Keywords: Embryo selection, Invasive, Noninvasive, Single embryo transfer

Introduction

Since the first birth via in vitro fertilization (IVF) in 1978 [1], considerable advances in assisted reproductive technologies (ART) have been made worldwide. Currently, approximately 1% to 4% of all newborns are conceived through ART [1]. Excessive controlled ovarian stimulation in women undergoing IVF often results in the formation of multiple embryos, yet this approach is still associated with low implantation rates [2]. While the simultaneous transfer of multiple embryos improves pregnancy rates, it also increases the risk of multiple pregnancies, which can lead to preterm birth, low birth weight, and an elevated likelihood of certain congenital anomalies [3]. In many European countries, legal restrictions cap the number of embryos that can be transferred in a single cycle, but such regulations are not common elsewhere in the world. Consequently, a pressing need exists for straightforward strategies to identify a competent embryo, thereby improving implantation rates and the chance of a successful live birth. Such an approach could reduce the incidence of multiple births and alleviate the associated financial costs, as well as the emotional distress linked to anxiety, depression, and stress following unsuccessful IVF treatments.

In recent decades, the selection of embryos has relied on morphological assessment [4]. However, this approach has limitations, including subjectivity, considerable variability among embryologists, suboptimal culture conditions, and a weak correlation with embryo implantation potential. Thus, concerted efforts have been made to improve the evaluation of embryo quality [5]. These advancements encompass both invasive and noninvasive methods, each with a set of advantages and disadvantages (Tables 1 and 2). Time-lapse systems (TLSs) that employ artificial intelligence (AI) and analyze cell kinetics have been developed to better assess embryo quality [6]. Preimplantation genetic testing (PGT) enables the prediction of chromosomal abnormalities prior to embryo transfer. Nevertheless, the invasiveness of the involved biopsy, the risk of mosaicism, and the associated high costs have prompted researchers to explore alternative strategies for embryo selection [7].

Noninvasive techniques that preserve embryo integrity have attracted attention. These methods are focused on analyzing the culture medium of individual embryos, including the assessment of biomolecule production or release. Noninvasive techniques encompass metabolic, proteomic, genomic, and transcriptomic profiling, as well as the measurement of oxygen consumption and oxidative stress status in the embryo culture medium [8]. However, these technologies face several challenges, such as the complex mixture of molecular components in the culture medium, difficulty in detecting key embryonic molecules present at very low concentrations [9], the cost of equipment, and the methodological complexity. As a result, these techniques are not yet suitable for routine clinical application. To improve the outcomes of IVF, embryo evaluation methods used in clinical settings must be user-friendly and readily available in the laboratory. Accordingly, the assessment of embryo quality has advanced considerably in recent years and has become a focal point of research. This article highlights current strategies for embryo selection and explores some of the most promising research areas that aim to improve the rates of single embryo transfer (SET) and increase the likelihood of achieving a full-term, healthy birth.

Invasive methods of embryo selection

Invasive methods can be helpful in selecting a competent embryo [10]. One of the most commonly used invasive tests is PGT, with the first pregnancies resulting from PGT reported in 1990 [11]. PGT involves taking a cellular biopsy from a human oocyte or embryo during an IVF cycle, analyzing its genetic composition, and using this information to select the most suitable embryos for subsequent uterine transfer [12]. Currently, PGT is employed to evaluate chromosomal aneuploidy (PGT-A), structural rearrangements (PGT-SR), and monogenic (single-gene) disorders (PGT-M). PGT-A detects embryos with de novo aneuploidy, such as subchromosomal deletions and duplications, in couples with chromosomally normal embryos. In theory, by not transferring these embryos, the risk of miscarriage and complications associated with pregnancy failure is reduced, and the likelihood of a successful pregnancy is increased [13]. This review will focus on the development of PGT-A for embryo selection, a technique that has been adopted by fertility clinics (Figure 1).

