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\begin{document}

\title{Assessment of left ventricular myocardial systolic dysfunction in
premature ovarian insufficiency using echocardiographic layer-specific
myocardial strain imaging}



\author[1]{Yu-lin Wang}%
\author[1]{Lixue Yin}%
\author[1]{Mei Li}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Objective: To explore the myocardial systolic dysfunction in premature
ovarian insufficiency (POI) patients with normal left ventricular
ejection fraction (LVEF) using echocardiographic layer-specific
myocardial strain imaging. Methods: Forty-eight newly diagnosed,
untreated POI patients with normal LVEF (POI group) and fifty healthy
female volunteers with matching age, height and weight (controls group)
were enrolled. Both groups underwent standard transthoracic
echocardiography to obtain conventional left ventricular and
layer-specific strain parameters, the layer-specific strain parameters
including the subendomyocardial global longitudinal strain (GLSendo),
the mid-layer myocardial global longitudinal strain (GLSmid), the
subepimyocardial global longitudinal strain (GLSepi), the
subendomyocardial global circumferential strain (GCSendo), the mid-layer
myocardial global circumferential strain (GCSmid), and the
subepimyocardial global circumferential strain (GCSepi). Results: The
end-diastolic interventricular septal thickness (IVST) in the POI group
was significantly thicker than that in the controls group (P=0.008), and
The E, E/A, lateral e' in POI group were significantly lower than those
in the controls group (P\textless{}0.05). The GLSendo, GLSmid, GLSepi,
GCSendo, GCSmid, and GCSepi in the POI group were significant lower than
those in the controls group (P<0.05). Receiver operating characteristic
(ROC) curve showed that the area under the curve (AUC) of GLSepi was the
largest in diagnosing impaired left ventricular systolic function in POI
patients (AUC=0.706, P=0.000). Conclusions: POI patients with normal
LVEF may suffer with subclinical left ventricular myocardial systolic
dysfunction. The echocardiographic layer-specific myocardial strain
could be more sensitive to be used to detect the subclinical impairment
of left ventricular systolic function in POI patients.%
\end{abstract}%



\sloppy


\textbf{1 INTRODUCTION}

Premature ovarian insufficiency (POI) refers to a clinical syndrome
caused by loss of ovarian activity in women before the age of 40 years.
POI is mainly manifested as menstrual abnormalities (oligomenorrhea or
amenorrhea), increased gonadotrophins such as follicle stimulating
hormone (FSH\textgreater{}25IU/L), decreased estrogen level, and the
population prevalence rate is about 1\%.\textsuperscript{1}

The etiology of POI is complex, which can be caused by genetic defects
(sex chromosomes and autosomes), immune system diseases, thyroid
diseases, environmental factors, and iatrogenic factors. However, there
are still some patients with unknown causes.\textsuperscript{1}

POI can make adverse effects on women's multiple systems, and the harm
to the cardiovascular system will reduce life
expectancy.\textsuperscript{1} POI can be used as an independent
intermediate risk factor for ischemic heart disease (IHD) and
cardiovascular diseases (CVD),\textsuperscript{2} and its
pathophysiological mechanism affecting the cardiovascular system is
mainly a series of changes caused by the lack of estrogen.

Most women with POI have no obvious precordial discomfort in the early
stage, and they often ignore changes in the cardiovascular system. In
2016, the European Society of Human Reproduction and Embryology (ESHER)
recommended that all women with POI should assess the risk of CVD and
receive early hormone replacement therapy (HRT) to control future CVD
risk, and POI patients with special types of Turner Syndrome (TS) who
have sex chromosomal abnormalities, once diagnosed, should be assessed
the CVD risk and CVD-related risk factors annually.\textsuperscript{1}

The emerging layer-specific myocardial strain imaging in recent years is
based on two-dimensional speckle tracking echocardiography (2D-STE),
which can quantify the myocardial strain parameters of each layer,
provide more accurate myocardial deformation informations, and provide
more guidances for clinical precision treatment. This study intended to
use layer-specific myocardial strain to quantitatively assess the
subclinical myocardial systolic impairment of the left ventricle in POI
women with preserved left ventricular ejection fraction (LVEF-pr),
expecting to provide a new non-invasive method for clinical evaluation
of early changes in the left ventricular myocardial systolic function in
POI women for more precise treatment..

