Treatment of infertility in women with multiple treatment failures using assisted reproductive technologies with co-culture of embryos with autologous follicular fluid-derived extracellular vesicles
Akhmedova Z.F., Sysoeva A.P., Gavrilov M.Yu., Zingerenko B.V., Shevtsova Yu.A., Silachev D.N., Nepsha O.S., Makarova N.P., Kulakova E.V., Kalinina E.A.
Relevance: The primary goal of infertility treatment using assisted reproductive technologies (ART) is to increase the rates of embryo implantation and the birth of healthy children. Scientific research has focused on enhancing culture conditions, particularly the co-culture of embryos at various developmental stages with somatic cells and extracellular vesicles (EVs). Experimental animal models have demonstrated that adding EVs derived from follicular fluid (FF) during in vitro culture can modulate embryo development and improve implantation rates. FF-derived EVs were specifically selected because of their proven roles in regulating oocyte quality and subsequent post-implantation embryo development.
Objective: To evaluate the clinical efficacy of ART for optimizing the embryological stage of infertility treatment by co-culturing FF-derived EVs with embryos in patients who have experienced multiple unsuccessful IVF attempts.
Materials and methods: This study involved married couples with a history of more than two unsuccessful IVF attempts, a normal karyotype, and mild pathozoospermia. A total of 76 women were included in this study. As part of the standard ART protocol, FF was collected on the day of transvaginal puncture from which EVs were isolated through sequential ultracentrifugation. After fertilization, oocytes were randomly divided into two groups: group 1 underwent co-culture of zygotes with FF-derived EVs for 24 h, while group 2 underwent classical culture. The addition of FF-derived EVs was performed as follows: in a well of a 4-well plate containing 0.3 ml of culture medium and post-ICSI oocytes, 2 μl of the medium with FF-derived EVs was added and incubated for 24 h. On the first day of culture, embryos at the zygote stage from group 1 were transferred to classical culture medium to continue developing to the blastocyst stage. Embryological and clinical outcomes were assessed, with the primary endpoint being pregnancy rate after transferring one embryo into the uterine cavity. The level of statistical significance was set at p<0.05.
Results: Assessment of early embryogenesis parameters revealed a statistically significant increase in fertilization rate for the co-culture group with FF-derived EVs: 88.1% compared to 77.3% in the classical culture group (p<0.001, chi-square test). Additionally, group 1 exhibited a significantly higher frequency of excellent- and good-quality blastocyst formation (55.0% vs. 42.6 %, p<0.01). When fertilized post-ICSI oocytes were co-cultured with FF-derived EVs, there were significantly more blastocysts suitable for transfer into the uterine cavity and cryopreservation, with a median of 1 [0; 1.25] in group 1 compared to 0 [0; 1] in group 2. Clinical data analysis indicated a pregnancy rate of 27.2% in group 2, while group 1 (EV FF) achieved clinical pregnancy in 14 of 45 transfers, with two biochemical pregnancies. The clinical pregnancy rate in group 1 was 31.1%. Notably, no statistically significant differences in clinical parameters were found across the overall cohort of women with a history of multiple IVF failures. However, there was a tendency for an increased pregnancy rate with co-culture of FF-derived EVs in younger women, in contrast to the near-complete absence of positive outcomes in patients of advanced reproductive age.
Conclusion: The results suggest that co-culture with FF-derived EVs may significantly enhance the embryological stage of infertility treatment programs in women with a history of multiple ART failures. Further studies are needed to establish statistically significant differences in clinical outcomes.
Authors' contributions: Akhmedova Z.F., Sysoeva A.P., Makarova N.P. – conception and design of the study; Akhmedova Z.F., Sysoeva A.P., Makarova N.P., Kulakova E.V. – drafting and editing of the manuscript; Akhmedova Z.F., Nepsha O.S., Gavrilov M.Yu. – statistical analysis; Akhmedova Z.F., Sysoeva A.P., Zingerenko B.V. – collection of biological material; Shevtsova Yu.A., Silachev D.N. – laboratory stage; Makarova N.P., Kulakova E.V., Kalinina E.A. – approval of the manuscript for submission.
Conflicts of interest: The authors have no conflicts of interest to declare.
Funding: The study was conducted within the framework of the initiative scientific project "Study of the influence of extracellular vesicles of biological fluids of reproductive organs and tissues on gametes, the process of fertilization and early human embryogenesis and implantation" (2025-2027, supervisor N.P. Makarova) of V.I. Kulakov NMRC for OG&P, Ministry of Health of Russia.
