State of the art in assisted reproductive technologies for patients with advanced maternal age

According to the World Health Organization, the female reproductive age lasts up to 49 years, but problems with the realization of women's reproductive rights may arise much earlier. Significant numbers of factors affect the state of reproductive health: socioeconomic, ecological, lifestyle features, the level of medical literacy, and the state of the organization and medical care quality. Among the reasons for fertility decline in advanced reproductive age are the loss of cellular receptors for gonadotropins, an increase in the threshold of sensitivity of the hypothalamic-pituitary system to the action of hormones and their metabolites, and many others. Furthermore, negative changes accumulate in the oocyte genome, reducing the possibility of fertilization, normal development and implantation of the embryo and healthy offspring birth. Another theory of ageing causing changes in oocytes is the mitochondrial free radical theory of ageing. Taking into account all these age-related changes in gametogenesis, this review considers modern technologies aimed at the preservation and realization of female fertility. Among the existing approaches, two main ones can be distinguished: methods allowing the preservation of reproductive cells at a younger age using ART intervention and cryobanking, as well as methods aimed at improving the basic functional state of advanced-age women's oocytes and embryos

Embryological Characteristics and Preimplantation Genetic Testing for Aneuploidy of Embryos Derived from Cryopreserved Oocytes of Women of Different Reproductive Ages

Oocyte vitrification is widely used for female fertility preservation. However, the efficacy of this procedure may depend on the women's age. The aim of the study was to compare the morphology, viability of cryopreserved oocytes, and their fertilization outcomes (fertilization, blastulation rate, level of embryo chromosomal aneuploidy-preimplantation genetic testing for aneuploidy [PGT-A]) in women of different reproductive ages. The studied oocytes were divided into groups depending on the age of patients: up to 30 years (group 1), 30-35 years (group 2), 36-40 years (group 3), and older than 40 years (group 4). It has been shown that in women of older reproductive age, the number of oocytes with polymorphism of endo- and extracytoplasmic structures was higher compared with younger patients. This could reflect on their cryosurvival rate, which was the highest in group 1 (98.1%) and the lowest was in group 4 (47.4%). With increasing age, the fertilization rate of cryopreserved oocytes and subsequent blastulation was decreased. However, the number of embryos with an aneuploid chromosome set number was increased. The chromosome set number euploidy rate of the embryos obtained from cryopreserved oocytes of advanced age women (group 4) did not differ from the fresh group with the same age (31.2% vs. 24.4%, p > 0.05), but the number of euploid embryos per patient was less than one (0.8 ± 0.1). Therefore, the decision to cryopreserve the oocytes of a patient of older reproductive age should be made individually for each situation, taking into account the prospects of obtaining full-fledged embryos and the chances of pregnancy