\textbf{2 METHODS}

2.1 Study population

Forty-eight patients, who were untreated with HRT, and newly diagnosed
as POI, from the Menopausal Gynecological Endocrinology Clinic of our
hospital were included in the POI group.

All subjects in the POI group underwent sex hormone testing, and
patients suspected TS underwent chromosome testing to confirm the
diagnosis. And inclusion criteria: Meet the POI diagnostic criteria
proposed in the 2016 by ESHER\textsuperscript{1}: oligomenorrhea /
amenorrhea at least 4 months, and an elevated FSH \textgreater{}25IU/L
on two occasions \textgreater{}4 weeks apart. No clinically diagnosed
severe cardiovascular diseases, and no obvious clinical manifestations
of heart involvement such as chest pain, chest tightness, and
palpitations.

Exclusion criteria: Age\textgreater{}40 years old, those who have
undergone HRT, those who have abnormal menstruation caused by
radiotherapy and chemotherapy for malignant tumors. Congenital and
acquired heart diseases, hypertension, diabetes, thyroid disease, blood
system disease, autoimmune system disease. Patients with abnormal LVEF
(LVEF\textless{}54\%)\textsuperscript{3} or those with poor image
quality.

Fifty healthy female volunteers who came to physical examination during
the same period and with matched age, height, and weight were selected
as the controls group. Inclusion criteria: sinus rhythm, no
hypertension, no hyperlipidemia, no diabetes, and normal transthoracic
echocardiography. Exclusion criteria: routine transthoracic
echocardiography revealed abnormalities or poor image quality.

This study was reviewed and approved by the hospital ethics committee
(batch number: No. 265), and all subjects signed an informed consent.

2.2 Procedures

All subjects rested peacefully for 10 minutes and then were measured the
blood pressure. After that, making them take the left side lying
position, breath calmly, be connected to the electrocardiogram, and be
performed a complete echocardiography. Using Vivid E9 (GE
Healthcare,US), with M5S (probe frequency:1.4-4.6MHz). The M5S probe was
used to collect two-dimensional dynamic images of at least three cardiac
cycles from the parasternal left ventricle long axis view, apical views,
and left ventricular short-axis views. Activate the pulsed wave Doppler
(PW) and tissue Doppler imaging (TDI) to collect the mitral valve
anterior blood flow spectrum and the velocity of the mitral annulus on
the apical 4-chamber view.

EHCO PAC 202 workstation was employed for image postprocessing and
measurement of the end-diastolic diameter of left ventricle (LVEDD),
end-diastolic thickness of left ventricular posterior wall (LVPWT), and
end-diastolic interventricular septal thickness (IVST) at the
parasternal left ventricular long axis view. The formulas as below were
used to calculate relative wall thickness (RWT) =2*LVPWT/LVEDD, left
ventricular mass (LVM) =0.8*1.04*{[}(LVEDD+IVST+LVPWT)\selectlanguage{ngerman}³-LVEDD³{]}+0.6,
body surface area (BSA) =0.0061*height (cm)+0.0128*weight (kg)-0.1529,
body mass index (BMI)=weight (kg)/height(m)\textsuperscript{2}, and left
ventricular mass index (LVMI) =LVM/BSA.

