Systems Biology in Reproductive Medicine

ISSN: 1939-6368 (Print) 1939-6376 (Online) Journal homepage: https://www.tandfonline.com/loi/iaan20

Do serum androgens influence blastocysts ploidy

in karyotypically normal women?

Michał Kunicki, Patrycja Skowrońska, Ewa Pastuszek, Grzegorz Jakiel, Roman

Smolarczyk & Krzysztof Łukaszuk

To cite this article: Michał Kunicki, Patrycja Skowrońska, Ewa Pastuszek, Grzegorz Jakiel, Roman
Smolarczyk & Krzysztof Łukaszuk (2019) Do serum androgens influence blastocysts ploidy in
karyotypically normal women?, Systems Biology in Reproductive Medicine, 65:4, 281-287, DOI:
10.1080/19396368.2019.1601295

To link to this article: https://doi.org/10.1080/19396368.2019.1601295

Published online: 17 Apr 2019.

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SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE
2019, VOL. 65, NO. 4, 281–287
https://doi.org/10.1080/19396368.2019.1601295

RESEARCH ARTICLE

Do serum androgens influence blastocysts ploidy in karyotypically normal

women?
Michał Kunickia,b, Patrycja Skowrońskac, Ewa Pastuszekc,d, Grzegorz Jakiela,e, Roman Smolarczykb,
and Krzysztof Łukaszuka,b,c,d
a
INVICTA Fertility and Reproductive Center, Warsaw, Poland; bDepartment of Gynecological Endocrinology, Medical University of Warsaw,
Warsaw, Poland; cDepartment of Obstetrics and Gynecological Nursing, Faculty of Health Sciences, Medical University of Gdansk, Gdańsk,
Poland; dINVICTA Fertility and Reproductive Center, Gdansk, Poland; eDepartment of Obstetrics and Gynecology, The Centre of Postgraduate
Medical Education, Warsaw, Poland

ABSTRACT ARTICLE HISTORY

The aim of the study was to determine if serum testosterone (T) and dehydroepiandrosterone Received 25 September 2018
(DHEAS) levels are a factor in determining increased risk for embryonic aneuploidy in karyotypi- Revised 3 March 2019
cally normal women undergoing in vitro fertilization (IVF) and preimplantation genetic testing Accepted 22 March 2019
screening for aneuploidy (PGT-A). This is a retrospective cohort study of IVF cycles with PGT-A KEYWORDS
performed during 2015–2016. A total of 256 cycles with 725 embryos were initially considered for Testosterone;
inclusion. A total of 208 cycles and 595 embryos determined to be either euploid or aneuploid dehydroepiandrosterone;
were included in the analysis. The mean age of women was 37.4 ± 4.4 years. There were 193 blastocysts ploidy;
(32.44%) euploid, and 338 (56.81%) aneuploid blastocysts. Sixty-four (10.76%) had ‘no diagnosis’ androgens
after PGT-A. The 32 embryos with ‘no diagnosis’ after first PGT-A were biopsied again and after
the second analysis, 7 were found to be euploid and 3 aneuploid. The remaining 32 embryos were
not reanalyzed due to the lack of patients’ consent for the second biopsy. The relationship
between embryo ploidy and levels of serum testosterone and dehydroepiandrosterone sulfate
was assessed using ordinal multivariable regression analysis. The model, adjusted for both anti-
Mullerian hormone (AMH) and age, showed no association between ploidy status and serum
levels of the two hormones. We concluded that the serum levels of testosterone and DHEAS do
not influence embryo ploidy in karyotypically normal women undergoing IVF.

Abbreviations: T: testosterone; DHEAS: dehydroepiandrosterone; IVF: in vitro fertilization; PGT-A:

preimplantation genetic testing screening for aneuploidy; AMH: anti-Mullerian hormone; FSH:
follicle-stimulating hormone; LH: luteinizing hormone; E2: oestradiol; P: progesterone

