An attempt was made to rejuvenate cumulus cells (CCs) of women of advanced maternal age (AMA) using extracellular vesicles (EV) isolated from the follicular fluid (FF) of young donors. FF samples were taken from healthy women aged 23-26 during the ART program. CCs from patients of advanced maternal age (36-47 years) were co-cultured with EV of young donors. The target genes were selected based on their functions in CC and early embryogenesis (PFKP, EREG, UBE2T, HAS2, VCAN, CYP19A, GREM1, STS, BAX, PTGS2, BCL2, SPSB2, AREG, CCNB1, EGFR). Comparative analysis of all women without stratification by the type of final follicle maturation drug (hCG or gonadotropin-releasing hormone agonist, GnRHa) revealed no statistically significant difference in the expression of the selected genes. In the group treated with GnRHa, a statistically significant increase in the expression of HAS2 (by 1.8 times, p = 0.04), GREM1 (by 1.4 times, p = 0.08), and BAX (by 1.4 times, p = 0.04) in CCs was observed after their co-culturing with FF EVs from young donors. The findings suggest the potential for the "rejuvenation" of CC in women of advanced maternal age when aGnRH is used in combination with FF EVs from young women, because the expression of apoptosis genes decreases and the expression cell proliferation gene increases during co-culturing of CC with FF EVs.

We studied the influence of extracellular vesicles from the follicular fluid of a young donor on gene expression (MKI67, MYBL2, CCNB1, CCND1, CCNE1, CALM2, BAX, NDRG1, TP53I3, VEGF, VCAN, HAS2, CTSL2, PIBF1, RPL37, PFKP, GPX3, and AQP3) in embryos of women of different ages. According to nanoparticle tracking analysis data, the concentration of extracellular vesicles was 3.75±0.47×1011 particles/ml and the mean particle size was 138.78±9.90 nm. During co-culturing of the follicular fluid extracellular vesicles with blastocysts of young women, we observed significantly increased expression of mRNA for genes CTSL2, CCND1, CCNE1, VEGF and reduced expression of BAX gene mRNA in comparison with embryos in women of late reproductive age. We hypothesized that addition of extracellular vesicles of the oocyte follicular fluid from a young donor to the culture medium of embryos could slow down apoptosis process typical of blastocyst cells in women above 36 years.

Introduction. Progesterone levels are a critical factor for embryo implantation in in vitro fertilization (IVF) programs, including thawed embryo transfer programs. Overweight/obese patients often have lower blood progesterone concentrations in the luteal phase during IVF cycles, which requires an individual approach to planning luteal support in this category of patients.Aim. To compare the pregnancy ratе and miscarriage rates up to 12 weeks of pregnancy in overweight/obese patients who underwent a program of transfer of thawed embryos into the uterine cavity, depending on the type and route of administration of gestagens.Materials and methods. The study included 76 overweight or obese patients. All patients were treated for infertility using a program for transferring a thawed embryo into the uterine cavity; luteal phase support was carried out either using micronized progesterone, identical to natural, – the drug Utrogestan in a daily dose of 800 mg intravaginally (40 patients), or combination therapy was prescribed – vaginal gel with progesterone 90 mg in combination with the synthetic progestin dydrogesterone in a daily dose of 30 mg, taken orally (36 patients).Results. The rates of pregnancy and miscarriage in the first trimester were comparable in both groups and did not depend on differences in luteal support.Conclusions. The formation of the luteal phase of the cycle in patients with overweight/obesity is equally effective using various forms of gestagens. The use of micronized progesterone may be preferable in patients with overweight/obesity in terms of the ability to monitor blood progesterone levels, a more favorable metabolic profile when used vaginally, and the possibility of switching to oral administration of the drug in the event of the development of adverse local events.

