Embryo-secreted microRNAs enable non-invasive assessment of chromosomal status and viability in in vitro fertilization

Abstract

Selecting embryos with the highest developmental potential remains a decisive step for achieving successful in vitro fertilization outcomes. Conventional evaluation methods such as morphological grading, time-lapse imaging, and preimplantation genetic testing for aneuploidy provide valuable information but are limited by subjectivity, invasiveness, and cost. These challenges have driven increasing interest in non-invasive biomarkers capable of improving the precision and safety of embryo assessment. Emerging evidence indicates that human embryos actively release microRNAs into their surrounding environment, particularly into the spent culture medium and blastocoel fluid. These extracellular microRNAs regulate pathways involved in cell cycle progression, apoptosis, differentiation, and embryo-endometrium communication. Distinct expression signatures, including miR-21-5p, miR-661, and members of the miR-17∼92 cluster, correlate with chromosomal integrity and implantation competence, suggesting their potential as complementary or alternative biomarkers to trophectoderm biopsy. Comparative analyses across digital PCR, quantitative RT-PCR, microarray, and next-generation sequencing platforms reveal both methodological advances and unresolved challenges, such as low RNA yield, contamination risk, and inconsistent normalization strategies. Embryo-derived microRNAs thus represent a promising avenue for non-invasive embryo evaluation, with future clinical translation hinging on robust multicenter validation and integration with imaging, metabolomics, and artificial intelligence–based analytics.

1 Introduction

In assisted reproductive technology (ART), embryo selection remains one of the most crucial determinants of IVF success, directly influencing implantation and live birth rates. Traditional methods, such as morphological grading and time-lapse imaging, provide useful morphological insights but have limited predictive accuracy and cannot reliably distinguish euploid from aneuploid embryos (Wang et al., 2023; Zou et al., 2024; Sciorio et al., 2025). Preimplantation genetic testing for aneuploidy (PGT-A) offers direct chromosomal assessment and has improved selection efficiency; however, it is invasive, costly, and occasionally discordant with the embryo’s true genetic state due to mosaicism (Navarro-Sánchez et al., 2022; Cinnioglu et al., 2023; Huang et al., 2023). These limitations have prompted the search for non-invasive biomarkers that can complement or even replace invasive testing. Among the candidates, microRNAs (miRNAs) have recently attracted considerable attention. MiRNAs are short, non-coding RNA molecules (∼22 nucleotides) that regulate gene expression post-transcriptionally by targeting messenger RNAs, thereby controlling essential cellular processes such as proliferation, apoptosis, and differentiation (Esmaeilivand et al., 2024). Importantly, preimplantation embryos secrete miRNAs into their surrounding microenvironment, including spent culture medium (SCM) and blastocoel fluid (BF), making these molecules accessible for non-invasive analysis (Esmaeilivand et al., 2022b).

Several studies have reported variable expression of embryo-derived miRNAs associated with chromosomal status and implantation potential. For example, certain miRNA signatures detected in spent culture medium (SCM) have been correlated with blastocyst viability and developmental competence (Esmaeilivand et al., 2022a; Omes et al., 2024; Caponnetto et al., 2025) while profiling of BF has revealed distinct expression patterns related to chromosomal abnormalities (Esmaeilivand et al., 2022b). Despite these promising findings, significant challenges remain, including low RNA yield, contamination from maternal cells or culture reagents, and the absence of standardized analytical protocols (Jusic et al., 2024; Mukerjee et al., 2025). Therefore, a comprehensive synthesis of current evidence is warranted to clarify the clinical potential of embryo-secreted miRNAs. Relevant literature was identified through searches in PubMed, Scopus, and Web of Science (supplemented by manual searches in Google Scholar). The search covered January 2014 to September 2025 and included studies focusing on embryo-derived microRNAs in spent culture medium, blastocoel fluid, or trophectoderm biopsy associated with aneuploidy, embryo viability, or implantation outcomes. After screening and eligibility assessment, 42 studies were included in this review.

This review aims to summarize the role of embryo-secreted miRNAs as non-invasive biomarkers of embryo viability and chromosomal integrity, highlight recent methodological advances, and outline future directions toward their integration into routine IVF practice.

2 Sources of embryo-secreted microRNAs

MicroRNAs (miRNAs) from the embryo have been discovered to be secreted from a variety of biological compartments associated with in vitro fertilization (IVF), providing a range of non-invasive research opportunities (Esmaeilivand et al., 2022a). Spent culture medium (SCM) and blastocoel fluid (BF), which are direct secretions from the developing embryo, are two sources that have been widely explored (Esmaeilivand et al., 2022b; Campos and Nel-Themaat, 2024; Caponnetto et al., 2025). Additional sources, such as trophectoderm (TE) biopsy samples, follicular fluid, and endometrial or uterine fluid, have also been explored, though these are either invasive or indirectly reflect embryo physiology (Ibañez-Perez et al., 2022; Aoki et al., 2024; Esmaeilivand et al., 2024). The relative invasiveness and diagnostic value of these biological sources are illustrated in Figure 1.

