2011). Therefore, further studies
should be carried out so that the long-term effect of AB treatment could be fully elucidated. It would
be of great importance to distinguish ABs by their classification, duration and timing of the treatment
24
in further study designs to clarify, whether the incongruent data regarding the effect of ABs could be
explained. However, while it is justified to use ABs against serious bacterial infections, it would be
of great importance if narrow spectrum ABs could be used judiciously to target specific pathogens
and minimize the perturbations regarding the gut microbiota development. The premise for such
proposition comes from previous studies. However, more effort should be put on research studying
the possible relationship between immunomodulatory outcomes and selected ABs so that more
detailed data regarding this matter could be recorded.
The infant gut microbiome has come to prominence as the mechanism postulated to imprint the
relationship between several perinatal exposures and allergy (Lodge and Dharmage, 2016). It was
observed that BF for 4 to 9 months increased, while BF for 10 months or longer decreased the
stimulated cytokine secretion at adolescence, thus supporting the initially postulated effect of BF on
immune functions in teenage. Statistically significant associations recorded between BF for 4 to 9
months and increased regulatory cytokine levels, were supported by previous research, thus
reinforcing the critical role of BF in the subsequent child immune development (Lang, 2015).
Therefore, the important role of distinct microbes found in breast milk previously stands out as an
essential step in establishment of the healthy gut microbiome (Lodge and Dharmage, 2016). This is
important especially among offspring born by the CS (especially preterm and elective CS), since the
immunological priming during the crucial period is delayed. As discussed, this deviant intestinal
colonization may prolong postnatal immunological immaturity and prevent appropriate
immunological priming, which leads to predisposition to increased risk for immune diseases later in
life (Cho & Norman, 2013; Kranich et al, 2011; McLoughlin & Mills, 2011). Despite the fact, that
the clinical significance of these obstetric factors remains unknown these observations may be of
serious implications in clinics, since a substantial number of infants are subjected to insufficient or
lacking BF and interventions during pregnancy, labour or directly in the immediate neonatal period.
Present outcome supported the WHO recommendation of exclusive BF for the first 6 months after
labour and highlighted the need for renewed effort to educate mothers about the benefits of BF.
Finally, means to support healthy microbial contact in neonates and infants requiring AB or NICU
treatment are needed.
As it was hypothesised, birth weight and maternal smoking associated with the long-term immune
functions. Neonatal weight of less than 4000 g appeared to associate with increased spontaneous and
POLYI:C-stimulated regulatory IL-2 cytokine secretion at adolescence in contrary to BMI.
Previously, a relationship is found between high birth weight (> 4000 g) and increased risk for obesity
25
from childhood to early adulthood. In addition, low birth weight infants are more prone to NICU
admission, which suggest that more attention should be addressed towards factors affecting neonatal
weight (Harju et al, 2016; Lodge & Dharmage, 2016). These results suggest that birth weight may
serve as a mediator between prenatal influences and immune functions later. Previous data are
consistent with the present assumption suggesting that birth weight as well as prenatal exposure to
smoking may have a detrimental effect on infants’ immune system also independently. Since maternal
smoking affects fetal growth, it is increasing the risks of low birth weight infants and preterm birth,
which in turn are also known to predispose to asthma. Supported by the previous findings, the
production of cytokines, especially proinflammatory TNFα was decreased among children of
smoking mothers. In addition to TNFα, also IL-6 and IL-10 secretion has been observed to decrease
previously, but these results were obtained from cord blood monocyte cell line (Noakes et al, 2006).
In the present study, 11 % of the mothers were smoking at some point of the pregnancy. However,
the fact that 4 % of the study parturients were smoking throughout the entire pregnancy, highlighted
the need for renewed effort to educate mothers about adverse effects of smoking and to educate them
about the positive effects of the cessation of smoking, which is shown to have protective effect against
asthma in offspring.
