MOLECULAR-MORPHOLOGICAL DISSOCIATION IN VITRIFIED-WARMED HUMAN EMBRYOS: RETHINKING EPIGENETIC RISK BEYOND MORPHOLOGY

Öz

Although vitrification has transformed assisted reproductive technology, it occurs during a critical developmental window characterized by extensive epigenetic remodeling and transcriptional recalibration; freeze-thaw procedures expose gametes and embryos to osmotic, cryoprotectant, oxidative, and mitochondrial stress, raising concerns about molecular effects beyond morphology. This structured narrative review, conducted without a predefined protocol, evaluates whether vitrification induces meaningful epigenetic disruption by integrating current human methylome, transcriptomic, and clinical outcome data. English-language literature was searched in PubMed/MEDLINE and Scopus, supplemented by manual screening of the reference lists of retrieved articles. Priority was given to human preimplantation embryo studies published up to January 2026, while mechanistic inferences were drawn selectively from oocyte, sperm, and animal-model data where direct human evidence was limited. Evidence derived from a limited number of primary human embryo studies generally suggests preservation of global DNA methylation architecture and overall transcriptional stability under contemporary protocols. Reported molecular alterations are predominantly localized and modest in magnitude, while available clinical data do not show a reproducible reduction in implantation or live birth rates associated with vitrification. The interaction between cryobiological stress and gamete-derived epigenetic architecture (paternal DNA methylation, chromatin organization, and small non-coding RNA cargo) may further influence embryonic sensitivity. Vitrification appears to be a biologically active yet developmentally buffered intervention; available data suggest that detectable molecular variation is generally limited rather than indicative of global epigenomic destabilization. Future stage-specific multi-omics studies integrated with longitudinal outcomes are required to define thresholds of biological relevance.

Anahtar Kelimeler

• vitrification
• cryopreservation
• DNA methylation
• gamete-derived epigenetics
• embryonic plasticity

VİTRİFİYE-ÇÖZÜLMÜŞ İNSAN EMBRİYOLARINDA MOLEKÜLER-MORFOLOJİK AYRIŞMA: EPİGENETİK RİSKİN MORFOLOJİNİN ÖTESİNDE YENİDEN DEĞERLENDİRİLMESİ

Öz

Vitrifikasyon yardımcı üreme teknolojisini dönüştürmüş olsa da, kapsamlı epigenetik yeniden şekillenme ve transkripsiyonel yeniden kalibrasyonla karakterize kritik bir gelişim penceresinde gerçekleşir; dondurma-çözme işlemleri gamet ve embriyoları ozmotik, kriyoprotektan, oksidatif ve mitokondriyal strese maruz bırakarak morfolojinin ötesinde moleküler etki kaygıları doğurur. Önceden tanımlanmış bir protokole dayanmayan bu yapılandırılmış anlatı derlemesi, vitrifikasyonun anlamlı epigenetik bozulmaya yol açıp açmadığını mevcut insan metilom, transkriptomik ve klinik sonuç verileriyle değerlendirmeyi amaçlamaktadır. İngilizce literatür PubMed/MEDLINE ve Scopus'ta tarandı; alınan makalelerin kaynakçaları elle gözden geçirildi. Ocak 2026'ya kadar yayımlanan insan implantasyon öncesi embriyo çalışmalarına öncelik verildi; doğrudan insan kanıtının sınırlı olduğu durumlarda oosit, sperm ve hayvan modeli verilerinden seçici çıkarımlar yapıldı. Sınırlı sayıda birincil insan embriyosu çalışmasına dayanan kanıtlar, çağdaş protokoller altında global DNA metilasyon mimarisinin ve genel transkripsiyonel stabilitenin büyük ölçüde korunduğunu düşündürmektedir. Bildirilen moleküler değişiklikler çoğunlukla lokalize ve mütevazı büyüklüktedir; mevcut klinik veriler ise implantasyon veya canlı doğum oranlarında vitrifikasyonla ilişkili tekrarlanabilir bir azalma göstermemektedir. Kriyobiyolojik stres ile gamet kaynaklı epigenetik mimari (baba kökenli DNA metilasyonu, kromatin organizasyonu ve küçük kodlayıcı olmayan RNA yükü) arasındaki etkileşim de embriyonik duyarlılığı etkileyebilir. Vitrifikasyon biyolojik olarak aktif ancak gelişimsel olarak tamponlanmış bir müdahale gibi görünmektedir; mevcut veriler, saptanabilir moleküler varyasyonun genellikle sınırlı olduğunu ve global epigenomik istikrarsızlaşmayı göstermediğini düşündürmektedir. Biyolojik önem eşiklerini tanımlamak için uzunlamasına sonuçlarla entegre, aşamaya özgü çoklu-omik çalışmalar gereklidir.