LVEF and left atrial volume (LAV) were measured by the biplane Simpson's
method, and left ventricular volume index (LAVI) was calculated by
LAV/BSA.\textsuperscript{3}

The apical views, left ventricular short-axis views were selected in
turn, starting the Q-analysis system of EHCO PAC 202 workstation,
entering the 2D-strain to manually trace the boundary of the endocardium
and epicardium, determining the region of interest (ROI), and the
software would automatically track the movement trajectory of the
myocardial spots in each layer, and get the subendomyocardial global
longitudinal strain (GLSendo), the mid-layer myocardial global
longitudinal strain (GLSmid), the subepimyocardial global longitudinal
strain (GLSepi), the subendomyocardial global circumferential strain
(GCSendo), the mid-layer myocardial global circumferential strain
(GCSmid), and the subepimyocardial global circumferential strain
(GCSepi) (Figure 1).

The evaluation of left ventricular diastolic function was based on the
evaluation guideline for left ventricular diastolic function proposed by
ASE/EACI in 2016.\textsuperscript{4}

Using simple random sampling method, 10 cases were randomly selected
from POI patients for intraobserver reproducibility test, and another 10
cases were randomly selected for interobserver reproducibility test.

2.3 \selectlanguage{english}\textbf{ } Statistical analysis

SPSS 25.0 was used for statistical analysis in this study,and the datas
were expressed as mean +- standard deviation. K-S test was used to
determine the distribution type of continuous variables, and Levene test
was used to test the homogeneity of variance.

If the datas from two geoups satisfied both normal distribution and
homogeneity of variance, a two independent samples t-test was performed;
otherwise, the Mann-Whitney U test of two independent samples was used.
Receiver operating characteristic (ROC) curve was used to study the
diagnostic efficacy of layer-specific strain parameters on left
ventricular systolic function impairment in POI patients. Intraclass
correlation coefficient (ICC) was used for the reproducibility test.

A \emph{P} -value\textless{}0.05 was regarded as statistically
significant for all analyses.

\textbf{3 RESULTS}

3.1 Routine clinical data

Among the forty-eight cases in the POI group, the estrogen levels of
twenty-two cases were below 37pmol/L (the bottom line of serum sex
hormone testing equipment was 37pmol/l), and the estrogen level of other
twenty-six cases was 88.37+-48.68pmol/l, and the FSH level of the POI
group was 66.70+-27.89mIU/ml.

There were five cases of TS patients in the POI group, of which three
cases had chromosomes: 45, X, and the other two cases respectively were:
45,X/46,XX/47,XXX; 45,X/46,X,r(X). Among the POI patients, four cases
were primary amenorrhea, and the abnormal menstruation history of
remaining forty-four cases was 13.07+-9.33 months.

No significant differences in age, BSA, BMI, systolic blood pressure,
diastolic blood pressure, and heart rate between the two groups were
found (all \emph{P} \textgreater{}0.05) (Table 1).

3.2 Standard echocardiography

The value of IVST in the POI group was significantly larger than that in
the controls group (\emph{P} =0.008), but no statistical differences
between the two groups in LVPWT, LVEDD, RWT, LVM, and LVMI (all\emph{P}
\textgreater{}0.05). The values of E, E/A, lateral e\selectlanguage{english}' in POI group were
significantly lower than those in controls group (all\emph{P}
\textless{}0.05), while no differences in A, septal e\selectlanguage{english}', average E/e\selectlanguage{english}' and
LVEF between the two groups were found (\emph{P} \textgreater{}0.05)
(Table 2).

According to the left ventricular diastolic function assessment
guidelines proposed by ASE/EACI in 2016,\textsuperscript{4} the left
ventricular diastolic function in all subjects remained normal, but
individual abnormal parameters in the POI group (E, E/A, lateral e\selectlanguage{english}')
still suggested that there were adverse changes in left ventricular
diastolic function in POI patients.

3.3 Layer-spcific myocardial strain

GLSendo, GLSmid, GLSepi, GCSendo, GCSmid and GCSepi in the POI group
were all lower than those in the controls group (\emph{P} \selectlanguage{english}<0.05
or\emph{P} \selectlanguage{english}<0.001), the longitudinal strain and circumferential strain
parameters of the two groups all followed the decreasing trend from the
subendocardium to the subepicardium (Table 3) (Figure 2, Figure 3).

