RU2515475C1 - Method for stimulation of endometrial decidua formation experimentally - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: suspension containing 300 thousand endometrial stem cells recovered from woman's menstrual blood is introduced experimentally into a pseudopregnant rat's uterine wall. That is followed by subcutaneous administration of progesterone 2 mg per gram of animal's weight.

EFFECT: method provides stimulating the endometrial decidua formation thereby modelling a control of a decidual reaction by stem cell transplantation.

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Description

The invention relates to the field of experimental medicine, namely to the creation of new methods for the treatment of miscarriage and other diseases of the uterus associated with endometrial failure, with a view to their subsequent clinical use.

The relentless interest in the problem of miscarriage is determined by the relative constancy of the frequency of this pathology (10-30%) in almost all countries of the world (A.V. Mironov et al., 2012). Reproductive losses, which are an irreplaceable loss of the life potential of a population, are fundamentally different from the mortality of other age groups, since unborn children and dead babies are an irrevocable loss of human capital. The total reproductive losses for the entire post-Soviet period (1991-2010) amounted to 5515667 fetuses (pregnancies) in Russia as a whole, which was 28.1656559 born during these years to 19.1 per 100 live births. In the structure of total reproductive losses, feto-infantile losses amounted to 10.9% (dead children of the first year of life 7.4%, stillbirths - 3.5%), prenatal losses before 28 weeks of gestation - 89.1% (spontaneous abortions - 65.3% , abortions for medical reasons - 15.4%, for social reasons - 8.4%) (Starodubov V.I. et al., 2012).

Implantation and the normal development of pregnancy depends on the functional usefulness of the endometrium. The morphological basis of spontaneous abortions and non-developing pregnancies is a combination of morphological and functional signs of chronic productive inflammation (microabscess in the uteroplacental region, decidual cell necrosis in the centers of abscesses and in the area of inflammatory ridges) in combination with local signs of hemostasis in the parietal endometrium and d.basalis with stratified and thrombosis before the formation of a retroplacental hematoma, despite adequate decidualization of the endometrium and sufficient gestation restructuring of the spiral arteries. The determination of pathological changes in the body's immunoreactivity, cytokine status, platelet and plasma hemostasis, AMHF (alpha-2-microglobulin fertility) in blood serum are prognostic indicators of structural and functional changes in the endometrium during miscarriage. In order to restore structural and functional disorders of the endometrium in women with miscarriage, a set of measures has been developed, including: immunotherapy, hyposensitizing therapy, improved microcirculation and trophism, restoration of the biphasic menstrual cycle, physiotherapeutic treatment and pregnancy planning.

Physiotherapeutic effects are carried out in various ways, for example, by intravenous administration of an ozonized physiological solution for 5 days (RU 208624, Torchinov AM et al., 2009), introduction of an acid fraction of electrochemically active water from pH 3.3-4.1 to coagulation of the contents of the uterine cavity (RU 2138270), acupuncture at certain points using microneedles (RU 213267), vibration and electric current (RU 92007560), using infrared radiation of minimum power (RU 2112444) or electric bipolar and the impulse of abdominally, vaginally (RU 2223798). To improve the condition of the endometrium, it is proposed to act with an abdominal and intravaginal traveling alternating magnetic field (RU 2302885). Microneedles can be administered while taking Wobenzym (RU 2159126). For the treatment of antiphospholipid syndrome with habitual miscarriage, it is proposed to carry out percutaneous low-intensity laser irradiation of blood in the infrared region of the spectrum before conception in combination with a constant magnetic field of projection of the subclavian, ulnar, femoral and popliteal arteries (RU 2188680). However, these methods of treating pregnancy loss with self-administration are of low effectiveness and therefore are used as an addition to the main drug treatment in order to reduce the dose of the drug, thereby reducing the harmful side effects of the drugs. For the same purpose, it is proposed to use homeopathic medicines (RU 2321403) and medicines that affect immunity (RU 2232028, RU 2452481, RU 2007126885, RU 2283653). A known method of preventing spontaneous or habitual miscarriage, based on the introduction of a woman one or more immunologically active exogenous gestogenic drugs, starting from the moment of ovulation. The drug (exogenous gestogen), a human progesterone receptor agonist, is selected from the following: desogestrel; dimethisterone; dydrogesterone; ethynodiol; ethynodiol diacetate; etono-gestrel; gestodene; hydroxyprogesterone; hydroxyprogesterone caproate; keto-desogestrel; lynestrenol; medrogestone; medroxyprogesterone; medroxyprogesterone acetate; megestrol; megestrol acetate; nestorone; nomegestrol; nomegestrol acetate; norethynodrel; norgestimate; norgestrienone; promegestone; quingestanol; quingestanol acetate and trimegestone, dydrogesterone. Treatment begins with a diagnostic step, when MCPCs (mononuclear cells) are isolated from a peripheral blood sample, they are cultured with protein extract of trophoblast, the concentrations of Th1 and Th2 cytokines in the supernatant of the MCCM culture are analyzed, and the Th1 / Th2 ratio for each patient is calculated. Depending on the magnitude of this ratio, one or another drug is prescribed. Oral administration is recommended, but intravaginal and transdermal administration is possible. Doses: 2.5-100 mg / day, usually 5-60 mg / day, depending on the condition, individual sensitivity, etc.