Anahtar Kelimeler

• vitrifikasyon
• kriyoprezervasyon
• DNA metilasyonu
• gamet kaynaklı epigenetik
• embriyonik plastisite

Kaynakça

1. Sciorio R, Manna C, Fauque P, Rinaudo P. Can cryopreservation in assisted reproductive technology (ART) induce epigenetic changes to gametes and embryos? J Clin Med. 2023;12(13):4444. doi:10.3390/jcm12134444.
2. Estudillo E, Jiménez A, Bustamante-Nieves PE, Palacios-Reyes C, Velasco I, López-Ornelas A. Cryopreservation of gametes and embryos and their molecular changes. Int J Mol Sci. 2021;22(19):10864. doi:10.3390/ijms221910864.
3. Saeedinia M, Chen J, Kohandel Gargari O, et al. Epigenetics modification among vitrified oocytes and early embryos derived from them: a narrative review. Cell Mol Biol (Noisy-le-grand). 2024;70(7):22-28. doi:10.14715/cmb/2024.70.7.4.
4. Chen H, Zhang L, Meng L, Liang L, Zhang C. Advantages of vitrification preservation in assisted reproduction and potential influences on imprinted genes. Clin Epigenetics. 2022;14(1):141. doi:10.1186/s13148-022-01355-y.
5. Osman E, Franasiak J, Scott R. Oocyte and embryo manipulation and epigenetics. Semin Reprod Med. 2018;36(3-4):e1-e9. doi:10.1055/s-0039-1688801.
6. Sciorio R, Campos G, Tramontano L, Bulletti FM, Baldini GM, Vinciguerra M. Exploring the effect of cryopreservation in assisted reproductive technology and potential epigenetic risk. Zygote. 2023;31(5):420-432. doi:10.1017/S0967199423000345.
7. Zhu P, Guo H, Ren Y, et al. Single-cell DNA methylome sequencing of human preimplantation embryos. Nat Genet. 2018;50(1):12-19. doi:10.1038/s41588-017-0007-6.
8. Li L, Guo F, Gao Y, et al. Single-cell multi-omics sequencing of human early embryos. Nat Cell Biol. 2018;20(7):847-858. doi:10.1038/s41556-018-0123-2.

