Keywords
Depression - Anxiety - Childbirth - Postpartum - Bariatric surgery - Obesity
Introduction
In patients seeking bariatric surgery (BS), psychiatric disorders are common, with a
prevalence of 19% for depression being more than twice as high as in the general
population [1]. Available evidence has
shown an improvement in mental health status after surgery, but recent meta-analyses
suggest deterioration several years postoperatively [2]
[3].
The majority of patients with BS are women, many of them of reproductive age [4], whose fertility usually improves after
surgery [5].
Postpartum depression (PPD) is one of the most common and disabling complications of
childbirth [6]. PPD has substantial
comorbidity with anxiety disorders, which are also common in the postpartum period
and merit clinical attention [7].
Recent meta-analyses revealed higher rates of postpartum depressive symptoms for
women with overweight (OR 1.14) and even higher rates for women with obesity (OR
1.39) as compared to women with normal weight or overweight [8]. Due to limited data, the association
between body mass index (BMI) and postpartum anxiety disorders remains uncertain
[8].
Data on the perinatal mental health of women following BS is scarce. Jans et al.
(2017) found increased anxiety but no differences in depressive symptoms in 54 women
after BS as compared to 25 pregnant women with obesity without BS [9]. Since 2021, four studies have
addressed antenatal depression and anxiety reporting higher rates in women after BS
than in pregnant women with or without obesity [10]
[11]
[12]
[13].
Only one study focused on postpartum mental health in patients following BS [10]. Records of 132 Brazilian women were
reviewed for diagnoses of pharmacologically treated depression and anxiety during
pregnancy and a prevalence of 13.6% was reported. Additionally, newly diagnosed PPD
(clinically diagnosed or self-reported) was found in 7.6% of women.
Despite potentially severe consequences of PPD, the postpartum mental health of women
following BS has not yet been examined with standardized instruments. i. e.,
standardized questionnaires or interviews.
The “SPOtMom study” is a pilot study examining PPD and anxiety with the goal of
establishing a prospective registry of women with pregnancy after BS in the future.
SPOtMom examines (1) the prevalence of depression diagnoses using a diagnostic
interview and (2) the prevalence of symptoms of depression and anxiety using
standardized questionnaires.
Methods
Study design and participants
Data were collected from January to May 2020 at two certified obesity centers
(Würzburg and Heide, Germany). Women of at least 18 years of age with BS prior
to pregnancy were eligible if childbirth dated back at least 6 months. The
survey only covered the first pregnancy following BS while subsequent
pregnancies were not analyzed. The study protocol was approved by the Ethics
Committee of the University of Würzburg (#62/19) and complied with the
principles of the Declaration of Helsinki. The trial was registered at
clinicaltrials.gov (NCT04297956). Written informed consent was obtained from all
participants prior to any study-related investigation.
Study course
Patients meeting the inclusion criteria were identified and an appointment for
the survey was scheduled. Patients were asked for demographic data and their
current mental health status (T2) as well as their recalled mental health status
in the first 4–6 weeks after giving birth (T1). Before answering questionnaires
retrospectively, patients were interviewed by a psychologist with a detailed
discussion of the postpartum period to facilitate recall of past symptoms.
Initially, the survey was implemented during post-bariatric aftercare visits.
After the coronavirus disease 2019 (COVID-19) outbreak in Germany at the end of
February 2020, face-to-face data collection was canceled and questionnaires were
sent by mail and patients were interviewed by phone.
Variables and measurements
Demographic data were obtained at T2. Patients were asked for their current
weight, weight at the time of BS, dates of BS, and childbirth. BMI, change in
BMI, and time intervals were calculated.
Standardized questionnaires were used to screen for depressive and anxiety
disorders and assess the severity of symptoms. To examine current symptoms of
depression, patients were asked to complete three different questionnaires
(Patient Health Questionnaire 9 (PHQ-9) [14], Edinburgh Postnatal Depression Scale (EPDS) [15], Beck Depression Inventory II
(BDI-II) [16]). For T2, patients were
only defined as having symptoms of depression when reaching the cutoff in at
least two out of the three depression questionnaires. In order not to overstrain
patients, there was only one questionnaire for current and a retrospective
symptoms of anxiety [General Anxiety Disorder 7 (GAD-7) [17] and the retrospective
investigation of depressive symptoms (PHQ-9). For retrospective assessment,
instructions of the questionnaires were modified: Instead of asking for symptoms
in the previous two weeks, patients were asked for symptoms within 4 to 6 weeks
after giving birth. Cutoff scores were determined according to literature
recommendations. To achieve a high sensitivity to detect patients at risk,
cutoff scores of ≥ 10 were defined for PHQ-9 [14]
[18], EPDS [15] and GAD-7 [17] and ≥ 13 for BDI-II [19].