Introduction aneuploidy where lower AMH concentrations have been

shown to correlate with increased aneuploidy rates in
The successful outcome of in vitro fertilization (IVF)
embryos (Katz-Jaffe et al. 2013; La Marca et al. 2017).
procedure depends on a variety of factors. Two of the
It has been established that androgens play an
most important ones, and independently influencing the
important role in folliculogenesis (Vendola et al.
results, are female age and her serum level of the anti-
1998). They act via androgen receptor and influence
Mullerian hormone (AMH) (Broekmans et al. 2006; La
the recruitment and growth of follicles during the early
Marca et al. 2011; Lehmann et al. 2014). Embryos
stages of the folliculogenesis (Ferrario et al. 2015). This
obtained from the IVF procedure are transferred to the
action is in some extent achieved via increasing of IGF-
uterus in either fresh or frozen cycle. It has been estab-
1 (Weil et al. 1999). They also increase FSH receptor
lished that ploidy of embryos influences both clinical
mRNA in granulosa cells and in that way exerts syner-
pregnancy and live birth ratios (Franasiak et al. 2014;
gistic effect with FSH on folliculogenesis (González-
Minasi et al. 2016; La Marca et al. 2017). In older
Comadran et al. 2012). That leads to diminished
women (over 42 years old) aneuploidy rates reach close
amount of FSH during controlled ovarian hyperstimu-
to 90% (Minasi et al. 2016). This age-dependent increase
lation-COH.
is mainly related to the lower quality of oocytes (Battaglia
The most potent androgens in serum are DHEAS
et al. 1996; Lim and Tsakok 1997; Pellestor et al. 2003).
and testosterone. They are predominantly synthetized
AMH level is also an independent factor related to

CONTACT Patrycja Skowrońska patrycja.kulwikowska@gmail.com Department of Obstetrics and Gynecological Nursing, Faculty of Health Sciences,
Medical University of Gdansk, Dębinki 7, Gdańsk 80-211, Poland
© 2019 Informa UK Limited, trading as Taylor & Francis Group
282 M. KUNICKI ET AL.
in ovaries and suprarenal glands. The enzyme steroid
sulphatase catalyzes the conversion of DHEAS to
DHEA (Bonser et al. 2000). There are studies showing
positive influence of serum androgen concentration on
the IVF outcome (Dickerson et al. 2010; Ferrario et al.
2015; Sathyapalan et al. 2017). It has also been well
established that supplementation of testosterone and
DHEA in women with low ovarian reserve and inade-
quate androgen concentrations diminishes miscarriage
rates and positively influences IVF outcome (González-
Comadran et al. 2012). That effect has not been
observed in women with normal response to stimula-
tion (Yeung et al. 2016).
There are a limited number of studies that investi-
gated the association between female serum androgens
and embryo aneuploidy rates. The results come from
either small number of women or from women supple-
mented with dehydroepiandrosterone (Gleicher et al.
2010, 2015) or women with polycystic ovary syndrome
(Weghofer et al. 2007). Additionally, in some studies
data regarding possible confounding factors are miss-
ing, i.e., information about parental karyotype or pater-
nal age. The question regarding the influence of
androgens on embryo ploidy remains open and thus
we aimed to investigate it in karyotypically normal
couples undergoing in vitro fertilization treatment.
Results
From the initial group of 207 patients (256 cycles)
168 patients with 208 cycles were included in the final
analysis. The total number of blastocysts was 595. The
