Liquid Biopsy as a Screening Tool in Recurrent Implantation Failure During IVF: A Narrative Review
Abstract
Objective: Recurrent implantation failure (RIF) remains a multifaceted challenge in assisted reproductive technology (ART), affecting a subset of in vitro fertilization (IVF) patients despite transfer of high-quality embryos. Conventional diagnostics often require invasive procedures that may disturb endometrial receptivity. This narrative review critically synthesizes the evidence on liquid-biopsy strategies for RIF screening, emphasizing biofluids such as uterine fluid (UF), spent embryo culture medium (SCM), peripheral blood, cervicovaginal fluid (CVF), and seminal plasma.
Materials and methods: Literature searches covered PubMed, Scopus, and Web of Science through August 2025, with inclusion of pivotal pre-2022 studies. Eligible works included observational studies, clinical trials, multi-omics analyses, and systematic reviews reporting molecular biomarkers relevant to endometrial receptivity or embryo competence.
Results: UF-derived microRNAs (miRNAs) and extracellular vesicle (EV) cargo during the window of implantation (WOI) consistently differentiate receptive from nonreceptive endometrium, with predictive accuracies (AUC 0.75–0.90) in small validation studies. SCM-based noninvasive PGT-A (niPGT-A) shows moderate concordance (~65%) with trophectoderm biopsy PGT-A, constrained by contamination and low cfDNA yield; recent advances include blastocoel fluid integration and methylation-based decontamination. Peripheral blood, CVF, and seminal plasma biomarkers remain exploratory due to lower specificity. Pre-analytical standardization and harmonized outcome definitions are critical for translation.
Conclusion: UF-miRNA/EV profiling holds the strongest promise for noninvasive RIF screening, whereas niPGT-A may serve as an adjunct for embryo competence assessment when biopsy is not feasible. Both approaches require multicenter validation and prospective trials demonstrating improved live-birth rates before routine clinical adoption.
1. Cimadomo D, de Los Santos MJ, Griesinger G, Lainas G, Le Clef N, McLernon DJ, et al. ESHRE good practice recommendations on recurrent implantation failure. Hum Reprod Open. 2023;2023(3):hoad023.
2. Pirtea P, Cedars MI, Devine K, Ata B, Franasiak J, Racowsky C, et al. Recurrent implantation failure: reality or a statistical mirage?: Consensus statement from the July 1, 2022 Lugano Workshop on recurrent implantation failure. Fertil Steril. 2023;120(1):45-59.
3. Wang J, Wang D, Zhang Y, Sun P, Yi L, Han A, et al. Extracellular vesicles in reproductive biology and disorders: a comprehensive review. Front Endocrinol (Lausanne). 2025;16:1550068.
4. Sui C, Liao Z, Bai J, Hu D, Yue J, Yang S. Current knowledge on the role of extracellular vesicles in endometrial receptivity. Eur J Med Res. 2023;28(1):471.
5. Pirtea P, De Ziegler D, Tao X, Sun L, Zhan Y, Ayoubi JM, et al. Rate of true recurrent implantation failure is low: results of three successive frozen euploid single embryo transfers. Fertil Steril. 2021;115(1):45-53.
6. Gill P, Ata B, Arnanz A, Cimadomo D, Vaiarelli A,
Fatemi HM, et al. Does recurrent implantation failure exist? Prevalence and outcomes of five consecutive euploid blastocyst transfers in 123,987 patients. Hum Reprod. 2024;39(5):974-80.
7. Meier M, di Giacopo SCM, Stucki P, Birchler K, Leeners B. Women's experiences and coping strategies for dealing with recurrent implantation failure in IVF/ICSI: an interview study. Gynecol Obstet Open Access. 2024;8:226.
8. Sun B, Yeh J. Non-invasive and mechanism-based molecular assessment of endometrial receptivity during the window of implantation: current concepts and future prospective testing directions. Front Reprod Health. 2022;4:863173.
9. Wu C, Sun Y, Yang D, Peng H. Advances in endometrial receptivity and embryo implantation by multi-omics techniques. Animals (Basel). 2025. [Epub ahead of print].
10. Voros C, Varthaliti A, Athanasiou D, Mavrogianni D, Bananis K, Athanasiou A, et al. MicroRNA signatures in endometrial receptivity—unlocking their role in embryo implantation and IVF success: a systematic review. Biomedicines. 2025;13(5). [In press].