To formally establish the diagnosis of depression, modules A-D of the clinical
version of the Structured Clinical Interview for DSM-5 (SCID-5) [20] were conducted. SCID captures
current diagnosis and can measure lifetime prevalence asking for the worst
episode of symptoms in the past. For this study, a question was added to
determine the prevalence of depressive disorders within the first 4 weeks
postpartum.
Statistical analysis
Solely descriptive statistics was used to present the demographics and survey
responses. Due to the small sample size, demographic data are presented as
median with interquartile range (IQR). Questionnaire scores are primarily
presented as mean with SD for comparability across studies.
Results
Sample description
The sample comprised n=22 women (Würzburg n=19, Heide n=3, median age 36.0 years,
IQR 30.3–38.0). Median current BMI was 36.2 (29.3–39.5) kg/m2 and
preoperative BMI was 54.7 (47.5–56.5) kg/m2. Further patients’
characteristics are depicted in [Table
1].
Table 1 Patient characteristics at T2
(n=22).
Age (years)
|
36.0 (30.3–38.0)
|
Current BMI (kg/ m²)
|
36.2 (29.3–39.5)
|
German nationality
|
22 (100.0)
|
Obesity center Würzburg / Heide
|
19 (86.4) / 3 (24.6)
|
Marital status (n=22)
|
|
Married
|
15 (68.2)
|
Being allied
|
4 (18.3)
|
Divorced
|
2 (9.1)
|
Separated
|
1 (4.5)
|
Employment status (n=20)
|
|
Employed
|
10 (50.0)
|
Parental leave
|
6 (30.0)
|
Housewife
|
2 (10.0)
|
Seeking work
|
2 (10.0)
|
Pregnancy and birth (n=22)
|
|
Primiparous women*
|
15 (71.4)
|
History of miscarriage
|
7 (31.8)
|
Planned pregnancy
|
14 (63.6)
|
In-vitro fertilization
|
2 (9.1)
|
Singleton pregnancy
|
22 (100.0)
|
Pregnancy complications
|
10 (45.5)
|
Gestational diabetes
|
3 (13.6)
|
Preeclampsia
|
0 (0.0)
|
Hyperemesis
|
2 (9.1)
|
Preterm birth
|
1 (4.5)
|
Vaginal bleeding
|
3 (13.6)
|
Hypertension
|
1 (4.5)
|
Other
|
4 (18.2)
|
Birth complications
|
5 (22.7)
|
Cesarian section
|
10 (45.5)
|
Breastfeeding
|
14 (63.6)
|
Health status
|
|
Current diseases*
|
9 (42.9)**
|
|
0
|
|
0
|
|
1 (4.5)
|
Medication intake*
|
11 (52.4)
|
|
0
|
|
0
|
|
1 (4.8)
|
|
1 (4.8)
|
Diseases remitted after BS*
|
|
|
5 (23.8)
|
|
7 (33.3)
|
|
2 (9.5)
|
Type of primary surgery (n=22)
|
|
Roux-en-Y gastric bypass
|
12 (54.5)
|
One anastomosis gastric bypass
|
1 (4.5)
|
Sleeve gastrectomy
|
9 (40.9)
|
Secondary BS before pregnancy
|
3 (14.3)
|
Time intervals (n=22)
|
|
Interval surgery – childbirth (months)
|
29.3 (20.7–45.5)
|
Interval surgery – survey (months)
|
62.2 (43.7–85.9)
|
Interval childbirth – survey (months)
|
21.1 (14.1–42.9)
|
Weight trajectory (n=20)
|
|
BMI (kg/ m²)
|
|
Preoperative
|
54.7 (47.5–56.5)
|
|
52.0 (±6.0)
|
T1
|
33.9 (31.7–37.6)
|
|
34.9 (±6.7)
|
T2
|
36.2 (29.3–39.5)
|
|
35.7 (±7.3)
|
Change in BMI (kg/ m²)
|
|
Preoperative vs. T1
|
− 17.9 (− 12.9 – − 22.7)
|
|
− 17.2 (±8.2)
|
T1 vs. T2
|
0.8 (− 0.5–2–0)
|
|
0.8 (±2.3)
|
%TWL (%)
|
|
Preoperative vs. T1
|
33.6 (27.9–39.5)
|
|
32.1 (±14.1)
|
Annotation 1: Variables are presented as median (interquartile range) or
n (%). For weight trajectory mean and SD are presented additionally. BS:
bariatric surgery; BMI: body mass index; TWL: total weight loss; *
information for “primiparous woman”, “existing diseases”, “remitted
diseases”, and “medication intake” was only available in 21 women; **
lipedema (2), bronchial asthma (2), multiple endocrine neoplasia 1
(MEN 1) (1), hypothyroidism (1), polycystic ovary syndrome (1),
post-traumatic stress disorder (1), gestational diabetes
(1).