number of euploid embryos was 193 (32.44%), aneu-
ploid 338 (56.81%), and 64 (10.77%) had ‘no diagnosis’
after PGT-A. Thirty-two embryos with ‘no diagnosis’
after first PGT-A were biopsied again and after the next
analysis, 7 were found to be euploid and 3 aneuploid.
The remaining 32 embryos were not reanalyzed as
patients did not consent to the second biopsy.
Baseline characteristics and ploidy of embryos in the
subgroups, defined as below and over median for
serum testosterone and DHEAS levels, are presented
in Table 1.
In the subgroup with serum DHEAS below median
the women were older (38.6 ± 3.5 vs. 36.2 ± 4.8; p =
0.006) and in the subgroup with testosterone over
median the serum AMH and LH were significantly
higher (2.8 vs. 1.6; p = 0.002 and 7.3 vs. 5.8; p =
0.041, respectively) and FSH significantly lower (5.9
vs. 6.5; p = 0.027).
Thus, it was necessary that our further analysis of
embryo ploidy was conducted with adjustment for age
and AMH. The data are presented in Table 2. However,
we found that neither serum testosterone nor DHEA-S
influences aneuploidy rates in embryos (p = 0.312 and
p = 0.286, respectively). All analyzed correlations are
presented in Figure 1.
Discussion
The cumulative pregnancy rate after both fresh and
frozen embryo transfers depends on the number of
euploid blastocysts (Capalbo et al. 2014). Embryo
ploidy is known to be dependent on female age,
serum AMH, and the number of mature oocytes
(Battaglia et al. 1996; Lim and Tsakok 1997; Pellestor
et al. 2003; La Marca et al. 2017). There is less informa-
tion available in the literature regarding the serum
androgen concentrations and their relationships with
the ploidy of embryos (Gleicher et al. 2009, 2010, 2015;
La Marca et al. 2017).
There have been different techniques used in the
embryo aneuploidy screening. The genomic hybridiza-
tion arrays have been often used in many centers but
have some limitations such as inability to detect
changes in DNA sequences like: point mutations, intra-
genic insertions or deletions, triplet repeat expansion
(Harper and Harton 2010). The next step in embryo
analysis was the introduction of next-generation
sequencing (NGS). This new technique gives more reli-
able and reproducible results in embryo ploidy testing
which is important from the clinical point of view
(Kuliev and Verlinsky 2004; Brezina et al. 2016).
In this study, we present the largest cohort of
women (cycles) for which we investigate the association
of androgens with embryo ploidy. We found that in the
below median DHEA-S subgroup women were older
and in the over the median testosterone subgroup
serum AMH was significantly higher, which can be
explained by the fact that DHEA-S levels in women
decrease with age (Labrie et al. 1997).
We found that neither serum testosterone nor
DHEA-S influences aneuploidy rates in embryos. The
same conclusion was true when the model was adjusted
for female age and serum AMH level. We have also
attempted to create other models. First, we included
each androgen variable separately. This model did not
show the influence of androgens on embryo ploidy. In
our patient cohort, over 40% of the women had no
euploid embryos. Therefore, next we modeled the bin-
ary outcome defined as having at least one euploid
embryo or no euploid embryos. This model failed to
show any association of androgens with such defined
outcome (data not shown). Finally, even adding the
number of mature oocytes (MII) as a variable did not
change our conclusions.
 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 283

