Clinical Endocrinology (2015) 83, 774–778
12853
ORIGINAL ARTICLE
The morning and late-night salivary cortisol ranges for healthy
women may be used in pregnancy
Urszula Ambroziak, Agnieszka Kondracka, Zbigniew Bartoszewicz, Małgorzata Krasnodez bska-Kiljan
Tomasz Bednarczuk
Department of Internal Medicine and Endocrinology, Medical University of Warsaw, Warsaw, Poland
Summary
Objectives The diagnosis of adrenal dysfunction in pregnancy
and in women taking oral contraceptives remains a diagnostic
challenge. Salivary cortisol seems to be a useful tool for the diag-
nosis of Cushing’s syndrome and adrenal insufficiency. However,
the changes in salivary cortisol concentration in healthy preg-
nancy are not clearly defined.
Design The aim of our study was to compare diurnal changes
in salivary cortisol in healthy pregnant women, healthy controls
and women on oral contraceptives.
Patients The study groups consisted of (i) 41 healthy pregnant
women, (ii) 42 healthy women and (iii) 12 healthy women on
oral contraceptives.
Measurements Serum and salivary cortisol in the morning and
salivary late-night cortisol were measured with Roche ECLIA
cortisol test (Elecsys 2010) in each trimester and postpartum.
Results Despite the elevation of morning serum cortisol in the
second and third trimesters of pregnancy, the morning salivary
values as well as late-night salivary cortisol throughout all trime-
sters were not significantly different from control values
(P > 0
5). In the postpartum period, the morning and late-night
salivary cortisol values were significantly lower than in late preg-
nancy. The morning and late-night salivary cortisol values in
women on contraceptives were also not different from those in
the healthy women group.
Conclusion The results of our study suggest that reference val-
ues for salivary cortisol established for a healthy adult popula-
tion can be used for pregnant women and women on oral
contraceptives in the initial diagnostic testing for Cushing’s syn-
drome and adrenal insufficiency.
(Received 28 April 2015; returned for revision 5 May 2015; finally
revised 2 July 2015; accepted 10 July 2015)
Correspondence: Urszula Ambroziak, Department of Internal Medicine
and Endocrinology, Medical University of Warsaw, Banacha 1 a street,
02-097 Warsaw, Poland. Tel.: 0048225992975; Fax: 0048225991975;
E-mail: uambroziak@wum.edu.pl
774
doi: 10.1111/cen.
 ska and
Introduction
Adrenal disorders in pregnancy are not common, but both
Cushing’s syndrome (CS) and adrenal insufficiency (AI) repre-
sent a serious risk for pregnancy outcome, so early diagnosis is
imperative.1 The diagnosis of adrenal dysfunction in pregnancy,
however, remains a diagnostic challenge. This is due to an over-
lapping clinical picture between CS and pregnancy, physiological
changes of the hypothalamic–pituitary–adrenal axis during preg-
nancy and hepatic overproduction of cortisol-binding globulin
(CBG) stimulated by oestrogens.2 Additionally, cortisol is dis-
placed from CBG by progesterone during pregnancy, so we can
observe an increase in free and total cortisol levels.3
Initial screening for CS in nonpregnant individuals includes
assessment of urinary free cortisol (UFC), overnight dexametha-
sone suppression test and late-night salivary cortisol (LNSC). For
pregnancy, UFC has been advised as a screening tool.4 A combina-
tion of UFC and midnight salivary cortisol was also proposed for
screening of CS in pregnancy without precise interpretation of
salivary cortisol results.5 The first step in diagnosis of AI is morn-
ing serum cortisol measurement confirmed when needed by an
acute ACTH stimulation test.6 There are no clear guidelines about
the diagnosis of AI in pregnancy, but in case of clinical suspicion,
morning serum cortisol has been proposed as a screening test.1
Recently, basal morning and ACTH-stimulated serum and salivary
cortisol levels in healthy pregnant women have been established.7
Morning and LNSC measurements seem to be useful in both
clinical situations, as during pregnancy diurnal ACTH and corti-
sol rhythm are maintained and are not influenced by CBG. Pre-
liminary results confirm the role of salivary cortisol
measurement in the diagnosis of adrenal dysfunction in preg-
nancy and in women taking oral contraceptives (OC).8,9
As changes in saliva cortisol concentration in healthy preg-
nancy are not yet clearly described, the aim of our study was to
compare diurnal changes in salivary cortisol in healthy pregnant
women, healthy controls and in women on OC.
Materials and methods
Subjects
Forty-one healthy pregnant women, nonshift workers, with a
median age of 30 (range 25–38) were enrolled in the study.
© 2015 John Wiley & Sons Ltd
 Salivary cortisol in pregnancy 775