11. Salmasi S, Heidar MS, Mahabady MK, Rashidi B, Mirzaei H. MicroRNAs, endometrial receptivity and molecular pathways. Reprod Biol Endocrinol. 2024;22(1):139.
12. von Grothusen C, Frisendahl C, Modhukur V, Lalitkumar PG, Peters M, Faridani OR, et al. Uterine fluid microRNAs are dysregulated in women with recurrent implantation failure. Hum Reprod. 2022;37(4):734-46.
13. Franasiak JM, Alecsandru D, Forman EJ, Gemmell LC, Goldberg JM, Llarena N, et al. A review of the pathophysiology of recurrent implantation failure. Fertil Steril. 2021;116(6):1436-48.
14. Bulletti FM, Sciorio R, Conforti A, De Luca R, Bulletti C, Palagiano A, et al. Causes of embryo implantation failure: a systematic review and meta-analysis of procedures to increase embryo implantation potential. Front Endocrinol (Lausanne). 2025;15:2024.
15. Garneau AS, Young SL. Defining recurrent implantation failure: a profusion of confusion or simply an illusion? Fertil Steril. 2021;116(6):1432-5.
16. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281-97.
17. He L, Hannon GJ. MicroRNAs: small RNAs with a
big role in gene regulation. Nat Rev Genet. 2004;5(7):522-31.
18. Liang J, Wang S, Wang Z. Role of microRNAs in embryo implantation. Reprod Biol Endocrinol.
2017;15(1):90.
19. Galliano D, Pellicer A. MicroRNA and implantation. Fertil Steril. 2014;101(6):1531-44.
20. Shekibi M, Heng S, Nie G. MicroRNAs in the regulation of endometrial receptivity for embryo implantation. Int J Mol Sci. 2022;23(11):6210.
21. Sluijter JPG, Davidson SM, Boulanger CM, Buzás EI, de Kleijn DPV, Engel FB, et al. Extracellular vesicles in diagnostics and therapy of the ischaemic heart: position paper from the Working Group on Cellular Biology of the Heart of the European Society of Cardiology. Cardiovasc Res. 2018;114(1):19-34.
22. Wu HM, Lo TC, Tsai CL, Chen LH, Huang HY, Wang HS, et al. Extracellular vesicle-associated microRNA-138-5p regulates embryo implantation and early pregnancy by adjusting GPR124. Pharmaceutics. 2022;14(6):1213.
23. Abu-Halima M, Häusler S, Backes C, Fehlmann T, Staib C, Nestel S, et al. Micro-ribonucleic acids and extracellular vesicles repertoire in the spent culture media is altered in women undergoing in vitro fertilization. Sci Rep. 2017;7(1):13525.
24. Nakamura K, Kusama K, Suda Y, Fujiwara H, Hori M, Imakawa K. Emerging role of extracellular vesicles in embryo-maternal communication throughout implantation processes. Int J Mol Sci. 2020;21(15):5523.
25. Kurian NK, Modi D. Extracellular vesicle mediated embryo-endometrial cross talk during implantation and in pregnancy. J Assist Reprod Genet. 2019;36(2):189-98.
26. Liu C, Li L, Wang M, Shui S, Yao H, Sui C, et al. Endometrial extracellular vesicles of recurrent implantation failure patients inhibit the proliferation, migration, and invasion of HTR8/SVneo cells. J Assist Reprod Genet. 2021;38(4):825-33.
27. Zhou W, Dimitriadis E. Secreted microRNA to predict embryo implantation outcome: from research to clinical diagnostic application. Front Cell Dev Biol. 2020;8:586.
28. Goharitaban S, Abedelahi A, Hamdi K, Khazaei M, Esmaeilivand M, Niknafs B. Role of endometrial microRNAs in repeated implantation failure (mini-review). Front Cell Dev Biol. 2022;10:936173.
29. Chen CH, Lu F, Yang WJ, Yang PE, Chen WM, Kang ST, et al. A novel platform for discovery of differentially expressed microRNAs in patients with repeated implantation failure. Fertil Steril. 2021;116(1):181-8.