Table 1. Baseline characteristics of the study population and differences between patients with low and high level of Testosterone
and DHEA-S.
Testosterone DHEA-S <
Total Testosterone <0.8 ≥ 0.8 176 DHEA-S ≥ 176
N = 208 N = 98 N = 110 p N = 104 N = 104 p
Age of women (years) 37.4 ± 4.4 37.7 ± 3.3 37.0 ± 5.0 0.636 38.6 ± 3.5 36.2 ± 4.8 0.006
Age of men (years) 38 (35–41) 39 (37–42.5) 38.59 ± 4.7 0.4031 39 (36–42) 39 (34.5–41) 0.5566
BMI (kg/m2) 22.9 ± 2.8 22.9 ± 2.5 23.0 ± 3.1 0.899 22.9 ± 2.2 22.9 ± 3.7 0.731
Primary infertility diagnosis 74% 68% 81% 0.097 72% 78% 0.399
Secondary infertility diagnosis 26% 32% 19% 28% 22%
AMH (ng/mL) 2.1 (1.1–3.6) 1.6 (0.9–3) 2.8 (1.3–4.5) 0.002 2.0 (1.1–3.8) 2.2 (1.1–3.4) 0.854
FSH (mIU/L) 6.3 (4.8–7.7) 6.5 (5.4–8.9) 5.9 (4.1–7.1) 0.027 6.5 (5.4–7.9) 6.0 (4.3–7.5) 0.460
LH (mIU/L) 6.6 (5.1–7.9) 5.8 (4.4–7.3) 7.3 (5.6–8.4) 0.041 6.4 (5.1–7.8) 6.7 (5.4–8.8) 0.265
Testosterone (nmol/l) 0.8 (0.5–1.1) - - - 0.6 (0.4–0.8) 1.1 (0.8–1.4) <0.001
DHEA-S (mg/dL) 169 (126–259) 146 (108.5–170.5) 230 (144–288) <0.001 - - -
E2 (pg/ml) 38 (29–55) 38 (29–54) 43 (33–100) 0.335 38 (33.5–55) 38 (24–82) 0.763
E2 on 8 ds. (pg/ml) 1653 1630 (1115–2249) 1590 0.942 1656 1621.5 0.667
(1148–2342) (1148–2319) (1110–2189) (1226–2484)
Duration of infertility (years) 4 (2–7) 4 (3–7) 4 (2–8) 0.939 4 (2–7) 4 (2–8) 0.714
Stimulation length (days) 9.5 (8–11) 9 (8–10) 10 (9–11) 0.006 9 (8–10.5) 10 (9–11) 0.298
Antral follicle count (AFC) 11 (7–17) 10 (7–15) 12 (8–18) 0.152 10 (7–17) 11 (8–17) 0.604
Total gonadotropin dose (units) 2294.4 ± 556.3 2236.3 ± 471.1 2282.8 ± 617.3 0.653 2314.6 ± 461.3 2274.2 ± 640.7 0.693
Number of cumulus 9 (6–13) 8 (6–14) 9 (6–12.5) 0.734 9 (6–13.5) 9 (5.5–13) 0.918
No of mature oocytes 7 (4–9.5) 7 (4–10) 6 (4.5–8.5) 0.945 7 (5–10) 6 (4–9) 0.553
Number of available embryos 5 (3–7) 4 (3–6) 5 (2–6) 0.665 5 (3–6.5) 4 (3–7) 0.432
Blastocysts biopsied 2 (1–3) 2 (1–3) 2 (1–3.5) 0.933 2.5 (1.5–4) 2 (1–3) 0.082
Euploid blastocysts % 40 (0–66.7) 50 (0–66.7) 33.3 (0–66.7) 0.565 50 (0–66.7) 50 (0–100) 0.510
Aneuploid blastocysts % 60 (33.3–100) 50 (33.3–100) 66.7 0.565 50 (33.3–100) 50 (0–100) 0.510
(33.3–100)
No. of subjects with indicated cause of
infertility
History of miscarriage 36 19 17 19 17
Advanced maternal age (set at 35) 70 33 37 0.952 32 38 0.091
Male factor 70 31 39 41 29
Anovulation 2 1 1 2 0
Unexplained 30 14 16 10 20
BMI: body mass index; FSH: follicle-stimulating hormone; AMH: anti-Müllerian hormone; LH: luteinizing hormone; E2: oestradiol; ds: day of stimulation
Data with not normal distribution are presented as: median (25th–75th)
Data with normal distribution as a mean ± standard deviation
P value for statistically significant results is shown in bold