Psychopathology
Patients were defined as showing signs of depression or anxiety or both
(“patients with dep/anx”) when reaching the predefined questionnaire cutoff
value in PHQ-9 (T1) / GAD-7 (T1 or T2) or in at least 2 out of 3 depression
questionnaires at T2 or obtaining a diagnosis of any depressive disorder
according to SCID-interview.
Overall, 8 of 22 patients (36%) showed signs of depression/ anxiety: four of them
at both T1 and T2, one patient only at T1 and three patients only at T2.
Concerning the type of psychopathology, six patients reported signs of
depression as well as anxiety across T1 and T2, one patient only showed
depressive symptoms and one patient suffered at least from depression, while for
anxiety, a questionnaire was missing.
Depressive disorders
At T1, 3 patients (14%) showed depressive symptoms in PHQ-9. Two of them were
also diagnosed with major depressive disorder (MDD) according to the SCID ([Table 2]). One more patient was
diagnosed with “other depressive disorder” as the criteria for MDD were not
fulfilled. Her questionnaire data are missing.
Table 2 Depression and anxiety: interview and
questionnaire results.
|
|
Depression
|
|
|
|
Anxiety
|
|
|
SCID
|
PHQ-9
|
BDI-II
|
EPDS
|
GAD-7
|
T1
|
Total, n
|
22
|
21
|
/
|
/
|
21
|
|
Pathologic, n (%)
|
3 (13.6)
|
3 (14.3)
|
/
|
/
|
3 (14.3)
|
|
Score, mean ±SD
|
/
|
7.1±6.3
|
/
|
/
|
5.1±4.8
|
|
Score, median (IQR)
|
/
|
6.0 (3.0–8.0)
|
/
|
/
|
4,0 (2.0–7.0)
|
T2
|
Total, n
|
22
|
22
|
21
|
22
|
22
|
|
Pathologic, n (%)
|
1 (4.5)
|
7 (31.8)
|
9 (42.9)
|
6 (27.3)
|
6 (27.3)
|
|
Score, mean ±SD
|
/
|
8.2±5.9
|
13.5±13.1
|
7.0±7.3
|
6.4±6,5
|
|
Score, median (IQR)
|
/
|
6.5 (3.3–11.5)
|
8.0 (3.0–22.0)
|
4,5 (2.0–11.3)
|
4.0 (1.3–10.0)
|
IQR: interquartile range; SD: standard deviation; SCID: Structured
Clinical Interview for DSM; PHQ: Patient Health Questionnaire; BDI-II:
Beck Depression Inventory II; EPDS: Edinburgh Postnatal Depression
Scale; GAD-7: General Anxiety Disorder 7.
At T2, between 27% and 43% of patients showed symptoms of depression, depending
on the questionnaire used. One of those patients (5%) was also diagnosed with
MDD at T2 ([Fig. 1]).
Fig. 1 Depression. Patients showing pathological results for
depression
Using the SCID interviews, 12 patients were diagnosed with at least one
additional depressive lifetime episode, corresponding to a lifetime prevalence
of 55%. Asked for the worst depressive episode apart from T1 or T2, most of the
patients reported preoperative episodes. Depressive episodes apart from T1 and
T2 are minimum numbers, and further episodes may have occurred at other time
points.
Anxiety
At T1, three patients (14%) showed high levels of anxiety symptoms. With three
more patients reaching the cutoff at T2, six patients (27%) showed pathological
results.
