The Effects of Hormonal Contraceptives on

Cortisol Responders to Psychosocial Stress

Matt London, Angela Mapanao, Brissa Ortega, Carlos Almeida,

Michael Namekata, and Cheryl Chancellor-Freeland, Ph.D.

San Jose State University

April 24, 2014

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Abstract

Cortisol, an adrenocortical glucocorticoid representing a physiological stress response

(Lovell et al., 2011), is frequently instigated by the Trier Social Stress Test (TSST; Kirschbaum

et al., 1993), a psychosocial stress inducing procedure, which has proven to be a reliable cortisol

inciting protocol (Campbell & Ehlert, 2012). Collection and analysis of salivary cortisol costs

>$30 in supplies and >5 work hours/participant. Non-responding participants (identified by

<15.5% cortisol increase from baseline; Miller et al., 2013) are not identified until after funds

and labor have been spent to collect and analyze the data. Consequently, this data is obsolete and

unacceptable for the purposes of the study. For over five years the International Neuroeconomics

Institute (INI), at San Jose State University (SJSU) has been analyzing salivary cortisol activity

in response to psychosocial stress, instigated by the TSST, and have been faced with 20% of

their participants being classified as cortisol non-responders. Hormonal birth control has been

demonstrated to be correlated with attenuated cortisol response (Kirschbaum et al., 1995;

Kirschaum et al., 1999). However, it had not been determined whether it is related to the

proportion of psychosocial stress induced cortisol responders. This study compared the TSST

induced salivary cortisol response of 16 females who use hormonal birth control (Mage = 18.9,

SD = 1.41) to 18 who do not (Mage = 18.8, SD = 1.14), revealing that females who use hormonal

birth control exhibit a lower percentage of responders (56%), compared to non-birth control

users (84%), and are more likely to be classified as cortisol non-responders (X2(1, 65) = 5.11, p =

.02). Due to this finding, we advocate the exclusion of females who use hormonal birth control in

studies that utilize psychosocial stress inducing factors, specifically the TSST.. Doing so will

reduce the number of cortisol non-responders and, therefore, retain research funds.

Keywords: cortisol, corticosteroid-binding-globulins, Trier Social Stress Test, hormonal

birth control, attenuated response, psychosocial stress, cortisol non-responders

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Introduction

Cortisol, a glucocorticoid, serves as a negative feedback mechanism within the

hypothalamic-pituitary-adrenal axis. Psychosocial stress incites the hypothalamus to release

corticotropin releasing hormone (CRH) to the anterior pituitary, which in turn releases

adrenocorticotropic hormone (ACTH) to the adrenal cortex. Within the zona fasciculata of the

adrenal cortex, cortisol is produced and released to the hypothalamus and the anterior pituitary,

within a negative feedback loop, to initiate inhibitory actions on these glands and to modulate

stress and inflammation pathways. Only 2-15% of the released cortisol is not bound to carriers

such as corticosteroid-binding globulin (CBG), albumin, and erythrocytes. The unbound (“free,”

bioactive) proportion of cortisol is able to cause genomic cortisol effects in peripheral tissues and

the brain, as opposed to the bound cortisol that cannot cause such effects. Bio-active and bound

cortisol is found in the blood, while only bio-active cortisol in found in the saliva. For this

reason, salivary cortisol collection is preferred as a more efficient and cost-effective method than

blood cortisol analysis, which can often prove to be counterproductive when the act of

venipuncture, itself, incites significant HPA activation and, therefore, an undesired cortisol

response (Kirschbaum & Hellhammer, 2000).

Typically, salivary cortisol follows a standard circadian rhythm where it is boosted upon

awaking in the morning and increases 50-100% until it peaks after 30 min., where after it

decreases until, in response to substantial amounts of food at lunch time, it peaks at about 150%

and steadily decreases throughout the afternoon and into the evening without another major

secretory instance (Kirschbaum & Hellhammer, 2000). Considering that a typical cortisol

secretory episode initiates a 55.2 nmol/l increase of total plasma cortisol and that salivary

cortisol constitutes 2% to 5% of plasma cortisol Kirschbaum et al. (1992) and Wust et al. (2000)

established a 2.5 nmol/l salivary cortisol increase to be valid classification criterion for a

significant cortisol response.

