Accepted Manuscript

Title: In pregnancy increased maternal STAI trait stress score

shows decreased insulin sensitivity and increased stress
hormones

Authors: Georgios Valsamakis, Dimitrios C. Papatheodorou,

Nikolaos Chalarakis, N. Vrachnis, Elpida J. Sidiropoulou,
Maria Manolikaki, Aimilia Mantzou, Alexandra Margeli,
Ioannis Papassotiriou, George P. Chrousos, George
Mastorakos

PII: S0306-4530(17)30043-4
DOI: http://dx.doi.org/doi:10.1016/j.psyneuen.2017.06.008
Reference: PNEC 3652

To appear in:

Received date: 15-1-2017

Revised date: 23-4-2017
Accepted date: 10-6-2017

Please cite this article as: {http://dx.doi.org/

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G Valsamakis[Type text] [Type text] [Type text]

In Pregnancy Increased Maternal STAI Trait Stress Score Shows Decreased Insulin Sensitivity

And Increased Stress Hormones

Short title: stress and insulin sensitivity in pregnancy

Georgios Valsamakis, MD1* mastorakg@ath.forthnet.gr , Dimitrios C Papatheodorou, MD1, Nikolaos

Chalarakis, MD1, N Vrachnis MD1, Elpida J Sidiropoulou, MD1, Maria Manolikaki, MD1, Aimilia

Mantzou, PhD2, Alexandra Margeli, PhD3, Ioannis Papassotiriou, PhD3, George P. Chrousos, MD3,

George Mastorakos MD1

1
Endocrine Unit, 2nd Department of Obstetrics and Gynecology and Pathology Department,

Aretaieion University Hospital, Athens Medical School, National and Kapodistrian University of

Athens, Athens, Greece,

Department of Clinical Biochemistry, “Aghia Sophia” Children’s Hospital, National and

2

Kapodistrian University of Athens, Athens, Greece,

3
Endocrinology Unit, National and Kapodistrian University of Athens, Athens, Greece,

*
Corresponding author: Dr. George Mastorakos

3, Neofytou Vamva str

10674 Athens, Greece

tel: 00302106977698009

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G Valsamakis[Type text] [Type text] [Type text]

Main Highlights

1) women with higher STAI trait scores (≥40) had greater serum cortisol and CRH
concentrations
2) women with higher STAI trait scores (≥40) had lower insulin sensitivity index values
(ISI)
3) CRH concentrations correlated positively with maternal STAI state score, HOMAR
(insulin resistance), 1st and 2nd phase insulin secretion and negatively with ISI.
4) STAI state score correlated positively with HOMAR

Abstract

Introduction: Chronic or acute stressors influence maternal and fetal Hypothalamus-

Pituitary-Adrenal Axes (HPA) during pregnancy. In this study, the effect of maternal stress into

maternal insulin sensitivity was investigated during pregnancy.

Materials and Methods: Eighty-two pregnant women [aged 27.1±2.5 (mean±SD) yrs;

BMI=25±2.2 kg/m2] had at the 2nd and 3rd trimesters anthropometry, fasting blood samples (cortisol,

Corticotropin Releasing Hormone (CRH), active amylin, Interleukin IL6), Oral Glucose Tolerance

Test (OGTT) for (glucose, insulin), state-trait anxiety inventory (STAI) trait and state questionnaires

(for stress assessment).

Results: Maternal cortisol, CRH and STAI state score increased significantly from 2nd to 3rd

trimester. At these trimesters women with STAI trait scores ≥40 had greater serum cortisol and CRH

concentrations and lower insulin sensitivity index (ISI) values than those with scores <40 while STAI

trait score predicted negatively ISI. At the 2nd trimester maternal CRH concentrations correlated

positively with maternal STAI state, Homeostatic Model Assessment Insulin Resistance (HOMAR),

1st and 2nd phase insulin secretion and negatively with ISI. STAI trait correlated negatively with ISI.

