Kseniia V. Obedkova, Julian R. Ryzhov, Kseniia V. Storozheva, Vladislava V. Khalenko, Galina Kh. Safarian, Alevtina M. Savicheva, Olga V. Budilovskaya, Anna A. Krysanova, Aleksandr M. Gzgzyan, Olesya N. Bespalova, Natalya I. Tapilskaya

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductive Medicine, St. Petersburg, Russia

Повторная имплантационная недостаточность и повторная потеря беременности являются значительными проблемами в репродуктивной медицине, часто связанными с дисфункцией эндометрия и хроническим эндометритом. Современные методы лечения, включая антибактериальную терапию, не всегда эффективны. В Статье приведены данные исследования по оценке клинической эффективности секретома мононуклеарных клеток периферической крови (PBMC) в повышении восприимчивости эндометрия и улучшении показателей наступления беременности у пациенток с RIF, связанным с хроническим эндометритом.

Одним из перспективных вариантов является введение секретома мононуклеарных клеток периферической крови (PBMC), содержащего различные биологически активные вещества, включая белки, внеклеточные везикулы, антимикробные пептиды, нуклеотиды, липиды, интерлейкины и цитокины, необходимые для повышения восприимчивости эндометрия у пациенток с хроническим эндометритом и бесплодием. В исследование было включено 54 женщины с длительным бесплодием, которые были разделены на две группы: основная группа, получавшая секретом PBMC, и группа сравнения, проходившая стандартную антибактериальную терапию. Секретом PBMC был приготовлен из культивированных PBMC, и была оценена его биологическая активность. Результаты исследования оценивали по частоте наступления беременности у пациенток с длительным бесплодием после введения секретома PBMC была значительно выше по сравнению с женщинами после антибактериальной терапии.

Были сделаны следующие выводы: применение секретома PBMC, по-видимому, улучшает репродуктивные результаты у женщин с повторными потерями беременности из-за ХЭ. Эти результаты подтверждают потенциал секретома PBMC как нового терапевтического варианта для улучшения здоровья эндометрия и требуют дальнейшего изучения в более крупных клинических испытаниях.

Abstract. Background and aim: Recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) are significant challenges in reproductive medicine, often linked to endometrial dysfunction and chronic endometritis (CE). Current treatments, including antibacterial therapies, are not always effective. This study aims to evaluate the clinical efficacy of peripheral blood mononuclear cell (PBMC) secretome in enhancing endometrial receptivity and improving pregnancy rates in patients with RIF associated with CE. Methods: At present, the research for alternative agents for the complex treatment of chronic endometritis are conducted. One of the promising options is the introduction of peripheral blood mononuclear cell (PBMC) secretome containing a variety of biologically active substances, including proteins, extracellular vesicles, antimicrobial peptides, nucleotides, lipids, interleukins and cytokines necessary for endometrial receptivity enhancement in patients with chronic endometritis and infertility. A total of 54 women with long-term infertility were enrolled and divided into two groups: the main group receiving PBMC secretome and a comparison group undergoing standard antibacterial therapy. The PBMC secretome was prepared from cultured PBMCs, and its biological activity was assessed. Results: In our study, the pregnancy rate in patients with long-term infertility after PBMC secretome administration was significantly higher when compared to women after antibacterial therapy. Conclusions: The application of PBMC secretome appears to enhance reproductive outcomes in women with RIF due to CE. These findings support the potential of PBMC secretome as a novel therapeutic option for improving endometrial health and warrant further investigation in larger clinical trials. (www.actabiomedica.it)

Introduction

Recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) represent a detrimental reason for multiple medical interventions in women desiring to conceive, especially when the etiology of infertility remains unclear. According to ESHRE (European Society of Human Reproduction and Embryology) implantation failure is believed being one of the major unresolved issues in reproductive medicine. It has been established that endometrial dysfunction and reduced endometrial receptivity, caused by inflammatory or immunological processes, are associated with both RIF and RPL.

