Keywords 3D ultrasound - junctional zone - reproducibility - infertility - associated factors
Palavras-chave ecografia 3D - zona juncional - reprodutibilidade - infertilidade - factores associados
Introduction
The junctional zone (JZ) is a hormone-dependent zone that corresponds to the inner
layer of the myometrium, which derives from the paramesonephric ducts. In contrast,
the outer myometrium has a different embryonic origin – a mesenchymal one.[1 ] The JZ contains myocytes as the outer myometrium, but with different morphologic
characteristics. Junctional-zone myocytes have a higher nuclear-cytoplasmic ratio,
reduced extracellular matrix, low content of water, and a concentric architectural
arrangement.[1 ] These characteristics enable the identification of the JZ on ultrasound.[1 ]
The JZ plays an important role in reproduction and pregnancy. Uterine contractility
in non-pregnant women originated exclusively in the JZ changes in orientation, amplitude,
and frequency according to the phase of the menstrual cycle.[2 ]
[3 ] These contractions increase and take the direction of the cervix to the uterine
fundus in the follicular and periovulatory phase, facilitating sperm transport.[2 ]
[4 ]
[5 ] Peristaltic activity decreases in the luteal phase, facilitating the implantation,
oxygenation, and nutrition of the blastocyst.[2 ]
[6 ]
When uterine contractility is excessive and uncoordinated, it is associated with lower
pregnancy rates both in natural and assisted reproductive technology cycles.[7 ]
[8 ] Studies[2 ]
[9 ]
[10 ] show that a dysfunctional JZ plays an important role in the physiopathology of endometriosis,
facilitating retrograde menstruation and implantation of endometrial cells in the
pelvis. On the other hand, JZ characteristics observed by three-dimensional (3D) ultrasound
and magnetic resonance imaging (MRI), such as increased thickness and disruption,
are associated with endometriosis, but also with adenomyosis.[11 ]
[12 ]
Several studies[1 ]
[13 ]
[14 ] suggest that the assessment of the JZ by 3D transvaginal ultrasound should be included
in routine uterine ultrasound. Through the coronal plane, it is possible to delimit
the JZ at the fundus and lateral walls, which is observed as a thin and regular layer
with hypoechogenic characteristics between the basal layer of the endometrium and
the outer myometrium.[1 ]
[15 ] Despite growing research involving the evaluation of the JZ by 3D ultrasound, there
is no universally accepted methodology.
Due to the cost-benefit ratio, convenience and availability of 3D ultrasound, and
the importance of the JZ, particularly in infertile women, we aim to evaluate the
interobserver and intraobserver reproducibility of the visualization and continuity
of the JZ by 3D ultrasound as well as to identify the sociodemographic and physiological
factors that affect the JZ.
Methods
A prospective observational study was conducted with women followed at the Assisted
Reproductive Technology Unit of Hospital Senhora da Oliveira, in the city of Guimarães,
Portugal, from July to October of 2018. The exclusion criteria were serum follicle-stimulating
hormone levels greater than 25UI/L, women older than 40 years of age, and cases with
incomplete data on the ultrasound evaluation (absence of 2 volumes per case). One
of the authors (VS) acquired the planes and volumes through transvaginal ultrasound
(with the woman in the lithotomy position and an empty bladder), using a Samsung (Seoul,
South Korea) HS50 ultrasound with a 5–9 MHz 2D/3D endocavitary probe. All evaluations
were performed using a standardized methodology, as explained below.
For the uterine assessment, two-dimensional (2D) transverse and longitudinal planes
were performed. The uterus was centered on the screen. In the longitudinal plane,
the endometrial thickness was measured (in the double layer, in its thickest zone)
parallel to the anteroposterior diameter of the uterus. In the presence of intracavitary
fluid, the endometrial thickness was obtained by subtracting the thickness of the
intracavitary fluid from the total thickness of the endometrial cavity.[16 ] The anomalies observed, such as myomas or adenomyosis, were identified according
to established diagnostic criteria.[17 ] The pelvic cavity was explored for signs of endometriosis (endometriomas or deep
endometriosis with the involvement of the bladder, bowel, or rectovaginal septum).[18 ] After the 2D ultrasound, a 3D ultrasound was performed. In the longitudinal plane,
the uterus was centered on the screen so that the cervical canal and the myometrium
were fully visible. The 3D acquisition box was adjusted to include a margin around
the uterus, which allowed delimitation of the myometrium. A maximal image quality
and an acquisition angle (90°) were selected. The probe was held firmly, and the women
were asked to hold their breath during acquisition to minimize the artifacts. After
the acquisition, a summary evaluation of the volume was performed, and whenever it
turned out to be of poor quality (a volume that did not enable the complete assessment
of the uterine volume or the presence of movement artifacts), new ones were obtained.