Medical examination and blood and saliva sampling were carried
out in the first, second and third trimester (T1, T2 and T3) and
9–15 weeks postpartum (PP, n = 32) in an ambulatory setting.
Control groups consisted of (i) healthy women (HW), n = 42, age
31 (22–40) and (ii) healthy women on OC, n = 12, age 32
(23–50). Oral contraceptive pill contained 20 lg of ethiny-
loestradiol. The study group was partially described in Bar-
toszewicz et al.10 and Krasnodez bska-Kilja
nska et al.11 The clinical
characteristics of studied groups are presented in Table 1.
The project has been approved by the Bioethical Committee
of the Medical University of Warsaw, and written informed con-
sent for participation in this study was obtained from all partici-
pants.
Study design
Two morning and two evening saliva samples were obtained
from all subjects on two separate days. Salivetteâ device (Sarst-
edt, Germany) with cotton swabs was used for saliva collection.
Written instructions for saliva sampling were given to each per-
son collecting saliva at home. Morning serum and saliva cortisol
samples were collected at 0700–0900 h from both pregnant
women and control groups. Evening saliva samples were obtained
in both groups at 2230–2330 h before sleep. All subjects were
asked to repeat saliva sampling at home: one morning and one
evening sample in the control group and one morning and two
evening samples collected on separate days for pregnant women.
Morning saliva samples were obtained fasting without brushing
teeth, and evening samples at least 2 h after a meal. 5 min before
sampling, subjects were asked to rinse the mouth with water.
Home samples were refrigerated and transported to the labora-
tory no longer than 2 days after sampling, or kept frozen if trans-
ported later. Serum and saliva samples were kept frozen at
20°C until assayed. 94% and 72% of home samples were
returned for analysis from the pregnant and control groups,
respectively. Less than 2% of swab devices were dry or contained
too little saliva to complete the cortisol test. Home saliva samples
reported to be completed outside the requested time window
(for both morning and evening samples) were excluded (3%).
Assays
Serum and salivary cortisol was measured using Elecsys 2010
(Hitachi, Japan) analyzer and Roche Diagnostics (Germany) test
designed for both serum and saliva samples. Measurements were
performed prospectively during routine analyses, to consider
interassay variation. Randomly selected samples were also mea-
sured by Salimetrics, LLC (USA). Correlation of the Roche test
with Salimetrics was measured (n = 257): r = 0
817 P < 0
001.
To ensure quality control of salivary cortisol measurement,
pooled saliva with low, medium and high cortisol concentrations
was used as a complement to routine inter- and intralaboratory
quality control designed for serum samples.
Characteristics of the tests. Roche Diagnostics: analytic sensitivity
0
5 nmol/l (no data for functional sensitivity), repeatability and
precision within the concentration range for saliva 1
5–6
1% and
4
1–33
4% respectively, cross-reactivity with cortisone 0
3%,
sample volume 20 ll.
Salimetrics: analytic sensitivity 0
19 nmol/l (no data for func-
tional sensitivity), repeatability and precision 4
2–5
3% and 6
9–
10
6% respectively, cross-reactivity with cortisone 0
31%, sample
volume 25 ll.
Statistical analysis
Statistical analyses were performed using Statistica version 10
(StatSoft, Tulsa, OK, USA).
Data are expressed as mean and range. For demographic data,
the range minimal-to-maximal value is used. For cortisol con-