Ethical Approval: The study was reviewed and approved by the Research Ethics Committee of the V.I. Kulakov NMRC for OG&P (Ref. No: 10 of October 20, 2022).
Patient Consent for Publication: All patients provided informed consent for the publication of their data.
Authors' Data Sharing Statement: The data supporting the findings of this study are available upon request from the corresponding author after approval from the principal investigator.
For citation: Akhmedova Z.F., Sysoeva A.P., Gavrilov M.Yu., Zingerenko B.V., Shevtsova Yu.A., Silachev D.N., Nepsha O.S., Makarova N.P., Kulakova E.V., Kalinina E.A. Treatment of infertility in women with
multiple treatment failures using assisted reproductive technologies with co-culture of
embryos with autologous follicular fluid-derived extracellular vesicles.
Akusherstvo i Ginekologiya/Obstetrics and Gynecology. 2025; (5): 66-77 (in Russian)
https://dx.doi.org/10.18565/aig.2025.85
Keywords
ART
infertility
co-culture
follicular fluid
extracellular vesicles
IVF
embryo
References
- Назаренко Т.А., ред. Бесплодный брак: клинические задачи и их решение. Пособие для врачей. М.: МЕДпресс-информ; 2024. 144 с. [Nazarenko T.A., ed. Infertile marriage: clinical problems and their solution. Manual for doctors. Moscow: MEDpress-inform; 2024. 144 p. (in Russian)].
- Shingshetty L., Cameron N.J., Mclernon D.J., Bhattacharya S. Predictors of success after in vitro fertilization. Fertil. Steril. 2024; 121(5): 742-51. https://dx.doi.org/10.1016/j.fertnstert.2024.03.003
- Wu C., Cai H., Pu Q., Yu L., Goswami A., Mo Z. Investigating the role of oviductal mucosa-endometrial co-culture in modulating factors relevant to embryo implantation. Open Med. (Wars.). 2024; 19(1): 20241077. https://dx.doi.org/10.1515/med-2024-1077
- Lorenzo M.S., Teplitz G.M., Luchetti C.G., Cruzans P.R., Bertonazzi A., Lombardo D.M. The coculture of in vitro produced porcine embryos and oviductal epithelial cells improves blastocyst formation and modify embryo quality. Theriogenology. 2024; 226: 141-50. https://dx.doi.org/10.1016/j.theriogenology.2024.06.007
- Le Saint C., Crespo K., Bourdiec A., Bissonnette F., Buzaglo K., Couturier B. et al. Autologous endometrial cell co-culture improves human embryo development to high-quality blastocysts: a randomized controlled trial. Reprod. Biomed. Online. 2019; 38(3): 321-9. https://dx.doi.org/10.1016/j.rbmo.2018.12.039
- Carles M., Lefranc E., Bosquet D., Capelle S., Scheffler F., Copin H. et al. In vitro maturation of oocytes from stimulated IVF-ICSI cycles using autologous cumulus cell co-culture: A preliminary study. Morphologie. 2023; 107(356): 28-37. https://dx.doi.org/10.1016/j.morpho.2022.02.002
- Асфарова Г.Р., Смольникова В.Ю., Макарова Н.П., Бобров М.Ю., Эльдаров Ч.М., Зингеренко Б.В., Калинина Е.А. Клинические и молекулярные аспекты аутологичного сокультивирования эмбрионов с клетками кумулюса в программах экстракорпорального оплодотворения. Акушерство и гинекология. 2023; 4: 97-110. [Asfarova G.R., Smolnikova V.Yu., Makarova N.P., Bobrov M.Yu., Eldarov Ch.M., Zingerenko B.V., Kalinina E.A. Clinical and molecular aspects of autologous embryo-cumulus cells co-culture in IVF programs. Obstetrics and Gynecology. 2023; (4): 97-110 (in Russian)]. https://dx.doi.org/10.18565/aig.2022.306
- da Silveira J.C., Andrade G.M., Del Collado M., Sampaio R.V., Sangalli J.R., Silva L.A. et al. Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development. PLoS One. 2017; 12(6): e0179451. https://dx.doi.org/10.1371/journal.pone.0179451
- Довгань А.А., Ахмедова З.Ф., Сысоева А.П., Зингеренко Б.В., Романов Е.А., Силачев Д.Н., Макарова Н.П., Калинина Е.А. Внеклеточные везикулы фолликулярной жидкости: клинические аспекты и молекулярная биология. Акушерство и гинекология. 2023; 6: 38-43. [Dovgan A.A., Akhmedova Z.F., Sysoeva A.P., Zingerenko B.V., Romanov E.A., Silachev D.N., Makarova N.P., Kalinina E.A. Extracellular vesicles in follicular fluid: clinical aspects and molecular biology. Obstetrics and Gynecology. 2023; (6): 38-43 (in Russian)]. https://dx.doi.org/10.18565/aig.2022.320
- Duval C., Wyse B.A., Tsang B.K., Librach C.L. Extracellular vesicles and their content in the context of polycystic ovarian syndrome and endometriosis: a review. J. Ovarian. Res. 2024; 17(1): 160. https://dx.doi.org/10.1186/s13048-024-01480-7
- Aoki S., Inoue Y., Hara S., Itou J., Shirasuna K., Iwata H. microRNAs associated with the quality of follicular fluids affect oocyte and early embryonic development. Reprod. Med. Biol. 2024; 23(1): e12559. https://dx.doi.org/10.1002/rmb2.12559.