30. Handayani N, Aubry D, Boediono A, Bowolaksono A, Sini I, Haq NMD, et al. Non-invasive pre-implantation genetic testing's reliability for aneuploidy using cell-free DNA in embryo culture media. J Gynecol Obstet Hum Reprod. 2024;53(8):102808.
31. Zhang Z, Qiao J, Chen Y, Zhou P. Genetic Deconvolution of Embryonic and Maternal Cell-Free DNA in Spent Culture Medium of Human Preimplantation Embryo Through Deep Learning. Adv Sci (Weinh). 2025;12(34):e12660.
32. Vilella F, Moreno-Moya JM, Balaguer N, Grasso A, Herrero M, Martínez S, et al. Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome. Development. 2015;142(18):3210-21.
33. Cannon S, Fagan S, DeVane A, et al. Small RNA sequencing of extracellular vesicles from uterine aspirates identifies candidate biomarkers for implantation success. J Assist Reprod Genet. 2021;38(Suppl 1):S78.
34. Rai A, Fang H, Clarizia L, et al. Proteomic profiling of uterine fluid extracellular vesicles reveals dynamic changes associated with the window of implantation. Hum Reprod. 2021;36(Suppl 1):deab130.092.
35. Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750.
36. Shitara A, Saito T, Horikawa T, et al. Non-invasive preimplantation genetic testing for aneuploidy using cell-free DNA in spent embryo culture medium: A prospective study. J Assist Reprod Genet. 2021;38(10):2567-75.
37. Lledó B, Morales R, Ortiz JA, et al. Technical
challenges and standardization in non-invasive PGT-A using spent culture medium. Hum Reprod Update. 2023;29(3):345-61.
38. Chen Y, Zhang Z, Qiao J, Zhou P. Methylation-based deconvolution of cell-free DNA in spent embryo culture medium improves non-invasive PGT-A accuracy. Hum Reprod. 2025;40(2):325-36.
39. Xu J, Fang R, Chen L, et al. Combined analysis of spent culture medium and blastocoel fluid improves non-invasive preimplantation genetic testing. Reprod Biomed Online. 2024;48(1):103478.
40. Martínez-Zamudio R, Rizo JM, Flores-Herrera H, et al. Circulating exosomal microRNAs as potential biomarkers for recurrent implantation failure. Int J Mol Sci. 2023;24(5):4456.
41. Ahmadi M, Rahimi S, Shamsi M, et al. Exosomal miRNAs in peripheral blood of RIF patients: a systematic review. J Reprod Immunol. 2024;162:104198.
42. Lamon S, Le Carré J, Petito G, Duong HP, Luthi F, Hiam D, et al. The effect of the menstrual cycle on the circulating microRNA pool in human plasma: a pilot study. Hum Reprod. 2023 Jan 5;38(1):46-56.
43. Kanannejad Z, Kheshtchin N, Nabavizadeh H. The immunomodulatory role of seminal plasma in endometrial receptivity and embryo implantation. Am
J Clin Exp Immunol. 2025;14(4):167-184.
2. Pirtea P, Cedars MI, Devine K, Ata B, Franasiak J, Racowsky C, et al. Recurrent implantation failure: reality or a statistical mirage?: Consensus statement from the July 1, 2022 Lugano Workshop on recurrent implantation failure. Fertil Steril. 2023;120(1):45-59.
3. Wang J, Wang D, Zhang Y, Sun P, Yi L, Han A, et al. Extracellular vesicles in reproductive biology and disorders: a comprehensive review. Front Endocrinol (Lausanne). 2025;16:1550068.
4. Sui C, Liao Z, Bai J, Hu D, Yue J, Yang S. Current knowledge on the role of extracellular vesicles in endometrial receptivity. Eur J Med Res. 2023;28(1):471.
5. Pirtea P, De Ziegler D, Tao X, Sun L, Zhan Y, Ayoubi JM, et al. Rate of true recurrent implantation failure is low: results of three successive frozen euploid single embryo transfers. Fertil Steril. 2021;115(1):45-53.
6. Gill P, Ata B, Arnanz A, Cimadomo D, Vaiarelli A,
Fatemi HM, et al. Does recurrent implantation failure exist? Prevalence and outcomes of five consecutive euploid blastocyst transfers in 123,987 patients. Hum Reprod. 2024;39(5):974-80.