1. Biopsy procedures in the IVF laboratory

1. Biopsy procedures in the IVF laboratory

1) Blastomere biopsy

1) Blastomere biopsy

Most normally growing embryos undergo a blastomere biopsy (involving one to two blastomeres) on the third day after fertilization, when they have reached the six- to eight-cell stage. While two-cell biopsy is more accurate, it may compromise embryo survival because it involves removing approximately 30% of the embryo’s total cell mass. In contrast, single-cell biopsy can lead to false or inaccurate diagnoses [14]. During the cleavage stage, only a limited amount of cellular material is available for genetic analysis. However, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) technologies have been well established for single-cell analysis. FISH was the first technique used for PGT in couples with an X-linked disorder [15]. This method is employed to evaluate structural and numerical chromosome abnormalities, through the screening of only eight chromosomes, in day-3 embryos for both clinical PGT and research purposes [16]. Mosaicism at the cleavage stage remains a substantial challenge for chromosomal aneuploidy analysis, increasing the risk of both false negatives and false positives [17]. In two randomized controlled trials conducted in 2007 and 2011 using FISH, the findings indicated that FISH did not improve the live birth rate. In fact, the pregnancy rate in the PGT-FISH group was lower than that in the control group (32% vs. 42%) [18,19]. Due to the limitations of FISH, comparative genomic hybridization (CGH) has been successfully employed to identify a range of unbalanced chromosomal complements in prenatal samples, leading to its first successful live birth being reported in 2001 [20]. CGH offers an advantage over FISH in that it can assess copy number variation across all chromosomes, whereas FISH is limited to detecting a specific subset of chromosomal locations. However, CGH cannot detect triploidy/tetraploidy or balanced structural rearrangements [21,22].

In recent years, the emergence of next-generation sequencing (NGS) technologies has helped advance PGT. After embryo biopsy and whole-genome amplification, the amplified DNA is fragmented into millions of small pieces, which are then sequenced in parallel and aligned to the reference sequence of the human genome [23]. The number of sequenced fragments from each chromosome provides a precise measure of the sample’s copy number. NGS can detect balanced translocations, small deletions, and mosaic embryos [24]. In one study, cleavage-stage embryo biopsies with NGS were conducted in a total of 45 patients. The findings showed that the clinical pregnancy rate per transfer was higher in the NGS group than among control participants (84.4% vs. 41.5%). Additionally, the implantation rate was greater in the NGS group (61.5% vs. 34.8%), while the miscarriage rates between the two groups were comparable (2.8% vs. 4.6%) [25]. Ye et al. [26] explored the impact of NGS on IVF outcomes across 169 cycles in patients with reduced ovarian reserve and a limited number of embryos. The study revealed higher pregnancy and live birth rates in the group with NGS-evaluated cleavage-stage embryos compared to the control group, though the implantation rates were similar [26]. NGS has become widely adopted in contemporary PGT laboratories due to its high accuracy, rapid turnaround, cost-effectiveness, and broad compatibility [21]. However, studies of pregnancy outcomes following the transfer of mosaic embryos have yielded varying results. A 2017 study reported that the miscarriage rate for euploid embryos (identified by NGS) was lower than that for mosaic embryos, and mosaic embryos with 40% to 80% abnormal cells exhibited a higher implantation rate than those with less than 40% abnormal cells [27]. Conversely, a 2018 study found no significant difference in ongoing pregnancy or miscarriage rates between mosaic embryo transfers at any aneuploidy threshold [28]. While the literature clearly indicates that euploid embryos result in better pregnancy outcomes than mosaic embryos, it remains uncertain whether the degree of mosaicism in trophectoderm (TE) biopsy can predict ongoing pregnancy. Another notable point from the literature is the potential for euploid embryos to be misclassified as mosaic, which may then self-correct and develop normally. Conversely, some embryos identified as mosaic may not truly exhibit mosaicism, and this diagnostic inaccuracy could stem from technical issues [29]. Consequently, concern exists regarding the possibility of discarding normal embryos due to the misinterpretation of these results.

2) Blastocyst biopsy

2) Blastocyst biopsy

The genome of a blastocyst stage embryo is already activated, enabling precise examination [30]. Typically, analysis involves the removal of five to eight TE cells, a process that does not endanger the survival of the blastocyst.