To date the world community is actively working to optimize the approaches to determining chromosomal abnormalities in embryos. The study was aimed to assess the possibility of using noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) through analysis of cell-free DNA in spent culture medium (SCM). We conducted niPGT-A of aneuploid embryos by analysis of cell-free DNA in SCM. All blastocysts were considered to be aneuploid based on the results of previous preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy. The study involved 11 embryos from seven couples. All the embryos were warmed and individually cultured in the 10 µL drops for 9 h. All SCM was collected and analyzed by niPGT-A. The results obtained were tested for concordance with previous PGT-A data. A total of 12 SCM samples were assessed: 11 samples, in which the embryos were cultured, and one control sample. Chaotic niPGT-A results not allowing the karyotype diagnosis were obtained in one case (9.1%) out of 11. Full concordance of the PGT-A and niPGT-A results was revealed in seven cases out of 10 (70%), while clinical concordance was found in nine cases out of 10 (90%). In one case (10%), the blastocyst was considered to have euploid karyotype based on the niPGT-A data. It has been concluded that niPGT-A can be a promising method of preimplantation embryonal chromosomal status diagnosis that requires no biopsy.

V nastoyashchee vremya v mire idet aktivnaya rabota po optimizacii primeneniya podhodov k opredeleniyu hromosomnoj patologii embrionov. Cel'yu issledovaniya bylo ocenit' vozmozhnost' ispol'zovaniya neinvazivnogo preimplantacionnogo geneticheskogo testirovaniya na aneuploidii (niPGT-A) putem analiza vnekletochnoj DNK v otrabotannoj kul'tural'noj srede (OKS). Provedeno niPGT-A aneuploidnyh embrionov putem analiza vnekletochnoj DNK v OKS. Vse blastocisty byli aneuploidnymi po rezul'tatam predshestvuyushchego preimplantacionnogo geneticheskogo testirovaniya na aneuploidii (PGT-A) s biopsiej trofektodermy (TFE). V issledovanie bylo vklyucheno 11 embrionov ot semi supruzheskih par. Vse embriony razmorazhivali i kul'tivirovali v kaplyah po 10 mkl v techenie 9 ch. Ves' ob"em OKS sobirali i analizirovali putem niPGT-A. Poluchennye rezul'taty sravnivali na sootvetstvie s predshestvuyushchimi dannymi po PGT-A. Summarno vypolnili analiz 12 obrazcov OKS: 11 obrazcov, v kotoryh byli kul'tivirovany embriony, i odin kontrol'nyj obrazec. V odnom sluchae (9,1%) iz 11 byli polucheny haotichnye rezul'taty po dannym niPGT-A, ne pozvolyayushchie provesti diagnostiku sostoyaniya kariotipa. Polnoe sootvetstvie rezul'tatov PGT-A i niPGT-A polucheno v semi sluchayah iz 10 (70%), klinicheskoe sootvetstvie rezul'tatov — v devyati sluchayah iz 10 (90%). V odnom sluchae (10%) po dannym niPGT-A kariotip blastocisty byl diagnostirovan kak euploidnyj. Vyvod: niPGT-A mozhet byt' perspektivnym, ne trebuyushchim biopsii, metodom diagnostiki hromosomnogo statusa preimplantacionnyh embrionov.

Introduction. A comparative analysis of urinary and recombinant gonadotropins is relevant for the enhancement of the effectiveness of assisted reproductive technologies (ART) programs. Of particular interest is the analysis of the pharmacoeconomic efficiency of highly purified human menopausal gonadotropins (HMG) and the identification of individual groups of patients for whom the use of these drugs for ovarian stimulation in the ART program is most feasible and economically justified.Aim. To conduct a cost-effectiveness analysis of the use of Menopur Multidose at a dosage of 1200 IU for ovarian function stimulation in the standard ART protocols with gonadotropin-releasing hormone agonists and antagonists (GnRH-a and GnRH-ant).Materials and methods. The retrospective study included 4080 women aged 20 to 43 years. Stimulation of ovarian function in the ART program was carried out according to the GnRH antagonist protocol in 65.8% of patients and according to the GnRH-a protocol using Menopur Multidose at a dosage of 1200 IU supplemented with Menopur at a dosage of 75 IU used for its indications in 34.2% of women.Results and discussion. The clinical and laboratory data of the patients, stimulated cycle parameters, characteristics of the embryological stage, and the results of the ART program were analysed. The characteristics of the embryological stage met the KPI criteria, reflecting the key performance indicators of the embryology laboratory. Pharmacoeconomic analysis showed high involvement and adherence of patients to the treatment. In addition, it was found that the use of HMG makes efficient use of the budget within the average compulsory health insurance (CHI) tariff and accounts for 12% of the total tariff.Conclusion. Prescription of highly purified HMG (Menopur Multidose 1200 IU) to stimulate ovarian function in patients of different ages is accompanied by an adequate number of mature oocytes, good quality blastocysts, as well as satisfactory indicators of clinical pregnancy. A cost-effectiveness analysis of the use of Menopur Multidose at a dosage of 1200 IU showed that the prescription of this drug is clinically justified and economically feasible in patients of different age groups.