The use of antibodies is suggested for the treatment of infertility and the prevention of miscarriage caused by brucellosis (JP 2008222590). A method for reducing the risk of miscarriage is proposed, based on the use of drugs that inhibit the expression of tissue factor (TF, for example statins) and / or the activity of neutrophils and / or monocytes (e.g. statins), as well as diagnostic methods for individuals with an increased risk of miscarriage (US 20090312411). Therapy begins before pregnancy (for example, 1 month before the planned pregnancy), or after it begins and the entire period of risk of miscarriage continues (for example, the 1st trimester), then stops, or throughout the pregnancy, before childbirth. Dosage forms: usually in the form of organic or inorganic salts. Forms of administration: injectable, oral, nasal, buccal (for simvastatin: 80 mg / day). The method has a relatively low efficiency.

Combined therapy aimed at preventing the pathology of newborns, miscarriages and infertility in women by normalizing hormones to the level of young women and therapy with aromatase inhibitors (US 20090137478). To do this, they regulate the levels of FSH, estrogen, LH, estradiol and progesterone activity, bringing them to the indicator of healthy young women and inject an aromatase enzyme inhibitor that regulates estradiol levels. The following drugs are used: gonadotropin-releasing hormone (GnRH), an agonist and / or antagonist thereof; FSH; LH and / or chorionic gonadotropin, progesterone, an aromatase enzyme inhibitor. Duration of therapy: 2-4 to 34 months. The positive effect due to the method is as follows: prevention of female infertility, normalization of maturation of follicles and oocytes, stimulation of correct segregation of chromosomes in oocytes and in early embryogenesis. The disadvantages of the method include a very high variability of the result. It is unlikely that such therapy will help to effectively reduce the risk of developing chromosomal abnormalities.

In the presence of insufficiency of the function of the corpus luteum, preference is given to chorionic gonadotropin, since after 8 weeks of pregnancy it stimulates the synthesis of endogenous progesterone by the corpus luteum and trophoblast. Additionally, there is a direct stimulating effect on the implantation process of the ovum, and the production of relaxin, a hormone necessary for the growth and relaxation of the uterus, is also enhanced. For the treatment of early spontaneous miscarriage, both symptomatic (semi / bed rest, physical and sexual rest, psychological support) therapy are widely used, as well as the appointment of sedatives, antispasmodics, vitamin therapy, hormonal drugs. Currently, estrogens are used very carefully, which are indicated only in the case of confirmed hypoestrogenia in early pregnancy, but should be completely canceled by 9-10 weeks. In order to maintain pregnancy during the first 20 weeks, it is advisable and effective to prescribe progesterones (progesterone and its synthetic analogues, progestogens). The most pathogenetically substantiated method of treating spontaneous abortion is the use of beta-adrenergic agonists (tocolytics) from the 16th week of pregnancy. Tocolytics, acting on beta-adrenergic receptors, lead to a decrease in tone and contractile activity of the myometrium. Parenteral administration of magnesium sulfate, non-steroidal anti-inflammatory drugs and non-dihydroperidine calcium channel blockers (Tetelyutina F.K. et al., 2010) is possible as a complex therapy for a threatening late spontaneous miscarriage.

For the treatment of endometrial hyperplastic processes in patients with metabolic syndrome, metformin (RU 2438662) is prescribed along with progesterone (progesterone and progestogen). In order to increase the effectiveness of the treatment of infertility caused by small forms of external genital endometriosis, synthetic androgenic drugs are used (RU 2273481).