Two volumes per case were obtained from each participant, and the volumes were stored
in the ultrasound memory. An analysis of the 3D volumes was performed posteriorly,
using the Samsung Medison 5D Viewer software. The methodology of 3D image postprocessing
and classification of JZ visualization and continuity was initially standardized between
the authors (VS and FR) in a set of volumes not included in the study sample. The
multiplanar image was freely adjusted for the JZ assessment to obtain a coronal plane
with adequate visualization of the endometrial cavity, the external contour of the
uterus, and the interstitial portions of the fallopian tube. A region of interest
(ROI) box with any rendering mode combination was used to optimize the visualization
of the JZ. The best reconstructed coronal image was chosen for each participant from
the two volumes acquired per operator. The visualization of the JZ was classified
as proposed by Naftalin et al[14 ] ([Fig. 1 ]):
Optimal: JZ clearly visible and evaluable in its entirety.
Satisfactory: most, but not all of the JZ can be clearly observed
Unsatisfactory: a great portion of the JZ could not be clearly observed.[14 ]
Fig. 1 Three-dimensional ultrasound imaging of optimal (A ), satisfactory (B ) and unsatisfactory (C ) visualizations of the JZ in the coronal plane.
The continuity of the JZ was classified as proposed by Van den Bosch et al[17 ] ([Fig. 2 ]):
Fig. 2 Three-dimensional ultrasound imaging of continuous (A ) and discontinuous (B ) JZ in the coronal plane. Note the discontinuous JZ on the coronal section, marked
in (B ) by the red circle.
Two authors (VS and FR) performed the postprocessing and analysis of the 3D volumes
independently and blindly. Both authors classified the best coronal plane of the two
volumes acquired per participant to determine the interobserver agreement. Four months
after the first analysis, VS reassessed the volumes (two volumes per case, choosing
the best reconstructed coronal image from the two volumes acquired) to determine the
intraobserver agreement. The intraobserver and interobserver agreements were assessed
for visualization and continuity of the JZ.
Demographic and physiological (hormonal and structural) variables, as well as gynecological
antecedents were collected by consulting the participants' electronic clinical process
and 2D/3D transvaginal ultrasound. Then their effects on the visualization and continuity
of the JZ were evaluated.
Data analysis was performed using the Statistical Package for Social Sciences (SPSS,
IBM Corp., Armonk, NY, US), version 25.0. The interobserver and intraobserver agreements
were assessed using the Cohen k, which was interpreted as poor (k < 0.20), reasonable
(k between 0.21 and 0.40), moderate (k between 0.41 and 0.60), good (k between 0.61
and 0.80), and very good (k between 0.81 and 1.00).[19 ] Rejection of the null hypothesis was based on the significance level of p < 0.05. The cases of unsatisfactory visualization were excluded from the assessment
of continuity of the JZ, and most cases of disagreement in the classification of visualization
of the JZ (unsatisfactory versus satisfactory/optimal) were solved. The unsolved cases
of disagreement were excluded from the analysis.
A descriptive analysis was performed taking into account the symmetry of the distribution
of the continuous variables. Based on the result of the Shapiro-Wilk test, parametric
or nonparametric statistics were used according to p > 0.05 or p < 0.05. The Chi-squared (χ2 ) test or the Fisher exact test were used to evaluate the association between visualization
or continuity of the JZ and the categorical variables. Analysis of variance (ANOVA)
and t -tests were used to analyze the continuous variables with normal distribution. The
continuous variables without normal distribution were compared using the Kruskall-Wallis
and Mann-Whitney tests. Additionally, the effect on the visualization and continuity
of the JZ was evaluated through univariate and multivariate analyses. Cases of intraobserver
and interobserver disagreement were excluded from the analysis. Cases of unsatisfactory
visualization were excluded to evaluate the factors that influence the continuity
of the JZ.