We studied the effect of co-culturing of extracellular vesicles in the follicular fluid of young women and women of advanced maternal age on sperm motility. Vesicles were obtained by differential centrifugation. The sperm fraction was isolated from the seminal fluid of 18 patients (age 28-36 years). The spermatozoa were incubated with vesicles (1:2 ratio) for 60 or 120 min at 37°C in a CO2 incubator. A fraction of spermatozoa incubated without vesicles served as the control. After the incubation, the sperm samples were sedimented by centrifugation, fixed in 2.5% glutaraldehyde, and analyzed by transmission electron microscopy. RNA was isolated from the follicular fluid vesicles by column method followed by cDNA synthesis in a reaction mixture according to miScript II RT Kit protocol (Qiagen). After 60-min incubation with extracellular vesicles from the follicular fluid of women of advanced maternal age, the sperm motility and hyperactivation slightly changed in comparison with the group where incubation was performed with follicular fluid vesicles from young women and control group. Follicular fluid miRNA profiles in women of different ages varied, which suggests different functional compositions and effects of follicular fluid vesicles of different age groups on sperm characteristics. Transmission electron microscopy revealed differences in the interaction of follicular fluid vesicles from women of different age groups with spermatozoa. Further study of the effect of extracellular vesicles from the follicular fluid and analysis of their transcriptomic, proteomic, and metabolomic composition on sperm mobility and fertilizing ability will improve the effectiveness of assisted reproductive technology programs in patients with male infertility.

Проведение преимплантационного генетического тестирования на определение анеуплоидий (ПГТ-А) при экстракорпоральном оплодотворении (ЭКО) является одним из методов первичной профилактики некоторых наследственных заболеваний. Следовательно, процедура ПГТ-А может способствовать повышению востребованности программ вспомогательных репродуктивных технологий (ВРТ) и быть экономически оправданной. ЦЕЛЬ ИССЛЕДОВАНИЯ Оценить значение и возможности применения ПГТ-А у супружеских пар разных возрастных групп при лечении бесплодия методом ВРТ. МАТЕРИАЛ И МЕТОДЫ За период 2016—2021 гг. проанализировано 1 108 протоколов ЭКО, включающих интрацитоплазматические инъекции сперматозоида в ооцит (ЭКО/ИКСИ) и процедуры ПГТ-А эмбрионов супружеских пар трех возрастных групп: 22—30, 31—36 и 37—46 лет. РЕЗУЛЬТАТЫ Самыми частыми причинами проведения ПГТ-А в программах ВРТ были старший, 37 лет и более, репродуктивный возраст женщин (48%), нарушения сперматогенеза (35%), множественные неудачные попытки ЭКО в анамнезе (20%), а также наличие экстрагенитального эндометриоза у женщин старшего репродуктивного возраста (15%). За период наблюдения количество выполненных комплексов процедур ЭКО/ИКСИ с проведением ПГТ-А увеличилось в 2,1 раза. Эффективность программы ЭКО/ИКСИ с проведением ПГТ-А эмбриона была сопоставима в разных возрастных группах супружеских пар и находилась в пределах 39—41%. ЗАКЛЮЧЕНИЕ Проведение преимплантационного генетического тестирования эмбриона на определение анеуплоидий является экономически оправданной процедурой в определенных клинических ситуациях, повышая тем самым востребованность программ ВРТ. Сопоставимая эффективность выполненных протоколов, включающих ЭКО/ИКСИ и ПГТ-А эмбриона, в различных возрастных группах супружеских пар может быть связана с особенностями критериев включения пациентов в данное исследование, а также свидетельствовать о важной, определяющей, роли качества эмбриона в исходах таких программ.