For the prevention of preterm labor and miscarriage associated with spontaneous or bradykinin-induced (but not oxytocin- or prostoglandin F2-alpha-induced) contractions of the myometrium, preparations containing the adrenomedullin peptide (in several compositional options) are proposed. Adrenomedullin is obtained from pheochromocytoma tissue or synthesized (EP 1205185, US 20070161555). For these purposes, the drugs were not effective enough and found application mainly before cesarean section and for the treatment of dysmenorrhea. A method for stimulating placental growth and overcoming problems associated with placental development disorders by measuring the level of macrophage inhibitory cytokine-1 (MITs-1) or a similar protein in biological fluids is proposed. Then, the overall level of MITs-1 in the placental extract (i.e., endogenous and injected) is adjusted to 15-70 ng / ml, preferably to 30-50 ng / ml, and this level is maintained throughout the pregnancy. The method of administration is any: oral, nasal, injection, into the uterus. Perhaps the use of viral vectors (genetic engineering). This reduces the risk of miscarriage or premature birth (EP 1914554). However, this method is quite time-consuming and expensive, since it requires regular laboratory diagnostic tests.

To date, sufficient material has been accumulated indicating the important role of progesterone in ensuring the physiological course of pregnancy. On the basis of progesterone, effective steroid compounds have been created that have a progestagenic effect when administered orally (acetomepregenol, utrozhestan, duphaston, etc.). However, the search for highly effective and safe synthetic analogues of progesterone continues. As experimental studies have shown, the ability to support the development of pregnancy in ovariectomized rabbits provides an insufficiently high percentage of blastocyst conservation. With the introduction of isomethylene acetate, the number of stored blastocysts amounted to 47.6% of the number of promoted follicles; with the introduction of butamepregenol - 56.14%. Duphaston, widely used in obstetric practice, retained only 28% of the blastocysts of the number of follicles that promoted (Korhov V.V. et al., 2008). An analysis of the English-language publications on the use of progestogens in the treatment of miscarriage presented in the MEDLINE and Embase databases over a long period of time (1966-2011) concluded that there is currently no convincing evidence in favor of choosing progestogens to reduce cases of abortion (Likis FE et al., 2012).

A known method for restoring the endometrium, selected by us as a prototype (Nagori et al., 2011). An isolated case is described - a woman of 33 years with primary infertility (8 years married). Unsuccessful in vitro fertilization (a severe case of Asherman syndrome after curettage of the endometrium). The generally accepted method of treatment, when a special contraceptive device was placed in the uterus, which conducted cyclic hormone therapy for 6 months for the development of the endometrium, was ineffective. To restore the endometrial tissue of the uterus, stem cells were isolated from this woman’s bone marrow (i.e., autologous). After the puncture, bone marrow was aspirated in an amount of 45 ml. Stem cells were then isolated using magnetic sorting on CD9, CD44, and CD90. 39 million positive cells were obtained for these markers, which were diluted in phosphate-buffered saline (0.7 ml) with 2% autologous serum. On the 2nd day of the menstrual cycle, under the supervision of an ultrasound scan, the cannula was inserted into the uterine cavity (0.5 cm from the bottom), and the cell suspension was introduced into the uterine cavity (from the bottom to the middle) on the reverse movement of the cannula. Hormone therapy was carried out for 25 days from the day of transplantation according to the scheme: estradiol valerate, 6 mg / day from the day of transplantation for 25 days and medroxyprogesterone acetate 10 mg / day from the 20th to the 25th day. The endometrium was periodically studied on an ultrasonic device. After 4.5 months, when reaching an endometrial thickness of 8 mm and good vascularization (development of spiral vessels), in vitro fertilization and embryo transfer were performed. After fertilization, hormone therapy was performed according to the following scheme: progesterone vaginal gel (Crinone 8%) twice a day and ethinyl estradiol 6 mg / day. This led to a successful pregnancy, which was confirmed by the fetal membranes (including the decidual membrane), the yolk sac of the embryo and the germinal field with cardiac activity during ultrasound examination.

Thus, endometrial angiogenetic stem cells isolated from autologous adult bone marrow stem cells. could regenerate a damaged endometrium that does not respond to conventional treatment for Asherman's syndrome. However, this method is rather traumatic (puncture of bone tissue, which can take 2-3 hours, and is associated with the risk of complications) and time-consuming.