FIGURE 1

2.1 Spent culture medium (SCM)

Embryos, as living organisms, are capable of secreting and releasing a variety of biological molecules, including microRNAs (miRNAs), into their surrounding culture medium. Several studies have demonstrated that the miRNA profile in spent culture medium (SCM) is linked to blastocyst morphology, developmental competence, and implantation potential. Therefore, determining the miRNA pattern in spent culture medium (SCM) can be used as a non-invasive biomarker to assess embryo quality in vitro fertilization (IVF) processes (Abu-Halima et al., 2020; Ovčar and Kovačič, 2024). However, the amount of RNA released into the culture medium is very low, which makes its extraction and accurate identification challenging. On the other hand, the possibility of sample contamination with RNA from peripheral cells, such as cumulus cells surrounding the oocyte or embryo, as well as compounds present in laboratory reagents, can lead to inaccurate or ambiguous results. Therefore, the precise isolation of embryo-specific miRNAs from other sources must be carefully performed to ensure reliable analyses (Voros et al., 2025c). Different biological sources of embryo-secreted miRNAs, together with their main advantages, limitations, and clinical relevance, are summarized in Table 1.

2.2 Blastocoel fluid (BF)

During blastocyst development, the blastocoel cavity enlarges, and the fluid within it contains extracellular vesicles enriched with miRNAs. These vesicles play a crucial role in cellular signaling and gene expression regulation during early embryonic development, indicating the presence of complex regulatory mechanisms that are initiated by the embryo (Rule et al., 2018; Esmaeilivand et al., 2022b; Campos and Nel-Themaat, 2024). Studies have shown that miRNA profiles in BF can show significant differences between euploid and aneuploid embryos. These differences imply the potential of BF to provide reliable and non-invasive biomarkers for the detection of chromosomal status and embryo competence (Battaglia et al., 2019; Esmaeilivand et al., 2022b). While the extraction and analysis of miRNA and DNA from blastocoel fluid (BF) is challenging due to the small sample size and the need for sensitive and precise methods, with the advancement of molecular technologies, this pathway has been proposed as a new strategy in assisted reproductive technology (ART) to improve the selection of high-quality embryos and increase the implantation success rate (Frisendahl et al., 2025).

2.3 Trophectoderm (TE) biopsy

Trophectoderm (TE) biopsy is used for preimplantation genetic testing (PGT-A). Although it is an invasive procedure, it remains the gold standard for genetic health screening and detection of chromosomal abnormalities in IVF (Tomic et al., 2022; Voros et al., 2025a). Some studies have shown that microRNA and mRNA can be extracted from trophectoderm samples, and specific molecular patterns associated with aneuploidy and implantation success can be identified (Esmaeilivand et al., 2024). This provides important biological evidence in confirming embryo-derived microRNAs detected in non-invasive samples such as blastocoel fluid (BF) or culture medium. This means that molecular findings from TE biopsy provide biological validity to data obtained from less invasive samples (Voros et al., 2025a). However, TE biopsy itself, due to its invasive nature, cannot replace non-invasive methods in clinical practice (Tomic et al., 2022).

2.4 Other sources

Follicular fluid is the environment in which the oocyte grows and develops and contains a collection of microRNAs. These miRNAs regulate gene expression in various pathways that control oocyte maturation, health, and fertility (Feng et al., 2015; Aoki et al., 2024). In diseases such as polycystic ovary syndrome, the microRNA profile in follicular fluid is altered, which can lead to reduced oocyte quality (Butler et al., 2019). In addition, the cumulus cells that surround the oocyte also produce and secrete microRNAs and extracellular vesicles containing these RNAs, which act as molecular messengers for communication with the oocyte and help regulate signaling pathways related to oocyte growth, maturation, and fertilization (Da Broi et al., 2018). Endometrial fluid is another important source of microRNAs that provide the uterine environment for embryo implantation. miRNAs in this fluid may play a role in the regulatory processes of implantation and embryo maintenance in the uterus; changes in the expression of these miRNAs may affect implantation success and pregnancy health (Voros et al., 2025c). While these fluids provide valuable insights into the reproductive microenvironment, they represent a mixture of maternal and embryonic contributions, which limits their specificity as embryo-derived biomarkers (Da Broi et al., 2018; Voros et al., 2025c). Nevertheless, these non-embryo-specific sources may hold promise in multi-omics strategies that integrate both embryonic and maternal signals for a more comprehensive assessment of implantation potential (Voros et al., 2025c).

