RU2809667C1 - Method of treating patients with infertility caused by fibrosclerotica or hypoplastic transformation of tendometria with history of repeated implantation failures in art cycles - Google Patents

Abstract

FIELD: medicine; reproductology; IVF methods.

SUBSTANCE: invention can be used to treat patients with infertility caused by fibrosclerotic or hypoplastic transformation of the endometrium with a history of repeated implantation failures in ART cycles. Against the background of hormonal therapy, on the 5th–6th day of the menstrual cycle, a complex transplantation of a cellular product is performed based on autologous bone marrow mesenchymal stem cells (BMSCs) obtained from an iliac crest biopsy. A 100 ml volume of cell product containing BM MSCs immobilized in a gelatin-based transport solution is administered intravenously over 30–40 minutes at the rate of 1 million cells per kg of the patient’s body weight in 100 ml of solution. After this, intrauterine injection of the cell product of 1 ml volume containing 10 million BM MSCs in a similar solution is performed under ultrasound control by irrigation.

EFFECT: method ensures increased effectiveness of treatment, promotes positive dynamics of endometrial growth and its structure, and improves the results of clinical pregnancy in patients.

1 cl, 2 ex, 5 tbl

Description

The invention relates to the field of medicine, namely to reproductology and IVF methods, and can be used to treat patients with infertility caused by fibrosclerotic or hypoplastic transformation of the endometrium with a history of repeated implantation failures in ART cycles.

Currently, reproductive specialists around the world are paying great attention to the problem of “thin endometrium” associated with hypoplastic endometrium. The prevalence of “thin endometrial” syndrome in ART protocols is at least 2.4% (1). Thin endometrium refers to an echographically determined endometrial thickness of less than 7 mm. A decrease in endometrial thickness to less than 7 mm reduces the implantation rate by 2 times (2). Regardless of the nature of the damaging factor, the endometrial reaction is universal; alteration of the structural components of the endometrium is accompanied by stromal fibrosis, a deficiency of specialized cells and the development of functional endometrial failure. Currently existing therapeutic approaches, including complex surgical and conservative, combined hormonal methods, often do not provide complete restoration of the functional activity of the endometrium. For such patients, the only way to overcome infertility is the surrogacy program, which, although it allows solving the problem that has arisen in some patients, can hardly be considered as a full-fledged treatment approach in the presence of uterine factor infertility, since it does not restore the lost function of the damaged organ.

Mesenchymal stromal (stem) cells (MSCs) are one of the most promising objects in regenerative medicine and cell therapy of human diseases. It has been established that MSC transplantation stimulates tissue regeneration, including bone tissue, skin, myocardium, peripheral nervous system, skeletal muscle, liver tissue, and also serve as a source of growth factors and cytokines (3).

Studies of stem cell transplantation from bone marrow and adipose tissue in experimental models of endometrial injury showed that the injected cells migrated to the damaged uterus compared to the lack of effect in the placebo group, regardless of the route of administration of the cell product (intrauterine or tail vein) [4, 5]. The concept of the effectiveness of intravenous administration of stem cells is based on the homing effect using the example of a model of introducing cardiomyocyte progenitor cells to improve blood supply and myocardial contractility in patients with coronary heart disease. The homing effect is based on increased migration of MSCs and progenitor cells differentiated towards cardiomyocytes instead of the damaged myocardium. The intravenous (systemic) route of administration is the simplest and least invasive method, which does not require surgical and/or endoscopic interventions.

Positive preliminary results of clinical and experimental studies conducted in various countries substantiate the relevance, prospects and sufficient safety of the method of transplantation of autologous MSCs in the treatment of patients with thin endometrial syndrome with repeated implantation failures in assisted reproductive technology (ART) programs.

There is a known method of transplanting a cellular product based on MSCs of bone marrow (BM) origin by introducing it into the uterine arteries using an approach similar to that used for embolization of the uterine arteries. The use of this approach was tested in 16 patients with Asherman's syndrome. In 15 of them, as a result of the treatment, the menstrual cycle was restored. 7 patients became pregnant. Three spontaneously, four after cryoprotocols.