The aim of the invention is to reduce the morbidity and runtime of the method. This goal is achieved by the fact that in a female rat a state of pseudopregnancy was caused, then stem cells isolated from the human endometrium were implanted into the uterine wall. This made it possible to significantly simplify and reduce the morbidity of stimulation of the formation of the decidual endometrial sheath. This is due to the relative ease of obtaining endometrial tissue from menstrual blood and the small cost of isolating stem cells from endometrial tissue.

The method is as follows. Menstrual blood containing endometrial fragments was obtained from women who were preparing for in vitro fertilization at the International Center for Reproductive Medicine (St. Petersburg). The age of the patients was 30-40 years. All menstrual cycle was regular 27-30 days. Menstrual blood was taken on the 2nd day of the menstrual cycle. Blood was collected with an Ipas MVA plus aspirator with a nozzle 4 mm in diameter inserted into the cervical canal to the internal pharynx. On average, 1-2 ml was obtained from each patient. The resulting sample was transferred to phosphate-buffered saline (PBS) with sodium citrate and a 10% mixture of antibiotic and antimycotic (a mixture of penicillin, streptomycin and amphotericin). In this solution, at a temperature of 4 ° C, blood was delivered to the laboratory within 2-18 hours. A centrifuged pellet containing endometrial fragments was resuspended in PBS with the addition of a 10% mixture of antibiotic and antimycotic and incubated for 1 h at 37 ° C. Then, centrifuged endometrial-menstrual cells were transferred into 6 cm Petri dishes. To obtain an adhesive cell population, cells were cultured in DMEM / F12 medium containing 10% bovine fetal serum, 1% mixture of antibiotic and antimycotic, 1% glutamax. Cells were cultured for 3-7 days. During this time, the environment was changed several times. The resulting adhesive population of cells (4–9 million) was subcultured using a 0.05% trypsin solution and EDTA in a ratio of 1: 4 to the 5th passage 2 times in 1 week, then in a ratio of 1: 3 and frozen. For cultivation used bottles of T25 and T75. The density at reseeding was 14-15 thousand cells / cm ² .

The cultivation of endometrial cells in preparation for transplantation was performed using DMEM / F12 medium supplemented with fetal bovine serum and glutamine. On the third and fourth day after reseeding, when the cell culture reached a monolayer, the cells were removed from the substrate and transferred to suspension. A mixture of trypsin and EDTA was used to detach the cells. Since the enzymatic effect of trypsin can damage the cell membranes, which would lead to a decrease in their viability during transplantation, trypsin of low concentration (0.05%) and, possibly, a shorter period (within 5 minutes) was used. For the fast action of trypsin, after removing the growth medium, the cell monolayer was washed several times with PBS. After cell detachment, the action of trypsin was inactivated by adding a sufficient amount of growth medium with 10% serum to the suspension. To achieve homogeneity, the suspension was carefully pipetted. After the preparation of the unicellular suspension, the cells were counted in the Goryaev chamber. Further, the culture medium was replaced by PBS. For this, the cell suspension was centrifuged for 5 min at 1500 rpm, the supernatant was drained and the pellet was resuspended in PBS, bringing the number of cells to the desired concentration by dilution. Then a suspension of cells was collected in syringes in the required quantity for further transplantation. All manipulations to avoid infection of the animal were carried out in compliance with sterility.

Before surgery on an animal by the method of studying vaginal smears, an outbred white rat in the oestrus stage was selected. To induce pseudopregnancy, the vagina was irritated with a faradic electric current of 1 Hz, voltage of 10 mV for 40 seconds. After the manipulations, the animal was given rest. On the fifth day, under sterile conditions, under ether anesthesia, the animal was fixed on the operating table in the dorsal position. The surgical field was carefully shaved and treated twice with an antiseptic. A 2-3 cm incision was made along the midline of the abdomen from the pelvis up. After opening the abdominal cavity from the lateral sinuses into the surgical wound, the uterine horns were removed. Both horns (right and left) were fixed with tweezers and stitched with a thread of the 40th size in two places, near the ovary and near the vaginal end of the horn. A suspension of 300,000 cells of a human endometrial stem cell (SCE) was transplanted into the suture area near the ovary of the right horn and into the uterine wall. Further, all internal organs were laid in place and the wound was sutured in layers. After transplantation, the animal received daily subcutaneous injections of progesterone (2 mg per gram of weight). After 6 days, the rat was withdrawn from the experiment by an overdose of ether anesthesia and both uterine horns were removed for morphological examination.