3 miRNAs and chromosomal aneuploidy

Recent research has highlighted embryo-secreted microRNAs as promising indicators of chromosomal status (Almutlaq et al., 2024; Caponnetto et al., 2025; Toporcerová et al., 2025). Rosenbluth et al. (2014) reported that miR-191 levels were increased in the culture medium of aneuploid embryos. This miRNA has been associated with activation of the PI3K/AKT and MAPK pathways in other cellular models, suggesting a possible role in promoting cell survival and reducing apoptosis, which may allow chromosomally abnormal embryos to persist rather than undergo natural elimination. (Rosenbluth et al., 2014). Esmaeilivand et al. (2022a) reported differential expression of miR-661 and miR-20a between euploid and aneuploid blastocysts in blastocoel fluid (BF) (Esmaeilivand et al., 2022b). In addition, Omes et al. (2024) reported specific miRNAs, such as miR-21-5p and miR-661, in spent culture media that were linked not only to embryo developmental potential but also suggested a possible link to chromosomal constitution (Omes et al., 2024). Studies suggest that miR-20a plays an important role in G1/S progression by modulating the E2F/Cyclin D1 axis; its increased expression may lead to accelerated and insufficient passage through cell-cycle checkpoints (Trompeter et al., 2011). miR-661 has been linked to reduced cell adhesion and induction of epithelial-mesenchymal transition (EMT) in several cellular models, reflecting cellular instability and stress responses that may parallel chromosomal imbalance in embryos (Cuman et al., 2015). In addition, miR-21-5p may influence apoptosis-related signaling by targeting PTEN and modulating the PI3K/AKT pathway, as reported in other cellular contexts, which could support cell survival in embryos with chromosomal abnormalities (Zhang et al., 2015).

Also, Almutlaq et al. (2024) in their systematic review, reported that miR-19b, miR-517c, miR-518e, miR-522, miR-92a, and miR-106a were consistently downregulated in aneuploid blastocysts, reinforcing the significant association between miRNA patterns and chromosomal errors (Aoki et al., 2024). Among these, miR-19b, miR-92a, and miR-106a are members of the miR-17∼92 family, which has been suggested to regulate cell-cycle progression by modulating Cyclins and CDKs, as well as TGF-β and Wnt signaling pathways. Reduced expression of these miRNAs may impair checkpoint control and contribute to irregular chromosomal segregation (Concepcion et al., 2012). Esmaeilivand et al. (2024) analyzed trophectoderm biopsies and showed that miR-30c and miR-372 were downregulated in aneuploid blastocysts. miR-30c has been suggested to modulate the Wnt/β-catenin pathway involved in blastocyst development and differentiation, and its reduced expression may disrupt the coordination of cell division. On the other hand, miR-372, which belongs to the RAS/MAPK and TGF-β signaling axes, has been suggested to participate in maintaining the pluripotency of embryonic cells. Its reduced expression in aneuploid embryos may indicate a loss of the natural balance between pluripotency and differentiation (Esmaeilivand et al., 2024). The embryo-secreted microRNAs associated with chromosomal aneuploidy and checkpoint dysregulation are shown in Figure 2. Key miRNAs implicated in aneuploidy, their signaling pathways, and reported biological effects are listed in Table 2.

FIGURE 2

4 miRNAs and implantation potential

Successful implantation depends on a complex interplay between embryo competence and endometrial receptivity. Even chromosomally normal embryos may fail to implant due to inadequate molecular signaling or impaired embryo–endometrium crosstalk (Kim and Kim, 2017; Lacconi et al., 2024). Recent studies suggest that microRNAs (miRNAs) secreted by embryos and present in maternal reproductive fluids may serve as biomarkers of implantation potential (Mutia et al., 2023; Qi et al., 2023).

Rosenbluth et al. (2014) demonstrated that human embryos release miR-191 into the culture medium and that its levels are associated with implantation outcomes. This miRNA has been linked to activation of the PI3K/AKT and MAPK pathways, which promote cell survival and inhibit apoptosis, potentially supporting the persistence of embryos capable of implantation (Rosenbluth et al., 2014). In addition, Battaglia et al. (2019) showed that miRNAs in blastocoel fluid (BF) are connected to the Wnt/β-catenin and RAS/MAPK pathways and that changes in their expression can disrupt the balance between pluripotency and differentiation and affect implantation capacity (Battaglia et al., 2019).