Moreover, as it was expected, an association was recorded between the season of birth, especially
winter and increased unstimulated proinflammatory cytokines and overall decrease of TLRstimulated cytokine production in teenagers when compared to children born during autumn. This
result is supported by the previously recorded variations in cytokine responses and asthma risk
(Keski-Nisula et al, 2010a). The effect of seasons, especially winter or spring may result from
different exposures to sunlight and pollen during gestation, which is postulated to drive the maternal,
and further, infant’s cytokine environment towards Th2-biased, thus altering the promotion of Treg
expansion. The premise for such assumption comes from previous studies and corroborates with the
hypothesized need of vitamin D supplementation to mothers as potential form of preventive therapy.
In addition, first childbirth seemed to lead to increased cytokine secretion among teenagers in general
when compared to children whose mothers have been in labour previously. These observations were
in respect with the previously reported association between primiparity of advanced age mothers and
increased risk of CS and preterm delivery, low birthweight and NICU admission (Kalayci et al, 2016).
Maternal parity may be a potent modulator of immune development among offspring especially
among mothers of advanced age. However, the present data are hard to decipher since no previous
data discussing the associations between maternal parity and immune responses among adolescence
26
has been published. Noteworthy, decreased proinflammatory cytokine production at teenage was
observed among girls as expected. Despite that no statistically significant associations were observed
among all cytokines, they are in line with the previous studies where gender is acknowledged as a
potential factor that affects immune development (Keski-Nisula et al, 2009a; Keski-Nisula et al,
2010a; Markle et al, 2013). This could be explained by the already hypothesized capability of sex
hormone levels and microbiota to exert potent effects on autoimmune disease development in
genetically susceptible individuals with a higher incidence of autoimmunity in female gender.
However, further studies are needed to fully establish the gender- and parity-associated conclusions
regarding their possible immunomodulatory potent.
The early life microbial exposures have been noted to be able to protect against asthma and allergy
previously. This process has been suggested to be mediated by the initiation of early activation of the
innate immune system and development of regulatory immune responses (Smits et al, 2016). Atopy
and asthma have been linked to aberrant immune functions by measuring cytokine responses from
different biological fluids. Herein, the hypothesized contribution of obstetric factors in affecting the
immune development is in agreement with previous findings regarding their immunomodulatory
potent. Although the sample size was relatively small, aberrant immune functions associated with
doctor diagnosed atopy and asthma. Multiple factors, including the CS itself, are involved in selection
of a microbiota that may lack the diversity and resilience necessary for development of balanced
immune responses. It has a fundamental role on the induction, training and function of the host
immune system. Therefore, optimal immune system–microbiota alliance allows the protective
responses to pathogens and the maintenance of regulatory pathways involved in the maintenance of
tolerance to innocuous antigens. This phenomenon regarding the middling priming of innate immune
system is proposed to be accounted for some of the rise in autoimmune and inflammatory prevalence
thus acknowledging the possible implications also by the present study not only from the scientific
point of view, but also in future health care.
In this birth cohort, we had a rather unique study population, which provided an opportunity to
investigate and elucidate the impact of intrauterine microbes, infections and other obstetric factors on
immune responses like never before. In addition, used multiplex immunoassay method and
longitudinal approach provided this thesis with the opportunity to study the long-term effects of
studied factors by measuring several cytokines simultaneously. What comes to the methodological
considerations, the real theoretical limitations of the present study deserve discussion. Cytokine
concentrations from biological fluids and cells are widely used biomarkers to investigate the
27
activation of immunological processes and measurement of functions of immune system. In addition,
the use of fresh samples would have been ideal despite of the fact that used thawing method is proved
to be appropriate and restore the cells well (Kääriö et al, 2016a; Kääriö et al, 2016b). Furthermore,
our study was limited to the relatively small population. When interpreting the results, it is important
to consider the large number of studied associations, possibly leading to statistically significant
findings due to chance. In the present study, studied obstetric factors were selected a priori as the
exposures of interest. Furthermore, our findings are supported by biological plausibility and similar
patterns following different innate stimuli. Thus, multiple testing correction was not carried out in the
present thesis. In the future, similar approach with fresh samples and with larger sample size would
be of great benefit to further unravel the role of CS as a potent modulator of both innate and adult
immune system. Furthermore, even longer stimulation time would be preferable to ensure the optimal
stimulation time for all secreted cytokines if possible. Additional focus should be placed on the role
of gut microbiota, host genetics, environmental factors and their fine interplay possibly by
incorporating different technical approaches, like high-throughput sequencing, which would greatly
facilitate this task.