Table 1. Characteristics of pregnant women in the first (T1), second (T2) and third trimester (T3), postpartum (PP), healthy women (HW) and
women on oral contraceptives (OC)

Healthy women Women on OC

T1 (n = 41) T2 (n = 41) T3 (n = 41) PP (n = 32) (HW) (n = 42) (OC) (n = 12)

Age (year) 30 (25–38) 31 (22–40) 32 (23–50)

Weeks of 10 (5–13) 21 (15–26) 31 (27–36) 12 (9–15) N/A N/A
gestation or
postpartum
BMI (kg/m2) 22
5 (18
4–32
1) N/A N/A 23
7 (17
4–31
9) 23
9 (17
5–36
0) 21
8 (18
4–36
1)
Weight gain (kg) 2
5 ( 4–20) 7
1 ( 3–26) 12
0 ( 8–23) N/A N/A N/A
SBP (mm Hg) 113 (90–140) 114 (100–150) 117 (100–140) 118 (100–140) 115 (90–140) 98 (90–110)
DBP (mm Hg) 75 (60–90) 73 (50–90) 76 (60–90) 77 (50–90) 74 (60–90) 62 (60–70)
Medication Multivitamin, Multivitamin, Multivitamin, Multivitamin, No No
iodinesupplem- iodinesupplem- iodinesupplem- iodinesupplem-
entation entation entation entation
Comorbidities No No No No No No

Values are presented as mean and range. No statistically significant differences between HW, OC and pregnant women parameters were detected (ANOVA
–Scheffe test).
N/A – not applicable.

© 2015 John Wiley & Sons Ltd

Clinical Endocrinology (2015), 83, 774–778
776 A. Urszula et al.

centration, the range between 2
5 and 97
5 percentile is reported.
Results below the detection limit of the assay for salivary cortisol
concentrations (<0
49 nmol/l), for statistical purposes, were set
to the limit of detection value (1
4% of data). ANOVA–post hoc
Scheffe test was used to assess the significance of differences
between subgroups.
(P = 0
99). There was no difference between HW and OC
groups. Noteworthy, LNSC in the OC group also was not differ-
ent from T1, T2 and T3 (P = 1
0, P = 1
0 and P = 0
99, respec-
tively). Three months postpartum, LNSC was lower compared
to T3 (Fig. 1c, P = 0
0001). The difference was also seen
between LNSC 3 months PP and HW (Fig. 1c, P = 0
04)

