Cryopreserved Placental Derivatives Increase Survival of Mice With Cyclophosphamide-Induced Ovarian Failure

Received 22, November, 2022 Accepted 27, February, 2023 Тривалість та якість життя жінки значною мірою залежить від її репродуктивного періоду та рівня продукції статевих гормонів [4, 5], на які суттєвий вплив має передчасне згасання функції яєчників, у тому числі внаслідок онкологічних захворювань та їх терапії [2, 5]. Ефективним методом для відновлення функції яєчників після хіміотерапії є застосування стовбурових клітин різного походження [1, 2, 8]. Похідні плаценти, що містять характерні для стовбурових клітин фактори, ряд біологічно активних речовин і сполук, можуть бути потенційно ефективними у подоланні наслідків хіміотерапії. На даний час загальноприйнятою тваринною моделлю передчасної оваріальної недостатності (ОН) після хіміотерапії є використання комбінації циклофосфаміду та бусульфану. Така модель дозволяє у короткі терміни формувати ознаки ОН, які відповідають клінічному діагнозу, при цьому викликає меншу кількість побічних ефектів та демонструє високу результативність і стабільність [7, 8, 10]. У попередніх роботах нами була показана ефективність різних видів тканинної терапії на моделі циклофосфамід-індукованої оваріальної недостатності (МЦІОН), а також збільшення під впливом кріоконсервованих похідних Women’s life expectancy and its quality largely depend on the duration of reproductive period and the sex hormones production [4, 5], signifi cantly aff ected by premature ovarian failure, as well as resulted from oncology diseases and their therapy [2, 5]. An eff ective method for restoring ovarian function after chemotherapy is the use of stem cells of various origins [1, 2, 8]. Placental derivatives containing both stem cell-specifi c factors and a number of biologically active substances as well as compounds can be potentially eff ective in overcoming the chemotherapy eff ects. Currently, the generally accepted animal model of premature ovarian failure (OF) after chemotherapy uses a combination of cyclophosphamide and busulfan. This model allows the most rapid formation of OF signs, corresponding to the clinical diagnosis, but with fewer side eff ects, and demonstrates high effi ciency and stability [7, 8, 10]. Our previous studies have shown the eff ectiveness of various types of tissue therapy in the model of cyclophosphamide-induced ovarian failure (MCIOF), as well as an increased lifespan of male mice and the duration of reproductive period of females under the infl uence of cryopreserved placental derivatives [4, 5]. This suggests that the use of placental deriva-

Thus, the aim of our work was to determine the eff ect of cryopreserved cells, extract and explants of human placenta on the lifespan of female mice in a model of cyclophosphamide-induced ovarian failure.
The experiments were carried out in compliance with the requirements of the Bioethics Committee of the Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine (protocol № 1 of February 26, 2019), consistent with the provisions of the 'European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientifi c Purposes' (Strasbourg, 1986) and the provisions of Declaration of Helsinki adopted by the General Assembly of the World Medical Association.
Cyclophosphamide-induced OF was simulated according to the previously described method by intraperitoneal administration of 200 mg/kg cyclophosphamide (Sandoz, Austria) and 20 mg/kg busulfan (Accord Healthcare Polska Sp. z o. o., Poland) [7,8].Ovarian insuffi ciency in female mice was confi rmed by the presence of atrophic ovaries/uterus, and the absence of the estrous cycle and estrogen saturation of the body.Placentas were obtained after caesarean section with the informed consent of the women.Placental derivatives were isolated, cryopreserved, dosed and administered according to previously described methods.Two weeks after the confi rmation of OF, the CPE was used at the rate of 10 μl per animal, CPF -10 mg per animal, CPC -at the rate of 100,000 cells per animal intraperitoneally [4][5][6].
У тварин групи 2 спостерігали різке збільшення частоти смертності протягом перших місяців після введення хіміопрепаратів, після чого відбувалася стабілізація даного показника (рисунок).Це може бути пов'язане з гострою токсичністю застосованих препаратів -пошкоджуюча дія на організм тварин була найбільш вираженою одразу після введення.Загальний стан тварин, які, незважаючи на наявність токсичних проявів, переживали цей період, відновлювався до вихідного, у подальшому ймовірність їх виживання підвищувалася.Порівняння кривих виживання Каплана-Майєра за Log-rank тестом показало значущу відмінність між собою.Се-the age of 18-20 months, which corresponded to an active reproductive period when the protective eff ect of estrogen took place; a sharp increase in the frequency of their mortality after the age of 16-18 months, when ovarian function declined and the synthesis of steroid sex hormones decreased (fi gure) [9].The average lifespan was (21.16 ± ± 0.05) months, the median survival rate was 21.80%, the survival rate and the maximum survival rate made 18 and 30 months, respectively.Animals of group 2 showed a sharp increase in the mortality rate during the fi rst months after the administration of chemotherapy drugs, afterwards this index stabilized (Figure ).This may be due to the acute toxicity of the drugs used, i. e. the damaging eff ect on the animal organism was most pronounced immediately after administration.The general condition of the animals, which survived during this period, despite the manifestations of toxic eff ects, was restored to the initial level; the probability of their further survival increased.Comparison of Kaplan-Meier survival curves according to the Logrank test showed a signifi cant diff erence between them.The average lifespan of animals was (10.2 ± 0.09) months, the median survival rate was 10.8%, the survival rate and the maximum survival rate made 6 and 19 months, respectively.
of the animals was (16.8 ± 0.06) months, the median survival rate was 17.8%, the survival rate the survival rate and the maximum survival rate made 14 and 24 months, correspondingly.In animals of group 4, the Kaplan-Meier curve diff ered signifi cantly from the curves of animals of groups 1 and 2 and did not diff er from the curves of animals of groups 3 and 5.A certain decrease in the mortality rate of female mice after CPF administration was noted, however, it was less pronounced compared with the use of CPE.This eff ect can be explained by faster absorption of the placental extract components by binding to specifi c receptors present on the surface of target cells, followed by stimulation of damaged cells, which ensured tissue repair and regeneration [3].The average lifespan of the animals was (15.6 ± 0.06) months, the median survival rate was 18.8%, the survival rate and the maximum survival rate made 10 and 24 months, respectively.In animals of group 5, the Kaplan-Meier curve diff ered signifi cantly from the curves of groups 1 and 2, had no diff erences compared to the curves of groups 3 and 4 (fi gure).The average lifespan of female mice was (14.36 ± 0.07) months, the median survival rate was 17.8%, the survival rate and the maximum survival rate amounted to 8 and 23 months, respectively.
A feature of the model of cyclophosphamide-induced ovarian failure is a sharp increase in animal mortality from acute toxicity during the fi rst two months after administration of chemotherapy drugs.The use of cryopreserved placental cells, extract or fragments increased the lifespan and survival of female mice in a model of cyclophosphamide-induced ovarian failure.The highest rates of survival and lifespan of female mice with cyclophosphamide-induced ovarian failure after administration of cryopreserved placental derivatives have been shown for the placental extract.
3.15 software (University of Oslo, Norway), the critical signifi cance level was taken at p ≤ 0.05.An analysis of the survival curve of animals in the control group showed that it had a shape typical for females and represented the characteristics of sex: a low mortality rate in female mice under проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 33, №/issue 1, 2023