Present data raises a question about the possible differences regarding the immunomodulatory
capabilities between different factors, such as preterm and full term CS and birth phases at the time
of the CS. Noteworthy, present data highlights the importance of taking all possible confounding
factors into concern. Moreover, the establishment of a causal relationship between CS and long-term
immune development presents a great challenge due to the difficulties in controlling the relevant
factors occurring between CS and adolescence or adulthood and in ensuring no relevant factors have
escaped consideration in estimating these associations. Furthermore, if we assume that these unknown
and confounding factors serve as critical points it is of great importance to recognize them as critical
points and adjust them to avoid the biased and further invalid estimates of the effect of intrauterine
and obstetric factors on immune functions among adolescents.
The speculations regarding the capability of the birth process itself as well as the obstetric and
intrauterine factors in modifying development of the immune system have emerged. Unlike it has
been thought, these observations are not disturbing immune maturation just between CS and vaginal
delivery, but also within the Caesarean deliveries. Therefore, when making the clinical decisions that
can potentially lead to compromised immune maturation, all relevant factors should be considered by
obstetricians. Moreover, since the clinical decision making plays a critical role in immune
28
development among offspring born by the means of CS, it should be carried out in respect to current
knowledge and balance the experience with the gathered information in a proper manner.
As stated previously, clinical studies have highlighted the potent of intestinal microbiota to modulate
the development of immune cells. Therefore, possible intervention methods in restoring the
microbiota of infants born by CS should be considered in future health care. Determination of the
keystone microbial species, which should be received by the infant during normal delivery plays a
critical role in replicating the beneficial microbial load in possible future restoration procedures in
clinics. However, the individual differences in microbiota composition poses a critical role in
development of future therapeutics. It is suggested that vaginal microbial transfer is capable to
partially restore the microbiota of infants born by CS. Moreover, also the transplantation of fecal flora
from genetically profiled healthy donor establishes genetically identical and stable flora in the
recipient and could therefore be used in restoration. Also, therapies that can enhance protective Treg
cell responses are implicated in protection against inflammatory and autoimmune diseases
(Dominguez-Bello et al, 2016; McLoughlin & Mills, 2011). However, the long-term health
consequences of suggested therapy methods and related initiatives remain unclear and require further
investigation. The critical point is now to ascertain the dialogue between CS, interventions on
microbiota and long-term immune development of the individual subjects.
In summary, given results enhance the scientific basis for future studies aiming to elucidate long-term
immunomodulatory effects of obstetric factors, including intrauterine factors and their relationship
with immune diseases. Noteworthy, especially NICU admission and the lack of natural processes of
birth, that is, CS before the onset of labour, seem to lead to impaired cytokine responses in
adolescence, which postulates their role not only in an early-life immunomodulation, but also
potential long-term effects up to early adulthood. The epidemic nature of CS and constantly
accumulating evidence of the critical role of microbiota in shaping innate and adaptive immune
responses have been under a big question mark. A greater emphasis should be placed on studying
whether obstetric factors and CS cause long-term effects on the immune system of the offspring that
further leads to compromised immune functions. Despite the fact these associations are being
investigated by constantly increasing amounts of studies, they are still unclear and remain to be
understood. Therefore, all findings discussing this compelling puzzle can be considered instrumental
in design and implementation of novel preventive and therapeutic interventions, hence improving the
public health care. The present findings necessitate novel implications in enhancing the understanding
29
the full scope of underlying mechanisms in the background of the compromised immune system and
long-term effects of CS.