9. Ferreux L, Ducreux B, Firmin J, et al. Transcript profiling and gene regulation of the human pre-implantation embryo: parental effects and impact of ARTs. Hum Reprod Update. 2026;32(1):33-57. doi:10.1093/humupd/dmaf022.
10. Mateo-Otero Y, Yeste M, Damato A, Giaretta E. Cryopreservation and oxidative stress in porcine oocytes. Res Vet Sci. 2021;135:20-26. doi:10.1016/j.rvsc.2020.12.024.
11. Shi H, Li QY, Li H, et al. ROS-induced oxidative stress is a major contributor to sperm cryoinjury. Hum Reprod. 2024;39(2):310-325. doi:10.1093/humrep/dead250.
12. Zhang C, Zhao M, Xue Y, Tang X, Shi D, Wang X. Effects of vitrification on mitochondrial ultrastructure and membrane potential and its distribution in mouse oocytes. Cryo Letters. 2024;45(5):301-308. doi:10.54680/fr24510110212.
13. Barberet J, Barry F, Choux C, et al. What impact does oocyte vitrification have on epigenetics and gene expression? Clin Epigenetics. 2020;12(1):121. doi:10.1186/s13148-020-00911-8.
14. Chatzimeletiou K, Sioga A, Petrogiannis N, et al. Viability assessment using fluorescent markers and ultrastructure of human biopsied embryos vitrified in open and closed systems. Reprod Biomed Online. 2021;43(5):833-842. doi:10.1016/j.rbmo.2021.05.011.
15. Pérez-Sánchez M, Pardiñas ML, Díez-Juan A, et al. The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles. J Assist Reprod Genet. 2023;40(11):2577-2589. doi:10.1007/s10815-023-02952-3.
16. Zhu L, Sun L, Liu W, Han W, Huang G, Li J. Long-term storage does not affect the DNA methylation profiles of vitrified-warmed human embryos. Mol Reprod Dev. 2024;91(8):e23713. doi:10.1002/mrd.23713.
17. Li J, Zhu L, Huang J, Liu W, Han W, Huang G. Long-term storage does not affect the expression profiles of mRNA and long non-coding RNA in vitrified-warmed human embryos. Front Genet. 2022;12:751467. doi:10.3389/fgene.2021.751467.
18. Azizi E, Ghaffari Novin M, Naji M, Amidi F, Hosseinirad H, Shams Mofarahe Z. Effect of vitrification on biogenesis pathway and expression of development-related microRNAs in preimplantation mouse embryos. Cell Tissue Bank. 2021;22(1):103-114. doi:10.1007/s10561-020-09870-z.
19. Barberet J, Ducreux B, Bruno C, et al. Comparison of oocyte vitrification using a semi-automated or a manual closed system in human siblings: survival and transcriptomic analyses. J Ovarian Res. 2022;15(1):128. doi:10.1186/s13048-022-01064-3.
20. Li L, Bi X, Wu X, Chen Z, Cao Y, Zhao G. Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins. Hum Reprod. 2024;39(6):1275-1290. doi:10.1093/humrep/deae065.
21. Hosseini S, Hosseini S, Aligholi H, Salehi M. Embryo vitrification impacts learning and spatial memory by altering the imprinting genes expression level in the mouse offspring hippocampus. Sci Rep. 2025;15(1):5419. doi:10.1038/s41598-025-89857-6.
22. Li S, Zhang Q, Zhang L, et al. Lycopene improves the developmental competence of vitrified-thawed oocytes by reestablishing redox balance and modulating Rock1 expression. J Assist Reprod Genet. 2025;42(10):3505-3517. doi:10.1007/s10815-025-03640-0.
23. Zmuidinaite R, Sharara FI, Iles RK. Current advancements in noninvasive profiling of the embryo culture media secretome. Int J Mol Sci. 2021;22(5):2513. doi:10.3390/ijms22052513.
24. Al Bijou AA, Ilfergani JA, Bader TS, et al. Cryopreservation increases sperm DNA fragmentation in normozoospermic Libyan men: the role of oxidative stress and the protective effect of melatonin. Libyan J Med. 2025;20(1):2569151. doi:10.1080/19932820.2025.2569151.
25. Sciorio R, Cantatore C, D'Amato G, Smith GD. Cryopreservation, cryoprotectants, and potential risk of epigenetic alteration. J Assist Reprod Genet. 2024;41(11):2953-2967. doi:10.1007/s10815-024-03287-3.