The primary limitation of blastocyst biopsy is that only a small percentage of embryos reach the blastocyst stage in vitro. Consequently, very few embryos are typically available for biopsy. Additionally, the precision of the diagnosis can be compromised if the TE cells, from which the biopsy is taken, exhibit genetic differences from the inner cell mass (ICM). This discrepancy has been observed in 1% of gestations; in such cases, the chromosomal status of the embryo differs from that of the placenta, a condition known as confined placental mosaicism [31].

A 2013 study reported that cleavage-stage biopsy resulted in a significant reduction in the implantation rate compared to the control group (30% vs. 50%). Additionally, the implantation rate for blastocyst embryos was similar to that of control embryos (51% vs. 54%) [32]. These findings suggest that cleavage-stage biopsy may negatively impact the growth potential of the embryo. Consequently, TE biopsy has become the preferred method for performing PGT. In a 2018 study involving over 900 cycles, pregnancy and live birth rates were significantly higher for embryo transfers using NGS compared to those employing array CGH [33]. A 2019 study employed NGS to evaluate the blastocysts of 247 patients undergoing IVF who were under 35 years old. The results indicated that the clinical pregnancy rate in the NGS group was 71.7%, compared to 52.1% in the group evaluated based on morphology. Thus, NGS appears to represent an effective and high-throughput technology for selecting single blastocysts for transfer, with positive impacts on the clinical and ongoing pregnancy outcomes of SET in patients undergoing IVF [34]. While the primary goal of PGT-A is to improve live birth rates by selecting euploid embryos, consistent results across studies using PGT-A for all couples undergoing IVF—including women aged 35 to 40 years—have not been reported. Based on data from various randomized trials, its broad applicability to all patient groups is still unclear [35,36].

3) Polar body biopsy

3) Polar body biopsy

The polar body biopsy technique was first utilized to diagnose chromosomal abnormalities by Verlinsky et al. [37] in the 1990s. This method involves removing the first and/or second polar bodies to indirectly infer the genetic composition of the oocyte from the polar bodies. Polar body biopsy is ineffective if the mother is homozygous for an autosomal recessive disorder and the father is a carrier, as the mutation will be present in all of the mother’s oocytes and polar bodies. If only one recombination event occurs within the specific gene region, the single chromosome will not possess both alleles. This problem can be addressed by isolating both polar bodies and analyzing them. However, since polar body analysis cannot detect anomalies that arise after the fusion of male and female pronuclei, it has limited utility in predicting the genetic condition of the embryo, such as mitotic anomalies. Polar body biopsy performed after meiosis I can also detect meiotic aneuploidies that may self-correct following meiosis II [38].

To assess the impact of polar body analysis on live birth rates, Feichtinger et al. [39] utilized CGH-PGT. The live birth rates achieved in the CGH-PGT group were 26.4% per embryo and 35.7% per patient, significantly exceeding those observed in the control group. These findings underscore the benefits of CGH for couples undergoing IVF, as well as the effectiveness and appropriateness of the polar body pooling strategy [39]. By selecting euploid eggs, it is possible to improve the likelihood of a live birth following IVF, even in cases with poor prognosis.

4) Blastocoel fluid biopsy

4) Blastocoel fluid biopsy

Due to the inherently invasive nature of PGT-A, many researchers have begun to explore the potential of less invasive or noninvasive PGT (ni-OGT) methods. The blastocoelic fluid (BF) is a potential source of embryonic DNA. It can be extracted from the blastocyst before vitrification to protect the embryo from the formation of ice crystals, which can damage the cell membrane [40]. Notably, the aspiration of activated BF does not affect the structure of the embryo, resulting in high survival rates for embryos with good and poor morphology alike [41]. Furthermore, it appears to be compatible with common IVF laboratory processes. Since the discovery of cell-free amplifiable DNA in BF, numerous studies have suggested that the BF can be analyzed using CGH or NGS for detailed chromosome analysis [42]. In a 2018 study, researchers investigated the combination of BF and culture medium to assess the aneuploidy of blastocyst embryos [43]. This combined approach demonstrated a high level of total chromosome copy number concordance when compared to whole embryo biopsy using NGS. Although ni-PGT may represent a less burdensome and more cost-effective testing method, further research is necessary to confirm the accuracy of the results, as well as to determine the associated pregnancy and live birth rates.