Berkhout et al. (2020) reported that high-quality embryos secrete miR-320a, this miRNA enhances the migration of endometrial stromal cells (hESCs) and prepares the receptive field for implantation through cytoskeletal rearrangement and activation of genes related to cell motility (Berkhout et al., 2020). Wang et al. (2021) identified three miRNAs—miR-199a-5p, miR-483-5p, and miR-432-5p—in the spent culture medium (SCM) of human embryos, whose expression was associated with successful implantation. Pathway analysis suggested that these miRNAs were involved in cell-cycle regulation and apoptosis, indicating their potential as markers of blastocyst growth and viability (Wang et al., 2021). Kamijo et al. (2022) analyzed more than 60 blastocysts and detected 53 distinct miRNAs. Eight miRNAs, including miR-191-5p, miR-320a, miR-92a-3p, miR-509-3p, miR-378a-3p, miR-28-3p, miR-512-5p, and miR-181a-5p, showed the strongest association with implantation outcomes. Pathway enrichment analysis indicated that these miRNAs are involved in PI3K/AKT, MAPK/ERK, cell-cycle, and survival pathways, suggesting that their coordinated activity may influence implantation potential (Kamijo et al., 2022). Recently, Caponnetto et al. (2025) compared miRNAs derived from blastocoel fluid (BF) and spent culture medium (SCM), revealing distinct expression patterns between the two sources. Some miRNAs specific to spent culture medium (SCM) were associated with endometrial receptivity, adhesion, and invasion pathways (Caponnetto et al., 2025).

Additionally, studies have shown that uterine fluid miRNA profiles are disrupted in women with recurrent implantation failure, suggesting a disruption in the embryo-endometrial communication (von Grothusen et al., 2022). Similarly, specific miRNA expression in endometrial fluid can non-invasively identify an implanting (receptive) endometrium (Ibañez-Perez et al., 2022). In addition, distinct plasma miRNA patterns during the preimplantation period are associated with biochemical pregnancy loss after embryo transfer, reinforcing the role of systemic signaling of miRNAs in early pregnancy outcomes (Shen et al., 2024). However, the molecular overlap between implantation-related miRNAs and those linked to general embryo stress complicates their interpretation. For example, Soczewski et al. (2023) reported that miR-21-5p (among others) is involved in both stress/inflammatory pathways and implantation-associated signaling, highlighting this overlap(Soczewski et al., 2023). A schematic representation of the main embryo and endometrium-derived miRNAs and their signaling interactions during implantation is shown in Figure 3. miRNAs associated with implantation potential, their roles, and sample origins are presented in Table 3.

FIGURE 3

5 Methods of miRNA profiling in IVF

Accurate detection and quantification of embryo-derived microRNAs in non-invasive samples such as spent culture medium (SCM) or blastocoel fluid (BF) remain challenging because of the extremely low RNA content and limited sample volume (Roest et al., 2021). To overcome these limitations, several analytical platforms have been developed, each offering distinct advantages and constraints. Among them, qRT-PCR remains the most widely used because of its high sensitivity, specificity, and relatively low cost. This approach enables detailed analysis of known miRNAs but has limited capacity to identify novel or low-abundance species (Kroh et al., 2010). Microarray technology allows simultaneous screening of hundreds of miRNAs and offers broader coverage than qRT-PCR, although its lower sensitivity and background hybridization noise can affect data quality (Wang et al., 2012). Next-generation sequencing (NGS) provides the most comprehensive overview, enabling unbiased discovery of known and novel miRNAs, detection of isoforms, and identification of sequence variants. However, it requires specialized bioinformatics expertise, high-quality input material, and remains expensive. Building on these approaches, digital PCR (dPCR) has recently been introduced for absolute quantification of embryo-derived miRNAs, offering higher precision than conventional qRT-PCR (Whale et al., 2012). Although not yet widely adopted in reproductive medicine, dPCR shows strong potential for rapid and clinically scalable applications (Busato et al., 2025).

Regardless of the platform used, challenges such as low RNA extraction efficiency, risk of contamination, lack of consensus on normalization, and absence of standardized laboratory protocols still hinder reproducibility and inter-study comparisons (Moldovan et al., 2014; Roest et al., 2021). Overall, developing standardized procedures for isolation, quantification, and analysis of miRNA data is a key prerequisite for integrating these technologies into routine IVF practice. The advantages, limitations, and recommended applications of the major miRNA profiling methods are summarized in Table 4.

6 Limitations and challenges

Although microRNAs have emerged as promising non-invasive biomarkers for assessing embryo viability and chromosomal status, several obstacles still prevent their entry into routine use in IVF. Embryo-derived miRNAs are present at extremely low levels, often near the detection limits of current assays (Kroh et al., 2010; Moldovan et al., 2014; Roest et al., 2021). Variability in RNA extraction efficiency, reverse transcription, and amplification introduces technical noise and compromises reproducibility (Moldovan et al., 2014). Spent culture medium (SCM) and blastocoel fluid (BF) are often contaminated by maternal cells, sperm remnants, or culture reagents, so distinguishing embryo-specific miRNAs from background signals therefore remains a critical challenge (Rosenbluth et al., 2013; Roest et al., 2021).