Table 2. Ordinal multivariable logistic regression model for In our study, the age of women correlated signifi-
embryo ploidy. cantly with number of euploid embryos. This is in agree-
OR 95% CI for OR p ment with many other studies showing an increase in
Aneuploid blastocysts Testosterone 0.396 0.066–2.387 0.312 aneuploidy rates with woman’s age up to 90% for women
DHEA-S 1.004 0.996–1.013 0.286
All (euploid and Testosterone 0.277 0.042–1.838 0.184 older than 42 years (La Marca et al. 2017). In compar-
aneuploid) blastocysts DHEA-S 0.647 0.993–1.011 0.647 ison, we did not show a significant correlation between
Adjusted by AMH and age
embryo ploidy and serum AMH levels. This is
a surprising finding worth to point out given conflicting
Our results are in agreement with few studies in reports on the influence of ovarian reserve on embryo
which the relationship between serum androgens and ploidy (La Marca et al. 2017; Jiang et al. 2018).
ploidy was investigated (Weghofer et al. 2007; Gleicher It is important to note that we included in the
et al. 2009, 2010, 2015). It is important to note that the analysis only couples with normal karyotypes (both
previously published studies regarding androgens and partners). We aimed to include only couples with nor-
ploidy of embryos included only patients who were mal karyotype as abnormal parental karyotype
supplemented with DHEA. Authors concluded that it increases the risk of embryonic aneuploidy. We have
could diminish aneuploidy and miscarriage rates also excluded embryos that did not receive a PGT-A
(Gleicher et al. 2009, 2010). result (classified as ‘no diagnosis’ after PGT-A). Our
In our study the number of women with extremely goal was to obtain the study group that was as homo-
low AMH <0.4 (and supplemented with DHEA) was genous as possible.
four (with five cycles) and we do not think it could The strength of this study is the strict inclusion
have impact on the final results. Additionally, supple- criteria. We assessed karyotypes in all included couples
mentation with DHEA was stopped before the begin- (this is performed routinely in our clinic) and analyzed
ning of ovarian stimulation. our data with respect to confounders such as age and
284 M. KUNICKI ET AL.
Figure 1. Correlations among clinical and embryological variables.
Circles represent strength (size and color) and direction (color) of the correlation. Non statistically significant correlations are crossed out.
AMH. We have also taken into consideration the men’s
age as a factor which possibly has an impact on the
blastocyst ploidy (García-Ferreyra et al. 2018). In our
study, the men’s age was similar in all subgroups.
Additionally, all IVF cycles and PGT were performed
in a single center. This seems to be an important factor
as having different laboratories that use various meth-
ods of aneuploidy screening could affect the results
(Mateo et al. 2017; Munné et al. 2017; Grati et al.
2018; Spinella et al. 2018).
We have also excluded obese women as obesity can
influence androgen levels. Our method of screening
embryos was NGS which is routinely used in our clinic
and currently considered to be the most reliable and
accurate method of assessment of embryo ploidy.
The limitation could be the retrospective nature of
the study, but we believe that the strict inclusion cri-
teria outweighed this bias.
Finally, we have not classified embryos according to
the current PGDIS criteria (PGDIS Position Statement
on Chromosome Mosaicism and Preimplantation
Aneuploidy Testing at the Blastocyst Stage 2016) as
most of our data were collected before its publication.
The issue of mosaicism is still under debate and various
clinical, methodological and laboratory factors could
influence the results (Scott et al. 2012; Treff et al.
2012; Wells et al. 2014; Maxwell and Grifo 2018;
Munné 2018; Palmerola et al. 2019; Victor et al. 2019).
Conclusion
In conclusion, our data provide support for the
assumption that the serum concentrations of testoster-
one and DHEA-S do not affect the risk for embryonic
chromosomal abnormalities.
Materials and methods
We retrospectively analyzed data obtained from couples
who underwent their cycle of IVF treatment with pre-
implantation genetic testing for aneuploidy at the
Invicta Fertility Clinic, between January 2015 and
December 2016.
During that period 207 patients had 256 cycles with
the total number of 725 analyzed blastocysts. All pro-
cedures performed in this study were in accordance
with 1964 Helsinki declaration, and its later amend-
ments and the study protocol was approved by the
appropriate Institutional Review Board and written
informed consent was given by all participating
patients.