The disadvantages of the described method include the need to use angiographic equipment, complex access with puncture of the uterine arteries, which prevents the wide reproducibility of this method and is an expensive technique (6).

There is a known method of introducing stem cells, described by Tan et al (7). It is based on the isolation of stem cells from menstrual blood, their cultivation and subsequent transfer of the cellular product into the uterine cavity. The method was tested in 7 patients with Asherman's syndrome. After the isolation of stem cells and cultivation, the suspension with the resulting cell product was introduced into the uterine cavity of the patients after scratching, and then the thickness of the endometrium was monitored. According to the results, out of 7 patients, 1 had a spontaneous pregnancy, and 2 after the transfer of cryopreserved embryos. It is reported that in one case there was a biochemical pregnancy, in the second a tubal pregnancy, spontaneous pregnancy was developing at the time of publication of the article.

The disadvantages of the described method include its low efficiency and testing on a small number of patients.

The technical result of the invention is to increase the efficiency of the method: positive dynamics of endometrial growth and its structure, increasing the results of clinical pregnancy in patients.

The specified technical result is achieved in a method for treating patients with infertility caused by fibrosclerotic or hypoplastic transformation of the endometrium with repeated failures of implantation in ART cycles in history by transplantation into the uterine cavity of a cellular product based on autologous mesenchymal stem cells (MSCs) against the background of hormonal therapy, which differs the fact that transplantation of a cellular product based on bone marrow MSCs (BM MSCs), obtained from iliac crest biopsy, is carried out comprehensively on the 5-6th day of the menstrual cycle, intravenously for 30-40 minutes. a cellular product with a volume of 100 ml is introduced containing BM MSCs immobilized in a transport solution based on gelatin at the rate of 1 million cells per kg of the patient’s body weight in 100 ml of solution, after which intrauterine administration of the cellular product is carried out under ultrasound control by irrigation, in a volume of 1 ml, containing 10 million BM MSCs in a similar solution.

The study on which the invention is based included three phases and was carried out in accordance with the principles of the World Association of Helsinki Declaration of Ethical Principles for Scientific and Medical Research Involving Human Subjects, current Practices and Standards of Care and other applicable regulatory requirements for clinical research and observational programs in the Russian Federation. The patient observation protocol and examination program were approved by the local ethics committee.

At the preparatory stage, the cell product was prepared. A bone marrow sample was obtained by a transfusiologist using a standard technique during biopsy of the iliac crest under local anesthesia.

The criteria for inclusion in the study at the preparatory stage were:

- Age from 20 to 45 years inclusive at the time of inclusion in the study;

- Patients with repeated unsuccessful IVF attempts and cryoprotocols in the presence of high-quality embryos;

- The thickness of the endometrium according to ultrasound data during the use of hormone replacement therapy in the preparation protocol for embryo transfer is no more than 6 mm in at least two attempts;

- The presence of a fibrosclerotic process in the uterine cavity according to hysteroscopic examination and the absence of positive dynamics as a result of replacement therapy for 3 months after surgical treatment;

- Absence of infertility or impaired fertility of any other origin.

Exclusion criteria were:

- The presence of infertility or impaired fertility of another etiology, including tubo-peritoneal infertility, anovulatory infertility, male infertility, including subfertile spermogram results;

- Contraindications to pregnancy;

- Lack of high quality cryopreserved embryos stored in the OVRT cryobank;

- Diabetes mellitus and other carbohydrate metabolism disorders;

- Uncompensated dysfunction of the thyroid gland and other endocrine glands (pituitary gland, adrenal glands, parathyroid glands, ovaries), including those requiring hormone replacement therapy;

- Psychiatric diseases, alcoholism, drug addiction;

- Malignant neoplasms, including intraepithelial dysplasia of any location, and/or lymphoproliferative diseases in history or identified during the study;

- Broca body mass index more than 29.9 kg/m2, less than 19.0 kg/m2;