Історія допоможних репродуктивних технологій: від заборони до визнання

The birth of children after infertility treatment of married couples with the help of assisted reproductive technologies has become a reality after many years of basic research on the physiology of reproductive system, development of oocyte’s in vitro fertilization methods and cultivation of embryos at pre-implantation stages. Given the widespread use of assisted reproductive technologies in modern medical practice and the great interest of society to this problem, the aim of the study was to trace the main stages and key events of assisted reproductive technologies in the world and in Ukraine, as well as to highlight the activities of outstanding scientists of domestic and world science who were at the origins of the development of this area. The paper used historical methods to study and interpret the texts of primary sources and present scientific historical events. In addition, the current trends in assisted reproductive technologies are covered based on the results of our own, more than 30 years of experience in the field of reproductive biology and medicine, and the achievements of world scientists. As a result of the work, it has been shown that despite certain ethical and social biases, the discovery of individual predecessor scientists became the basis for the efforts of Robert Edwards and Patrick Steptoe to ensure birth of the world's first child, whose conception occurred outside the mother's body. There are also historical facts and unique photos from our own archive, which confirm the fact of the first successful oocyte in vitro fertilization and the birth of a child after the use of assisted reproductive technologies in Ukraine. Over the last 20 years, assisted reproductive technologies have continued to grow, addressing many other issues of reproductive potential preservation and infertility treatment. State of the art methods of assisted reproductive technologies include the development of  cryopreservation method of gametes and embryos by vitrification, genetic screening of embryos in order to prevent the hereditary diseases transmission and embryo transfer with chromosomal abnormalities, the birth of a child “from three parents” in severe cases of mutations in the mitochondrial genome, etc.Рождения детей после лечения бесплодия семейных пар с помощью вспомогательных репродуктивных технологий стало реальностью после многих лет фундаментальных исследований  физиологии репродуктивной системы, разработки методов оплодотворения in vitro ооцитов и культивирование эмбрионов доимплантационных стадий развития. Учитывая широкое применение вспомогательных репродуктивных технологий в современной медицинской практике и большой интерес общества к этой проблеме целью исследования было проследить основные этапы и ключевые события становления вспомогательных репродуктивных технологий в мире и в Украине, а также осветить деятельность выдающихся ученых отечественной и мировой науки, которые стояли у истоков развития этого направления. В работе использовали исторические методы для изучения и интерпретации текстов первоисточников и представления научных исторических событий. Кроме того, на основе результатов собственного, более чем 30-летнего опыта работы в сфере репродуктивной биологии и медицины и достижений ученых мировой науки, освещены современные направления развития вспомогательных репродуктивных технологий. В результате работы показано, что, несмотря на определенные этические и социальные предубеждения, открытие отдельных ученых ‒ предшественников создали фундамент того, что усилиями Роберта Эдвардса и Патрика Стептоу родился первый в мире ребенок, зачатие которого произошло вне организма матери. Также представлены исторические факты и уникальные фотографии из собственного архива, подтверждающие факт первого успешного оплодотворения ооцита in vitro и рождения ребенка после применения вспомогательных репродуктивных технологий в Украине. Последние 20 лет вспомогательных репродуктивных технологий продолжают неустанно развиваться, решая многие проблемы сохранения репродуктивного потенциала и лечения бесплодия. Среди современных методов вспомогательных репродуктивных технологий можно выделить разработку способа криоконсервирование гамет и эмбрионов путем витрификации, проведение генетического скрининга эмбрионов с целью предотвращения передачи наследственных болезней, и переноса эмбрионов с хромосомными аномалиями, рождение ребенка “от трех родителей” при тяжелых случаях мутаций митохондриального генома и многое другое.Народження дітей після лікування безпліддя сімейних пар за допомогою допоміжних репродуктивних технологій стало реальністю після багатьох років фундаментальних досліджень щодо фізіології репродуктивної системи, розробки методів запліднення in vitro ооцитів та культивування ембріонів передімплантаційних стадій розвитку. Враховуючи широке застосування допоміжних репродуктивних технологій у сучасній медичній практиці та великий інтерес суспільства до цієї проблеми метою дослідження було прослідкувати основні етапи та ключові події становлення допоміжних репродуктивних технологій в світі та в Україні, а також висвітлити діяльність видатних вчених вітчизняної та світової науки, які стояли у витоків розвитку цього напряму. В роботі використовувались історичні методи для вивчення та інтерпретації текстів першоджерел та представлення наукових історичних подій. Крім того, на  основі результатів власного, більш ніж 30-ти річного досвіду роботи у сфері репродуктивної біології і медицини та досягнень вчених світової науки, висвітлено сучасні напрямки розвитку допоміжних репродуктивних технологій. В результаті  роботи показано, що, не дивлячись на певні етичні та соціальні упередження, відкриття окремих вчених-попередників стали підґрунтя для того, щоб зусиллями Роберта Едвардса та Патрика Стептоу народилася перша в світі дитина, зачаття якої відбулося поза організмом матері. Також представлено історичні факти та унікальні фотографії з власного архіву, які підтверджують факт першого успішного запліднення ооцита in vitro та народження дитини після застосування допоміжних репродуктивних технологій в Україні. Останні 20 років допоміжних репродуктивних технологій продовжують невпинно розвиватися, вирішуючи багато інших проблем збереження репродуктивного потенціалу та лікування безпліддя. Серед сучасних методів допоміжних репродуктивних технологій можна виділити розробку способу кріоконсервування гамет та ембріонів шляхом вітрифікації, проведення генетичного скринінгу  ембріонів з метою запобігання  передачі спадкових хвороб, та переносу ембріонів з хромосомними аномаліями, народження дитини “від трьох батьків” при тяжких випадках мутацій мітохондріального геному та багато іншого

Using Dextran Instead of Egg Yolk in Extender for Cryopreservation of Spermatozoa of Dogs of Different Ages

Egg yolk is a very common supplement of extenders aimed to protect sperm from cryoinjury, but due to their biological risks and difficulties with media standardization, there is a search for alternative. In addition, sperm cryoresistance can be affected by the initial decrease of their functional characteristics caused by age. The aim of this work was to evaluate the efficiency of using dextran (molecular weight 500 kDa) in the extenders instead of egg yolk for the cryopreservation of spermatozoa of dogs (Chinese Crested breed) of different ages. The obtained ejaculates were divided into three groups depending on the animal’s age: 1–3, 4–6 and 7–10 years old. Sperm was cryopreserved by using 7% glycerol and 20% egg yolk, or 20% dextran. The cryoresistance of spermatozoa of the oldest age category was dramatically decreased, which was manifested in their morphology, motility, and DNA fragmentation rate. There were no differences between the cryoprotectant effect of the dextran-based extender on spermatozoa and the egg yolk-based extender in all age categories of dogs. However, given the benefits of dextran-containing media, its use for the cryopreservation of canine spermatozoa has potential benefits that need to be confirmed by sperm fertilization outcomes