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A lack of systematic evaluation studies of the 2.5 nmol/l criterion has led many

experimenters to utilize 30% (Kimura et al., 2013) and 40% (Kunz-Ebrecht et al., 2003) levels of

significance for classification of cortisol responders (i.e. participants who exhibit high levels of

stress induced cortisol) and non-responders (i.e. participants who exhibit low levels of stress

induced cortisol). Binary median splits were not utilized due to a high number of participants

who were highly indistinct in terms of their cortisol response profile. Since its inception, the 2.5

nmol/l criterion has resulted in a high rate of 16.5% false-negative classifications, where non-

responders were classified as responders. Miller et al. (2013) conducted a study to develop a

cortisol response classification criterion that was more accurate than the 2.5 nmol/l criterion. As

a result, Miller et al. (2013) found that a 1.5 nmol/l cortisol increase criterion for classification of

responders resulted in 39.3% less misclassifications. A 15.5% baseline-to-peak increase criterion

also proved to be an accurate appraisal of cortisol response, exhibiting 26.7% less

misclassifications.

A study on the effects of the functional integrity of the HPA axis, on outcomes of patients

with brain injury, classified cortisol non-responders as participants who exhibited a <500 nmol/l

plasma cortisol response [Considering that a typical cortisol secretory episode initiates a 55.2

nmol/l increase of total plasma cortisol and that salivary cortisol constitutes 2% to 5% of plasma

cortisol Kirschbaum et al. (1992) and Wust et al. (2000) established a 2.5 nmol/l salivary cortisol

increase to be valid classification criterion for a significant cortisol response. Although, 55.2 =

11.04 % of 500. ] to a low-dose stimulation test, comprising an injection of 1 μg of tetracosactrin

(Dimopoulou et al., 2004). 30 min post-dose, a blood sample was collected and measured for

cortisol. A typical response to this test, by participants who maintain healthy HPA axis

functionality, is 18 micrograms/dl (i.e. 500 nmol/l). Based on this response threshold, 13% of

brain injured participants showed blunted cortisol responses. A limitation of the study was that

corticosteroid-binding-globulins (CBG’s) were not measured. Brain injured participants typically

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exhibit a significant decrease of CBG’s immediately following the trauma. CBG levels return to

normal within 7 days after the trauma. 75% of brain injured participants were analyzed after the

7 day time period wherein CBG’s typically return to normal levels. Participants on estrogen

therapy were excluded due to its tendency to increase plasma CBG concentrations. Women who

were taking oral contraceptives were excluded due to its tendency to increase CBG’s.

Psychosocial stressors have been used to incite a psychobiological stress response, which

can be measured by a salivary cortisol response compared against the typical cortisol circadian

rhythm (Balodis, Wynne-Edwards, & Olmstead, 2010; Campbell & Ehlert, 2012; Dickerson et

al., 2004; Kirschbaum et al., 1999). Numerous studies have utilized public speaking (Bassett,

Marshall, & Spillane, 1987) or mental arithmetic (Frederickson, Tuomisto, Bergman-Losman,

1991; Jorgensen et al., 1990; Trestman et al., 1991) or both (Kemmer et al., 1986), although they

were not standardized into one reliable protocol, to induce psychobiological stress responses.

Due to the insignificant cortisol responses and lack of reliability of these previous

protocols, Kirschbaum et al. (1993) created the Trier Social Stress Test (TSST), which consists

of a protocol that utilizes both tasks to elicit a valid and reliable psychosocial stress inducing

salivary cortisol response. These researchers revealed a high reproducibility across five separate

studies, in which the psychosocial stress of the TSST induced salivary cortisol levels to peak 10

min after TSST cessation at 5.3 to 8.2 nmol/l above baseline levels of 4 to 9 nmol/l. Since these

findings, many studies have used the TSST and concluded that upon termination of psychosocial

stress, the salivary cortisol response becomes evident at 5-20 min and peaks at 10-30 min

(Kirschbaum & Hellhammer, 2000).