Characterization of patients with depression and/or anxiety
The characteristics of patients with vs. without dep/anx are shown in [Table 3]. Patients with dep/anx were
older, had a higher BMI, and experienced less weight loss as compared to
pre-surgery than patients without dep/anx. Moreover, patients with dep/anx had
more often a history of miscarriage and complications during pregnancy.
Table 3 Sample characteristics of patients with vs.
without signs of depression/ anxiety.
Variables
|
Patients without dep/anx (n=14)
|
Patients with dep/anx (n=8)
|
Age (years)
|
32.5 (30.0–38.0)
|
37.0 (34.8–38.0)
|
Current BMI (kg/ m²)
|
31.8 (28.4–38.0)
|
39.5 (35.0–43.0)
|
Obesity center Würzburg
|
12 (85.7)
|
7 (87.5)
|
Marital status
|
|
|
Married
|
8 (57.1)
|
7 (87.5)
|
Being allied
|
4 (28.6)
|
0 (0)
|
Divorced
|
1 (7.1)
|
1 (12.5)
|
Separated
|
1 (7.1)
|
0 (0)
|
Employment status
|
|
*
|
Employed
|
7 (50.0)
|
3 (42.9)
|
Parental leave
|
4 (28.6)
|
2 (28.6)
|
Housewife
|
1 (7.1)
|
1 (14.3)
|
Seeking work
|
1 (7.1)
|
1 (14.3)
|
Pregnancy and birth
|
|
|
Primiparous women
|
11 (78.6)
|
4 (57.1)*
|
History of miscarriage
|
2 (14.3)
|
5 (62.5)
|
Planned pregnancy
|
9 (64.3)
|
5 (62.5)
|
In-vitro fertilization
|
0
|
2 (25.0)
|
Pregnancy complications
|
4 (28.6)
|
6 (75.0)
|
Gestational diabetes
|
1 (7.1)
|
2 (25.0)
|
Hyperemesis
|
1 (7.1)
|
1 (12.5)
|
Preterm birth
|
1 (7.1)
|
0
|
Bleeding
|
1 (7.1)
|
2 (25.0)
|
Hypertension
|
1 (7.1)
|
0
|
Other
|
1 (7.1)
|
3 (37.5)
|
Birth complications
|
3 (21.4)
|
2 (25.0)
|
Cesarian section
|
6 (42.9)
|
4 (50.0)
|
Breastfeeding
|
8 (57.1)
|
6 (75.0)
|
Health status (n)
|
**
|
|
Current diseases***
|
5 (38.5)
|
4 (50.0)
|
|
0
|
0
|
|
0
|
0
|
|
0
|
1 (12.5)
|
Current medication intake
|
5 (38.5)
|
6 (75.0)
|
|
0
|
0
|
|
0
|
0
|
|
0
|
1 (12.5)
|
Diseases remitted after BS*
|
|
|
|
1 (7.1)
|
3 (37.5)
|
|
4 (28.6)
|
4 (50)
|
|
1 (7.1)
|
1 (12.5)
|
Type of primary surgery
|
|
|
Roux-en-Y gastric bypass
|
8 (57.1)
|
4 (50.0)
|
One anastomosis gastric bypass
|
1 (7.1)
|
0 (0.0)
|
Sleeve gastrectomy
|
5 (35.7)
|
4 (50.0)
|
Secondary BS before pregnancy
|
1 (7.7)
|
2 (25.0)
|
Weight trajectory
|
**
|
*
|
BMI (kg/ m²)
|
|
|
Preoperative
|
54.6 (49.0–56.3)
|
55.8 (47.3–56.8)
|
|
51.2 (±6.9)
|
53.6 (±7.9)
|
T1
|
34.0 (29.8–37.5)
|
33.9 (33.6–39.4)
|
|
33.5 (±6.7)
|
37.4 (±6.4)
|
T2
|
34.3 (28.4–38.3)
|
39.4 (34.6–41.7)
|
|
33.8 (±7.2)
|
39.4 (±6.4)
|
Change in BMI (kg/ m²)
|
|
|
Preoperative vs. T1
|
− 19.1 (− 14.5 – − 23.7)
|
− 13.9 (− 12.7 – − 20.6)
|
|
− 17.6 (±9.2)
|
− 16.2 (±6.5)
|
T1 vs. T2
|
0.6 (− 1.4–1.4)
|
1.9 (0.4–2.3)
|
|
0.2 (±1.8)
|
1.9 (±2.8)
|
%TWL (%)
|
|
|
Preoperative vs. T1
|
37.3 (31.2–43.1)
|
29.1 (27.8–35.9)
|
|
33.3 (±16.4)
|
29.8 (±9.2)
|
Annotation: Variables are presented as median (interquartile range) or n
(%). For weight trajectory mean and SD are presented additionally. BS:
bariatric surgery; dep/ anx: depression/ anxiety; BMI: body mass index;
TWL: total weight loss *for “employment status”, “primiparous women”,
and “weight trajectory” only data of seven patients available; ** for
“health status” and “weight trajectory” only data of 13 patients
available; *** lipedema (2), bronchial asthma (2), multiple endocrine
neoplasia 1 (MEN 1) (1), hypothyroidism (1), polycystic ovary syndrome
(1), post-traumatic stress disorder (1), gestational diabetes (1).