STAI state correlated positively with maternal systolic blood pressure and HOMAR. At the 3rd

trimester STAI trait correlated negatively and positively with ISI and STAI state, respectively, while

STAI state correlated positively with HOMAR. In women with STAI state scores ≥ 40, these scores

correlated positively with maternal CRH.

Conclusions: In normal pregnant women, enhanced long-term stress is associated with

decreased insulin sensitivity. Both long- and short- term stress are associated with enhanced

maternal HPA axis and increased placental CRH secretion.

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Keywords: long-term stress, pregnancy, insulin sensitivity, CRH, cortisol

1.0 Introduction

‘‘Stress’’ is defined as a state of disharmony or threatened homeostasis. The main

components of the stress system response are the hypothalamic-pituitary-adrenal (HPA) axis and the

locus ceruleus - norepinephrine (LC/NE) - autonomic nervous systems (Chrousos and Gold, 1992).

Pregnancy is a transient period of relative hypercortisolism. During pregnancy, placenta-derived

CRH progressively increases in the maternal circulation, especially during the third trimester, while

hypothalamic maternal CRH secretion is suppressed following the relatively increased circulating

concentrations of maternal cortisol (Mastorakos ansd Ilias, 2000). During late gestation, the activity

of the fetal HPA axis is enhanced, while premature activation of this axis may result from an adverse

intra-uterine environment, such as hypoxemia and/or inflammation (Challis et al., 2001). Placenta is

considered a stress-sensitive organ, while placental CRH and an activated fetal HPA axis may trigger

labor. Increased, normal or decreased placental CRH concentrations have been associated with pre-

term, term, or post-term labor, respectively (Mastorakos and Ilias, 2000, Magiakou et al., 1996 a).

Decreased insulin sensitivity is a feature of normal pregnancy. It has been previously

proposed that it reflects an adaptive phenomenon aiming at diverting maternal glucose towards fetal

needs (Mastorakos et al., 2007). To date, studies have shown that in humans the exposure of the

mother to prenatal psychosocial stress could be associated with decreased insulin sensitivity in the

offspring assessed during young adulthood (Entringer et al., 2008), while in rats it was associated

with IUGR and glucose intolerance (Lesage et al., 2004). To our knowledge, there are no studies

showing a direct effect of maternal stress (chronic or acute) during pregnancy on maternal insulin

sensitivity.

Feelings of anxiety may occur in stressful situations. The state-trait anxiety inventory (STAI)

is a well-standardized, 40 item questionnaire, designed as a self-report instrument for the evaluation

of both state and trait anxiety. State anxiety can be defined as fear, nervousness, discomfort, and

arousal of the autonomic nervous system induced temporarily by situations perceived as dangerous

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(i.e. how a person is feeling at the time of a perceived threat), while trait anxiety can be defined as a

relatively enduring disposition to feel stress, worry, and discomfort (Spielberger and Sydeman,

1994). STAI trait and state have been validated in pregnancy for stress assessment (Rondo et al.,

2003; Di Pietro et al., 2006).

The aim of this study was to investigate the association between stress (assessed by STAI

scores and stress hormones) and insulin sensitivity in the second and third trimesters of

gestation.

2.0 Materials and Methods

2.1 Subjects

This study was approved by the institutional ethical committee, functioning according to the

4th edition of the Guidelines on the Practice of Ethical Committees in Medical Research issued by the

Royal College of Physicians of London (Royal College of Physicians, 2007). Consent has been

obtained from each patient after full explanation of the purpose and nature of all procedures used.

Ninety-five pregnant primigravidae caucasian women were recruited during the first trimester.

