Kalimatova D.M., Department of Obstetrics and Gynecology, N.I.Pirogov Russian National Research Medical University - Russian Federation, Dobrokhotova Yu.E., Department of Obstetrics and Gynecology, N.I.Pirogov Russian National Research Medical University - Russian Federation

Email address: 9227707@gmail.com (Kalimatova D.M.), pr.dobrohotova@mail.ru (Dobrokhotova Yu.E.)
To cite this article: Kalimatova D.M., Dobrokhotova Yu.E.

Abstract: The study was conducted to search for relationships between the severity of endothelial dysfunction, hemostasis disorders and ovarian reserve in patients with endometriosis who had a coronavirus infection. 156 patients with infertility and ovarian endometriomas were examined and divided into 2 groups: - group 1 - 72 women who had a laboratory-confirmed infection with SARS-CoV-2 within 6 months before the study; group 2 - 84 patients who had no history of SARS-CoV-2 infection. In these patients the ovarian reserve was assessed - the levels of follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH), the state of the coagulation system. The indicators of endothelial dysfunction were also studied: plasma levels of endothelin 1, endothelial protein C receptor (PROCR), and vascular endothelial growth factor (VEGF). The determination of significant endometriosis-associated predictors of reduced fertility in patients with endometriosis who underwent COVID-19 infection was carried out by the method of correlation analysis. It has been established that women with endometriosis who have had an infection caused by the SARS-CoV-2 virus have a decrease in ovarian reserve and signs of endothelial dysfunction, which are combined with changes in the hemostasis system. At the same time, significant correlations were found between the severity of changes in the blood coagulation system and the degree of endothelial dysfunction in these women.

Keywords: Endometriosis, coronavirus infection, markers of endothelial dysfunction, ovarian reserve

Introduction. Endometriosis is a complex syndrome, the pathogenesis and clinical manifestations of which are based on an estrogen-dependent chronic inflammatory process that primarily affects the pelvic organs, foremost the ovaries [1, 2].

Endometriosis is the most common cause of chronic pelvic pain in women and one of the most crucial factors of infertility [1, 3]. In 44% of women suffering from endometriosis, endometrioid ovarian cysts (EOC) are detected, which are often combined with tubal sterility. EOC is the most particular manifestation of genital endometriosis and occurs mainly in women of childbearing age [3, 4].

It is believed that endothelial dysfunction observed in this disease plays an important role in the pathogenesis of changes in the function of the hemostatic system [5-7]. The most significant complication of COVID-19 is venous thromboembolism, the frequency of which in hospitalized patients reaches up to 10% [6-8]. Prolonged immobilization during illness, dehydration, acute inflammatory process, risk factors of cardiovascular diseases (hypertension, diabetes, obesity) or cardio-vascular diseases, as well as classical genetic thrombophilia (for example, heterozygous mutation of Factor V Leiden) - all of these factors are concomitant diseases that potentially increase the risk of venous thromboembolic complications in patients hospitalized with COVID-19 [9, 10]. Activation/damage of endothelial cells when the virus binds to ACE-2 also increases the risk of developing these complications [11, 12].

Activation of the congenital immune system and the release of a large number of BAS involved in the process of inflammation in the walls of blood vessels also contribute to an increase in the damaging effect of the causative agent SARS-CoV-2. The vicious circle of angiotensin II (Ang II) production and suppression of hACE2-R receptor activity substantially affects the state of the arterial and venous endothelium. This ensures the formation of an environment contributing to the development of disseminated intravascular coagulation processes [7, 10].

The virus penetrates into the vascular endothelium, reducing the density of hACE2-R receptors and generating a chain of events (as a result of the inflammatory action of Ang II), which induces a pro-adhesive environment for aggregation and migration of inflammatory cells of macrophages, leukocytes and lymphocytes. These cells produce gamma interferon, tumor necrosis factor-alpha, interleukins-1, 6 and profibrotic factors such as tissue factor, plasminogen activation factor-1 and von Willebrand factor [13-15].