More of the patients with dep/anx stated having ever been treated because of
depressive symptoms (75% vs. 36%) or having family members suffering from
depression (63% vs. 50%). Based on SCID, more patients with dep/anx had a
depression diagnosis prior to T1 (63% vs. 36%).
Time intervals were analyzed separately for patients with dep/anx at T1 or T2.
Patients with dep/anx at T1 gave birth more often within 2 years after BS (60%
vs. 24%).
In patients with pathologic results at T2, more time had passed since childbirth
[3.3 years (2.8–5.2) vs. 1.7 years (0.9–2.5)] and fewer patients were within the
first year postpartum (14% vs. 27%) or with their child in early infancy up to 3
years of age (29% vs. 73%). The median time since surgery was similar for
patients with [5.3 years (4.6–7.4)] as compared to patients without dep/anx [5.0
years (3.2–7.1)].
For all patients with dep/anx, surgery dated back more than 3.5 years, while
60.0% of patients without dep/anx were within the first 3.5 years
postoperatively. 13.3% of patients without dep/anx were within the first 3.0
years following surgery.
Discussion
To our knowledge, this is the first study using standardized instruments to examine
mental health after childbirth in women with previous BS. In the immediate
postpartum period (T1), 14% of patients were diagnosed with depression and showed
symptoms of depression or anxiety, whereas 21.1 (14.1–42.9) months after childbirth
(T2) a depressive disorder could be diagnosed in only 5% of patients. However,
symptoms also covering subclinical depression assessed with questionnaires increased
to 32%. Anxiety symptoms increased to 27% and were highly comorbid with depressive
symptoms.
There is still an ongoing debate about the effects of BMS on depression as well as a
possible increase in self-harm and suicide after BS. Several authors reported an
increased hazard ratio when comparing patients with their preoperative state or with
matched controls [21]. A potential risk
was even postulated for patients treated with GLP-1 receptor agonists [22], but while an increase in depressive
symptoms and suicide ideation was described in comparison to other antidiabetic
treatments, statistically increased risk for suicide attempts or completed suicide
has not been confirmed when examining large databases [23]
[24]. However, it is generally accepted that the average course of
psychological well-being after BS is characterized by an initial “honeymoon” phase
with improvement of symptoms of depression and anxiety, while in the long term, a
renewed deterioration of psychopathology has been described in systematic reviews,
mostly based on self-reported data [2]
[3].
Concerning T2, for patients with dep/anx, more time had passed since childbirth and
fewer women were within the first year postpartum or having infant children.
Therefore, in these patients, birth and postpartum do not seem to explain elevated
levels of depression or anxiety. Increased symptom levels could be rather due to
longer time intervals since BS. In our study, no patient with dep/anx at T2 was
within 3 years postoperatively, and almost half of the women without dep/anx were
within 3.5 years postoperatively.
However, high symptoms of depression and anxiety at T2 could also be a reaction to
the global COVID-19 crisis and its first wave of infections in Germany [25]. On the other hand, symptomatology at
T1 may be underestimated due to recall bias, as data collection took place at T2 and
the postpartum period was assessed retrospectively [26].