Ten women among them were not included in the protocol because they presented with

miscarriages. Thus, finally, eighty-five women (age, mean±SD: 27.73.6 years; pre-pregnancy

BMI: 26±2.1 kg/m²) continued in the study protocol which was initiated during the second

trimester. The recruitment of all women was done in an Obstetrics and Gynecology outpatient

clinic of a university hospital between May 2015 and May 2016. Exclusion criteria included non-

caucasian origin, BMI>30 kg/m2 before pregnancy, history of type 1 or type 2 diabetes mellitus or

gestational diabetes (GDM), glucose intolerance, multiple pregnancy, major vaginal bleeding,

hypertension, preeclampsia, urinary tract infection, fetal-placental abnormalities, such as congenital

anomalies, placenta previa and placental abruption, nephropathy, liver disease, previous or

current mental health diseases (such as severe depression, anxiety etc) and current smoking or

alcohol intake. All women were interviewed by a psychiatrist during the first trimester and

those with remarkable mental health problems were not included in the study. Women with

non-Caucasian origin were excluded from the study in an effort to study a homogeneous

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population regarding insulin resistance traits. To avoid bias, women were recruited based on a

computer software random numbers generator

2.2 Protocol

Women were seen once during each of the second and third trimesters of their pregnancy

between 24th-26th and 34th-36th week, respectively. Pregnant women had a basic dietetic advice at the

beginning without regular dietetic follow-up. At each visit, they were submitted to anthropometric

measurements; a fasting blood sampling for measurement of hormones (active amylin, insulin, CRH,

cortisol) and IL6; a 75gr oral glucose tolerance test (OGTT) with blood samples drawn at 0, 30, 60,

90 and 120 min time-points for measurement of glucose and insulin concentrations; a fetal ultrasound

and a session of STAI state and trait questionnaires. Diagnosis of GDM was based on the OGTT

according to the diagnostic criteria set by the HAPO study (Coustan et al., 2010). Three of the

recruited women were diagnosed with GDM and were excluded from the study. Age and BMI of the

remaining 82 women were 27.12.5 years and 25±2.2 kg/m², respectively. No woman among them

developed gestational diabetes during the study as indicated by the OGTT performed to all of

them at the 3rd trimester. Blood samples for measurement of hormones in the plasma were collected

in tubes with EDTA. After blood collection, Millipore’s serine protease inhibitor was added. Tubes

were inverted several times to mix and they were centrifuged immediately. After centrifugation,

plasma was collected, aliquoted and stored at –70° C until assayed.

2.3 Anthropometry and blood pressure measurements

All measurements of pregnant women were carried out by a single observer. For all women,

weight before pregnancy was retrieved from their records and height was measured to the nearest mm

using a stadiometer. At each visit, weight without shoes and light clothing was measured in kilograms

to the nearest 0.1 kg on a beam balance and BMI was calculated in kg/m2. Blood pressure was

measured after patient sitting for 10 minutes and taking the mean value of two measurements after

waiting for 5 minutes for the second measurement.

2.5 Stress Questionnaires

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The STAI trait questionnaire is composed of 20 questions about how the person usually feels.

The STAI state questionnaire is composed of 20 questions inquiring about how the individual feels at

the specific time period of assessment. To further analyze the effect of stress as assessed by the STAI

state and trait questionnaires into maternal and metabolic variables, women were divided into those

with high and low STAI trait and state scores (trait or state scores ≥40 and <40), respectively

(Spielberger and Sydeman, 1994; Powell et al., 2011)

2.6 Indices of carbohydrate metabolism

Insulin resistance was estimated by the homeostasis model assessment [HOMA-R=(insulin

at baseline, pmol/L x glucose at baseline, mmol/L)/135] (Matthews et al., 1985; Cohen et al., 2006).

Beta cell secretion of insulin was estimated by the following indices: Predicted index of first phase of

insulin secretion [1st PHIS=1283+(1.289 x insulin at 30 min in pmol/L)-(138.7 x glucose at 30 min in

mmol/L)+(3.772 x insulin at baseline in pmol/L)], and predicted index of second phase of insulin

secretion [2nd PHIS=287+ (0.4164 x insulin at 30 min in pmol/L)-(26.07 x glucose at 30 min in

mmol/L)+ (0.9226 x insulin at baseline in pmol/L)]. Insulin sensitivity was estimated by use of the

insulin sensitivity index (ISI) = 0.226 − [0.0032 × BMI] − [0.0000645 × insulin at 120 min (pmol/L)]

− [0.00375 × glucose at 90 min (mmol/L)] (Stumvoll et al., 2000)

2.7 Blood chemistry and hormone assays

Serum glucose levels were measured with the Siemens Advia 1800 Clinical Chemistry System

(Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Serum insulin and cortisol concentrations

were measured with electrochemiluminescence immunoassays with the Cobas e411

immunochemistry analyzer (Roche Diagnostics, Basel, CH) with intra- and inter- assay coefficients

of variation (CV) < 2.0% and <2.8%, respectively for both assays. Active amylin was measured in

plasma with a multiplex assay (MILLIPLEX Human Metabolic Hormone Panel, Millipore Corp. St.