In Kirschbaum, Wust, and Hellhammer’s (1992) study the stress induced cortisol

responses of 153 healthy participants, split across four different studies, contraceptive use was

not a criterion for exclusion. Females who used contraceptives were not excluded due to the

results of Kirschbaum and Hellhammer (1989) and unpublished results of the Kirschbaum
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(1992), which stated that low estrogen-containing contraceptives do not significantly manipulate

cortisol levels. The participants were split across four different studies. Studies 1, 2, and 3

exposed the participants to a psychosocial stress paradigm that is virtually identical to the TSST,

although it had not yet been named as such. Studies 1 and 2 resulted in no significant effect of

sex on baseline salivary cortisol (Male = 5.5 nmol/l; Female = 5.0 nmol/l). Although, there was

a significant sex effect on delta (total) salivary cortisol response to psychosocial stress (M = 9.2

nmol/l delta response, 14.7 +/- 1.1 nmol/l peak, 1.67 fold increase; F = 4.8 delta, 9.8 +/- 0.8, 0.96

fold increase. Study 3 administered synthetic corticotropin-releasing hormone (CRH) and a

bicycle ergometry test, in addition to the psychosocial stress paradigm. Study 4 only led the 18

participants (9 Female, Mage = 22.6) years to anticipate the stress of public speaking, yet did not

require them to perform the speech.

Kimura et al. (2013) defined cortisol reactivity as the change score from baseline to RE1

(10 min into the recovery period, 10 min after TSST conclusion, 48 minutes after experiment

onset). Following Kuntz-Ebrecht et al. (2003), Kimura et al. (2013) classified cortisol responders

as the upper 30% and non-responders as the lower 30% in terms of cortisol responsiveness. Non-

responders showed a mean cortisol baseline of 2.87 nmol/l below baseline of responders.

Responders showed a 133% baseline-to-peak cortisol increase percentage, while non-responders

exhibited a 20% decrease. None of the participants took oral contraceptives. To control for

unwanted influences of sex hormones on the HPA axis, females were included in the study only

if they were in the late luteal or early follicular phases of their menstrual cycles.

Kunz-Ebrecht et al. (2003) did not screen out females who were taking oral

contraceptives. Cortisol non-responders showed higher IL-6 levels and higher IL-1ra response to

stress, compared to cortisol responders. Cortisol responders exhibited a mean stress induced

cortisol increase of 3.45 +/- 2.7 nmol/l. Non-responders exhibited a mean cortisol decrease of

2.55 +/- 2.7 nmol/l.

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The International Neuroeconomics Institute (INI), led by Dr. Chancellor-Freeland at San

Jose State University, CA, primarily engages in research on the effects of psychosocial stress as

it impacts multiple domains in one’s life, including decision making, cognition, and physiology.

The INI utilizes the TSST to incite a psychobiological stress response, which can be assessed

with salivary cortisol. Throughout the time that the INI has been assessing TSST induced

salivary cortisol, over five years, 20% of their participants have been classified as cortisol non-

responders. A cortisol responder is defined as a participant who manifests a peak cortisol

increase of at least 15.5% from baseline (Miller et al., 2013). The cost of collecting, preparing,

and analyzing the increase percentage of salivary cortisol for each participant is >$30 in supplies

and >5 work hours. If a participant proves to be a cortisol non-responder, their data is invalid and

cannot be used in the study.

Numerous studies have concluded that women who take oral contraceptives manifest

abnormally increased CBG levels and, therefore, exhibiting lower levels of free cortisol than that

which would be displayed without the oral contraceptives. Kirschbaum et al. (1999) discovered

that females who use hormonal birth control exhibit “blunted or completely absent salivary

cortisol responses,” despite significant secretion of cortisol by the adrenal cortex (Kirschbaum et

al., 1999).

The study-wide effect of the oral contraceptive attenuated stress response is evident by

the oral contraceptive excluded cortisol responders of Kimura (2013) showing an average stress

induced baseline-to-peak cortisol response at 9.38 nmol/l (38%) higher than the oral

contraceptive included cortisol responders of Kunz-Ebrecht et al. (2003) and Buchanan & Tranel

(2008). Furthermore, oral contraceptive excluded cortisol non-responders of Kimura (2013)

showed a 1.07 nmol/l (44%) less baseline-to-peak cortisol decrease than the oral contraceptive

included cortisol non-responders of Kunz-Ebrecht et al. (2003) and Buchanan & Tranel (2008).

Therefore, this study hypothesized that females who use hormonal birth control are more
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likely to be classified as cortisol non-responders, exhibiting a cortisol increase of less than 15.5%

above baseline (Miller et al. 2013), in response to the psychosocial stress induced by the TSST.