- Nepsha O.S., Burmenskaya O.V., Akhmedova Z.F., Romanov E.A., Sysoeva A.P., Goryunov K.V. et al. Changes in the transcription of proliferation- and apoptosis-related genes in embryos in women of different ages under the influence of extracellular vesicles from donor follicular fluid in vitro. Bull. Exp. Biol. Med. 2024; 176(5): 658-65. https://dx.doi.org/10.1007/s10517-024-06087-y
- Российская Ассоциация Репродукции Человека. Секция «Клиническая эмбриология». Оценка ооцитов и эмбрионов в лаборатории ВРТ. Методические рекомендации. 2021. [Russian Association of Human Reproduction. Clinical Embryology Section. Evaluation of oocytes and embryos in the ART laboratory. Guidelines. 2021. (in Russian)].
- Sallem A., Santulli P., Barraud-Lange V., Le Foll N., Ferreux L., Maignien C. et al. Extended culture of poor-quality supernumerary embryos improves ART outcomes. J. Assist. Reprod. Genet. 2018; 35(2): 311-9. https://dx.doi.org/10.1007/s10815-017-1063-7
- Gabryś J., Gurgul A., Szmatoła T., Kij-Mitka B., Andronowska A., Karnas E. et al. Follicular fluid-derived extracellular vesicles influence on in vitro maturation of equine oocyte: impact on cumulus cell viability, expansion and transcriptome. Int. J. Mol. Sci. 2024; 25(6): 3262. https://dx.doi.org/10.3390/ijms25063262
- Soares M., Pinto M.M., Nobre R.J., de Almeida L.P., da Graça Rasteiro M., Almeida-Santos T. et al. Isolation of extracellular vesicles from human follicular fluid: size-exclusion chromatography versus ultracentrifugation. Biomolecules. 2023; 13(2): 278. https://dx.doi.org/10.3390/biom13020278
- Neyroud A.S., Chiechio R.M., Moulin G., Ducarre S., Heichette C., Dupont A. et al. Diversity of extracellular vesicles in human follicular fluid: morphological analysis and quantification. Int. J. Mol. Sci. 2022; 23(19): 11676. https://dx.doi.org/10.3390/ijms231911676
- Simon C., Greening D.W., Bolumar D., Balaguer N., Salamonsen L.A., Vilella F. Extracellular vesicles in human reproduction in health and disease. Endocr. Rev. 2018; 39(3): 292-32. https://dx.doi.org/10.1210/er.2017-00229
- Andronico F., Battaglia R., Ragusa M., Barbagallo D., Purrello M., Di Pietro C. Extracellular vesicles in human oogenesis and implantation. Int. J. Mol. Sci. 2019; 20(9): 2162. https://dx.doi.org/10.3390/ijms20092162
- Зингеренко Б.В., Бурменская О.В., Сысоева А.П., Шевцова Ю.А., Силачев Д.Н., Макарова Н.П., Калинина Е.А. Изменение транскрипции генов в клетках кумулюса возрастных женщин под действием внеклеточных везикул фолликулярной жидкости молодых доноров. Бюллетень экспериментальной биологии и медицины. 2024; 178(12): 767-73. [Zingerenko B.V., Burmenskaya O.V., Sysoeva A.P., Shevtsova Yu.A., Silachev D.N., Makarova N.P., Kalinina E.A. Gene transcription changes in advanced maternal aged women cumulus cells under influence of young donors follicular fluid extracellular vesicles. Bulletin of Experimental Biology and Medicine. 2024; 178(12): 767-73. (in Russian)]. https://dx.doi.org/10.47056/0365-9615-2024-178-12-767-773
- Fan W., Qi Y., Wang Y., Yan H., Li X., Zhang Y. Messenger roles of extracellular vesicles during fertilization of gametes, development and implantation: Recent advances. Front. Cell Dev. Biol. 2023; 10: 1079387. https://dx.doi.org/10.3389/fcell.2022.1079387
- Назаренко Т.А. Стимуляция функции яичников. 7-е изд. М.: МЕДпресс-информ; 2023. 268 с. [Nazarenko T.A. Stimulation of ovarian function. 7th ed. Moscow: MEDpress-inform; 2023. 268 p. (in Russian)].