- HIV-positive patients or patients with a high risk of infection, receiving prophylactic antiretroviral therapy, viral hepatitis B, C, including a history of infection;

- Anamnestic data on thromboembolic disorders and/or the presence of proven hereditary thrombophilia;

- The presence of myomatous nodes with a diameter of more than 4 cm or 2 or more myomatous nodes with a diameter of more than 2.5 cm;

- The presence of a nodular form of adenomyosis;

- Presence of ovarian cysts with a diameter of more than 4 cm;

- Reluctance of the patient to participate in the study and/or low compliance.

The patient's examination included routine serological and PNR testing for the presence of pathogens of infectious diseases: HCV, HBV, HIV 1/2, syphilis, CMV, Epstein-Barr virus, toxoplasmosis. The CM sample was placed in a sterile transport package and labeled. Then sowing, expansion, cryopreservation, and culture reinitiation were carried out. These steps were carried out in accordance with the standard operating procedures of Trans-Technologies LLC (St. Petersburg).

Expansion of BM MSCs (cultivation). Cells were incubated at 37°C in an atmosphere of 5% CO2 in an incubator. The culture medium was changed 100% every 4 days. Daily visual monitoring of cell growth was performed. Upon reaching 80% confluency, cells were subcultured (subculture density 10,000 cells/cm2).

Cryopreservation. After the first passage, MSCs were resuspended in culture medium at a concentration of 2×10(6)/ml. The cell suspension was packaged into cryovials in an amount of 10 million cells/vial (2 ml). The suspension was mixed with a pre-cooled cryoprotective mixture consisting of culture medium (80% by volume) and dimethyl sulfoxide (20% by volume). Cooled in a program freezer at a temperature reduction rate of 1°C/min to -80°C. After this, the cryovials were placed in a freezer at -80°C. If long-term storage was necessary, they were placed in liquid nitrogen.

Reinitiation of MSC CM culture. Cryovials were thawed in water preheated to 37°C. Cells were resuspended in culture medium and placed in a CO2 incubator. Daily visual monitoring of cell growth was performed. When 80% confluency was reached, cells were collected.

Preparation of the final MSC product. The resulting cells were washed 3 times with physiological solution by centrifugation and removal of the supernatant. Samples were taken for quality control. MSCs at a concentration of 10 million/ml were aseptically packaged in 1.8 ml vials. Transported in transport containers to the clinic for subsequent administration to the patient.

Before further use, the final product was evaluated according to the following criteria: 1. Technical characteristics Expression of surface markers (passage 2 cells):

- CD90+>90%;

- CD44+>90%;

- CD105+>90%;

- CD73+>90%.

Presence of contaminating hematopoietic cells (passage 2 cells):

- CD45+<1%;

- CD34+<0.5%;

Microbial contamination assessment:

- There is no growth of bacterial colonies when inoculated on standard media;

- Mycoplasma DNA: not detected Number of cells: 10 million;

- Cell viability (according to 7ADD): <10% of non-viable cells;

2. Characteristics of impurities:

In the final product of BM MSCs, the presence of traces of culture medium and other reagents used in the production could not be excluded. The minimum dilution factor for auxiliary reagents was 8×106.

3. Stability, storage and transportation conditions:

The final product (MSC CM in storage solution) is stable for up to 6 hours from the moment of release and should be stored in a dark place at a temperature of 18°-22°.

At the next stage, the procedure of transplantation of BM MSCs into patients was carried out. At the same time, the patients received standard therapy with estrogen and gestagen drugs that modulate hormonal changes occurring in the natural menstrual cycle (estradnol valerate 4 mg/day from the 2nd to 25th day of the cycle, dydrogesterone 20 mg/day from the 15th to 25th day of the cycle). Treatment continued for 3 cycles. On the 3rd cycle of hormonal therapy, on the 20th day, a pipell biopsy of the endometrium was performed, followed by histological and immunohistochemical studies to determine the expression levels of estrogen and progesterone receptors. On days 9-10 and 19-21 of each cycle, an ultrasound of the pelvic organs was performed to determine the condition of the endometrium. Upon receipt of morphological and echographic indicators of the endometrium acceptable for preparation of the endometrium in the protocol for transfer of thawed embryos, the patient was included in the protocol for preparation of the endometrium for transfer of the embryo into the uterine cavity. In the absence of indicators that meet the eligibility criteria for the transfer of thawed embryos, the patient was offered a repeated procedure for using the cell product (no more than 3 times) - with an interval of 3 months.