The prevalence of chronic endometritis (CE) in women with RIF reaches 67.5%, in women with RPL – up to 67.6%. It is unclear whether these differences in prevalence are due to variations in the studied populations, the prevalence of different pathogens or methods of diagnostic evaluation and threshold values. Antibacterial or antiviral agents are used in the treatment of CE, but they are not always effective (1). Currently, the ongoing research are looking for the alternative agents that could be incorporated alongside antibacterial treatment in chronic endometritis management, as well as in the treatment of recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL). Medenica S et al report that novel cell and gene therapies, particularly mesenchymal stem cells, show great promise for the treatment of infertility and autoimmune diseases such as thyroid autoimmunity (2). Another approach involves using the secretome of peripheral blood mononuclear cells (PBMCs), which contain a variety of biomolecules including proteins, extracellular vesicles, antimicrobial peptides, nucleotides, lipids, interleukins, and cytokines necessary for improving endometrial receptivity in CE and infertility. In 2021, Gugerell et al. Reported on the use of secretome from stressed peripheral blood mononuclear cell secretome in patients with diabetic foot ulcers. The mixed extracellular secretome of peripheral blood mononuclear cells (APOSEC) and hydrogel was applied locally three times a week for 4 weeks. APOSEC is predominantly effective in tissue damage caused by hypoxia, modulating the immune system and enhancing angiogenesis, thereby positively affecting tissues through its antimicrobial and neuroregenerative abilities. This preparation also contains important cytokines: interleukin (IL)-8 (0–5214 pg/mL), epidermal growth factor (EGF; 25–226 pg/mL), and transforming growth factor β (TGF-β; 2575–21732 pg/mL) (3). The mentioned preparation turned out to be the closest analog to the medication we currently use. The secretome used in our research is of xenogeneic origin: from whole alogeneic blood. This pharmaceutical substance has an international non-patented name “Protein-peptide complex from blood leukocytes”, registered in the drug registry “PN002448/01-2003” since 2010 (4). The difference in obtaining PBMC secretome lies in the means of mononuclear cell (MNC) activation and origin. For the production of APOSEC, allogeneic whole human blood is used. Centrifugation is performed using LSM 1077 (medium for lymphocyte separation, Lonza, Switzerland). LSM is removed in two stages by washing with Dulbecco’s phosphatebuffered saline (Lonza, Switzerland). MNCs are suspended in CellGro GMP DC medium. The leukocyte concentration is adjusted to 25 × 10^6 cells/ml. Then irradiation with a dose of 60 Gy is performed, which induces MNC apoptosis. Cultivating apoptotic MNCs in CellGro GMP DC medium results in the secretome release. After 24 ± 2 hours of incubation, cells are removed by centrifugation, and the supernatant containing the secretome is sterile-filtered using a 0.22 μm pore size filter (5). The protein-peptide complex from alogeneic blood leukocytes is obtained as follows: peripheral blood is mixed in a 1:5 ratio with a 10% gelatin solution. The blood is allowed to settle for 30 minutes at a room temperature, followed by 30 minutes in a 37°C thermostat. Cells are washed twice with 0.9% NaCl solution, centrifuged for 10 minutes at 200g. The isolated cells are counted using a Goryaev chamber. The cell concentration is adjusted to 25×106 cells/ml in medium-199 containing gentamicin (40 μg/ml). To stimulate leukocytes, PHA (Phytohemagglutinin) is used at a dose of 10 – 15 μg/ml of cell suspension. Leukocyte stimulation is carried out for 3 hours at 37°C. After 3 hours, the stimulator is removed, and the cells are washed three times in 10-fold volume of 0.9% NaCl solution, followed by centrifugation (10 minutes at 200 g). The cells are then placed in clean flasks with fresh culture medium at a concentration of 25×106 cells/ml, cultivated at 37°C for 20 – 24 hours. After cultivation, the supernatant is separated from cells by centrifugation for 15 minutes at 400 g. Fractionation of the concentrated supernatants is carried out on a chromatographic column 2.5×100 cm in size (“Whatman”, UK) filled with Sephadex G-100, equilibrated with distilled water. Elution rate – 30 ml/h, fraction collection time – 10 minutes. Elution is done in an upward flow of water. The polypeptide fraction in the area of molecular masses less than or equal to 40 kDa is collected, lyophilized. The preparation is then diluted in distilled water (100 μg/ml), subjected to biological filtration (Millipore-QS filters, USA, 0.22 μm pore size), protein concentration is determined using the Lowry method, and biological activity is determined in assays such as macrophage migration inhibition test and macrophage chemiluminescence, and other tests of biological activity. Antiviral treatment of the final product is carried out (6). Thus, the present study aimed to evaluation of the clinical efficacy of peripheral blood mononuclear cell xenogeneic secretome application in patients with repeated implantation failure associated with uterine factor of infertility.