- Sysoeva A., Akhmedova Z., Nepsha O., Makarova N., Silachev D., Shevtsova Y. et al. Characteristics of the follicular fluid extracellular vesicle molecular profile in women in different age groups in ART programs. Life (Basel). 2024; 14(5): 541. https://dx.doi.org/10.3390/life14050541
- Battaglia R., Musumeci P., Ragusa M., Barbagallo D., Scalia M., Zimbone M. et al. Ovarian aging increases small extracellular vesicle CD81+ release in human follicular fluid and influences miRNA profiles. Aging (Albany NY). 2020; 12(12): 12324-41. https://dx.doi.org/10.18632/aging.103441
- Zhang H., Lin S., McElroy C.L., Wang B., Jin D., Uteshev V.V. et al. Circulating pro-inflammatory exosomes worsen stroke outcomes in aging. Circ. Res. 2021; 129(7): e121-e140. https://dx.doi.org/10.1161/CIRCRESAHA.121.318897
- Gu Y., Zhang X., Wang R., Wei Y., Peng H., Wang K. et al. Metabolomic profiling of exosomes reveals age-related changes in ovarian follicular fluid. Eur. J. Med. Res. 2024; 29(1): 4. https://dx.doi.org/10.1186/s40001-023-01586-6
Received 25.03.2025
Accepted 21.04.2025
About the Authors
Zumriiat F. Akhmedova, PhD student at the Prof. B.V. Leonov Department for Assisted Technologies in Infertility Treatment, Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4, zyuka-1997@mail.ru,https://orcid.org/0000-0002-4483-8820
Anastasia P. Sysoeva, PhD, Clinical Embryologist at the Prof. B.V. Leonov Department for Assisted Technologies in Infertility Treatment, Academician V.I. Kulakov
National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4,
a_sysoeva@oparina4.ru, https://orcid.org/0000-0002-6502-4498
Maxim Yu. Gavrilov, Junior Researcher at the Prof. B.V. Leonov Department of Assistive Technologies in Infertility Treatment, Academician V.I. Kulakov National
Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4, maxgavr67@gmail.com,
https://orcid.org/0000-0001-6189-0287
Boris V. Zingerenko, Junior Researcher at the Prof. B.V. Leonov Department of Assistive Technologies in Infertility Treatment, Academician V.I. Kulakov National
Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4, b_zingerenko@oparina4.ru,
https://orcid.org/0000-0002-8784-5502
Yulia A. Shevtsova, Junior Researcher at the Cell Technologies Laboratory, Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4, u_shevtsova@oparina4.ru
Denis N. Silachev, Dr. Bio. Sci., Head of the Cell Technologies Laboratory, Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4, d_silachev@oparina4.ru, https://orcid.org/0000-0003-0581-9755
Oksana S. Nepsha, PhD, Researcher at the Prof. B.V. Leonov Department of Assistive Technologies in Infertility Treatment, Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4, o_nepsha@oparina4.ru,
https://orcid.org/0000-0002-9988-2810
Natalya P. Makarova, Dr. Bio. Sci., Leading Researcher at the Prof. B.V. Leonov Department of Assistive Technologies in Infertility Treatment, Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4,
np_makarova@oparina4.ru, https://orcid.org/0000-0003-1396-7272
Elena V. Kulakova, Dr. Med. Sci., Senior Researcher at the Prof. B.V. Leonov Department of Assistive Technologies in Infertility Treatment, Academician V.I. Kulakov
National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4,
e_kulakova@oparina4.ru, https://orcid.org/0000-0002-4433-4163
Elena A. Kalinina, Dr. Med. Sci., Professor, Head of the Prof. B.V. Leonov Department of Assistive Technologies in Infertility Treatment, Academician V.I. Kulakov
National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia, 117997, Russia, Moscow, Ac. Oparin str., 4,
e_kalinina@oparina4.ru, https://orcid.org/0000-0002-8922-2878
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