Charles, Missouri, USA) on the Luminex-100 Integrated System (Luminex Corporation, Austin, TX,

USA) with intra- and inter- assay CVs <11% and <19 %, respectively, according to the

manufacturer. Serum IL-6 was measured by the quantitative sandwich enzyme immunoassay

technique (Quantikine, R&D Systems, Minneapolis, MN, USA) with the intra- and inter- assay CVs

ranging between 6.9 and 7.4% and between 6.5 and 9.6%, respectively. CRH was measured by a

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quantitative kit based on the principle of “competitive” enzyme immunoassay (Phoenix

Pharmaceuticals, Inc, Burlingame, CA, USA) with intra- and inter- assay CVs at <10% and <12%,

respectively, according to the manufacturer.

2.8 Statistical analysis

Data are described as mean  SD or median and interquartile range (25th-75th percentile) for

data not normally distributed. To test the change of each variable among different trimesters of

pregnancy the one-way repeated measures ANOVA test was used for normally distributed variables

and the non parametric Friedman ANOVA test for non-normally distributed variables. To test for

differences of ISI values between groups of pregnant women with STAI scores either >40 or

<40 within the same trimester one factor ANOVA repeated measures was used; to test for

differences of ISI values between groups of pregnant women with STAI scores either >40 or

<40 in the two the trimesters studied (2nd and 3rd) one factor ANOVA repeated measures was

used. To test for associations between different variables and to assess possible correlations between

differences the Spearmann correlation analysis was performed. In the 2nd and 3rd trimester,

stepwise multiple regression analysis was undertaken to define predictors of maternal insulin

sensitivity as calculated by the ISI index. To test whether the positive correlation of STAI state

with HOMAR is independent of maternal cortisol levels a backwards multiple regression model

analysis was performed. A p-value of <0.05 was considered significant. The SPSS statistical

software was used for statistical analysis (SPSS Inc., Chicago, IL, USA, 1999).

3.0 Results

3.1 Maternal weight, hormonal and metabolic variables, maternal STAI questionnaire

scores at the 2nd and 3rd trimesters of pregnancy.

Maternal weight values increased significantly from the 1st to the 2nd and 3rd trimesters

(p<0.05) with a median increase of 12.5 kg from the 1st to the 3rd trimester. Circulating maternal

fasting insulin and active amylin concentrations and systolic blood pressure levels increased

significantly from the 2nd to the 3rd trimester (p<0.05) (Table 1). HOMAR, PHIS1 and PHIS 2 values

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increased significantly (p<0.05), while ISI values decreased significantly (p<0.05) from the 2nd to the

3rd trimester (Table 1). Circulating maternal fasting cortisol and CRH concentrations increased

significantly (p<0.05) from the 2nd to the 3rd trimester, while IL6 concentrations did not (table 1).

STAI state scores increased significantly (p<0.05) from 2nd to 3rd trimester, while, as expected STAI

trait scores did not (Table 1).

During the 2nd trimester, women with STAI trait scores ≥40 had significantly lower ISI

values, higher serum fasting CRH and cortisol concentrations, than those with STAI trait

scores <40 (p<0.001, p<0.001 and p<0.001, respectively) (Figure 1, 2 and 3). In the 3rd trimester,

women with STAI trait scores ≥40 had significantly lower ISI values, higher serum fasting

CRH and cortisol concentrations, than those with STAI trait scores <40 (p<0.001, p<0.001 and

p<0.001, respectively), (Figure 1, 2 and 3).