3.4 Efficacy of layer-specific strain parameters in diagnosing the
impairment of left ventricular systolic function in POI patients.

ROC analysis showed that the area under the curve (AUC) of GLSepi was
the largest in diagnosing impairment of left ventricular systolic
function in POI patients (AUC=0.706, \emph{P} =0.000), and when the
cut-off value was -20.15\%, the sensitivity was 79.9\% and the
specificity was 54.7\% (Table 4) (Figure 4).

3.5 Reproducibility of layer-specific strains

Intraobserver and interobserver coefficients of GLSendo, GLSmid, GLSepi,
GCSendo, GCSmid, GCSepi were all greater than 0.900, and the
reproducibility of layer-specific strains was acceptable (Table 5).

\textbf{4 DISCUSSION}

The concept of premature ovarian failure (POF) appears before POI. POF
refers to women with amenorrhea, elevated gonadotropin levels, and
decreased estrogen levels before the age of 40, accompanied by various
perimenopausal related symptoms (such as hot flashes, insomnia, body
pain, etc.), and it is the final stage of POI.\textsuperscript{5} The
population prevalence rate of POI is about 1\%,\textsuperscript{1} the
incidence of POF before the age of 30 is about 1\selectlanguage{english}%0, the incidence of POF
before the age of 40 is about 1\%, the incidence of POF in patients with
secondary amenorrhea is about 4\%-18\%, and the incidence of POF in
patients with primary amenorrhea is about 10\%-28\%.\textsuperscript{6}

Due to the loss of functional follicles, women with POI are in a state
of low estrogen for a long time, causing them to face a series of
problems such as infertility and perimenopausal syndrome. In addition,
Cardiovascular markers in women with POI have deteriorated, women with
POI have a higher risk of IHD and CVD, POI can be used as an independent
intermediate risk factor for IHD and CVD.\textsuperscript{2,7}

Another study suggested that early menopause was associated with an
increased risk of heart failure.\textsuperscript{8} Since most women
with POI do not have chest tightness, chest pain and other CVD-related
clinical features in the early stage, CVD risk detection is often
ignored. LVEF is commonly used in clinical evaluation of left
ventricular systolic function, and myocardial strain can more
sensitively reflect the subclinical myocardial changes in the early
stage of the disease than LVEF.\textsuperscript{9} The layer-specific
myocardial strain can more accurately quantitatively analyze the
deformation of the endocardium, middle myocardium, and epicardium, and
has been more maturely used in the study of various
diseases.\textsuperscript{10-12} However, there is still a lack of study
reports on layer-specific myocardial strain in POI patients.

In this study, the IVST in the POI group was thicker than that in the
controls group (\emph{P} =0.008), suggesting that women with POI may
have left ventricular remodeling. The possible pathophysiological
mechanisms are: Estradiol (E\textsubscript{2}) as a fast activator of
endothelial nitric oxide synthase (NOs) can stimulate the production of
nitric oxide (NO), but women with POI continue to be in a low
E\textsubscript{2}status, resulting in reduced NO production by vascular
endothelial cells, decreased vascular elasticity and compliance, and
resulting in increased cardiac afterload, stimulating myocardial cell
hypertrophy, and promoting left ventricular remodeling and myocardial
hypertrophy;\textsuperscript{13-14} otherwise low E\textsubscript{2} can
lead to abnormalities in the renin-angiotensin system (RAS) in the heart
and systemic circulation, and enhance the activity of angiotensin
converting enzyme (ACE), leading to ventricular remodeling and cardiac
hypertrophy.\textsuperscript{15}