Methods

Participants

65 females were recruited from undergraduate Psychology courses, at an ethnically

diverse university located in Northern California, and were given course credit for their

participation in the study. Participants were notified of the opportunity to participate in the study

by their professor. Before entering the study, all participants provided written consent and

completed a medical questionnaire that screened for and excluded participants who had past or

current health problems and who had used prescription medication and/or nicotine in the past six

months. A demographics questionnaire revealed that 16 females (Mage = 18.9, SD = 1.41)

currently used hormonal birth control and 49 females (Mage = 18.8, SD = 1.14) currently did not

use hormonal birth control.

Procedure

All cortisol samples were salivary cortisol samples collected with Salimetrics SalivaBio

Oral Swabs (SOS’s). Four salivary cortisol samples were collected at 10-20 min intervals. The

first salivary cortisol sample was collected at 13 min following participant's arrival to the

laboratory, and a screening questionnaire, as a measure of baseline salivary cortisol. The second

salivary cortisol sample was collected immediately following the TSST, 28 min post-participant

arrival. The third cortisol sample was collected 10 min post-TSST cessation (38 min) post-

participant arrival as a measure of peak salivary cortisol onset. The fourth, and final, salivary

cortisol sample was collected at 30 min post-TSST cessation (20 min post-3rd sample collection,

58 min post-participant arrival) as a measure of peak salivary cortisol cessation. Figure 1

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displays the relative collection times of each of the four salivary cortisol samples. The first

cortisol sample was used to assess a salivary cortisol baseline. It should be noted that the 3rd

cortisol sample was intended to measure peak cortisol levels. Salivary Cortisol samples were

prepared with Salimetrics Cortisol Salivary Immunoassay Kit’s (ELISA/EIA), MTX Pipettes,

Model 614L Laboratory Centrifuge by the Druker Lab, and a Fisher Stirring Hotplate. Prepared

cortisol samples were analyzed with a Finstruments Microplate Reader by MTX Lab Systems

Inc. and DeltaSoft JV Data Template.

Figure 1: Task sequence and cortisol sampling in the procedure.

Results

A chi-squared test showed a significant difference in percentage of responders

depending on birth control use, (X2(1, 65) = 5.11, p = .02). Based on a 15.5% cortisol increase

from baseline to be classified as a responder, females who use hormonal birth control

demonstrated a lower percentage of responders (56%) compared to females who do not use

hormonal birth control (84%) (see Figure 2).

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Figure 2: Relative comparison of cortisol responders

Discussion

Numerous studies have found a significant negative correlation with hormonal

birth control use and salivary cortisol response to psychosocial stress, induced by the TSST. It

has been shown that the ethinyl estradiol in hormonal birth control does not decrease cortisol

levels. Instead, ethinyl estradiol increases CBG's, which reduces bioactive cortisol. Since saliva

is the only vehicle that is used for cortisol collection that exclusively contains bio-active cortisol,

studies that investigate total cortisol must take this factor into account. This study agrees with

Kirschbaum et al. (1999) that future studies that investigate cortisol responses should measure

both blood and salivary cortisol. By doing so, a total cortisol level will be exhibited in the blood

and strictly bioactive cortisol will be manifest in the saliva.

Affect has been negatively correlated with cortisol levels (Kirschbaum & Hellhammer,

2000). Future studies should investigate the effects that hormonal birth control has on affect. If
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hormonal birth control increases positive affect, then this may be a reason for why an attenuated

cortisol response is observed in females who use birth control. Further investigations into the

effects of affect on CBG, and other steroid carrier, levels may reveal that an increased positive

affect, induced by hormonal birth control (specifically ethinyl estradiol) has a more significant

effect on increased CBG levels and, therefore, an attenuated salivary cortisol response to

psychosocial stress.

Future studies should investigate how other psychosocial stress protocols, such as the

socially evaluated cold-pressor test (SECPT; Schwabe, Haddad, Schachinger, 2008), affect the

cortisol levels in females who use hormonal birth control.

This study revisits questions proposed by Kirschbaum et al. (1999). Is the attenuated

salivary cortisol response to psychosocial stress exhibited by females who use hormonal birth

control associated with an increased number of physical complaints and/or ailments? Or does

their physiology adapt to this endocrine phenomenon with intracellular processes designed to

manage this unique cortisol response?

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