7. Meier M, di Giacopo SCM, Stucki P, Birchler K, Leeners B. Women's experiences and coping strategies for dealing with recurrent implantation failure in IVF/ICSI: an interview study. Gynecol Obstet Open Access. 2024;8:226.
8. Sun B, Yeh J. Non-invasive and mechanism-based molecular assessment of endometrial receptivity during the window of implantation: current concepts and future prospective testing directions. Front Reprod Health. 2022;4:863173.
9. Wu C, Sun Y, Yang D, Peng H. Advances in endometrial receptivity and embryo implantation by multi-omics techniques. Animals (Basel). 2025. [Epub ahead of print].
10. Voros C, Varthaliti A, Athanasiou D, Mavrogianni D, Bananis K, Athanasiou A, et al. MicroRNA signatures in endometrial receptivity—unlocking their role in embryo implantation and IVF success: a systematic review. Biomedicines. 2025;13(5). [In press].
11. Salmasi S, Heidar MS, Mahabady MK, Rashidi B, Mirzaei H. MicroRNAs, endometrial receptivity and molecular pathways. Reprod Biol Endocrinol. 2024;22(1):139.
12. von Grothusen C, Frisendahl C, Modhukur V, Lalitkumar PG, Peters M, Faridani OR, et al. Uterine fluid microRNAs are dysregulated in women with recurrent implantation failure. Hum Reprod. 2022;37(4):734-46.
13. Franasiak JM, Alecsandru D, Forman EJ, Gemmell LC, Goldberg JM, Llarena N, et al. A review of the pathophysiology of recurrent implantation failure. Fertil Steril. 2021;116(6):1436-48.
14. Bulletti FM, Sciorio R, Conforti A, De Luca R, Bulletti C, Palagiano A, et al. Causes of embryo implantation failure: a systematic review and meta-analysis of procedures to increase embryo implantation potential. Front Endocrinol (Lausanne). 2025;15:2024.
15. Garneau AS, Young SL. Defining recurrent implantation failure: a profusion of confusion or simply an illusion? Fertil Steril. 2021;116(6):1432-5.
16. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281-97.
17. He L, Hannon GJ. MicroRNAs: small RNAs with a
big role in gene regulation. Nat Rev Genet. 2004;5(7):522-31.
18. Liang J, Wang S, Wang Z. Role of microRNAs in embryo implantation. Reprod Biol Endocrinol.
2017;15(1):90.
19. Galliano D, Pellicer A. MicroRNA and implantation. Fertil Steril. 2014;101(6):1531-44.
20. Shekibi M, Heng S, Nie G. MicroRNAs in the regulation of endometrial receptivity for embryo implantation. Int J Mol Sci. 2022;23(11):6210.
21. Sluijter JPG, Davidson SM, Boulanger CM, Buzás EI, de Kleijn DPV, Engel FB, et al. Extracellular vesicles in diagnostics and therapy of the ischaemic heart: position paper from the Working Group on Cellular Biology of the Heart of the European Society of Cardiology. Cardiovasc Res. 2018;114(1):19-34.
22. Wu HM, Lo TC, Tsai CL, Chen LH, Huang HY, Wang HS, et al. Extracellular vesicle-associated microRNA-138-5p regulates embryo implantation and early pregnancy by adjusting GPR124. Pharmaceutics. 2022;14(6):1213.
23. Abu-Halima M, Häusler S, Backes C, Fehlmann T, Staib C, Nestel S, et al. Micro-ribonucleic acids and extracellular vesicles repertoire in the spent culture media is altered in women undergoing in vitro fertilization. Sci Rep. 2017;7(1):13525.
24. Nakamura K, Kusama K, Suda Y, Fujiwara H, Hori M, Imakawa K. Emerging role of extracellular vesicles in embryo-maternal communication throughout implantation processes. Int J Mol Sci. 2020;21(15):5523.
25. Kurian NK, Modi D. Extracellular vesicle mediated embryo-endometrial cross talk during implantation and in pregnancy. J Assist Reprod Genet. 2019;36(2):189-98.
26. Liu C, Li L, Wang M, Shui S, Yao H, Sui C, et al. Endometrial extracellular vesicles of recurrent implantation failure patients inhibit the proliferation, migration, and invasion of HTR8/SVneo cells. J Assist Reprod Genet. 2021;38(4):825-33.