When we compared our results to community samples within the first 6 weeks postpartum
from literature using identical methods and cutoffs, we found – descriptively – a
higher rate of depressive psychopathology in our patients. In SPOtMom 9.1% of women
after BS were diagnosed with postpartum MDD by SCID in comparison to a prevalence of
2.9–5.9% [27]
[28]
[29]. Regarding depressive symptoms according to PHQ-9, literature data
showed similar (12.4% – 14.9%) [30]
[31]
[32] or lower (2.5% – 9.0%) [28]
[33] prevalence in the
general postpartum population than in our cohort (14.3%). However, the prevalence of
anxiety symptoms was not different between SPOtMom (14.3%) and the reported
prevalence of 10.0–15.0% in the community setting [34]
[35].
When comparing our data to that of a cohort also within 2–4 years after BS – which
corresponds to T1 – frequency of SCID diagnoses of MDD in the SPOtMom study (9.1%)
was similar to results from Hayden et al. reporting 7.3% prevalence of MDD
(including partial remission) and to the 4.4% to 9.7% of MDD diagnoses in
non-pregnant patients at different time points after RYGB in a study by Kalarchian
et al. [36]
[37] Questionnaire scores in our study
sample were also in the upper range of comparable published data: The mean score of
PHQ-9 in the SPOtMom study was 7.1, while literature results range from 4.3 to 6.7
[38]
[39]
[40]
[41]
[42]
[43]. The mean GAD-7 score of the SPOtMom study (5.1) was similar to
scores from literature data (3.4–5.1) [38]
[39]
[40]
[43].
As contraception is recommended for 12–24 months postoperatively according to
guidelines [44]
[45], pregnancy occurring in this period
may lead to additional psychological burden still apparent in the early postpartum.
This might have been the case in our cohort, as childbirth was more often within the
first two years after BS in patients with dep/anx in postpartum.
A lifetime prevalence of 55% for depressive disorders in our patients, mostly
emerging preoperatively, is in line with other studies from Germany reporting 50–60%
lifetime depressive disorders with SCID in BS candidates [46]
[47]. This high prevalence may reflect the given psychological burden of
patients presenting for BS [48]. With
obesity being accepted as a mediator for higher depression prevalence, future
investigations should include a BMI-matched control group.
Patients with dep/anx suffered more often from previous depressive episodes and
showed slightly higher rates of family history of depression, which is consistent
with literature data [6]
[49]
[50] and might help to identify patients at even higher risk for an
unfavorable course. These patients had also higher current BMI and worse weight loss
trajectories. This could be due to higher preoperative BMI [51] or postoperative depression possibly
being associated with poorer weight outcomes [52]. Trends of a more frequent history of miscarriage and higher rates of
pregnancy complications in patients with dep/anx should be evaluated in further
studies with a larger sample size.
Several limitations arising from the nature of the work as a pilot study exist. The
sample size was small and no control group was included. Therefore, rather than
inferring final conclusions, we aimed to describe preliminary trends and generate
hypotheses. Because of the retrospective nature, prevalence rates concerning the
early postpartum may be underestimated. As instruments were not validated for
retrospective use, with the exception of lifetime prevalence based on SCID, results
for T1 must be interpreted with caution. Furthermore, as anxiety disorders were not
captured in SCID, statements are only possible at the symptom level. However,
SPOtMom has also relevant strengths. It is the very first study to evaluate mental
health following childbirth in women with previous BS with standardized instruments.
It was possible to differentiate symptom level and valid depression diagnosis by use
of SCID interview as gold standard. Comparability was enhanced by a literature
search of studies that are consistent in timing and study instruments.
In conclusion, women following BS could have higher prevalence of depression
diagnosis and, by trend, higher depression symptomatology within the early
postpartum compared to women from the general population while no clear differences
in symptoms of anxiety were seen. In the long run, motherhood does not seem to
further deteriorate mental well-being when compared to a general postbariatric
cohort. The time interval from BS seems to be a more important factor, emphasizing
the necessity of long-term bariatric aftercare. Especially due to possible recall
bias underestimating postpartum depressive symptoms, further studies with
prospective assessment of depressive symptoms starting from pregnancy, larger sample
size, and BMI-matched control group are required for which now this pilot project
provides the basis.
Ethical Approval
All procedures performed in studies involving human participants were in accordance
with the ethical standards of the institutional and/or national research committee
and with the 1964 Helsinki declaration and its later amendments or comparable
ethical standards.
Informed consent
Informed consent was obtained from all individual participants included in the
study.