Studies employ different RNA isolation kits, input volumes, and normalization strategies. This heterogeneity limits comparability across studies, thereby preventing meta-analysis (Moldovan et al., 2014). Embryo-to-embryo heterogeneity and developmental stage–specific expression patterns further complicate interpretation (Esmaeilivand et al., 2022b). Moreover, some miRNAs associated with aneuploidy or implantation failure overlap with stress-induced miRNAs, undermining specificity (Soczewski et al., 2023). Most studies to date have small sample sizes and lack prospective validation (Caponnetto et al., 2025). Consequently, predictive accuracy remains moderate, and no consensus panel of miRNAs has yet been established. The integration of miRNA testing into IVF workflows would require robust, rapid, and cost-effective assays. Current technologies, such as NGS, are costly and time-consuming, thus posing barriers to clinical translation (Roest et al., 2021).

6.1 Normalization challenges in embryo-derived miRNA studies

Normalization of miRNA expression in embryo culture samples remains a key limitation when comparing results across studies. Variations in RNA extraction yield, reverse transcription efficiency, and amplification bias can introduce significant error, especially given the extremely low RNA content of embryo-derived media. Some researchers use exogenous spike-ins, such as cel-miR-39, to adjust for technical differences, while others rely on endogenous reference miRNAs, including miR-16 or miR-191, that appear relatively stable in preimplantation settings. For example, Hawke et al. (2023) evaluated miR-16, miR-191, and miR-106 as stable references for preimplantation mouse embryos (Hawke et al., 2023). In addition, several studies have applied global mean or multi-gene normalization strategies supported by algorithms like NormFinder and geNorm to improve reproducibility. Achieving consensus on standardized normalization approaches will be essential for future clinical translation of embryo-secreted miRNA analyses.

7 Future directions

Future research should prioritize the development of standardized protocols for sample collection, RNA isolation, normalization strategies, and data interpretation to improve reproducibility across embryo-derived microRNA studies. The lack of harmonized methodologies remains a major barrier to cross-study comparison and clinical translation, underscoring the need for consensus-driven technical guidelines (Moldovan et al., 2014; Roest et al., 2021).

Prospective, multicenter clinical studies with adequately powered cohorts are essential to validate candidate miRNA panels and to establish clinically actionable thresholds for embryo selection (Rosenbluth et al., 2014; Kamijo et al., 2022). Such validation frameworks will be critical for distinguishing biologically meaningful signals from stress-related or culture-induced miRNA expression.

Technological advances, particularly digital PCR and microfluidic-based platforms, offer promising opportunities for rapid and sensitive detection of low-abundance embryo-secreted miRNAs and may enable same-cycle embryo assessment in IVF laboratories (Whale et al., 2012; Caponnetto et al., 2025). In parallel, integrating miRNA profiling with complementary modalities such as time-lapse imaging, metabolomics, and artificial intelligence–based modeling may enhance predictive accuracy beyond single-parameter approaches (Siristatidis et al., 2021; Gao et al., 2025).

Finally, the translation of miRNA-based embryo assessment tools into routine IVF practice should be guided by robust regulatory frameworks and ethical oversight to ensure embryo safety, data integrity, and equitable clinical implementation (Voros et al., 2025b).

8 Conclusion

Accumulating evidence indicates that embryos actively secrete microRNAs into their surrounding environment, particularly into spent culture medium and blastocoel fluid, and that distinct miRNA signatures are associated with chromosomal status and implantation potential. These findings highlight embryo-derived miRNAs as promising non-invasive biomarkers that may complement existing embryo assessment strategies. However, current evidence remains largely observational, and technical challenges, including low RNA yield, contamination risk, and lack of standardized protocols, limit immediate clinical implementation. Continued methodological refinement and large-scale validation will be essential to define the clinical utility of miRNA-based embryo evaluation in IVF.

References

• 1

  Abu-HalimaM.KhaizaranZ. A.AyeshB. M.FischerU.KhaizaranS. A.Al-BattahF.et al (2020). MicroRNAs in combined spent culture media and sperm are associated with embryo quality and pregnancy outcome. Fertil. Steril.113 (5), 970–980.e972. 10.1016/j.fertnstert.2019.12.028

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 2

  AlmutlaqA.Viñals GonzalezX.SenGuptaS. (2024). Differentially expressed microRNAs in aneuploid preimplantation blastocysts: a systematic review. Front. Reprod. Health6, 1370341. 10.3389/frph.2024.1370341

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 3

  AokiS.InoueY.HaraS.ItouJ.ShirasunaK.IwataH. (2024). microRNAs associated with the quality of follicular fluids affect oocyte and early embryonic development. Reprod. Med. Biol.23 (1), e12559. 10.1002/rmb2.12559

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 4

  BattagliaR.PaliniS.VentoM. E.La FerlitaA.Lo FaroM. J.CaroppoE.et al (2019). Identification of extracellular vesicles and characterization of miRNA expression profiles in human blastocoel fluid. Sci. Rep.9 (1), 84. 10.1038/s41598-018-36452-7