Noninvasive methods for embryo selection

Addressing the deficiencies of invasive embryo selection, noninvasive methods enable the identification of competent embryos without the risk of impacts from the survey itself [44]. These new techniques utilize various criteria in determining the ploidy state and selecting the embryo with the greatest potential for live birth (Figure 2) [45]. Except for embryo morphological evaluation and time-lapse imaging, noninvasive methods have not yet been incorporated into routine clinical practice.

1. Morphology: appearances can be deceiving

1. Morphology: appearances can be deceiving

For more than 30 years, morphological evaluation has been the primary technique employed by embryologists to identify competent embryos. While morphological parameters are straightforward to assess, the European Society of Human Reproduction and Embryology, along with Alpha Scientists in Reproductive Medicine, have recently highlighted certain limitations in the morphological classification of oocytes and embryos [46,47]. According to their findings, the presence of smooth endoplasmic reticulum clusters is the only significant morphological feature at the oocyte stage [47,48]. Research has indeed demonstrated that the presence of smooth endoplasmic reticulum clusters is associated with poor pregnancy outcomes, as evidenced by increased rates of biochemical pregnancy loss and early miscarriage [48].

A major obstacle in morphological selection during cleavage stages is the variability encountered in embryology laboratories. These systems rely on blastomere number and embryo quality, with time being a critical factor [49]. While time assessment circumvents some of the issues associated with embryo grading, discrete temporal analyses necessitate removing the embryo from the incubator for microscopic examination, while offering only intermittent glimpses of the highly dynamic preimplantation development period. Even under the limitations of this time-restricted analysis, certain key morphological features of the embryo clearly appear linked to its developmental potential [50].

2. Culture selection: survival of the fittest

2. Culture selection: survival of the fittest

Selecting viable embryos through extended culture to the blastocyst stage is likely a beneficial approach, as many embryos undergo developmental arrest at early stages. The development of embryos in extended culture media until day 5 has been the focus of meta-analyses, with the evolution of blastocyst transfer thoroughly reviewed in articles within the Cochrane database between 2005 and 2012 [44]. A 2005 assessment found a significant advantage in pregnancy and live birth rates with blastocyst culture and transfer [51]. Glujovsky et al. [52] reported that transferring ‘fresh’ embryos at the blastocyst stage (day 5 to 6) may result in more live births compared to ‘fresh’ embryos transferred at the cleavage stage (day 2 to 3). This indicates that if 31% of women achieve live birth after ‘fresh’ cleavage-stage embryo transfer, then between 32% and 41% might do so after ‘fresh’ blastocyst stage transfer [52].

The use of blastocyst culturing as a standard treatment option for all patients remains a topic of debate. However, the concept of integrating extended culture media with additional diagnostic tests to improve outcomes or to facilitate the selection of a single embryo for transfer has been proposed [53].

3. Analysis of follicle vascularity and follicular fluid

3. Analysis of follicle vascularity and follicular fluid

The prediction of embryonic quality based on follicular characteristics has attracted considerable interest [44]. Techniques such as three-dimensional ultrasonography and power Doppler angiography have been employed to assess follicular vascularity [54]. However, the results have been too inconsistent to establish a robust model for clinical application. In 2018, Naredi et al. [55] reported that perifollicular perfusion and follicular oxygenation are associated with oocyte maturation, which in turn is indicative of embryo quality.

Multiple studies have investigated the levels of inhibin A in follicular fluid and its impact on the outcomes of IVF. Vural et al. [56] found that follicle vascularization was associated with relatively high levels of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) in the follicular fluid, good oocyte and embryo quality, vascularization of the endometrium, and a higher pregnancy rate. Additionally, perifollicular blood flow, follicular fluid insulin-like growth factor 1, and serum EG-VEGF may serve as independent predictors of pregnancy outcomes [56]. Although these biochemical markers are the subject of ongoing research, none are currently used in routine clinical practice.