Results
Characteristics of studied groups
Characteristics of the pregnant women and control groups are
presented in Table 1. In pregnant women, mean BMI before
pregnancy was 22
7 kg/m2 (range 18
1–32
2), normal BMI was
observed in 73% and overweight and obesity in 21% of subjects.
During pregnancy, no complications were noted, that is gesta-
tional diabetes or hypertension. All pregnancies were singleton;
61% were first pregnancies; 29% were second; 56% of newborns
were male; mean term of delivery was at 39
9 weeks (range 38–
42), newborn birthweight 3474 g (2470–4250 g), and average 9
8
Apgar points (9–10); and all children were delivered healthy by
natural labour.
Cortisol concentrations
In pregnant women, the morning serum cortisol increased sig-
nificantly during the course of gestation in T2 and T3 and
returned to the HW values after delivery (Table 2 and Fig. 1a).
Significant differences were also detected between the healthy
women group and healthy subjects on OC (Table 2).
No significant increase in the morning salivary T3 compared
to T1 was observed (Fig. 1b, P = 0
4). Moreover, no significant
differences in the morning salivary cortisol between T1, T2, T3
and HW were present. There were also no significant differences
between the HW and OC groups (P = 1
0) or between the OC
group and T1, T2, T3 (P = 1
0). Three months postpartum
(PP), the morning salivary cortisol was lower compared to T2
and T3 (Fig. 1b, P = 0
01, <0
0001, respectively).
No significant increase in LNSC was observed in T3 compared
to T1 (Fig. 1c, P = 0
6). LNSC was also not significantly differ-
ent from the HW group in the first, second or third trimester
Discussion
There is growing evidence that salivary cortisol may be used to
diagnose adrenal dysfunction in pregnancy and in women on
OC. However, the need for reference ranges for morning and
late-night salivary cortisol in pregnancy is frequently empha-
sized.1,5,12–14 The results of our study show that morning sali-
vary cortisol and late-night salivary cortisol throughout
pregnancy were not significantly different from healthy control
women values.
LNSC is considered one of the most sensitive tests in screen-
ing for CS in nonpregnant patients.15 Data about the usefulness
of this parameter in the diagnosis of CS in pregnancy are still
very scanty. Because of the limited number of sporadic CS cases
in pregnancy, the construction of cut-off values on the basis of
ROC curve is unattainable.
In our study, the LNSC range for healthy women was not
exceeded even in late pregnancy although both morning salivary
cortisol and LNSC concentration increased linearly during preg-
nancy by a mean 0
81 nmol/l and 0
26 nmol/l per month,
respectively. The elevation of 97
5 percentile value was seen only
in 1
4%, 2
7% and 9% of measurements in subsequent trime-
sters, but without statistical significance. Also, no difference was
seen between pregnant women and women on OC, and women
on OC and HW. The results of our study may suggest that pop-
ulation cut-off values of LNSC may be used to exclude CS in
pregnancy. On the other hand, we noticed an increase in LNSC
values by about 20% in the third trimester, which suggests that
further, more expansive studies are needed to determine refer-
ence values in the third trimester.
There are many reports that salivary cortisol concentration
rises throughout pregnancy, but they often lack control
groups.16–18 Manetti et al.8 showed that, when compared
with controls, no difference was observed in the morning and

Table 2. Cortisol concentration (nmol/l) in the morning serum, morning saliva and late-night saliva [mean and range (2
5 and 97
5 percentile)] in
studied groups: healthy women, women on oral contraceptives, pregnant women in the first, second, third trimester and postpartum

No of samples analysed Morning serum Morning saliva Late-night saliva

Healthy women 42/59/55 484
9 (274
8–721
4)** 18
5 (5
27–37
8) 4
6 (1
1–9
0)

Women on OC 12/11/13 1002
8 (766
9–1369
4)* 19
9 (9
9–26
2) 4
7 (3
1–7
9)
Pregnancy, 1st trimester 41/71/71 586
2 (281
7–954
8)** 18
4 (7
2–34
2) 4
4 (1
8–9
1)
Pregnancy, 2nd trimester 41/76/74 833
1 (456
8–1305
4)* 19
6 (6
9–36
4) 4
6 (1
7–9
8)
Pregnancy, 3rd trimester 41/76/77 973
5 (406
6–1464
5)* 21
9 (8
9–39
7) 5
6 (1
3–13
5)
Postpartum, 3 months 32/57/57 487
4 (293
8–706
2)** 13
8 (4
08–26
01) 3
0 (1
0–5
3)***

Marked values were significantly different compared to healthy women group: *P < 0
001 or to women on OC: **P < 0
001, ***P < 0
05 (ANOVA –
Scheffe test).