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 5

  BerkhoutR. P.KeijserR.ReppingS.LambalkC. B.AfinkG. B.MastenbroekS.et al (2020). High-quality human preimplantation embryos stimulate endometrial stromal cell migration via secretion of microRNA hsa-miR-320a. Hum. Reprod.35 (8), 1797–1807. 10.1093/humrep/deaa149

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 6

  BusatoF.UrsueguiS.DeleuzeJ. F.TostJ. (2025). Multiplex digital PCR for the simultaneous quantification of a miRNA panel. Anal. Chim. Acta1335, 343440. 10.1016/j.aca.2024.343440

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 7

  ButlerA. E.RamachandranV.HayatS.DarghamS. R.CunninghamT. K.BenurwarM.et al (2019). Expression of microRNA in follicular fluid in women with and without PCOS. Sci. Rep.9 (1), 16306. 10.1038/s41598-019-52856-5

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 8

  CamposG.Nel-ThemaatL. (2024). Blastocoel fluid as an alternative source of DNA for minimally invasive PGT and biomarker of embryo competence. Reprod. Biomed. Online49 (4), 104322. 10.1016/j.rbmo.2024.104322

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 9

  CaponnettoA.FerraraC.FazzioA.CarliL.BarbagalloC.StellaM.et al (2025). MicroRNAs secreted by the embryo in spent culture medium can regulate mRNAs involved in endometrial receptivity, embryo attachment, and invasion. Int. J. Mol. Sci.26 (18), 8879. 10.3390/ijms26188879

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 10

  CinniogluC.GlessnerH.JordanA.BunshaftS. (2023). A systematic review of noninvasive preimplantation genetic testing for aneuploidy. Fertil. Steril.120 (2), 235–239. 10.1016/j.fertnstert.2023.06.013

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 11

  ConcepcionC. P.BonettiC.VenturaA. (2012). The MicroRNA-17-92 family of MicroRNA clusters in development and disease. Cancer J.18 (3), 262–267. 10.1097/PPO.0b013e318258b60a

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 12

  CumanC.Van SinderenM.GantierM. P.RainczukK.SorbyK.RombautsL.et al (2015). Human blastocyst secreted microRNA regulate endometrial epithelial cell adhesion. eBioMedicine2 (10), 1528–1535. 10.1016/j.ebiom.2015.09.003

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 13

  Da BroiM. G.GiorgiV. S. I.WangF.KeefeD. L.AlbertiniD.NavarroP. A. (2018). Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications. J. Assist. Reprod. Genet.35 (5), 735–751. 10.1007/s10815-018-1143-3

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 14

  EsmaeilivandM.AbedelahiA.HamdiK.FarzadiL.GoharitabanS.FattahiA.et al (2022a). Role of miRNAs in preimplantation embryo development and their potential as embryo selection biomarkers. Reprod. Fertil. Dev.34 (8), 589–597. 10.1071/rd21274

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 15

  EsmaeilivandM.FattahiA.AbedelahiA.HamdiK.FarzadiL.GoharitabanS.et al (2022b). microRNAs in the blastocoel fluid as accessible indicators of chromosomal normality. Reprod. Biol.22 (4), 100695. 10.1016/j.repbio.2022.100695

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 16

  EsmaeilivandM.GhasemzadehA.NiknafsB.FattahiA. (2024). Association of trophectoderm mRNAs and MicroRNAs with chromosomal aneuploidy of embryo. Reprod. Sci.31 (4), 1028–1033. 10.1007/s43032-023-01381-y

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 17

  FengR.SangQ.ZhuY.FuW.LiuM.XuY.et al (2015). MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro. Sci. Rep.5, 8689. 10.1038/srep08689

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 18

  FrisendahlC.Gemzell-DanielssonK.NairK.KochF.MenezesJ.SheikiM.et al (2025). Blastocoel fluid RNA predicts pregnancy outcome in assisted reproduction. bioRxiv2025 (2003), 641486.

  • Google Scholar
  • View reference in article

• 19

  GaoY.YuanY.WangK.WangY.GaoT.YangY.et al (2025). Current progress and open challenges for applying artificial intelligence across the in vitro fertilization cycle. Patterns6, 101347. 10.1016/j.patter.2025.101347

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 20

  HawkeD. C.WatsonA. J.BettsD. H. (2023). Selecting normalizers for MicroRNA RT-qPCR expression analysis in murine preimplantation embryos and the associated conditioned culture media. J. Dev. Biol.11 (2), 17. 10.3390/jdb11020017

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 21

  HuangB.LuoX.WuR.QiuL.LinS.HuangX.et al (2023). Evaluation of non-invasive gene detection in preimplantation embryos: a systematic review and meta-analysis. J. Assist. Reprod. Genet.40 (6), 1243–1253. 10.1007/s10815-023-02760-9