4. Relationship of cumulus cells with embryo quality

4. Relationship of cumulus cells with embryo quality

Cumulus cell (CC) gene expression has been explored as a marker of oocyte developmental competence and embryo quality. Research has focused on a variety of genes associated with processes such as metabolism, steroidogenesis, signaling, and cumulus expansion. These genes have been examined in the context of oocyte maturation, fertilization, embryo quality on days 2 and 3, blastocyst development, and maternal aging [57]. Insights from these studies suggest that assessing the expression levels of specific genes in CCs could represent an additional method for embryo selection.

Transcriptome technologies, including reverse transcription-quantitative PCR (RT-qPCR), microarrays, and RNA-Seq, have been applied to the analysis of cumulus and granulosa cells [58]. Various studies have identified correlations between oocyte quality, embryo development, and the comparatively high expression of multiple genes in CCs, including growth differentiation factor-9 (GDF9) (as well as hyaluronan synthase 2 [HAS2], prostaglandin-endoperoxide synthase 2 [PTGS2], and gremlin-1 [GREM1]) [59], pentraxin-3 (PTX3) [60], bone morphogenetic protein 2 (BMP2) [61], anti-mullerian hormone receptor type 2 (AMHR2), and leukemia inhibitory factor (LIF) [62]. Additionally, research indicates that the expression levels of GDF9 and BMP15 messenger RNA are significantly higher in pregnant patients than in non-pregnant participants [63,64]. Le et al. [65] investigated the relative expression levels of long noncoding RNAs, AK124742, and proteasome 26s subunit, non-atpase 6 (PSMD6) in CCs, revealing significantly higher expression levels in the pregnant group compared to the non-pregnant participants. These findings suggest the potential of these species as biomarkers to assist in embryo selection. In summary, integrating cytokinetic methods with CC gene expression appears to improve the accuracy of embryo selection and the pregnancy rate in ART clinics.

5. TLSs in embryo selection

5. TLSs in embryo selection

Embryo selection is a critical component of the ART process and relies heavily on the morphological assessment of embryos. A TLS provides an uninterrupted culture environment, minimizes the need for embryo handling, and protects the embryos from exposure to non-incubator conditions [66]. In recent years, the TLS has emerged as a promising tool for improving embryo selection and facilitating the adoption of elective SET, which is increasingly employed to decrease the incidence of multiple pregnancies following IVF [67]. This technology enables the continuous observation of various processes, including the cytoplasmic movements of the oocyte during the resumption of meiosis, embryonic activities, fertilization events, the onset of the first mitosis, and the dynamics of blastocyst formation [68].

TLS offers a more cost-effective, faster, and less invasive approach for assessing embryo ploidy status compared to PGT-A. Nevertheless, integrating PGT-A with morphokinetic analysis may facilitate the selection of embryos with the highest implantation potential [69].

A 2017 meta-analysis provided growing evidence for the clinical benefits of using imaging systems in human IVF [70]. Chera-aree et al. [71] compared IVF pregnancy outcomes between embryos cultured in a TLS and those placed in a conventional incubator. The TLS group displayed significantly higher implantation rates (27.1% vs. 12.0%), clinical pregnancy rates (46.4% vs. 27.2%), and live birth rates (32.0% vs. 18.4%) than the conventional incubator group across different age categories [71]. Consequently, while standard morphological assessment should remain the gold standard for initiating embryo evaluation, this approach should be complemented with the detection of kinetic markers known to improve clinical outcomes. This integrated approach aims to enable embryologists to perform more accurate and objective embryo selection, thereby making the goal of SET increasingly achievable.