© 2015 John Wiley & Sons Ltd

Clinical Endocrinology (2015), 83, 774–778
 Salivary cortisol in pregnancy 777

during the second half of pregnancy.19 At the same time, how-

(a)
***
2000 *** *** ever, salivary cortisol concentration was constant throughout all
*** *** *** trimesters, but higher than postpartum. The conclusion that dur-
Morning serum cortisol nmol/l

ing pregnancy salivary cortisol concentration is elevated is often

1500
the result of comparison with postpartum concentrations.17,19 In
our study, the comparison between third trimester LNSC and the
postpartum period also showed a significant drop in cortisol con-
1000
centration. LNSC 3 months postpartum was even lower than HW
values, which suggests decreased activity of the HPA axis postpar-
500
tum along with increased activity during pregnancy. The lack of
difference that was observed in the morning serum cortisol
*** ***
*** between HW and PP also suggests the impact of elevated CBG
0
*** levels up to 3 months postpartum.20,21
HW OC T1 T2 T3 PP Morning salivary cortisol is considered less sensitive and
(b) specific for the diagnosis of adrenal insufficiency than serum
50
* cortisol in nonpregnant subjects.22 As demonstrated by Suri
***
Morning salivary cortisol nmol/l

et al., however, morning salivary cortisol is a reliable method of

40
AI diagnosis in pregnancy which is especially important in the
second and third trimesters. Numerous reports show that mea-
30 suring baseline serum cortisol concentration in late gestation can
be misleading.23 Our results confirm this data and suggest that,
20 when assessing pregnant woman for adrenal insufficiency using
morning serum cortisol level, one may rely on population refer-
10
ence ranges only in the first trimester. Morning salivary cortisol
in our pregnant women did not differ from controls either in
early or late pregnancy. This was also confirmed by others.14
0
HW OC T1 T2 T3 PP The elevation of the 97
5 percentile value of the control group
as compared to 97
5 percentile value in pregnancy was not
(c)
20 * observed in T1 and T2 and was seen in 5
3% of measurements
*** in T3. Values below the 2
5 percentile in the control group were
Late-night salivary cortisol nmol/l

seen in 1/71, 1/76 and 5/76 measurements as compared to T1,

15 T2 and T3, respectively.
Salivary cortisol measurements in women on oral contracep-
tives showed no difference as compared to values in healthy
10 women. The data are consistent with previous reports.8 This
would therefore be useful in the screening for adrenal dysfunc-
tion in women on OC.
5 Several important limitations of our study need to be consid-
ered: (i) relatively small study group; (ii) cortisol measurement
by immunoassay; and (iii) lack of pregnant women with CS or
0
HW OC T1 T2 T3 PP AI to confirm the usefulness of the tests.

Fig. 1 (a), Morning serum cortisol. (b), Morning salivary cortisol. (c),
Late-night salivary cortisol. Cortisol concentration (nmol/l) in healthy
women (HW), women on OC, pregnant women in the first (T1), second
(T2), third trimester (T3) and postpartum (PP). Number of samples:
morning serum cortisol (HW n = 42, OC n = 12, T1,T2,T3 n = 41, PP
n = 32), morning salivary cortisol (HW n = 59, OC n = 11, T1 n = 71,
T2,T3 n = 76, PP n = 57), late-night salivary cortisol (HW n = 55, OC
n = 13, T1 n = 71, T2 n = 74, T3 n = 77, PP n = 57). Significant
differences between parameters: ***P < 0.0001, *P < 0.05 (ANOVA- Scheffe
test)
Conclusions
The results of our study suggest that reference values for salivary
cortisol established for a healthy adult population can be used
for pregnant women and women on oral contraceptives in initial
testing for Cushing’s syndrome and adrenal insufficiency. Fur-
ther clinical trials are needed, assessing salivary cortisol in the
diagnosis and treatment of CS or AI in pregnant women or
women on OC.

afternoon samples, while midnight salivary cortisol differed

Acknowledgements
significantly from healthy subjects.
Maulenberg & Hofman showed that, during pregnancy, We would like to thank all participants of this study and Suzy
plasma cortisol concentration in the afternoon was increased Zurecka for English correction.

© 2015 John Wiley & Sons Ltd

Clinical Endocrinology (2015), 83, 774–778
778 A. Urszula et al.
Conflict of interest
The authors have nothing to declare.
Financial disclosure
The study was not financially supported.
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Clinical Endocrinology (2015), 83, 774–778