3.2 Correlations and predictors among maternal STAI questionnaire scores, hormonal,

metabolic, and anthropometric variables in the 2nd and 3rd trimesters.

For both the 2nd and the 3rd trimesters, statistically significant correlations among STAI

questionnaires scores (STAI state, STAI state ≥40; STAI trait, STAI trait ≥40), carbohydrate

metabolism indices (ISI, HOMAR, PHIS 1, PHIS 2 and amylin) and stress hormones (CRH,

cortisol) are presented in Table 2. In the 2nd trimester, to test whether the positive correlation of

STAI state with HOMAR is independent of maternal cortisol levels a backwards multiple

regression model analysis was performed with HOMAR as the dependent variable and STAI

state, maternal cortisol levels and weight as independent variables. This analysis revealed STAI

state (p=0.001, beta=0.340) as the best predictor among the other independent variables of

HOMAR. In the 3rd trimester, to test whether the positive correlation of STAI state with

HOMAR is independent of maternal cortisol levels a backwards multiple regression model

analysis was performed with HOMAR as the dependent variable and STAI state, maternal

cortisol levels and weight as independent variables. This analysis revealed STAI state (p=0.002,

beta=0.450) as the best predictor among the other independent variables of HOMAR.

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In addition, in the 2nd trimester, STAI state scores correlated positively (p<0.05) with STAI

trait scores (r=0.482), systolic blood pressure values (r=0.334) and maternal CRH (r=0.466)

concentrations. Also, maternal cortisol concentrations correlated positively (p<0.05) with maternal

weight (r=0.368), systolic blood pressure values (r=0.613) and IL6 concentrations (r=0.650);

maternal CRH concentrations correlated positively (p<0.05) with maternal weight (r=0.357) and

cortisol concentrations (r=0.329).

Furthermore, in the 3rd trimester, STAI state scores correlated positively (p<0.05) with STAI

trait scores (r=0.753); STAI trait score ≥40 correlated positively (p<0.05) with STAI state scores

(r=0.703) and maternal weight (r=0.700) values; STAI state score ≥40 correlated positively (p<0.05)

with STAI trait scores (r=0.542) and CRH concentrations (r=0.872); maternal cortisol concentrations

correlated positively (p<0.05) with maternal weight values (r=0.416) and IL6 concentrations

(r=0.857).

All 10 cases that scored over 70 in STAI state at the first trimester presented with

miscarriages standardized for any additional risk factors.

3.3 Predictors of ISI

In the 2nd trimester, STAI trait score was the best negative predictor of ISI values

(dependent variable) among maternal CRH, IL6 and cortisol concentrations, maternal pre-

pregnancy BMI, STAI state and trait scores employed as independent variables (Table 3). In

the 3rd trimester, STAI trait score was the best negative predictor of ISI values (dependent

variable) among STAI trait and state scores, maternal pre-pregnancy BMI, maternal cortisol,

CRH and IL6 concentrations employed as independent variables (Table 3).

4.0 Discussion

We found that STAI trait score was the best negative predictor of insulin sensitivity

during the 2nd and 3rd trimesters of pregnancy among maternal STAI trait score, STAI state

score, CRH, IL6 and cortisol concentrations, and pre-pregnancy BMI employed as independent

variables. Thus, it seems, that the clinical expression of stress during pregnancy is negatively

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associated with insulin sensitivity and it is affected by personality-related stress characteristics

rather than stressful events experienced in short-term by the person assessed. Furthermore, in

this study, maternal awareness of anxiety as assessed by the STAI trait questionnaire,

correlated negatively with maternal insulin sensitivity during the 2nd and 3rd trimesters, and

ISI was lower in women with STAI trait scores ≥40 than those with scores <40. Indeed, in the

past, type 2 diabetes mellitus has been associated with personality-related stress (Lane et al.,

2000), while anxiety trait has been associated with impaired glycemic control in non-diabetic

black women (Tsenkova et al., 2012). In addition, in this study, maternal STAI trait and state

scores in the 2nd trimester correlated positively with maternal active amylin concentrations, a

marker of insulin resistance and hyperinsulinemia (Hou et al., 2011), a correlation even more

intense in women with STAI trait scores ≥40. In the past, illness associated stress was suggested

to lead to stress hyperglycemia and increased long-term diabetes mellitus risk (McAllister et al.