However, according to adult echocardiographic quantitative measurement
guideline proposed by ASE/EACI in 2015 for left ventricular hypertrophy
(RWT\textgreater{}0.42, female
LVMI\textgreater{}95g/m\textsuperscript{2}),\textsuperscript{3} All
subjects in this study did not meet the criteria for left ventricular
hypertrophy, but fifteen cases in the POI group (34.25\% of the POI
group) reached the criteria for concentric remodeling of the left
ventricle (RWT\textgreater{} 0.42, female
LVMI\selectlanguage{english}[?]95g/m\textsuperscript{2}).\textsuperscript{3}

In addition, no statistical differences in LVPWT, LVEDD, RWT, LVM, LVMI
between the two groups were found (\emph{P} \textgreater{}0.05),
indicating that although POI women had local myocardial thickening, they
had not yet caused significant changes in the overall geometry of the
left ventricle.

Echocardiography can not only evaluate changes in left ventricular
geometry in women with POI, but also quantitatively evaluate changes in
left ventricular function.

This study used TDI to assess the changes in left ventricular diastolic
function in patients with POI. In this study, the E, E/A, and lateral e\selectlanguage{english}'
in POI group were lower those in the controls group (\emph{P}
\textless{}0.05 or \emph{P} \textless{}0.01), but no differences in
septal e\selectlanguage{english}', average E/e\selectlanguage{english}', LAVI were found between the two groups
(\emph{P} \textgreater{}0.05), but according to guidelines proposed by
the ASE/EACI in 2016 for left ventricular diastolic
function,\textsuperscript{4} the left ventricular diastolic function did
not decrease in both two groups in this study. And the above suggested
although there was no significant decrease in left ventricular diastolic
function in patients with POI, there were still some signs of decreased
diastolic function.

The reasons for the decline of left ventricular diastolic function due
to E\textsubscript{2} deficiency are more complicated, the main possible
reasons are: an animal experiment showed that MCP-1 and MCP-5 mRNAs in
the cardiomyocytes of ovariectomized rats, as the isoforms of chymase
forming angiotensin II (Ang II), participate in the loss of normal heart
function and structure caused by ovariectomy in rodents, including
decreased left ventricular diastolic function (increased E/e') and
increased LVEDD;\textsuperscript{16} And one other animal experiment
showed that E\textsubscript{2} could improve the antioxidant capacity of
cardiomyocytes in ovariectomized rats and play a protective effect on
myocardium,\textsuperscript{17} then suggesting that lack of
E\textsubscript{2} could lead to the reduction of myocardial antioxidant
capacity, aggravation of fibrosis, and further reduction of left
ventricular diastolic function; In addition, it had been reported that
E\textsubscript{2} deficiency would lead to reduced mitochondrial
function of cardiomyocytes and inhibit NOs signaling, thereby inhibiting
the GPR30 pathway, which together leaded to a decrease in left
ventricular diastolic function.\textsuperscript{14}

Changes in left ventricular diastolic function are often inseparable
from changes in systolic function. However, there was no obvious
abnormality in left ventricular systolic function when the left
ventricular diastolic function in the POI group in conventional
echocardiography was abnormal (there was no significant difference in
LVEF between the two groups), suggesting that the use of conventional
LVEF may not reflect the subclinical changes of left ventricular
systolic function early.