Patients and Methods

General study design

The study was approved by the ethics committee of “The Research Institute of Obstetrics, Gynecology and Reproductive medicine named after D. O. Ott” (protocol code 129 dated as of October 10, 2023) and performed at the Department of Assisted Reproductive Technologies. The recruitment period of participants was from February 2022 to May 2024. All participants gave informed written consent for participation. A prospective analysis of the peripheral blood mononuclear cell xenogeneic secretome clinical efficacy along with antibacterial therapy (Moxifloxacin) in patients with repeated implantation failure associated with uterine factor of infertility was carried out. The study included 54 women aged 29-42 y.o. Inclusion criteria were the following: history of long-term infertility (≥5 years) of uterine origin, multiple (3 or more) implantation failures in IVF cycles and/or 2 or more miscarriages, chronic endometritis diagnosed upon hysteroscopic examination, detection of microorganisms in the endometrium by Pipelle sampling, cryopreserved high-quality embryos, presence of a permanent sexual partner, absence of other significant somatic pathology affecting fertility. Exclusion criteria: couples with male or any other factor of infertility, thyroid pathology, hereditary or acquired thrombophilia, established deficiency of protein C, protein S and/or antithrombin, the presence of more than one risk factor (for women ≤ 35 years old) or one risk factor (for women > 35 years old) for the development of venous or arterial vascular thrombosis, diabetes mellitus, heart valve disease and atrial fibrillation, dyslipoproteinemia, family history of thrombosis, smoking, controlled arterial hypertension, BMI > 30 kg/m2, history of malignant tumors, history of any thrombotic event, systemic autoimmune diseases, psychiatric disorders, alcohol and/or drug abuse, HIV-positivity regardless of HAART, history of viral hepatitis B or C, low patient compliance, withdrawal of informed written consent.

Patient selection, examination, randomization,
and therapy assignment

At the pre-screening stage, patients were selected based on their medical history and previous examination data. During the screening stage, after obtaining the signed informed consent, patients were investigated for hereditary thrombophilia (F2 G20210A, F5 G1691A), markers of antiphospholipid syndrome (total antibodies to annexin V, β2-glycoprotein, cardiolipin) and homocysteine levels. Patients negative for hereditary and/or acquired thrombophilia underwent hysteroscopy with subsequent endometrial investigation for microbial and viral agents.

Patient randomization was carried out using a random number generator. As a result, patients were divided into two groups. The first group (main group) included patients who received antibacterial or antiviral therapy followed by intrauterine infusions of peripheral blood mononuclear cell secretome for 10 days. The second group (comparison group) consisted of patients receiving antibacterial and/or antiviral therapy. The volume of examination during patients’ selection is presented in Table 1.

Figure 1 contains the number of patients included in the study. Initially 111 infertile women were recruited. 28 women were excluded from the study: 9 couples suffered of male infertility, 3 women were diagnosed with thrombophilia, 4 patients had chromosomal aberrations, 4 patients had significant somatic conditions and gynecological pathology meeting the exclusion criteria was revealed in 6 women. Low patient compliance was noted in 2 women.

Thus, 83 women underwent hysteroscopy and uterine cavity examination, resulting in no signs of chronic endometritis in 21 women and lack of intrauterine microorganisms in another 8 patients. In total, 54 women meeting the inclusion criteria were recruited. According to randomization, 30 patients were assigned to the main group, while 24 patients comprised the comparison group.

Ознакомиться с материалом можно по ссылке: К.В. Обедкова, Ю. Р. Рыжов К. В. Сторожева, Н.И. Тапильская с соавт. «Клиническая эффективность применения секретома мононуклеаров периферической крови у пациентов с повторной неудачей имплантации» ж-л Acta Biomed 2024; Vol. 95, N. 5.