The transplantation procedure was carried out in a clean dressing clinic on days 5-6 of the menstrual cycle. After treating the external genitalia with speculum under aseptic conditions, a conductor from the embryo transfer catheter was inserted into the patient’s cervical canal to the area of the internal os. Then, a catheter with a pre-drawn suspension of 1 ml containing 10 million autologous BM-MSCs in a gelatin-based transport solution was inserted through a guide into the uterine cavity under ultrasound control, after which the suspension was injected into the uterine cavity with a syringe.

The group of patients at the intermediate stage of the study included 30 patients who underwent intrauterine injection of a cell product based on autologous BM MSCs.

At the final stage of the study, the patients received intrauterine and intravenous administration of a cellular product based on autologous mesenchymal bone marrow cells. The cell product containing BM MSCs immobilized in a gelatin-based transport solution was administered intravenously by drip for 30-40 minutes. at the rate of 1 million cells per kg of the patient’s body weight in 100 ml of solution, intrauterine administration of the cell product was carried out according to the method described above immediately after the end of intravenous administration.

The group of patients at the final stage of the study included: 32 patients who underwent intrauterine and intravenous administration of a cell product based on autologous mesenchymal bone marrow cells. Patients who, as a result of intrauterine insertion, were unable to achieve an M-echo of more than 6 mm in the second phase of the menstrual cycle were transferred to this group.

The average age of women was 37.3±0.5 years. 28% of patients had a history of primary infertility, 50% had a history of miscarriage, and in 31% of patients the pregnancy ended in artificial abortion. There was a history of chronic salpingoophoritis in 31%, chronic endometritis in 87.5% of patients. At the time of contacting the ART department, each patient had a history of several intrauterine interventions that were performed for diagnostic or therapeutic purposes; 24 patients had curettage of the uterine cavity. The duration of infertility of the patients included in the study at the time of contacting the clinic averaged 7.02±0.5 years. At the time of inclusion in the study, each patient already had experience in infertility treatment and, more often, using several methods.

All patients underwent a morphological examination of the endometrium before starting the protocol. According to histological examination, all women with repeated implantation failures included in the study were diagnosed with hypoplastic changes in the endometrium; 81% of women had a combination of hypoplastic changes and chronic endometritis. In all examined patients, there was a discrepancy between the morphological characteristics of the endometrium and the day of the menstrual cycle. Fibroplastic changes in the endometrial stroma in women of the study group manifested themselves in the form of both focal and diffuse stromal fibrosis (Table 1).

In addition to the histological analysis of the endometrium, the subpopulation composition of endometrial mononuclear cells was determined in all patients using the immunohistochemical method (Table 2).

Based on the combined data of histological and immunohistochemical studies, chronic endometritis was diagnosed in 68.8% of women with repeated implantation failures in ART protocols included in the study.

In addition, as part of the immunohistochemical study, an analysis of the receptor status was carried out, taking into account the correspondence of the studied endometrial biopsies to various stages of the secretion phase: early stage (n = 30 for women of the study group and the middle stage of the secretion phase. To conduct a comparative analysis of the receptor status of the endometrium, data from an immunohistochemical study of patients with repeated failures in IVF programs were compared with similar indicators of healthy women with male factor infertility (Table 3).

The vast majority of patients of the main group in IVF programs showed a decrease in the expression of PgR in the glands and stroma in the early stage of secretion (p <0.001) and a sharp (<100 points on HS) decrease in PgR in the stromal component in the middle stage of the secretion phase (p <0.001 )(Table 4).