2014), while the likelihood of concurrent anxiety disorders in diabetic patients was increased

(Kruse et al., 2003). Of note, the Monica/Kora Augsburg cohort study (5300 non-diabetic

employed individuals were followed over 13 years) reported that job strain-stress could

increase the long- term risk of type 2 diabetes mellitus (Huth et al., 2014) and in another study

perceived work stress was associated with increased blood glucose levels (Sancini A et al.,

2017).

Of note, in this study, maternal CRH and cortisol concentrations were greater in

women with STAI trait scores ≥40 than those with scores <40 in both the 2 nd and 3rd trimesters,

while both cortisol and CRH concentrations correlated positively with insulin resistance and

markers of insulin secretion (PHIS 1 and 2, active amylin) and negatively with insulin

sensitivity, as assessed by ISI. In addition, STAI state scores correlated positively with CRH

concentrations in all women in the 2nd trimester and in women with STAI state scores ≥40 in

the 3rd trimester of pregnancy. These findings suggest a possible direct and/or indirect

association of maternal stress with activation of the maternal HPA axis during normal

pregnancy. Maternal stress during pregnancy might be associated with stress-induced secretion

of catecholamines by the brain arousal and sympathetic systems (locus

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ceruleus/norepinephrine) (Goldstein, 2003). The latter can lead to maternal cortisol secretion in

a circadian and pulsatile fashion via AVP, a major ACTH secretagogue, secreted by

parvocellular neurons of the paraventricular hypothalamic nucleus (Magiakou et al., 1996 b).

Acute social stress, as assessed by the Trier public speaking stress task, increases cortisol

concentrations in obese adolescents (Verdejo-Garcia et al., 2015), whereas anxiety measured

with the brief symptom inventory (Derogaitis and Melissaratos, 1983) in black adolescents

increases salivary cortisol (Assari S et al., 2015; Vreeburg et al., 2010).

The positive associations found in this study between maternal anxiety and insulin

resistance during pregnancy could be attributed to possible parallel increase of catecholamines

secretion (Goldstein, 2003). Indeed, the multiple regression analysis models employed in the 2nd

and 3rd trimesters in this study revealed a stronger positive effect of STAI state scores upon

HOMAR than that of maternal cortisol concentrations while The stronger prediction of insulin

sensitivity by the STAI trait scores than that by CRH and maternal cortisol concentrations in

the two trimesters of pregnancy studied, both suggest a possible additional pathophysiological

role of catecholamines in the development of stress-related insulin resistance expressed via

sympathetic system (locus ceruleus, catecholamines) activation. In fact, during stressful events,

the stimulated maternal peripheral sympathetic nervous system could lead to enhanced

placental CRH secretion via reduction of uterine blood flow, explaining the positive correlation

of maternal stress state with placental CRH concentrations (Rakers et al., 2015; Jones et al.,

1989). The suggested involvement of catecholamines could further explain the positive

correlations in the 2nd trimester between maternal systolic blood pressure values and maternal

STAI state scores. In the past, the increase of CRH concentrations between the 2nd (18 to 20

weeks of gestation) and the 3rd trimester (28 to 30 weeks of gestation) were associated with

perceived stress level and state anxiety evaluated at the 2nd trimester (Hobel et al., 1990). In that

study the perception of situations in one’s life was assessed by an 8-item abbreviation of the

perceived stress scale administered to measure general feelings of stress during pregnancy,

while a shortened 10-item version of Spielberger’s State Anxiety Inventory was used to

measure subjective feelings of anxiety during pregnancy (Hobel et al., 1990). Thus, in the 2nd

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trimester assessment of maternal stress together with CRH concentrations have been suggested

as potential markers for women at risk of preterm birth (Hobel et al., 1990). Furthermore in

the 2nd trimester maternal CRH concentrations correlated positively with maternal cortisol

concentrations.