Introduction. Comparative analysis of urinary and recombinant gonadotropins is ongoing to improve the efficiency of assisted reproductive technology programs. Particular interest focused on the identification of individual groups of patients with maximal efficacy of using certain ovarian stimulation drugs in an assisted reproductive technology program.Objective. To review the application of Menopur Multidose 1200 IU for ovarian stimulation in assisted reproductive technology protocols with gonadotropin-releasing hormone agonists (GnRH) or gonadotropin-releasing hormone antagonists (GnRH antagonists) in the current clinical practice.Materials and methods. The study retrospectively enrolled 4,080 women aged 20-43 years. Ovarian stimulation in the Assisted Reproductive Technology program was performed using the GnRH antagonist protocol in 65.8% of patients and the GnRH agonist protocol with Menopur Multidose 1200 IU with or without the additional Menopur 75 IU in 34.2% of women.Results. Clinical and laboratory data of the patients, stimulated cycle parameters, characteristics of the embryological stage, and results of the assisted reproductive technology program were analyzed. The frequency of obtaining degenerated oocytes did not exceed 5%. The relative number of blastocysts of good quality was 59.9%, while for patients in the older age group, the figure was 54.6. The average pregnancy rate in patients in the “Menopur Multidose 1200 IU p/k” group was 39.3%, which corresponds to high-performance indicators of the assisted reproductive technology program.Conclusions. Administration of highly purified human menopausal gonadotropin (hMG) (Menopur Multidose 1200 IU) to stimulate ovarian function in patients of different ages is accompanied by an adequate number of mature oocytes, good quality blastocysts, and satisfactory clinical pregnancy rates. Thus, highly purified human menopausal gonadotropin is not inferior to recombinant follicle-stimulating hormone(r-FSH) concerning the efficacy of assisted reproductive technology programs.

Introduction. Despite the availability of general principles and regimens for ovarian stimulation on the assisted reproductive technology (ART) programs, an individual situational modification is required in each specific case.Objective. To optimize the ovarian stimulation protocols on the ART program based on the clinical and medical history records of the married couple.Materials and methods.The clinical and medical history records, as well as the stimulation cycle parameters of 60 married couples were analysed. The married couples enrolled in this study were diagnosed with infertility caused by tubo-peritoneal (44 married couples), male (5 married couples), combined factor (6 married couples), as well as external genital endometriosis combined with a male factor (3 married couples), and tubo-peritoneal factor (2 married couples). All married couples underwent IVF/ICSI procedures under the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol and ovarian stimulation on day 2 or 3 of the menstrual cycle.Results. All married couples were divided into two groups: 38 couples with a negative implantation result and 20 married couples with positive implantation results. The surgical removal of fallopian tubes has been shown to be inversely correlated with the duration of infertility. The use of hCG positively correlates to the ratio of mature oocytes and oocyte-cumulus complexes (OCC) and the onset of pregnancy. A reduced percentage of morphologically healthy sperm with an overall high concentration of sperm in the ejaculate can be interpreted as fertile sperm.Conclusions. During ovarian stimulation on the ART program, special attention should be paid to the duration of stimulation, the types of interventions for triggering final oocyte maturation and the total gonadotropin dose. A differentiated approach to conducting the surgical removal of fallopian tubes is required. The reduced percentage of morphologically healthy sperm with an overall high concentration of sperm in the ejaculate serves as a favourable prognostic factor for the outcome of ART programs.

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 106/ml and incubated with vesicles (1:2) at 37°C in a CO2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm.