The Health Ethics Committee of Hospital Senhora da Oliveira approved the research
project. The participants signed an informed consent form to participate in the study.
Results
Sample
In total, 65 women between 23 and 40 years of age were included in the present study
(mean [M] = 33.01; standard deviation [SD] = 4.27), and 24 (36.9%) had irregular cycles,
28 (43.1%) were in the follicular phase of the menstrual cycle, and 43 (66.2%) had
a trilaminar endometrial pattern. No cases of adenomyosis were reported, and 15 (23.1%)
of the participants had diagnosed or suspected endometriosis. The results of the remaining
descriptive analysis are in [Table 1 ].
Table 1
Descriptive analysis of the characteristics of the sample (n = 65)
Variables
Age (years)
33.01(4.27)
Body mass index (kg/m2 )
25.8(5.39)
Menarche (years)
12.66(1.66)
Parity
Nullipara
51(78.5)
Regularity of cycle
Irregular
24(36.9)
Regular
41(63.1)
Stage of the cycle
Proliferative
28(43.1)
Secretory
13(20.0)
Previous uterine surgery
3(4.6)
Follicle-stimulating hormone (IU/L)
7.20(3.77)
Luteinizing hormone (IU/L)
4.77(2.46)
Estradiol (pg/mL)
61.15(33.58)
Total testosterone (ng/dL)
26.09(10.61)
Prolactin (µg/L)
16.79(29.12)
Endometrium thickness (mm)
7.18(3.06)
Endometrium thickness (≤5 mm)
16(24.6)
Endometrial ultrasound characteristics
Trilaminar
43(66.2)
Heterogeneous
22(33.8)
Mioma
10(15.4)
Diagnosed or suspected endometriosis
16(24.6)
Adenomyosis
0(0)
Results presented as M (SD) and n(%).
Abbreviations: M, average; SD, standard deviation; n, absolute frequency; %, relative
frequency; dl, deciliter; IU, international units; L, liter; pg, picogram; mL, mililiter;
mm, milimeter.
Interobserver and Intraobserver Agreements Regarding JZ Visualization
Regarding the visualization of the JZ, the interobserver agreement was good, with
a Cohen k of 0.635. The total agreement was of 80.0%. The highest number of disagreements
occurred between cases of satisfactory and optimal visualization (17.2%). The intraobserver
agreement was very good, with a Cohen k of 0.884, and the total agreement was of 93.9%.
Again, the highest number of disagreements occurred between cases of satisfactory
and optimal visualization (4.6%). Details of the analysis are in [Tables 2 ] and [3 ].
Table 2
Visualization of the junctional zone and interobserver agreement (n = 65; observers: FR and VS)
FR
VS
Unsatisfactory n(%)
Satisfactory n(%)
Optimal n(%)
Unsatisfactory n(%)
6(9.2)
1(1.5)
0(0)
Satisfactory n(%)
2(3.1)
12(18.5)
3(4.6)
Optimal n(%)
0(0)
7(12.6)
34(52.3)
Absolute agreement
Cohen k = 0.635
Table 3
Visualization of the junctional zone and intraobserver agreement (n = 65)
2nd assessment
1st assessment
Unsatisfactory n(%)
Satisfactory n(%)
Optimal n(%)
Unsatisfactory n(%)
7(10.8)
0(0)
0(0)
Satisfactory n(%)
1(1.5)
15(23.1)
1(1.5)
Optimal n(%)
0(0)
2(3.1)
39(60.0)
Absolute agreement
Cohen k = 0.884
Interobserver and Intraobserver Agreements Regarding JZ Continuity
Regarding the continuity of the JZ, the interobserver agreement was good, with a Cohen
k of 0.753. The total agreement was of 88.1%. The intraobserver agreement was very
good, with a Cohen k of 0.816, and a total agreement of 91.2%. Details of the analysis
are in [Tables 4 ] and [5 ].