The method was tested on 30 sexually mature white outbred female rats weighing 200-220 g. To confirm the stimulating effect of human endometrial stem cells on the formation of the decidual membrane (decidua) in rat uterus, a microscopic study of rat endometrial tissue was performed. The control horn was the left horn of the uterus, which was only stitched with thread in two places similar to the right horn. Slices of the formed decidua with a thickness of 15 μm were prepared on a cryostat so that the plane of the slice was perpendicular or parallel to the long axis of the uterine horns. In this case, the decidua was oriented so that the cut also passed parallel to the uterine-antimesometrical mesometrical axis. The strict orientation of the decidua section planes allows representative slices to be studied for the regional differentiation of decidua cells (Mikhailov, 1998). Sections were fixed in a mixture of ethanol-methanol at -20 ° C for 2 minutes and stained with hematoxylin and eosin. Microscopic examination assessed the integrity of decidua and the absence of foci of inflammation or necrosis, as well as the degree of differentiation of decidua in the mesometric-antimesometric direction. It is known that the anti-mesometric part of rodent decidua is occupied by the so-called large decidual cells (BDK), analogues of human BDK, which make up the bulk of decidua cells in humans and rats (Mikhailov, 1998). The proportion of the anti-mesometric part ranges from 30 to 40% of the decidua cut-off area. The mesometric part of the decidua in contact with the mesometric triangle is formed by the so-called small decidual cells and granular cells of the endometrium (Mikhailov, 1998).

Analysis of the obtained sections showed that transplantation of human stem cells does not violate the differentiation of decidual cells and the organization of tissue, while maintaining the relative position of differentiated cells in the resulting decidual shell of rats.

According to morphometry, the decidua sections into which human SEC was transplanted were characterized by an increase in diameter in the meso-antimesometric direction by about 1.5–2 times with respect to the diameter of the decidual sections of the left, control horn. Since the decidua has an oval shape, measuring the diameter gives an idea of the dimensions of the decidua and allows you to compare the decidua with each other by their area. The measurement data are presented in table 1.

Table 1 Effect on the growth of decidua transplantation into the uterus horn of pseudopregnant rat endometrial stem cells (SEC) Decidua rats on the 11th day of pseudo-pregnancy Decidua diameter after transplantation of human SCE (mm) experienced decidua 4.3 ± 0.5 (p <0.05) control decidua 2.6 ± 0.5

To assess the effect of stem cell transplantation on the differentiation of BDK, as the most differentiated cells, the thickness of the BDK layer was determined and its share (%) in the total diameter of each decidua was calculated (Table 2).

table 2 Change in the fraction of the BDK zone in the total decidua thickness on the 11th day of pseudo-pregnancy after human SCE transplantation. Decidua rats on the 11th day of pseudo-pregnancy The share of the BDK zone in the total decidua thickness after transplantation of human SCE,% experienced decidua 35.4 ± 3.6 (p> 0.05) control decidua 31.8 ± 1.6

From the obtained data it follows that the proportion of the antimesometric (and, correspondingly, vice versa, mesometric) side of the decidua formed by the BDK (and, accordingly, small decidual cells of the mesometric side of the decidua) in experimental decidua does not differ from those of the control decidua that the cells did not inject. Thus, it was established that human SCE transplantation at the site of future rat decidua formation significantly stimulates decidua growth compared with the control uterine horn, while maintaining tissue organization and the relative position of differentiated endometrial cells. The data obtained demonstrate the stimulating effect of human SLE on the maturation and viability of rat endometrial tissue under conditions of pseudopregnancy in experimental animals.

Thus, our method can be used to develop the clinical use of stem cells for the treatment of miscarriage and other diseases associated with impaired endometrial differentiation and decidua insolvency during pregnancy.

Bibliography

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Claims (1)

A method of stimulating the formation of the decidual endometrial membrane in an experiment, including the introduction of a suspension containing 300 thousand endometrial stem cells isolated from a woman’s menstrual blood into the uterine wall of a rat, which is pseudopregnant, and subsequent subcutaneous administration of progesterone at a dose of 2 mg per gram of animal weight.