A sustained positive correlation of maternal IL-6 with maternal cortisol concentrations was

observed in the 2nd and 3rd trimesters. IL-6 is widely expressed in the female reproductive tract and

gestational tissues, and exerts regulatory functions in embryo implantation and placental

development, as well as during the immune adaptations required to tolerate the fetal allograft (Florio

et al., 2007). Plasma IL-6 increases in response to chronic reductions in uterine perfusion in pregnant

rats, while a comparable elevation in plasma IL-6 is associated with arterial pressure increase and

reduction of renal function in pregnant rats (Prins et al., 2012). Increased basal IL-6 concentrations

correlate positively with increased cortisol response to ACTH stimulation, while IL-6 even at low

concentrations and under physiologic conditions, stimulates AVP secretion and modulates adrenal

cortex response to ACTH (Mastorakos et al., 1994). In addition, in the past we demonstrated that

maternal pulsatile IL-6 leads to pulsatile placental CRH secretion during the active phase of human

labor (Papatheodorou et al., 2013).

CRH modulates glucose transporter proteins in placental tissue, suggesting a link between

CRH levels and fetal growth (Thomson, 2013). These findings imply that maternal awareness of

anxiety during the specific time period of pregnancy (STAI state) is related to increased placental

CRH secretion more than maternal awareness of her character-associated anxiety as assessed by

STAI trait. Interestingly, in 10 cases of miscarriages at the 1st trimester among our cohort of pregnant

women the mothers had the highest STAI state scores (more than 70) among all women studied at all

the trimesters. The study includes only women that were not obese, did not have any pregnancy

complications and were Caucasian. While this reduces potential confounding, it limits

generalization of the findings.

4.1 Conclusion

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In summary, during the 2nd and 3rd trimesters of normal pregnancy, long-term maternal

stress, as assessed by STAI trait score, decreases insulin sensitivity and increases maternal CRH and

cortisol concentrations. STAI trait score was the best negative predictor of insulin sensitivity during

pregnancy. Short-term maternal stress, as assessed by STAI state score, was positively correlated

with CRH and cortisol concentrations, suggesting a direct and/or indirect association of maternal

stress with maternal HPA and placental CRH axies.

Evidence suggests that stressful experiences during pregnancy exert long-term consequences on the

future wellbeing of both the mother and the fetus. Altered epigenetic regulation may potentially

influence fetal endocrine programming and brain development across several generations (Bowers

and Yehuda, 2016). Further studies are needed to elucidate the pathophysiologic mechanisms

functioning during stress and their epigenetic influence regarding insulin sensitivity in adult life.

Author contribution paragraph: “G.V. researched, interpreted data, contributed to the concept and

wrote the manuscript; D.P., NC, MM, EJS researched data; A.M., AM and I.P. performed blood

chemistry, bioarrays analyses and revised the manuscript; NV and GC interpreted data and revised

the manuscript; G.M. contributed to the concept, interpreted data and reviewed/edited the manuscript

Funding

Funding was received from Athens University to Prof George Mastorakos. The funding source

played no role in the study design; in the collection, analysis, and interpretation of data; in the writing

of the report; or in the decision to submit the report for publication.

Conflict of interest Declaration

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and

writing of the paper.

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Legend to the Figures

Figure 1. Comparison of maternal ISI values depending on STAI trait scores >40 and <40 in the 2nd

and 3rd trimesters of pregnancy. Symbol “” indicates statistically significant difference between

groups of pregnant women compared within the same trimester; Symbol “ “ indicates statistically

significant difference between groups of pregnant women with STAI scores either >40 or <40 in

different trimesters. Statistical significance was set at p<0.01. Boxes represent interquartile range;

perpendicular lines inside boxes represent median value; cross represents mean marker; whiskers

represent the lowest and highest observations, respectively.