There are suggestions that the SARS-CoV-2 virus may have adverse effects on the reproductive system. Some studies have shown that with this infection, there are changes in fertility and impaired reproductive function in women [16-18]. The findings suggest that this formation of the virus-ACE2 protein complex may affect women's reproductive functions, leading to menstrual irregularities, infertility, and fetal distress.

At the same time, it should be noted that so far there is no information on the potential impact of COVID-19 disease on fertility in women, as well as on the role of changes in hemostasis shifts in presumed reproductive disorders. Obviously, the study of the relationship between COVID-19 infection and subsequent disorders of hemostasis and the reproductive system will provide new data on the state of fertility in recovered patients with endometriosis. This, in turn, will improve the management of women with infertility and ECO who have had an infection caused by the SARS-CoV-2 virus.

The sources also lack reliable results on the impact of COVID-19 infection on ovarian reserve, in particular in women with endometriosis and infertility. Data on the severity of endothelial dysfunction in this category of patients with endometriosis are not presented, while endothelial dysfunction is currently considered as the most important factor in the pathogenesis of disorders detected in the body in individuals who have had an infection caused by the SARS-CoV-2 virus.

Aim. The aim of the study is to search for the correlations between the severity of signs of endothelial dysfunction, hemostasis disorders and ovarian reserve in patients with endometriosis who underwent coronavirus infection.

Materials and methods. A comprehensive clinical and laboratory examination of 156 patients with infertility and ovarian endometriomas, who subsequently underwent laparoscopic removal of endometrioma were carried out on the basis of the Gynecological department of the Moscow Clinical Hospital №1 named after N.I.Pirogov and Moscow City Clinical Hospital №52.

Inclusion criteria were:
  • age from 18 to 40 years;
  • the diagnosis of external endometriosis. Endometrioid ovarian cysts;
  • no pregnancy within 1 year of regular sexual activity;
  • fertile spermogram of the sexual partner

Exclusion criteria were:
  • the presence of signs of endometrial hyperplasia;
  • uterine fibroids with clinically significant size and location of nodes;
  • anomalies in the development of internal genital organs;
  • the presence of ovarian formations of a different etiology;
  • acute purulent-inflammatory processes, including urogenital infections in the active phase;
  • somatic and/or neuropsychiatric diseases in the stage of decompensation

Patients were divided into 2 groups: Group №1 consisted of 72 women who had a laboratory-confirmed SARS-CoV-2 infection within 6 months prior to enrollment in the present study; Group №2 included 84 women, who did not have clinical or laboratory evidence of SARS-CoV-2 infection in the past history.

All patients were examined, with an assessment of the state of the ovarian reserve: on the 2nd–5th day of the menstrual cycle, the level of follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH) was measured.

Laboratory research included studying the state of the hemostasis system by assessing the number of platelets, APTT, prothrombin time, fibrinogen, D-dimer, protein C and protein S concentrations.

The indicators of endothelial dysfunction in the groups of examined patients, were also compared, while determining the concentrations in blood serum samples of the following markers: endothelin 1, endothelial receptor for protein C (PROCR), vascular endothelial growth factor (VEGF). Quantitative determination of endothelin was carried out by immune-enzyme analysis, using BIOMEDICA test systems (BIOMEDICA GRUPPE, Germany); PROCR - using test systems manufactured by USCN Life Science Inc. (China), VEGF - using the test systems VEGF - ELISA - BEST (Vector-BEST, Russia), according to the manufacturer's instructions. Multiskan FS (Thermo Scientific, USA) was used as an ELISA- reader.

Analysis of the study results was performed using Statsoft software packages. STATISTICA 10 and Microsoft Excel 2016. The choice of the main characteristics and statistical criteria when comparing them was carried out after studying the distribution of the feature and comparing it with the Gaussian distribution according to the Kolmogorov-Smirnov criterion. Since the revealed distribution of signs differed from the normal one, nonparametric methods were used for further work with the obtained data.

Quantitative data were described as Me (Q25; Q75), where Me is the median; Q25 and Q75 are the lower and upper quartiles, respectively. Intergroup comparisons in terms of quantitative indicators were carried out using the rank-based non-parametric Mann-Whitney test.