Table 4
Continuity of the junctional zone and interobserver agreement (n = 59*; observers: VS and FR)
FR
VS
Continuous n(%)
Discontinuous n(%)
Continuous n(%)
20(33.9)
2(3.4)
Discontinuous n(%)
5(8.5)
32(54.2)
Absolute agreement
Cohen k = 0.753
Note: *Excluding cases of unsatisfactory assessment of the junctional zone (n = 6) and visualization disagreements (unsatisfactory versus satisfactory/optimal;
n = 0).
Table 5
Continuity of the junctional zone and intraobserver agreement (n = 57*)
2nd assessment
1st assessment
Continuous n(%)
Discontinuous n(%)
Continuous n(%)
20(35.1)
2(3.5)
Discontinuous n(%)
3(5.3)
32(56.1)
Absolute agreement
Cohen k = 0.820
Note: *Excluding cases of unsatisfactory assessment of the junctional zone(n = 6) and visualization disagreements (unsatisfactory versus satisfactory/optimal;
n = 2).
Factors Affecting JZ Visualization
We tried to identify variables that influenced the visualization of the JZ. In order
to do so, cases of interobserver and intraobserver disagreement were solved. We excluded
five cases without a final agreement. Regarding the visualization of the JZ among
the 60 remaining cases, 65% (39/60) of the women had optimal, 25% (15/60) had satisfactory,
and 10% (6/60) had unsatisfactory visualization. Serum estradiol levels, endometrial
ultrasound appearance, and diagnosed or suspected endometriosis influenced the visualization
of the JZ ([Table 6 ]). Higher estradiol levels were associated with better visualization of the JZ (satisfactory
and optimal JZ, p = 0.016), particularly in the comparison between unsatisfactory and optimal JZ assessment
(p = 0.013). On the other hand, the presence of the trilaminar endometrial pattern was
associated with optimal visualization (79.5% [31/39] p = 0.012). In total, 83.3% (5/6) of the cases of diagnosed or suspected endometriosis
had unsatisfactory visualization of the JZ (p < 0.001) ([Table 6 ]). Performing a univariate logistic regression model, considering the unsatisfactory
assessment of the JZ as an outcome, we found that the serum estradiol levels (p = 0.016) and diagnosed or suspected endometriosis (p = 0.004) were the only factors associated with JZ visualization. The joint effect
of the variables on the visualization of the JZ was evaluated by multivariate analysis.
The results suggest that an increase of 1 unit in the level of serum estradiol represents
a 9.9% decrease in the odds of unsatisfactory visualization (odds ratio [OR] = 0.9;
95% confidence interval [95%CI] = 0.814–0.996; p = 0.042). On the other hand, the presence of diagnosed or suspected endometriosis
increases the odds of unsatisfactory visualization of the JZ by 24 times (OR = 23.7;
95%CI = 1.262–437.057; p = 0.034).
Table 6
Factors affecting the visualization of the junctional zone (n = 60# )
Factors
Visualization of the junctional zone
p -value
Optimal (n = 39)
Satisfatory (n = 15)
Unsatisfatory (n = 6)
Estradiol (pg/mL)
59.0(46.0–75.0)
50.0(42.0–84.0)
24.0(20.0–39.0)
0.016* (a)
Endometrial ultrasound characteristics
0.012* (b)
Trilaminar
31(79.5)
6(40.0)
3(50.0)
Heterogeneous
8 (20.5)
9 (60.0)
3 (50.0)
Diagnosed or suspected endometriosis
2 (5.1)
6 (40.0)
5 (83.3)
< 0.001* (b)
Notes: # Excluding cases of interobserver and intraobserver disagreement in the visualization
of the junctional zone (n = 5); results presented as Mdn (P25 –P75 ) = Mdn, median; P, percentile; and n(%); *p < 0,05; (a) Kruskall-Wallis test, Dunn test with differences between unsatisfactory and optimal
visualization (p = 0,013); (b) Chi-squared test.
Factors Affecting JZ Continuity
We tried to identify the variables that influenced the continuity of the JZ. The interobserver
and intraobserver disagreements were solved, and the cases of unsatisfactory visualization
were excluded. After the exclusion, 60% (33/55) of the women had a discontinuous JZ.