This study used layer-specific myocardial strain and found that GLSendo,
GLSmid, GLSepi, GCSendo, GCSmid, and GCSepi in the POI group were lower
than those in the controls group, suggesting that there were reduced
left ventricular systolic function in POI group, and layer-specific
myocardial strain could more sensitively prompt the left ventricular
systolic function impairment in POI patients than conventional
two-dimensional LVEF. The possible mechanisms for the decrease in
layer-specific strains of the left ventricle in the POI group were: an
animal experiment had shown that E\textsubscript{2} enhanced cardiac
systolic function by regulating the expression of cAMP-L-type
Ca\textsubscript{2}\textsuperscript{+} channel (cAMP-LTCC) related
genes, and confered cardioprotection of against stress through the GPR30
non-genomic acute signaling pathway,\textsuperscript{18} so the lack of
E\textsubscript{2} might lead to decreased myocardial systolic function,
appearing the result of this study; In addition, Cheng and other
researches believed that there were estrogen receptors (ER) in the
arterial wall and myocardial tissue, including ER\selectlanguage{greek}α, \selectlanguage{english}ER\selectlanguage{greek}β, \selectlanguage{english}and G
protein-coupled estrogen receptor (GPER), E\textsubscript{2} could
directly act on cardiomyocytes, enhance myocardial systolic function and
reduce heart rate variability,\textsuperscript{19-21} then lack of
E\textsubscript{2} may directly lead to increased myocardial oxygen
consumption and energy requirements, and decrease myocardial systolic
function.

There is a certain gradient in the deformability of the three-layer
myocardial structure of the normal left ventricle, the deformation of
the myocardium from endocardium to epicardium is gradually
decreasing.\textsuperscript{22} This study also confirmed this
decreasing trend from endocardium to epicardium, both in the POI group
and the controls group.

Through the ROC curve to analyze the efficacy of the layer-specific
strain parameters in diagnosing the left ventricular systolic function
in POI patients, we found that compared with other parameters, the AUC
of GLSepi was largest (AUC=0.706, P=0.000), indicating that it might
have a higher diagnostic value for the detecting of left ventricular
myocardial systolic function impairment in patients with POI.

But this study showed while using -20.15\% as the cut-off value of
GLSepi, the sensitivity was 79.9\%, and the specificity was 54.7\%, the
specificity was still low, the further research was needed in the later
period.

4.1 Study limitations

Limitations of this study: (a) Since the hospital serum sex hormone
testing equipment had been set 37pmol/l as the bottom line of estrogen,
it was impossible to collect the E2 of all patients in the POI group; E2
and FSH levels of the normal group were not collected; (b) The study was
still a preliminary study, and the sample size can be increased to
further verify the results; (c) The follow-up study during HRT had not
been conducted.

\textbf{5 CONCLUSIONS}

In summary, POI patients with normal LVEF may have subclinical left
ventricular myocardial systolic impairment. The layer-specific
myocardial strain can more sensitively detect the subclinical left
ventricular systolic impairment in POI patients, and can be used as a
new non-invasive method for early evaluation of the left ventricular
systolic function impairment in POI patients.

\textbf{AUTHOR CONTRIBITIONS}

Li-xue Yin was responsible for study conceptualization, design, and
supervision ,Yu-lin Wang was responsible for performing
echocardiography, data analysis, and writing manuscript, Mei Li was
responsible for providing cases. All authors have revised and approved
the final submission of the manuscript.

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22.Huttin O, Girerd N, Coiro S, et al. Association Between
Layer-Specific Longitudinal Strain and Risk Factors of Heart Failure and
Dyspnea: A Population-Based Study. J Am Soc Echocardiogr.
2019;32(7):854-865.e8.

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\verb`FIGURE 1.eps`  available at \url{https://authorea.com/users/373759/articles/491360-assessment-of-left-ventricular-myocardial-systolic-dysfunction-in-premature-ovarian-insufficiency-using-echocardiographic-layer-specific-myocardial-strain-imaging}\selectlanguage{english}
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\verb`FIGURE 3.eps`  available at \url{https://authorea.com/users/373759/articles/491360-assessment-of-left-ventricular-myocardial-systolic-dysfunction-in-premature-ovarian-insufficiency-using-echocardiographic-layer-specific-myocardial-strain-imaging}

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\verb`FIGURE 4.eps`  available at \url{https://authorea.com/users/373759/articles/491360-assessment-of-left-ventricular-myocardial-systolic-dysfunction-in-premature-ovarian-insufficiency-using-echocardiographic-layer-specific-myocardial-strain-imaging}

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