26. Viana IGR, Vireque AA, Da Luz CM, Alberici LC, Navarro PA. Fatty acids and L-carnitine supplementation in vitrification media improves oocyte mitochondrial function and inner cell mass in mouse blastocysts: a pilot study. J Assist Reprod Genet. 2026;43(2):673-685. doi:10.1007/s10815-025-03760-7.
27. Diaz-Jimenez M, Wang M, Wang W, et al. Cryo-banking of human spermatozoa by aseptic cryoprotectants-free vitrification in liquid air: positive effect of elevated warming temperature. Cell Tissue Bank. 2022;23(1):17-29. doi:10.1007/s10561-021-09904-0.
28. Sirayapiwat P, Amorim CA, Sereepapong W, Tuntiviriyapun P, Suebthawinkul C, Thuwanut P. Application of fibrin-based biomaterial for human ovarian tissue encapsulation and cryopreservation as alternative approach for fertility preservation. Cryobiology. 2024;117:104955. doi:10.1016/j.cryobiol.2024.104955.
29. Pietrowski D, Mladek R, Frank M, Erber J, Marschalek J, Schneeberger C. Analyses of human granulosa cell vitality by fluorescence activated cell sorting after rapid cooling. Hum Fertil (Camb). 2022;25(3):478-486. doi:10.1080/14647273.2020.1817578.
30. Han S, Zhao L, Yang C, et al. Vitrification with microinjection of single seminiferous tubules: an efficient cryopreservation approach for limited testicular tissue. Reprod Biomed Online. 2021;43(4):687-699. doi:10.1016/j.rbmo.2021.06.026.
31. Mehta P, Singh R. The composition of human sperm sncRNAome: a cross-country small RNA profiling. Reprod Biol Endocrinol. 2025;23(1):36. doi:10.1186/s12958-025-01358-3.
32. Roque M, Haahr T, Geber S, Esteves SC, Humaidan P. Fresh versus elective frozen embryo transfer in IVF/ICSI cycles: a systematic review and meta-analysis of reproductive outcomes. Hum Reprod Update. 2019;25(1):2-14. doi:10.1093/humupd/dmy033.
33. Zaat T, Zagers M, Mol F, Goddijn M, van Wely M, Mastenbroek S. Fresh versus frozen embryo transfers in assisted reproduction. Cochrane Database Syst Rev. 2021;2(2):CD011184. doi:10.1002/14651858.CD011184.pub3.
34. Azizi Z, Soleimani Mehranjani M, Shariatzadeh MA, Najdi N, Azimi AS. Papaverine enhances sperm quality in asthenospermia by reducing oxidative stress and inflammation while regulating the expression of apoptosis-related genes during cryopreservation. Cryobiology. 2025;121:105329. doi:10.1016/j.cryobiol.2025.105329.
35. Vireque AA, Berteli TS, Stolakis V, Bertero M, Kofinas J. Impact of double versus single blastocyst biopsy and vitrification on clinical and neonatal outcomes in PGT cycles: a systematic review and meta-analysis of embryo retesting. Acta Obstet Gynecol Scand. 2026;105(1):30-49. doi:10.1111/aogs.70075.
36. Bickendorf K, Qi F, Peirce K, et al. Impacts of double biopsy and double vitrification on the clinical outcomes following euploid blastocyst transfer: a systematic review and meta-analysis. Hum Reprod. 2024;39(12):2674-2684. doi:10.1093/humrep/deae235.
37. Roque M, Valle M, Sampaio M, Geber S. Obstetric outcomes after fresh versus frozen-thawed embryo transfers: a systematic review and meta-analysis. JBRA Assist Reprod. 2018;22(3):253-260. doi:10.5935/1518-0557.20180049.
38. Epelboin S, Labrosse J, De Mouzon J, et al. Higher risk of pre-eclampsia and other vascular disorders with artificial cycle for frozen-thawed embryo transfer compared to ovulatory cycle or to fresh embryo transfer following in vitro fertilization. Front Endocrinol (Lausanne). 2023;14:1182148. doi:10.3389/fendo.2023.1182148.
39. Han Y, Liu C, Liu D, Wu L, Huang W. Pregnancy outcomes in freeze-all versus fresh embryo transfer cycles of women with adenomyosis and endometriosis: a systemic review and meta-analysis. Front Endocrinol (Lausanne). 2025;16:1507252. doi:10.3389/fendo.2025.1507252.
40. Pohjonen EM, Ahinko K, Huhtala H, et al. Clinical features of preeclampsia and hypertensive disorders in pregnancies after different frozen embryo transfer regimens. Pregnancy Hypertens. 2024;36:101123. doi:10.1016/j.preghy.2024.101123.