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus–oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs

New method for cryoprotectant-free freezing of human oligoasthenoteratozoospremic spermatozoa with high-molecular polymer

Data about cryoprotectant-free cryopreservation of human ICSI spermatozoa are limited. The aim of this investigation was to compare two technologies for cryopreservation of spermatozoa from men with oligoasthenoteratozoospermia: standard conventional freezing with 5% glycerol (freezing in glycerol) and cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone (PVP) (PVPfreezing). Capillaries with spermatozoa were cooled in vapor and then plunginged into liquid nitrogen. Head-, midpiece- and tail-abnormality of spermatozoa, mitochondrial membrane potential (MMP) and DNA fragmentation rates after cryopreservation were evaluated. After warming of spermatozoa, fertilization of oocytes (ICSI) was performed. It was detected the lower rate of morphological abnormalities of PVP-frozen spermatozoa in comparison with cells frozen with glycerol (34.6 +/- 4.1% vs. 20.7 +/- 4.7%, respectively) (P < 0.05). Quality of cells with high MMP after warming in spermatozoa frozen with glycerol was lower than in PVP-frozen spermatozoa (34.7 +/- 4.2 vs. 54.5 +/- 4.2%, respectively) (P < 0.05). It was established that the DNA fragmentation rate in PVP-frozen spermatozoa was significantly lower in comparison with spermatozoa frozen with glycerol (23.1 +/- 2.5% vs. 38.8 +/- 3.0%, respectively) (P 0.05). After fertilization (ICSI) of oocytes, it was established that cleavage and blastulation rates were higher in oocytes after fertilization with PVP-frozen spermatozoa than with spermatozoa frozen with glycerol. Fertilization-, development to 8-blastomeres-, and blastocyst-rates were for PVP-frozen and spermatozoa frozen with glycerol, respectively: 94.4 +/- 7.8 vs. 82.2 +/- 6.2% (P 0.1 with tendency to increasing), 90.0 +/- 4.6 vs. 69.5 +/- 5.1% (P < 0.05), and 45.4 +/- 4.1% vs. 30.9 +/- 3.3% (P < 0.05). It was concluded that permeable cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone can be applied successfully for cryopreservation of human oligoasthenoteratozoospremic spermatozoa

Translation of Cryobiological Techniques to Socially Economically Deprived Populations—Part 1: Cryogenic Preservation Strategies

Use of cold for preservation of biological materials, avoidance of food spoilage and to manage a variety of medical conditions has been known for centuries. The cryobiological science justified these applications in the 1960s increasing their use in expanding global activities. However, the engineering and technological aspects associated with cryobiology can be expensive and this raises questions about the abilities of resource-restricted low and middle income countries (LMICs) to benefit from the advances. This review was undertaken to understand where or how access to cryobiological advances currently exist and the constraints on their usage. The subject areas investigated were based on themes which commonly appear in the journal Cryobiology. This led in the final analysis for separating the review into two parts, with the first part dealing with cold applied for biopreservation of living cells and tissues in science, health care and agriculture, and the second part dealing with cold destruction of tissues in medicine. The limitations of the approaches used are recognized, but as a first attempt to address these topics surrounding access to cryobiology in LMICs, the review should pave the way for future more subject-specific assessments of the true global uptake of the benefits of cryobiology.Fil: Buriak, Iryna. National Academy of Sciences of Ukraine; UcraniaFil: Fleck, Roland. Kings College London; Reino UnidoFil: Goltsev, Anatoliy. National Academy of Sciences of Ukraine; UcraniaFil: Shevchenko, Nadiya. National Academy of Sciences of Ukraine; UcraniaFil: Petrushko, Maryna. National Academy of Sciences of Ukraine; UcraniaFil: Yurchuk, Taisiia. National Academy of Sciences of Ukraine; UcraniaFil: Puhovkin, Anton. National Academy of Sciences of Ukraine; UcraniaFil: Rozanova, Svitlana. National Academy of Sciences of Ukraine; UcraniaFil: Guibert, Edgardo Elvio. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Robert, María Celeste. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Juan de Paz, Leonardo. Universidad Nacional de Rosario. Secretaria de Ciencia y Técnica. Centro Binacional de Investigación en Criobiología Clínica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Powell Palm, Matthew J.. University of California at Berkeley; Estados UnidosFil: Fuller, Barry. University College London; Estados Unido