The presence of myomas and of cases of diagnosed or suspected endometriosis are associated
with discontinuous JZ (p = 0.034 and 0.016 respectively). It was not possible to determine the OR due to the
absence of observed frequencies of these variables in continuous JZs ([Table 7 ]).
Table 7
Factors affecting the continuity of the junctional zone (n = 55# )
JZ continuity
p -value
Discontinuous n(%)
Continuous n(%)
Myoma n(%)
7(21.2)
0(0)
0.034* (a)
Diagnosed or suspected endometriosis n(%)
8(24.2)
0(0)
0.016* (a)
Notes: # Excluding cases of unsatisfactory visualization of the junctional zone (n = 6) and interobserver and intraobserver disagreement regarding continuity of the
junctional zone (n = 4); results presented as n(%); *p < 0,05; (a) Chi-squared test.
Discussion
In the present study, the interobserver and intraobserver reproducibility of the JZ
visualization was substantial, with values of the Cohen k of 0.635 and 0.884 respectively.
Similar results were found in other studies,[14 ]
[20 ] which indicates that the assessment of the JZ by 3D ultrasound is reproducible enough
to be used in the clinical practice. Naftalin et al[14 ] proposed that the distinction between optimal and satisfactory visualization of
the JZ is subjective. This assumption is corroborated by the present study, in which
the largest discrepancies regarding JZ visualization occurred among the cases classified
as optimal and satisfactory. On the other hand, the high agreement in cases of JZ
visualization classified as unsatisfactory is a relevant result, because JZs with
unsatisfactory visualization should not be taken into account for other qualitative
or quantitative assessments. These results suggest that a JZ visualization classification
system in two categories (satisfactory versus unsatisfactory) is more reproducible.
The interobserver and intraobserver reproducibility of JZ continuity were substantial
with the values of Cohen k of 0.753 and 0.816 respectively. To the best of our knowledge,
the present was the first study that assessed the reproducibility of this characteristic
of the JZ by 3D transvaginal ultrasound. Our findings are relevant and open doors
to the inclusion of this evaluation in the clinical practice and to the investigation
of its relevance, as it will be discuss later.
The present study showed that estradiol levels and trilaminar endometrial pattern
facilitate JZ visualization. The JZ experiences estradiol-mediated cyclic changes
consistent with those occurring in the endometrium, and reaches maximal thickness
around the 8th and 16th days of the menstrual cycle.[3 ]
[21 ]
[22 ] Thus, higher estradiol levels appear to be associated with a thicker JZ, which facilitates
JZ visualization. This finding needs to be proven in future studies. Additionally,
estradiol influences the echogenicity of the endometrium, and its role as a confounder
cannot be excluded.[23 ] In fact, in the present study the trilaminar endometrial pattern was associated
with better visualization of the JZ, perhaps because it increases the contrast between
the endometrium and the myometrium, which facilitates the achievement of the ideal
coronal plane for JZ visualization.
Contrary to other studies,[14 ]
[24 ] endometrial thickness did not affect the visualization of the JZ. The prevalence
of thin endometria (< 5 mm) in our study was of only 24.6%, which may have contributed
to this result. As in the study by Naftalin el al,[14 ] in the present study we were unable to demonstrate that the menstrual cycle phase
affects JZ visualization. However, other studies[25 ]
[26 ] have shown that the JZ changes between the follicular and luteal phases. One explanation
for this result may be the low number of women in the luteal phase in the present
study.