The indications for PGT-A (Preimplantation genetic
testing for aneuploidy – abnormal number of chromo-
somes) included: advanced maternal age, repeated
implantation failure, recurrent miscarriage, and severe
male infertility. Until November 2015 patients could
also request PGT-A to check embryo ploidy even if they
did not present with any of the abovementioned
indications.
The inclusion criteria were: IVF cycle with PGT-A,
normal karyotype of both partners, AMH level mea-
sured with either VIDAS AMH (bioMerieux) or Elecsys
AMH (Roche Diagnostics) assay. It has been previously
verified that results obtained with VIDAS AMH assay
were well correlated and comparable with Elecsys assay
(Pastuszek et al. 2017).
We excluded 44 cycles due to abnormal karyotype of
one of the parents (female partner: 24 cycles, 22
patients, male partner: 20 cycles, 15 patients), 3 cycles
due to polycystic ovary syndrome according to
Rotterdam criteria (PCOS) (Rotterdam 2004) (1
patient), and 1 cycle due to obesity (1 patient).
Ultimately 208 cycles (168 women) were included in
the analysis. The total number of analyzed blastocysts
was 595.
This study was approved by the Institutional Review
Board, decision number KB-29/17.
Hormone measurements
Venous blood samples were collected, between day 1
and 3 of the menstrual cycle, prior to stimulation. The
serum levels of follicle-stimulating hormone (FSH),
luteinizing hormone (LH), oestradiol (E2), progester-
one (P), testosterone (T), were measured with an
electrochemiluminescence immunometric assays using
commercial kits (Cobas 6000, e601). The intra- and
inter-assay coefficients of variation (CV), respectively,
were 7.4% and 4.7% for E2, 2.5% and 2.3% for FSH,
2.2% and 2.4% for LH, 4.9% and 5% for T, and 3.3%
and 3.2% for DHEAS.
The reference intervals were as follows: serum, T,
0.1–0.9 ng/ml; SHBG and DHEAS, 98.8–340 µg/dl.
Serum AMH were measured by enzyme-linked immuno-
sorbent assay (ELISA) using VIDAS AMH (bioMerieux)
and Elecsys AMH assay (Roche Diagnostics).
Protocol
All patients began treatment in the early follicular
phase between day 1 and 5 of the menstrual cycle.
The long GnRH agonist protocol used in our center is
started with oral contraceptive with Ovulastan
(ADAMED, Poland). Starting on treatment day 16 or
SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 285
17, triptorelin (Ferring, Germany) was administered
every two days for 14 days. After pituitary suppression,
confirmed by low serum levels of estradiol (<50 pg/ml)
and the absence of ovarian cysts, human menopausal
gonadotropins (HMGs) (Menopur, Ferring, Germany)
were initiated for ovarian controlled hyperstimulation.
It was continued until at least three follicles ≥17 mm
were visualized. Follicular growth was monitored on
treatment day 8 with an ultrasonographic scan and
serum E2 measurement. Ovulation was then induced
by administering 5,000 IU hCG (Choragon, Ferring,
Germany). Oocyte retrieval was performed by transva-
ginal aspiration 36 h after the hCG administration
(Lukaszuk et al. 2005).
PGT-A
In vitro fertilization, embryo culture and blastocyst biopsy
techniques were performed as previously described
(Lukaszuk et al. 2005). Intracytoplasmic sperm injection
was performed routinely to remove potential sperm or
cumulus cell DNA contamination. Trophectoderm
biopsy was performed on all expanding or fully expanded
blastocysts on postretrieval day 5 or 6, depending on the
rate at which individual embryos reached this develop-
mental stage. Following the biopsy embryos were frozen
and their outcome is not discussed in the manuscript.
Each trophectoderm biopsy sample underwent compre-
hensive chromosome screening analysis performed at the
same laboratory using next-generation sequencing (NGS)
technique (Kuliev and Verlinsky 2004). Embryos with
over 50% of aneuploid cells for any chromosome were
classified as aneuploid, those with up to 50% of aneuploid
cells were reported as euploid. If we were to compare our
classification criteria to those published by PGDIS
(PGDIS Position Statement on Chromosome Mosaicism
and Preimplantation Aneuploidy Testing at the Blastocyst
Stage 2016) that suggest the ‘cut-off point for definition of
mosaicism is >20%, so lower levels should be treated as
normal (euploid), >80% abnormal (aneuploid), and the
remaining ones between 20% and 80% mosaic (euploid-
aneuploid mosaics)’, we would say that, in our clinic,
embryos with mosaicism in the range of 20–50% were
reported as euploid and embryos with mosaicism in the
range of >50% to 80% were labeled as aneuploid (Liss et al.
2018). Segmental aberrations are reported as aneuploid
when they cover over 50% of a chromosome.
Statistics
The results were presented separately in the low/high
testosterone and DHEA-S groups. The cut-off point for
both variables was the median value, which for
286 M. KUNICKI ET AL.
testosterone was 0.8 mg/ml and for DHEA 176 µg/dl.
Differences between continuous variables were calcu-
lated with the t-test for symmetrical distributions and
with the Mann–Whitney test for asymmetrical distribu-
tions. Differences between qualitative variables were
calculated using the chi-square test (Table 1).
Correlations between quantitative variables were eval-
uated using the Spearman’s rank correlation coefficient
(rS), along with its significance.
An ordinal multivariable logistic regression model
was created to predict outcome defined as cycle result-
ing in all euploid, mixed euploid and aneuploid, and all
aneuploid embryos. Variables included in the model
were testosterone and DHEA levels as well as AMH
and female age (Table 2). The statistical analysis was
performed using the IBM SPSS version 23 statistical
package. The significance level was set at α = 0.05.
Due to the retrospective nature of the study, we did
not have any missing data.
Disclosure statement
No potential conflict of interest was reported by the authors.
Authors’ contributions
Conception and design of the idea, data interpretation and
preparation of manuscript: MK, PS, EP, GJ, RS, KŁ.
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