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 22

  Ibañez-PerezJ.Díaz-NuñezM.Clos-GarcíaM.LainzL.IglesiasM.Díez-ZapirainM.et al (2022). microRNA-based signatures obtained from endometrial fluid identify implantative endometrium. Hum. Reprod.37 (10), 2375–2391. 10.1093/humrep/deac184

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 23

  JusicA.ErpapazoglouZ.DalgaardL. T.LakkistoP.de Gonzalo-CalvoD.BenczikB.et al (2024). Guidelines for mitochondrial RNA analysis. Mol. Ther. - Nucleic Acids35 (3), 102262. 10.1016/j.omtn.2024.102262

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 24

  KamijoS.HamataniT.SasakiH.SuzukiH.AbeA.InoueO.et al (2022). MicroRNAs secreted by human preimplantation embryos and IVF outcome. Reprod. Biol. Endocrinol.20 (1), 130. 10.1186/s12958-022-00989-0

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 25

  KimS. M.KimJ. S. (2017). A review of mechanisms of implantation. Dev. Reprod.21 (4), 351–359. 10.12717/dr.2017.21.4.351

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 26

  KrohE. M.ParkinR. K.MitchellP. S.TewariM. (2010). Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods50 (4), 298–301. 10.1016/j.ymeth.2010.01.032

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 27

  LacconiV.MassimianiM.CarrieroI.BiancoC.TicconiC.PavoneV.et al (2024). When the embryo meets the endometrium: identifying the features required for successful embryo implantation. Int. J. Mol. Sci.25 (5), 2834. 10.3390/ijms25052834

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 28

  MoldovanL.BatteK. E.TrgovcichJ.WislerJ.MarshC. B.PiperM. (2014). Methodological challenges in utilizing miRNAs as circulating biomarkers. J. Cell Mol. Med.18 (3), 371–390. 10.1111/jcmm.12236

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 29

  MukerjeeN.BhattacharyaA.MaitraS.KaurM.GanesanS.MishraS.et al (2025). Exosome isolation and characterization for advanced diagnostic and therapeutic applications. Mater Today Bio31, 101613. 10.1016/j.mtbio.2025.101613

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 30

  MutiaK.WiwekoB.AbinawantoA.DwirantiA.BowolaksonoA. (2023). microRNAs as A biomarker to predict embryo quality assessment in in vitro fertilization. Int. J. Fertil. Steril.17 (2), 85–91. 10.22074/ijfs.2022.551571.1285

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 31

  Navarro-SánchezL.García-PascualC.RubioC.SimónC. (2022). Non-invasive preimplantation genetic testing for aneuploidies: an update. Reprod. Biomed. Online44 (5), 817–828. 10.1016/j.rbmo.2022.01.012

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 32

  OmesC.ContiA.BenedettiL.TomasoniV.De MarchiD.NappiR. E.et al (2024). Expression of miRNA from spent pre-implantation embryos culture media. Reprod. Biol.24 (2), 100847. 10.1016/j.repbio.2023.100847

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 33

  OvčarA.KovačičB. (2024). Biogenesis of extracellular vesicles (EVs) and the potential use of embryo-derived EVs in medically assisted reproduction. Int. J. Mol. Sci.26 (1), 42. 10.3390/ijms26010042

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 34

  QiS.KodithuwakkuS. P.PangR. T. K.ChiuP. C. N.TangM. H. Y.LeeK.-F. (2023). Role of microRNAs in embryo–endometrial interactions: biological functions and clinical applications. Reproductive Dev. Med.7 (4), 238–251. 10.1097/rd9.0000000000000073

  • CrossRef
  • Google Scholar
  • View reference in article

• 35

  RoestH. P.IjzermansJ. N. M.van der LaanL. J. W. (2021). Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids. BMC Biotechnol.21 (1), 48. 10.1186/s12896-021-00706-6

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 36

  RosenbluthE. M.SheltonD. N.SparksA. E.DevorE.ChristensonL.Van VoorhisB. J. (2013). MicroRNA expression in the human blastocyst. Fertil. Steril.99 (3), 855–861.e853. 10.1016/j.fertnstert.2012.11.001

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 37

  RosenbluthE. M.SheltonD. N.SparksA. E.DevorE.ChristensonL.Van VoorhisB. J. (2014). Human embryos secrete microRNAs into culture media—A potential biomarker for implantation. Fertil. Steril.101 (5), 1493–1500. 10.1016/j.fertnstert.2014.01.058

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 38

  RuleK.ChosedR. J.Arthur ChangT.David WiningerJ.RoudebushW. E. (2018). Relationship between blastocoel cell-free DNA and day-5 blastocyst morphology. J. Assist. Reprod. Genet.35 (8), 1497–1501. 10.1007/s10815-018-1223-4

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 39

  SciorioR.TramontanoL.GulloG. (2025). Use of time-lapse technology and artificial intelligence in the embryology laboratory: an updated review. JBRA Assist. Reprod.29 (2), 338–350. 10.5935/1518-0557.20250019