The determination of significant endometriosis-associated predictors of reduced fertility in patients with COVID-19 infection was carried out using the method of data correlation analysis with the calculation of the Spearman correlation coefficient. Differences were considered statistically significant if the "p" threshold value of the level of statistical significance of the null hypothesis (alpha), equal to 0.05, was not reached.

Results.
The analysis of the concentrations of markers of endothelial dysfunction in patients with endometriosis showed that in Group №1 women who had SARS-CoV-2 infection during the last 6 months before inclusion in the study, the concentrations of endothelin 1 and vascular endothelial growth factor were statistically significantly higher ( p=0.018) than in the group of non-ill patients (Table 1). At the same time, the concentration of the endothelial receptor for protein C was reduced by more than 3.5 times (p<0.001) in patients who underwent COVID-19, compared with the corresponding value in the second group.

Table 1
Levels of markers of endothelial dysfunction among patients with endometriosis, depending on the past infection with COVID-19, Me (Q25; Q75)

Note: * - differences are statistically significant (at p<0.05) according to the Mann-Whitney test) compared with the corresponding indicator in Group №1.
Table 2 shows the indicators of the hormonal status of the examined patients with endometriosis. As can be seen, the level of AMH in the peripheral blood of patients who underwent COVID-19 was statistically significantly lower (p=0.025) than the corresponding level in group 2. The FSH concentration was also slightly lower in women who recovered from the infection compared to the value of this indicator in patients without signs of infection during the last 6 months, while the identified differences did not reach statistical significance (p=0.079).

Table 2
Levels of Anti-Mullerian and follicle-stimulating hormones in patients with endometriosis depending on the past infection with COVID-19, Me (Q25; Q75)

Note: * - differences are statistically significant (at p<0.05) according to the Mann-Whitney test) compared with the corresponding indicator in group 1.
The analysis of the state of the blood coagulation system of the patients included in the study indicated changes in a number of hemostasis parameters among women who had an infection caused by the SARS-CoV-2 virus. As presented in Table 3, these patients had statistically significantly higher levels of D-dimer (p=0.002), fibrinogen (p=0.019) and prothrombin time (p=0.009) than in the second group (Table 3).
At the same time, the values of APTT, protein C and S concentrations were significantly lower among recovered patients, than in group 2 (p=0.002, p=0.002 and p=0.002, respectively). For the number of platelets in women who underwent COVID-19, there was a tendency to increase this parameter relative to the level in group 2, although there were no statistically significant intergroup differences (p=0.156).

Table 3
Comparative characteristics of the parameters of the blood coagulation system in patients with endometriosis, depending on the past infection with COVID-19, Me (Q25; Q75)

Note: * - differences are statistically significant (at p<0.05) according to the Mann-Whitney test) compared with the corresponding indicator in group 1.
The correlation analysis showed the presence of several statistically significant multidirectional relationships of moderate strength of endothelial dysfunction indicators in patients who underwent COVID-19, on the one hand, with AMH levels and hemostasis parameters, on the other. As can be seen from Table 4, the level of endothelin 1 was negatively moderately associated with the concentration of AMH and such indicators of the coagulation system as APTT, proteins S and C. At the same time, this indicator of endothelial dysfunction positively and significantly correlated with the level of D-dimer and prothrombin time.
At the same time, the PROCR level was positively associated with the values of the APTT and protein C concentrations, but at the same time had inverse statistically significant correlations with the values of D-dimer and prothrombin time.
The level of vascular endothelial growth factor had an inverse negative relationship with the concentration of AMH, at the same time, it correlated positively and statistically significantly with the level of D-dimer.

Table 4
Correlation between indicators of endothelial dysfunction, hemostasis and ovarian reserve among patients with endometrial cysts after SARS-CoV-2 infection)
(Spearman correlation coefficients, r)

Discussion
The results of the study indicate that women with endometriosis who have had an infection caused by the SARS-CoV-2 virus have signs of endothelial dysfunction, in particular, a significant increase in plasma concentrations of endothelin-1 and vascular endothelial growth factor along with a decrease in the level of the protein C receptor compared to patients who have not had the disease.