When comparing the expression of ER and PgR in the glandular and stromal components of the endometrium before and after treatment, no statistically significant differences were found. During the treatment, no adverse events related to bone marrow collection or intrauterine administration of the cell product were recorded.

During the clinical study, it was found that as a result of treatment, in 8 (25%) there was not only an increase in thickness, but also a change in the structure of the endometrium (Table 5). Ultrasound visualization of a three-layer endometrium in the corresponding phase of the menstrual cycle in patients of the study group gives reason to believe that with the help of a new treatment method it is possible to create the conditions necessary for the transfer of thawed embryos.

11 patients in the study group experienced clinical pregnancy (34%), including: 2 ectopic pregnancies, 1 anembryony, 1 non-developing pregnancy, 3 progressive pregnancies, 4 live births at term. At the same time, in 1 patient, after giving birth at term, another pregnancy occurred, which ended in delivery at term. 4 pregnancies occurred after ART protocols, 7 pregnancies occurred independently in a natural cycle. The average time until pregnancy is 9 months.

The method is confirmed by the following clinical examples.

Example 1. Patient D, born in 1981, applied to the ART department in 2019 with complaints of infertility of uterine origin. From the anamnesis, recurrent miscarriage, removal of both fallopian tubes during laparoscopy for ectopic pregnancies, repeated hysteroscopy to remove intrauterine synechiae. From 2017 to 2019, 3 unsuccessful IVF programs and 4 cryopreserved embryo transfers were carried out. In 2020, an IVF program was carried out with cryopreservation of embryos and refusal to transfer embryos due to insufficient endometrial thickness - 4 mm. During the preparation of the endometrium there was no further effect on stimulation with estrogen preparations. The thickness of the endometrium did not exceed 4 mm, regardless of the duration and dose of estrogen preparations used.

According to the described protocol, a biopsy of the iliac crest was performed in June 2020 with the preparation of a cell product. In July 2020, the first procedure for introducing a cellular product into the uterine cavity was performed. After 3 months, during which hormonal therapy was carried out, in accordance with the presented protocol, an endometrial biopsy was performed. According to ultrasound data, the thickness of the endometrium during the entire treatment period did not exceed 5 mm; according to histological and IHC studies, there was also no improvement in morphological parameters. It was decided to refrain from transferring cryopreserved embryos.

In October 2020, the cell product was reintroduced using a combined method: on the 5th day of the menstrual cycle, a cell product containing BM MSCs immobilized in a gelatin-based transport solution was injected intravenously for 30-40 minutes. at the rate of 1 million cells per kg of the patient’s body weight in 100 ml of solution, and then intrauterine administration of the cell product under ultrasound control by irrigation, 1 ml volume containing 10 million BM MSCs in a similar solution.

In parallel, cyclic hormonal therapy was continued. According to the results of ultrasound, it was possible to achieve an endometrial thickness of 8 mm. A cryopreserved embryo was transferred, resulting in a pregnancy that progressed without complications and resulted in the birth of a live, full-term fetus 3450/52? With an Apgar score of 8 points.

Example 2. Patient R, born in 1991, complained of secondary infertility of uterine origin. There was a history of non-developing early pregnancy, curettage of the uterine cavity, after which scanty menstruation was noted; repeated curettage of the uterine cavity and cervical canal was performed, and in the same year hysteroresectoscopy with dissection of synechiae in the uterine cavity. In 2019, an IVF protocol was carried out with cryopreservation of all embryos; embryo transfer was not carried out due to the small thickness of the endometrium (5 mm). Next, an attempt was made to achieve acceptable endometrial thickness by conducting cyclic hormonal therapy with estrogen and gestagen preparations. There was no effect from the therapy.

In 2019, a biopsy of the iliac crest was performed; MSCs were obtained, according to the presented protocol; after preparing the cell product, it was administered intrauterinely. The subsequent use of cyclic hormonal therapy for six months did not produce a clinical effect, which was confirmed by a morphological study of an endometrial biopsy specimen at the 3rd month of therapy.