Acknowledgements
I would like to give special thanks to the participating families and to Ms. Raija Juntunen and Ms.
Reetta Tiihonen for fieldwork. I also thank my supervisors Marjut Roponen, PhD (Pharm), Title of
Docent, and Leea Keski-Nisula, PhD (Medicine), Title of Docent. I am grateful to Juha Savinainen,
PhD (Biochemistry), Title of Docent, for scientific review and evaluation of the Master’s thesis and
to Professor Jorma Palvimo, PhD (Biochemistry), for formal examination of the Master’s thesis
project. Finally, I would like to thank the members from the research group in The Inhalation
Toxicology Laboratory (UEF). This work was supported by The Academy of Finland (grants 132393
and 256375).
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Appendix
SUPPLEMENTAL INFORMATION
THE EFFECT OF INTRAUTERINE AND OBSTETRIC FACTORS ON IMMUNE
RESPONSES AMONG ADOLESCENTS BORN BY CAESAREAN SECTION
Hermina Jakupović, Master of Science thesis, Master’s Degree Programme in Biomedicine, School
of Medicine, Faculty of Health Sciences, University of Eastern Finland
Inventory of Supplemetary Information
Figures
Appendix Figure A1. Association between section type and the production of cytokines at
adolescence.
Appendix Figure A2. Association between induced labour and the production of cytokines at
adolescence.
Appendix Figure A3. Association between rupture of membranes (ROM) and the production of
cytokines at adolescence.
Appendix Figure A4. Association between histology of the amniotic membranes and the production
of cytokines at adolescence.
Appendix Figure A5. Association between quality of the amniotic fluid (AF) and the production of
cytokines at adolescence.
Appendix Figure A6. Association between culturable bacteria in amniotic fluid (AF) and the
production of cytokines at adolescence.
35
Appendix Figure A7. Association between neonatal antibiotic (AB) treatment and the production of
cytokines at adolescence.
Appendix Figure A8. Association between breastfeeding (BF) and the production of cytokines at
adolescence.
Appendix Figure A9. Association between maternal smoking during pregnancy and the production
of cytokines at adolescence.
Appendix Figure A10. Association between maternal antibiotic (AB) treatment during pregnancy
and the production of cytokines at adolescence.
Appendix Figure A11. Association between neonatal weight and the production of cytokines at
adolescence.
Appendix Figure A12. Association between neonatal BMI and the production of cytokines at
adolescence.
Appendix Figure A13. Association between maternal parity and the production of cytokines at
adolescence.
Appendix Figure A14. Association between season of the birth and the production of cytokines at
adolescence.
Appendix Figure A15. Association between neonatal gender and the production of cytokines at
adolescence.
Appendix Figure A16. Association between doctor diagnosed asthma and the production of
cytokines at adolescence.
Appendix Figure A17. Association between doctor diagnosed atopic rash and the production of
cytokines at adolescence.
36
Appendix Figure A1. Association between section type and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood mononuclear cells (PBMC),
(B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D) olyinosinic:polycytidylic acid
(POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated cytokine values relative to
unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in elective (N=28) compared with emergency
(N=18) and urgent (N=33) section types. Reference line: Elective section type. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
37
Appendix Figure A2. Association between induced labour and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood mononuclear cells
(PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D) olyinosinic:polycytidylic acid
(POLYI:C)stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated cytokine values relative to
unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in uninduced (N=61) compared with induced
(N=17) labour. Reference line: Uninduced labour. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
38
Appendix Figure A3. Association between rupture of membranes (ROM) and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in intact (N=30)
compared with ruptured (N=49) section types. Reference line: Intact membranes. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p <
0.05.
39
Appendix Figure A4. Association between histology of the amniotic membranes and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in
changes/inflammation (N=19) compared with normal (N=53). Reference line: Changes/inflammation. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF).
*p < 0.10; **p < 0.05.