It has been established that ACE2 is a receptor for SARS-CoV [19]. The study of ACE2 expression was evaluated in various human organs, such as the respiratory tract, heart, kidneys, ovaries, uterus, testicles, vagina and placenta, as well as in the gastrointestinal tract [16, 20], while it was shown that ACE2 expression is significantly represented in the ovaries [20, 21]. ACE2 receptors regulate follicle development and ovulation, regulate angiogenesis and macular degeneration, and also affect periodic changes in endometrial tissue and embryo development [8, 17].

Taking into account the above, it is logical to assume that the SARS-CoV-2 virus can disrupt fertility in women by attacking ovarian tissue and granulosa cells or damaging the membrane protein of endometrial epithelial cells Basigin (BSG), which is also one of the most important COVID-19 receptors and mediates the penetration of the virus into the host body [10].

Currently, it has been established that SARS-CoV-2/COVID-19 infection often causes hypercoagulation with inflammation, is accompanied by an increase in the level of blood clotting factors and a violation of the normal homeostasis of vascular endothelial cells, which leads to microangiopathy, local thrombus formation and systemic coagulation disorder [10]. COVID-19-associated coagulopathy manifests itself in the form of increased fibrinogen and D-dimer levels and some shifts in prothrombin time, APTT and platelet levels [6,21].

It is generally recognized that the vascular endothelium is considered as the most significant systems for regulating vascular tone and blood clotting. In recent years, there have been reports confirming the role of endothelial dysfunction in the development of gestosis, and for the first time a hypothesis about the role of maternal endothelial activation in the pathogenesis of this complication was put forward by Roberts J. and Cooper D. (2001) [22].

It has been established that in SARS-CoV-2 infection, platelets are activated by various pro-inflammatory cytokines, and the damaged endothelium easily binds to platelets. At the same time, the dysfunction of endothelial cells caused by inflammation further accelerates the thrombotic reaction. It is believed that the activation of endothelial cells may be caused by changes in the placenta or systemic vascular diseases of the mother, which are accompanied by activation of the platelet-vascular link of hemostasis, damage and dysfunction of erythrocytes, vasoconstriction and violation of uteroplacental blood flow [6]. The mechanism of endothelial dysfunction is based on hypoxia, which develops in the tissues of the uteroplacental system. It was found that local damage to the endothelium leads to the release of toxic endothelin, a decrease in the synthesis of vasodilators, cellular disaggregants (bradykinin, prostacyclin). In its turn, endothelial damage increases its sensitivity to vasoactive substances, hypercoagulation and generalized vasospasm, which is expressed in hypoxia and ischemia of vital organs [6].

Recently, there have been studies aimed at studying the significance of mediators of endothelial dysfunction, which are produced when the integrity of the endothelium is damaged.
As one of the methods for evaluation the severity of gestosis, the determination of concentrations of markers of endothelial dysfunction is considered. As such, the following are considered: Willebrand factor, prostacyclin, fibronectin, thromboxane A2, nitric oxide (NO), endothelial relaxation factor, thrombomodulin, VEGF (vascular-endothelial growth factor), intercellular adhesion molecules and interleukins [23].

Our results are consistent with the above data and confirm that patients with EOC who have had a history of COVID-19 infection show signs of a decrease in ovarian reserve, as evidenced by a decrease in the concentration of anti-muller hormone. The revealed violations occur against the background of a decrease in the concentration of FSH, and are also combined with changes in the coagulation system of hemostasis in these women.

The study showed the presence of significant changes in the hemostasis system in patients with endometriosis who had an infection caused by the SARS-CoV-2 virus. At the same time, the results of the correlation analysis showed the presence of a number of statistically significant associations of changes in the coagulation system with the severity of endothelial dysfunction in these women. Since the obtained data confirm that such changes are associated with a decrease in ovarian reserve in patients who have been ill, the results suggest that the identified shifts in hemostasis parameters and changes in endothelial characteristics can in the future be considered as statistically significant endometriosis-associated predictors of fertility changes in patients who have had an infection caused by the SARS-CoV-2 virus, which it should be taken into account in the treatment of endometriosis-associated infertility.

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