Расширение возможностей вспомогательных репродуктивных технологий, в частности проведение биопсии клеток трофобласта на 5-е и 6-е сутки культивирования и последующее преимплантационное генетическое тестирование, в настоящее время значительно актуализировало вопросы реализации репродуктивной функции у пациентов с носительством моногенных заболеваний, к которым принадлежит и муковисцидоз. Муковисцидоз является одним из наиболее часто встречающихся моногенных заболеваний в российской популяции. В связи с этим гинекологам и репродуктологам важно понимать правильный алгоритм ведения таких пациентов в рамках программ лечения бесплодия и с целью рождения здоровых детей. В статье описан клинический случай рождения двух здоровых детей у супружеской пары с гетерозиготным носительством генетического варианта F508del в гене CFTR. Рожденные дети физически здоровы, развиваются соответственно возрасту.

For in vitro fertilization technology, the quality of oocytes has a direct impact on the egg fertilization and developmental competence of early embryo. The morphological criteria are used for the estimation of oocyte quality before its fertilization in vitro. To date, only one method is known to determine the maturity of oocyte. This is the routine observation with a light microscope. The aim of this article was to adapt the noninvasive quantitative laser scanning microtomography (QLSM) for the investigation of morphological features of a human oocyte in vitro. This approach was used to accumulate the Z-stack gallery of optical sections of IVF oocyte. The layer-by-layer acquisition allows the fine cytoplasmic structure imaging. Applying the QLSM Z-stack of optical sections, the cellular volume was calculated with quantitative 3D reconstruction of a human oocyte. The volume value and intracellular structure were used as novel criteria to assess the oocyte state after the stress evoked by cryopreservation procedure.

For a long time, the role of seminal plasma during human fertilization remained underestimated. Numerous studies related to the development of different methods for human embryo in vitro cultivation were gener-ally concerned with the quality of male and female gametes. However, in recent years, the development of Omix technologies provided a new insight into great seminal plasma influence on the morphofunctional characteristics of spermatozoa. This is especially true for the regulatory function of extracellular vesicles secreted by male reproductive tract cells. In this work, we attempted to analyze current data on the influence of extracellular seminal plasma vesicles on the morphofunctional characteristics of spermatozoa to solve male infertility topical issues. The review includes studies by foreign and Russian research groups that werу conducted within the past 5 years and found in PubMed, Google Scholar, and Cochrane Library databases. Very few studies demonstrate that seminal plasma vesicles act as functional regulators of male fertility and their dysfunction may lead to infertility. The use of seminal plasma extracellular vesicles in clinical practice may significantly increase the success of IVF programs, especially in impaired spermatogenesis. Keywords: extracellular vesicles, exosomes, biomarkers, seminal plasma, spermatozoa, assisted reproductive technology, cell biology, morphology

Using a non-invasive approach, quantitative laser scanning microtomography (QLSM), the morphology of human oocyte was studied layer-by-layer. Then, the cell volume was computed based on 3D reconstruction of a stack of optical sections obtained by QLSM. The integrity of oocyte membrane after cryopreservation was assessed by measuring the changes in oocyte volume in response to hypotonic shock.

We analyzed the effect of cryopreservation on the expression of glutathione peroxidase (GPX1) and glutathione reductase (GSR) genes in human sperm cells (15 sperm samples from fertile donors and 10 samples from infertile patients). The relative expression of GPX1 and GSR genes was determined by real-time PCR. The rate of post-thaw recovery was 2.1 times higher in the group of fertile donors. A significant increase in the expression of GPX1, but not GSR, was observed in sperm samples from infertile patients, while in patients with infertility, GPX1 expression significantly decreased after cryopreservation/thawing, in samples from fertile donors after the same procedure it increased to the level observed in the sperm samples from infertile patients. A positive correlation was revealed between GPX1 expression and sperm cryotolerance.

We summarized the data on the role and efficiency of mechanical microvibration during embryo culturing in assisted reproduction protocols. The efficiency of culturing systems with microvibration in assisted reproduction programs and possible mechanisms of microvibration effect on the preimplantation development of human embryos are discussed.