Previous studies[12 ]
[24 ] show higher prevalence of abnormalities in the JZ in women with endometriosis. In
the present study, most women with suspected or diagnosed endometriosis had unsatisfactory
visualization of the JZ. In fact, the presence of this pathology increases the chance
of unsatisfactory visualization of the JZ by ∼ 24 times. Distortions of the normal
pelvic anatomy, including the uterus, associated with endometriosis make the acquisition
of the ideal coronal plane for the visualization of the JZ difficult, which justifies
these findings.[27 ]
[28 ] On the other hand, the disruption of the normal architecture of the JZ associated
with endometriosis makes visualization of the JZ difficult.[12 ]
[24 ]
The prevalence of discontinuous JZs was 60%. The implications of this finding in women
with infertility are uncertain. According to the literature,[2 ]
[9 ]
[10 ]
[11 ]
[27 ] the disruption of the normal architecture of the JZ inevitably alters the coordinated
peristaltic activity of the myometrium, interferes with sperm transport and implantation,
and affects fertility. Disruption of the JZ also seems to be associated with JZ hyperplasia,
endometriosis, and adenomyosis-infertility-related diseases.[12 ]
[27 ]
[28 ]
In the present study, discontinuous JZs were associated with suspected or diagnosed
endometriosis. Peristaltic hyperactivity of the myometrium, conditioned by JZ disruption,
seems to play an important role in the pathophysiology of endometriosis, facilitating
retrograde menstruation and implantation of endometrial cells in the pelvic cavity.[2 ]
[9 ]
[10 ] On the other hand, JZ disruption may enable the penetration of endometrial glands
into the myometrium and the development of adenomyosis.[9 ]
[24 ]
The prevalence of adenomyosis in women of reproductive age is heterogeneous, ranging
from 16% to 66%.[14 ]
[29 ] Different study methodologies, populations, and diagnostic criteria can explain
this disparity. In the present study, the prevalence of adenomyosis was null. This
prevalence draws attention to the underdiagnoses of adenomyosis in infertile women.
Puente et al[11 ] showed that only one in five infertile women had a diagnosis of adenomyosis before
the ultrasound evaluation in their study, which reinforces the underdiagnosis of adenomyosis
in infertile women. These results highlight the need to make physicians aware of adenomyosis
in infertile women and the need to establish reproducible diagnostic ultrasound criteria.[30 ]
The pathophysiological link between adenomyosis and endometriosis seens to be a disruption
of JZ.[11 ]
[12 ]
[24 ]
[27 ]
[31 ]
[32 ] Based on this, the theory of “endometrial-subendometrial myometrium unit disruption
disease” was proposed by Tocci et al.[31 ] According to this theory, a disruption in the architecture of the JZ seems to constitute
the triggering event (causing dysfunction in the uterine peristalsis), and endometriosis
and adenomyosis represent phenotypes of “endomyometrial dysfunction” and not distinct
pathologies.[31 ] Efforts should be made to better describe this entity through 3D transvaginal ultrasound
and to assess its impact on women's reproductive functions.
In the present study, the presence of uterine myomas was associated with the presence
of discontinuous JZ. In fact, all of the myomas described were classified as type
1 to 3 myomas according to the Fédération Internationale de Gynécologie et d'Obstétrique
(International Federation of Gynecology and Obstetrics, FIGO, in the French acronym)
classification, which justifies the findings and implications on fertility.[33 ]
The 3D ultrasound to assess the JZ is cost-effective and noninvasive. The obtained
results indicate that the assessment of the visualization and continuity of the JZ
by 3D ultrasound is reproducible enough to be used in the clinical practice. The presence
of disruption in the architecture of the JZ should alert the clinician to the possibility
of the presence of “endometrial-subendometrial myometrium unit disruption disease”
due to its association with endometriosis and adenomyosis and, consequently, its implications
for women's reproductive and obstetric health.[34 ]
The assessment of visualization and continuity of the JZ can be criticized regarding
its subjectivity, which is a limitation of the present study. However, the same methodology
has been used in other studies[14 ]
[35 ] to assess the reproducibility of JZ visualization, and similar results have been
obtained. Another limitation consists in the small sample obtained.
To the best of our knowledge, the present is the first study aiming to assess the
JZ through 3D ultrasound in infertile women in Portugal. The study raises new lines
of research. One point to develop is the assessment of the impact of JZ disruption
on fertility. Long-term follow-up of these women is necessary to evaluate the results
of assisted reproductive technology and to relate them to our results.
Thus, due to its reproducibility, the clinical information obtained, and the cost-effectiveness,
the evaluation of the JZ through 3D ultrasound should be part of the routine study
of infertile women.
Conclusion
Based on the results obtained from the present study, we emphasize the reproducibility
of transvaginal 3D ultrasound assessment of the JZ and the clinical benefits of its
inclusion in the routine evaluation of infertile women.