27. Zhou W, Dimitriadis E. Secreted microRNA to predict embryo implantation outcome: from research to clinical diagnostic application. Front Cell Dev Biol. 2020;8:586.
28. Goharitaban S, Abedelahi A, Hamdi K, Khazaei M, Esmaeilivand M, Niknafs B. Role of endometrial microRNAs in repeated implantation failure (mini-review). Front Cell Dev Biol. 2022;10:936173.
29. Chen CH, Lu F, Yang WJ, Yang PE, Chen WM, Kang ST, et al. A novel platform for discovery of differentially expressed microRNAs in patients with repeated implantation failure. Fertil Steril. 2021;116(1):181-8.
30. Handayani N, Aubry D, Boediono A, Bowolaksono A, Sini I, Haq NMD, et al. Non-invasive pre-implantation genetic testing's reliability for aneuploidy using cell-free DNA in embryo culture media. J Gynecol Obstet Hum Reprod. 2024;53(8):102808.
31. Zhang Z, Qiao J, Chen Y, Zhou P. Genetic Deconvolution of Embryonic and Maternal Cell-Free DNA in Spent Culture Medium of Human Preimplantation Embryo Through Deep Learning. Adv Sci (Weinh). 2025;12(34):e12660.
32. Vilella F, Moreno-Moya JM, Balaguer N, Grasso A, Herrero M, Martínez S, et al. Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome. Development. 2015;142(18):3210-21.
33. Cannon S, Fagan S, DeVane A, et al. Small RNA sequencing of extracellular vesicles from uterine aspirates identifies candidate biomarkers for implantation success. J Assist Reprod Genet. 2021;38(Suppl 1):S78.
34. Rai A, Fang H, Clarizia L, et al. Proteomic profiling of uterine fluid extracellular vesicles reveals dynamic changes associated with the window of implantation. Hum Reprod. 2021;36(Suppl 1):deab130.092.
35. Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750.
36. Shitara A, Saito T, Horikawa T, et al. Non-invasive preimplantation genetic testing for aneuploidy using cell-free DNA in spent embryo culture medium: A prospective study. J Assist Reprod Genet. 2021;38(10):2567-75.
37. Lledó B, Morales R, Ortiz JA, et al. Technical
challenges and standardization in non-invasive PGT-A using spent culture medium. Hum Reprod Update. 2023;29(3):345-61.
38. Chen Y, Zhang Z, Qiao J, Zhou P. Methylation-based deconvolution of cell-free DNA in spent embryo culture medium improves non-invasive PGT-A accuracy. Hum Reprod. 2025;40(2):325-36.
39. Xu J, Fang R, Chen L, et al. Combined analysis of spent culture medium and blastocoel fluid improves non-invasive preimplantation genetic testing. Reprod Biomed Online. 2024;48(1):103478.
40. Martínez-Zamudio R, Rizo JM, Flores-Herrera H, et al. Circulating exosomal microRNAs as potential biomarkers for recurrent implantation failure. Int J Mol Sci. 2023;24(5):4456.
41. Ahmadi M, Rahimi S, Shamsi M, et al. Exosomal miRNAs in peripheral blood of RIF patients: a systematic review. J Reprod Immunol. 2024;162:104198.
42. Lamon S, Le Carré J, Petito G, Duong HP, Luthi F, Hiam D, et al. The effect of the menstrual cycle on the circulating microRNA pool in human plasma: a pilot study. Hum Reprod. 2023 Jan 5;38(1):46-56.
43. Kanannejad Z, Kheshtchin N, Nabavizadeh H. The immunomodulatory role of seminal plasma in endometrial receptivity and embryo implantation. Am
J Clin Exp Immunol. 2025;14(4):167-184.
| Files | ||
| Issue | Vol 20, No 2 (June 2026) | |
| Section | Review Articles | |
| Keywords | ||
| Recurrent Implantation Failure Liquid Biopsy Uterine Fluid Extracellular Vesicles | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Saeedinia R, Hemmat A, Heidary Z. Liquid Biopsy as a Screening Tool in Recurrent Implantation Failure During IVF: A Narrative Review. J Family Reprod Health. 2026;20(2):94-100.
