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4 Contrast-enhanced magnetic resonance imaging of the pelvis

to assess uterine scar 6-12 months after conservative myomectomy (n=30)

Following 6-12 months after laparoscopic myomectomy, all patients underwent contrast-enhanced magnetic resonance imaging (MRI) of pelvic masses using SignaInfinity Echo Speed 1.5 T (GeneralElectric) MRI scanner.

Pelvic native MRI technique

Pelvic native MRI procedure was performed as follows: automatic pelvic organs tracking was used to obtain dynamic MR images based on 14 second fast gradient echo pulse sequences without breath-hold (TR 20 ms, TE 5 ms, FOV 400×400 mm, acquisition matrix 128×256, slice thickness 10 mm, number of slices 3). The resulting sagittal, coronal and axial images were used to accurately reconstruct the slices.

After topographic evaluation, FSE pulse sequence was applied to obtain T2-VI images in the sagittal plane (TR 7500 ms, TE 102 ms, FOV 24 mm, matrix 256×224, slice thickness 3 mm, ETL 24, BW 25).

The section block was installed to match femoral and iliac bone landmarks. To minimize movement-caused artifacts, the MR-signal pre-saturation zone (saturator) was applied to the abdominal wall and intestines.

Based on prior topographic evaluation the T1-weighted images perpendicular to anteflexed or anteverted uterine axis. Axial Т1-weighted images were obtained using FSE pulse sequence technique (TR 575 ms, ТЕ 9 ms, FOV 24 mm, matrix 2224×256, slice thickness 5/1 mm, BW 15.56). The prescribed scanning region extended from the L4 vertebrum to the bladder neck.

Sagittal images were used for subsequent positioning of T2-weighted images in other planes. Further MRI sequences were set up depending on the uterine anatomy.

Coronal images were obtained along the long and short axis of the uterine body. At the next stage FSE pulse sequence was employed to obtain T2-weighted

images in axial plane: (TR 7500 ms, TE 102 ms, FOV 24 mm, matrix 256×224, slice thickness 5/1.5 mm, ETL 24, BW 25).

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patients with elevated blood clotting or when anticoagulants could not be used

to prevent intravenous catheter thrombosis, the keep vein open function was enabled

with saline solution drip-fed through the catheter.

First, contrast solution bolus was injected at 5 ml/s rate, then a bolus

of a physiological (0.9%) sodium chloride solution was injected at a rate of 5 ml/s with a volume of up to 15-20 ml. Identical contrast and saline solution administration rates

ensured high contrast concentration with low dilution prior to entering the heart

chambers. Decreased contrast dilution was also achieved by higher saline solution volume of over 15 ml). Such saline solution volume allowed for the intravenous piston effect, with contrast agent flowing to the heart chambers at an unchanged velocity. The paramagnetic contrast agents included two gadolinium chelate containing drugs approved

for clinical administration in the Russian Federation: gadodiamide 0.5 mmol and dimeglumin gadopentetate 0.5 mol.

This completes the process of collecting information and proceeds to the stage of postprocessing processing and analysis of the received data.

A visual assessment was made based on digital subtraction and scrolling series

of images as a cine loop. The pre-contrast first series was subtracted from the postcontrast series, allowing for a clear view of hyperand avascular areas (scarring) on subtraction images as background signals were eliminated and no smoothing of contours and structure in hypervascularization areas occurred.

In all patients MRI evaluated the following parameters: uterine volume, presence of new myomatous nodes, myometrium thickness in the post-op scar area, the intact myometrium thickness of the uterine wall where the node was removed. To assess scar area vascularization, contrast accumulation in the removed myomatous node area was analyzed.

5 Immunohistochemical examination (n=40)

To assess myometrium in the scar area and tissue reparation following myomectomy, biopsied samples of the intact myometrium in scar area underwent

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The study analyzed the following markers:

–type II collagen (Dako 1:150);

–p53 protein (Dako, 1:150);

–p53 protein (Dako, 1:150);

–p21 protein (Dako, 1:150);

–p16 protein (Dako, 1:150);

–VEGF and VEGFR (Novocastra, 1:100).

Alexa 647 secondary antibody was used with DAPI nuclei staining. Morphometry was performed using Olympus infrared laser microscope.

Photo imaging was performed at 400× magnification (eyepiece 10×, lens 40×), open aperture, without condenser and in Photo mode, with exposure time 1/20 s, maximum camera sensitivity, image size 1280×1024 pixels, JPEG (normal) graphic image format. Further quantitative research was carried out using Morphology 5.0 (VideoTest, Russia) computer image analysis software. Relative expression area (S, %) was calculated as the ratio of immunopositive cells area to the total specimen area.

The average expression brightness was calculated in accordance with Booger- Lambert-Behr law formula:

absorbing layer thickness, cm; – molar absorption coefficient. Average expression brightness is a fundamental parameter of VideoTest-Morphology 5.0 software.

Key statements presented for defense:

1.PCNA expression assessment shows that proliferative potential of uterine scar cells remains unchanged throughout reproductive age, while apoptosis and cellular aging in this tissue increases 1.6-6.7 times with age;

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values across different groups (once the distribution complied with normal distribution law by Lilliefors and Shapiro-Wilk criteria and equal trait distribution variance according to Levene criterion) for independent sample. For multiple comparisons Bonferroni test was used as post-hoc testing. Correlation analysis was performed using Spearman rank correlation analysis. The critical significance level for statistical hypotheses testing was assumed equal to 0.05.

Publications presenting key provisions of the dissertation:

Key provisions of the dissertation, are presented in 11 research papers, including 5 paper in fellow-reviewed journals recommended to aspiring postgraduates by the Higher Attestation Commission of the Ministry of Science and Higher Education of the Russian Federation, 6 papers in other journals and collections, and 2 reports in conference proceedings.

Presentation of results to fellow experts

The thesis results were presented to the community of fellow experts at the following conferences:

1st IFSA Winter Conference on Automation, Robotics & Communications for Industry 4.0 (ARCI’ 2021), 2021, on-line; X International Congress on Photodynamic Therapy and Photodiagnostics. Biomedical photonics, 2021, online; VII International conference for young researcher: biophysicists, biotechnologists, molecular biologists and virologists within the OpenBio-2020 platform, Novosibirsk, 2020.

Defendant’s personal contribution

The defendant’s personal contribution includes the trial design, performance

of experiments, statistical data processing and analysis. The author took part in all studies, including histological staining of samples, immunohistochemistry, immunofluorescent staining, laser confocal microscopy, morphometry, data collection from pregnant women for lab diagnostics, MRI, and post-op uterine scar biometry

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CHAPTER 1 BACKGROUND

1.1 Uterine fibroids. Incidence rate. Pathogenesis. Localization

Uterine fibroids are benign tumours of the myometrium and a most common

disease of the female reproductive system. Figure 1.1 presents different myomatous

nodule locations in accordance with the International Federation of Gynecology and Obstetrics (FIGO), 2011 [95]. Uterine fibroids occur in 20-50% of female population;

diseases [37;48]. The average age for myoma detection is 32 years, therefore myoma in pregnant patients is increasingly relevant due to myoma on-set in younger patients, on the one hand, and late reproduction age, on the other [40]. Unfortunately, 60 to 96%

of all uterine myoma surgeries are still radical interventions, often resulting in loss

of reproductive and menstrual function. Women of reproductive age constitute a large group of patients undergoing such interventions [20;40;69].

Uterine fibroids occur in pregnancies in 0.5-6% of cases [35]. Among those uterine fibroids are more common in nulliparous women aged over 30 having familial

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