In 2020, a combined re-introduction of the cell product was carried out (intravenous and intrauterine): on the 6th day of the menstrual cycle, a cell product containing BM MSCs immobilized in a gelatin-based transport solution was injected intravenously for 30-40 minutes. at the rate of 1 million cells per kg of the patient’s body weight in 100 ml of solution, and then - intrauterine administration of a cell product under ultrasound control by irrigation, a volume of 1 ml containing 10 million BM MSCs in a similar solution, after which hormonal therapy was continued. After 6 months of therapy, hormonal therapy was discontinued and the thickness of the endometrium in the spontaneous cycle reached 7 mm. The patient was preparing for a cryoprotocol, but after 1 month a spontaneous pregnancy occurred, which proceeded normally and resulted in the birth of a healthy fetus at 39 weeks 3300/51-8 points on the Apgar scale

Thus, the proposed method of treating patients with repeated failures in ART programs caused by hypoplastic and/or fibrosclerotic changes in the endometrium is safe and effective. The effectiveness of this technique is confirmed by the positive dynamics of endometrial growth in 25% and the onset of clinical pregnancy in 34% of patients.

The inventive method increases the effectiveness of treatment, promotes the positive dynamics of endometrial growth and its structure, and improves the results of clinical pregnancy in patients.

Information sources:

1. Kasius A, Smit JG, Torrance HL, Eijkemans MJ, Mol BW, Opmeer VS, et al. Endometrial thickness and pregnancy rates after IVF: A systematic review and meta-analysis. Hum Reprod Update. 2014;20:530-41.

2. Mahajan N, Sharma S. The endometrium in assisted reproductive technology: How thin is thin? J Hum Reprod Sci. 2016 Jan-Mar;9(1):3-8. doi: 10.4103/0974-1208.178632. PMID: 27110071; PMCID: PMC4817285.

3. Askari A.T., Unzek S., Popovic Z.B., Goldman C.K., Forudi F., Kie-drowski M., Rovner A., Ellis S.G., Thomas J.D., DiCorleto P.E., Topol E.J., Perm M.S. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischemic cardiomyopathy // The Lancet. 2003 T. 362 No9385. - pp. 697-703.

4. Alawadhi F, Du H, Cakmak H, Taylor HS. Bone Marrow-Derived Stem Cell (BMDSC) transplantation improves fertility in a murine model of Asherman's syndrome. PLoS One 2014;9:e96662.

5. Cervello'I, Gil-Sanchis C, Santamar'ia X. Human CD133(p) bone marrow-derived stem cells promote endometrial proliferation in a murine model of Asherman syndrome. Fertil Steril. 2015; 104(6): 1552-1560.e3

6. Santamaria X, Cabanillas S, Cervelló I, Arbona C, Raga F, Ferro J, Palmero J, Remohl J, Pellicer A, Simon C. Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman's syndrome and endometrial atrophy : a pilot cohort study. Hum Reprod. 2016 May;31(5): 1087-96. doi: 10.1093/humrep/dew042. Epub 2016 Mar 22. PMID: 27005892.

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Claims (1)

A method of treating patients with infertility caused by fibrosclerotic or hypoplastic transformation of the endometrium with repeated implantation failures in ART cycles in history by transplantation into the uterine cavity of a cellular product based on autologous mesenchymal stem cells (MSCs) against the background of hormonal therapy, characterized in that the transplantation of cellular product based on bone marrow MSCs (BM MSCs), obtained from iliac crest biopsy, is carried out comprehensively on days 5-6 of the menstrual cycle, while a 100 ml volume of cell product containing BM MSCs immobilized in the transport solution based on gelatin at the rate of 1 million cells per kg of the patient’s body weight in 100 ml of solution, after which intrauterine administration of the cell product is carried out under ultrasound control by irrigation, a volume of 1 ml containing 10 million BM MSCs in a similar solution.