Figure 2. Comparison of maternal CRH concentrations depending on STAI trait scores >40 and <40

in the 2nd and 3rd trimesters of pregnancy. Symbol “” indicates statistically significant difference

between groups of pregnant women compared within the same trimester; Symbol ““ indicates

statistically significant difference between groups of pregnant women with STAI scores either >40 or

<40 in different trimesters. Statistical significance was set at p<0.01. Boxes represent interquartile

range; perpendicular lines inside boxes represent median value; cross represents mean marker;

whiskers represent the lowest and highest observations, respectively.

Figure 3. Comparison of maternal cortisol concentrations depending on STAI trait scores >40 and

<40 in the 2nd and 3rd trimesters of pregnancy. Symbol “” indicates statistically significant

difference between groups of pregnant women compared within the same trimester; Symbol “ “

indicates statistically significant difference between groups of pregnant women with STAI scores

either >40 or <40 in different trimesters. Statistical significance was set at p<0.01. Boxes represent

interquartile range; perpendicular lines inside boxes represent median value; cross represents mean

marker; whiskers represent the lowest and highest observations, respectively.

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Table 1. Maternal age, pre-pregnancy BMI, weight, hormonal and metabolic variables, maternal

STAI scores, at the 2nd and 3rd trimesters of pregnancy; Variables are expressed as mean±SD or

median (25th–75th interquartile range). The (*) and the (≠) denote statistically significant difference

from the 1st and the 2nd trimester, respectively (P<0.05).

PREGNANCY 2nd trimester 3rd trimester

N=82

Age=27.1±2.5 years

Pre-pregnancy BMI= 25±2.2 kg/m²

Weight (kg) 70±12.7* 79±14 * #

CRH (ng/mL) 2.2±0.4 2.7±0.5#

Cortisol (μg/dL) 22.2±6.7 28±7.3#

IL-6 (pg/mL) 1.9 (1.1-4) 1.6 (1.2-1.9)

Fasting active amylin (pg/mL) 546.5 (252-727.2) 591.4 (412.6-771.6) #

Fasting insulin (pmol/L) 56 (44-120) 122 (89-160) #

Fasting glucose (mmol/L) 4.5±0.3 4.6±0.4

HOMA-R 1.9 (1.8-3.8) 4.0 (3.0-5.0)#

ISI 0.1±0.01 0.06±0.02#

1st PHIS 2,597 (2,134-3,500) 3,998 (3,435-4,976) #

2nd PHIS 700 (590-895) 1,045 (980-1,300) #

Systolic blood pressure (mmHg) 107±12 114±12#

STAI state 33.9±6.8 39.2±8.4#

STAI trait 39.6±8.4 34.9±8.7

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Table 2. Significant correlations (Spearman rho-value) among maternal stress hormones, maternal

STAI state and trait scores, and maternal insulin sensitivity (ISI, HOMA, Amylin) and secretion

(PHIS 1, PHIS 2) parameters at the 2nd and 3rd trimester of pregnancy.

Variable Cortisol CRH Trait Trait State score State score Cortisol Trait State Trait

2nd 2nd score score ≥40 2nd Score score score

2nd 3rd
2 nd
≥40 2 nd
3rd
3rd ≥40 2nd

ISI 2nd -0.309 -0.399 -0.571 -0.743 -0.571

HOMAR 0.534 0.358 0.564 0.821

2nd

PHIS 1 2nd 0.508 0.371

PHIS 2 2nd 0.476 0.384

Amylin 2nd 0.406 0.567 0.664 0.591

ISI 3rd -0.45 -0.432 -0.674

HOMAR 0.653 0.342

3rd

PHIS 1 3rd 0.490

PHIS 2 3rd 0.486

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Table 3: Results of stepwise multiple regression model in the second and third trimesters with

maternal STAI trait score being the best negative predictor of maternal ISI (dependent variable) in

both trimesters among maternal STAI trait score, STAI state score, IL6, cortisol, CRH, and maternal

pre-pregnancy BMI as independent variables.

Variable beta p-value

STAI trait 2nd -0.852 0.001

STAI trait 3rd -0.760 0.013

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