40
Appendix Figure A5. Association between the quality of amniotic fluid (AF) during labour and the production of cytokines at adolescence in (A) unstimulated culture of peripheral
blood mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in not transparent
(N=19) compared with transparent (N=57). Reference line: Other than transparent. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p <
0.05.
41
Appendix Figure A6. Association between culturable bacteria in amniotic fluid (AF) and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in negative
(N=39) compared with apathogen (N=13) and pathogen (N=8). Reference line: Negative. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p
< 0.05.
42
Appendix Figure A7. Association between neonatal antibiotic (AB) treatment and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in no (N=66)
compared with yes (N=13). Reference line: No AB treatment. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
43
Appendix Figure A8. Association between duration of breastfeeding (BF) and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in nursing for 03 months (N=22) compared with 4-9 months (N=31) and ≥ 10 months (N=20). Reference line: BF for 0 to 3 months. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis
factor (TNF). *p < 0.10; **p < 0.05.
44
Appendix Figure A9. Association between maternal smoking during pregnancy and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in yes (N=11)
compared with no (N=68). Reference line: Smoking. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
45
Appendix Figure A10. Association between maternal antibiotic (AB) treatment during pregnancy and the production of cytokines at adolescence in (A) unstimulated culture of
peripheral blood mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐
stimulation, (D) olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each
stimulated cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in no
(N=46) compared with yes (N=28). Reference line: No AB treatment. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
46
Appendix Figure A11. Association between neonatal weight and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood mononuclear cells
(PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D) olyinosinic:polycytidylic acid
(POLYI:C)stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated cytokine values relative to
unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in weight of > 4000 g (N=20) compared with
3200-4000 g (N=36) and < 3200 g (N=23). Reference line: Birth weight > 4000 g. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
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Appendix Figure A12. Association between neonatal body mass index (BMI) and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in > 23 (N=18)
compared with 20-23 (N=32) and < 20 (N=24). Reference line: Neonatal BMI > 23. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p <
0.05.
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Appendix Figure A13. Association between maternal parity and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood mononuclear cells
(PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D) olyinosinic:polycytidylic acid
(POLYI:C)stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated cytokine values relative to
unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in yes (N=40) compared with no (N=39).
Reference line: Has had previous labours. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
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Appendix Figure A14. Association between season of birth and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood mononuclear cells
(PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D) olyinosinic:polycytidylic acid
(POLYI:C)stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated cytokine values relative to
unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in births given on Autumn (N=17) compared
with Winter (N=16), Spring (N=26) and Summer (N=20) seasons. Reference line: Autumn. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10;
**p < 0.05.
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Appendix Figure A15. Association between gender and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood mononuclear cells (PBMC), (B)
induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D) olyinosinic:polycytidylic acid (POLYI:C)‐
stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated cytokine values relative to unstimulated. The
figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in boy (N=47) compared with girl (N=32). Reference line: Boy.
Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
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Appendix Figure A16. Association between doctor diagnosed asthma and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood mononuclear
cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D) olyinosinic:polycytidylic
acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated cytokine values relative to
unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in no (N=64) compared with yes (N=14).
Reference line: No. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
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Appendix Figure A17. Association between doctor diagnosed atopic rash and the production of cytokines at adolescence in (A) unstimulated culture of peripheral blood
mononuclear cells (PBMC), (B) induced by lipopolysaccharide (LPS)‐stimulation, (C) combination of phorbol myristate acetate and ionomycin (PMAIO)‐stimulation, (D)
olyinosinic:polycytidylic acid (POLYI:C)‐stimulation and (E) peptidoglycan (PPG)‐stimulation (5h). Stimulated cytokines were calculated as fold differences of each stimulated
cytokine values relative to unstimulated. The figure shows non-adjusted ratios of the geometric means (GMRs) and 95 % confidence intervals (95 % CI) of cytokines in no (N=69)
compared with yes (N=10). Reference line: No. Abbreviations: Interleukin (IL), Interferon (IFN), Tumour necrosis factor (TNF). *p < 0.10; **p < 0.05.
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