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 40

  ShenL.ZengH.FuY.MaW.GuoX.LuoG.et al (2024). Specific plasma microRNA profiles could be potential non-invasive biomarkers for biochemical pregnancy loss following embryo transfer. BMC Pregnancy Childbirth24 (1), 351. 10.1186/s12884-024-06488-x

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 41

  SiristatidisC.StavrosS.DrakeleyA.BettocchiS.PouliakisA.DrakakisP.et al (2021). Omics and artificial intelligence to improve in vitro fertilization (IVF) success: a proposed protocol. Diagn. (Basel)11 (5), 743. 10.3390/diagnostics11050743

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 42

  SoczewskiE.Murrieta-CoxcaJ. M.MirandaL.Fuentes-ZacaríasP.Gutiérrez-SamudioR.GrassoE.et al (2023). miRNAs associated with endoplasmic reticulum stress and unfolded protein response during decidualization. Reprod. Biomed. Online47 (5), 103289. 10.1016/j.rbmo.2023.103289

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 43

  TomicM.Vrtacnik BokalE.StimpfelM. (2022). Non-invasive preimplantation genetic testing for aneuploidy and the mystery of genetic material: a review article. Int. J. Mol. Sci.23 (7), 3568. 10.3390/ijms23073568

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 44

  ToporcerováS.BadovskáZ.KrivákováE.MikulováV.MarekováM.AltmäeS.et al (2025). Embryo secretome in predicting embryo quality and IVF treatment outcome. Reprod. Biomed. Online51 (1), 104825. 10.1016/j.rbmo.2025.104825

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 45

  TrompeterH.-I.AbbadH.IwaniukK. M.HafnerM.RenwickN.TuschlT.et al (2011). MicroRNAs MiR-17, MiR-20a, and MiR-106b act in concert to modulate E2F activity on cell cycle arrest during neuronal lineage differentiation of USSC. PLOS ONE6 (1), e16138. 10.1371/journal.pone.0016138

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 46

  von GrothusenC.FrisendahlC.ModhukurV.LalitkumarP. G.PetersM.FaridaniO. R.et al (2022). Uterine fluid microRNAs are dysregulated in women with recurrent implantation failure. Hum. Reprod.37 (4), 734–746. 10.1093/humrep/deac019

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 47

  VorosC.DarlasM.AthanasiouD.AthanasiouA.AthanasiouA.BananisK.et al (2025a). Evaluation of the effectiveness and accuracy of non-invasive preimplantation genetic testing (niPGT) compared to invasive embryo biopsy. Biomedicines13 (8), 2010. 10.3390/biomedicines13082010

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 48

  VorosC.VarthalitiA.AthanasiouD.MavrogianniD.BananisK.AthanasiouA.et al (2025b). MicroRNA signatures in endometrial receptivity—unlocking their role in embryo implantation and IVF success: a systematic review. Biomedicines13 (5), 1189. 10.3390/biomedicines13051189

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 49

  VorosC.VarthalitiA.AthanasiouD.MavrogianniD.PapahliouA. M.BananisK.et al (2025c). The whisper of the follicle: a systematic review of micro ribonucleic acids as predictors of oocyte quality and in vitro fertilization outcomes. Cells14 (11), 787. 10.3390/cells14110787

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 50

  WangK.YuanY.ChoJ.-H.McClartyS.BaxterD.GalasD. J. (2012). Comparing the MicroRNA spectrum between serum and plasma. PLOS ONE7 (7), e41561. 10.1371/journal.pone.0041561

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 51

  WangS.ChenL.ZhuY.JiangW. (2021). Characterization of microRNAs in spent culture medium associated with human embryo quality and development. Ann. Transl. Med.9 (22), 1648. 10.21037/atm-21-5029

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 52

  WangJ.GuoY.ZhangN.LiT. (2023). Research progress of time-lapse imaging technology and embryonic development potential: a review. Med. Baltim.102 (38), e35203. 10.1097/md.0000000000035203

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 53

  WhaleA. S.HuggettJ. F.CowenS.SpeirsV.ShawJ.EllisonS.et al (2012). Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res.40 (11), e82. 10.1093/nar/gks203

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 54

  ZhangC.ShiY. R.LiuX. R.CaoY. C.ZhenD.JiaZ. Y.et al (2015). The anti-apoptotic role of berberine in preimplantation embryo in vitro development through regulation of miRNA-21. PLoS One10 (6), e0129527. 10.1371/journal.pone.0129527

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article

• 55

  ZouH.WangR.MorbeckD. E. (2024). Diagnostic or prognostic? Decoding the role of embryo selection on in vitro fertilization treatment outcomes. Fertil. Steril.121 (5), 730–736. 10.1016/j.fertnstert.2024.01.005

  • Pubmed Abstract
  • CrossRef
  • Google Scholar
  • View reference in article