6. Microfluidics for embryo selection

6. Microfluidics for embryo selection

Lab-on-a-chip platforms have revolutionized the field of miniature or portable chemical and biomolecular analytical systems [72]. These platforms have been instrumental in a range of key endeavors, such as sperm capacitation and selection, oocyte maturation and selection, and IVF and embryo development, as well as the development of ovary-, oviduct-, and testis-on-a-chip systems, full menstrual cycle-on-a-chip technology, and gamete and embryo cryopreservation [73,74]. In addition, several reports have described the use of microfluidics as a qualified platform for embryo biomarkers in performing noninvasive operations. Moreover, microfluidics has been recognized as a qualified platform for the noninvasive assessment of embryo biomarkers. O’Donovan et al. [75] developed and tested a respirometric microfluidic cartridge designed to monitor the oxygen consumption of mouse embryos at the two-cell and blastocyst stages. Similarly, Urbanski et al. [76] conducted proof-of-concept experiments to demonstrate the capacity of a microfluidic chip to measure embryo metabolism, including the uptake of glucose, pyruvate, and lactate, from sub-microliter volumes. Talebjedi et al. [77] elucidated the fundamental principles of microfluidic technologies in addressing the challenges associated with isolating embryonic extracellular vesicles.

Although microfluidic technology has been available for several decades, it has not yet become commonplace in clinics and IVF laboratories. This delay can be attributed to several factors: (1) the limited automation within these platforms; (2) the lack of robust validation for their application in human embryo technology; and (3) the psychological barrier that embryologists and practitioners face when considering the adoption and implementation of microfluidic technology [78]. Despite these challenges, we expect that microfluidic platforms will soon make a considerable impact on the field of ART and basic research. In the long term, they are poised to bring about a new generation of clinical tools.

7. Embryo culture media secretome

7. Embryo culture media secretome

The embryo culture media secretome (ECMS) is a rich source of biological materials and potential biomarkers that are influenced by the embryo. As such, it can serve as a reflection of embryo quality and a valuable indicator of embryo competency. When analyzing the ECMS, it is important to consider factors such as temperature, humidity, and the type of commercial culture medium used. These can impact epigenetics and, consequently, embryo morphology, growth kinetics, physiology, and metabolism [79]. However, the standardization of culture media protocols has minimized differences in biomarker identification, resulting in reduced variability [80]. Therefore, based on the type of biomarkers present in the culture medium—which may include genomics, transcriptomics, metabolomics, proteomics, oxygen consumption, and oxidative status indicators—the ECMS can be utilized in embryo selection.

1) Metabolomic assessment of the ECMS

1) Metabolomic assessment of the ECMS

Metabolomics is a noninvasive method that provides extensive information about the competence, physiology, and function of gametes and embryos. It also identifies both well-known and previously unknown metabolites present in the culture media of preimplantation embryos. Metabolites are the end products of metabolic processes, characterized by their low molecular weight and lack of protein content [81,82]. These metabolites encompass a diverse range of molecules, including adenosine triphosphate, pyruvate, glucose, amino acids, leptin, and various hormones (such as mineralocorticoids, glucocorticoids, and sex steroids), as well as acyl carnitines, hexoses, sphingolipids, glycerophospholipids, human leukocyte antigen-G (HLA-G), platelet-activating factor (PAF), interleukin 6 (IL-6), interferon, and stem cell factor (SCF) [83-85].

Human embryos that consume more glucose demonstrate a higher growth capacity, survival rate, and implantation potential compared to those that consume less glucose [86]. HLA-G is primarily secreted by extravillous trophoblasts and serves to protect the embryo from the maternal immune response [87,88]. Some studies have suggested that the secretion of HLA-G into the ECMS is associated with improved embryo development [89-91]. PAF is another soluble factor involved in platelet activation. Research has indicated that PAF plays a role in enhancing pregnancy potential and embryo survival [92,93]. Leptin, another factor, is found in follicular fluids, fallopian tubes, and uterine fluids, as well as in oocytes and embryonic cells [94,95].

Increased concentrations of ubiquitin and decreased concentrations of haptoglobin alpha-1 have been observed in the ECMS and are associated with the formation of blastocysts [96]. A variety of methods have been employed to analyze embryo metabolism [97], such as matrix-assisted laser desorption/ionization (MALDI) coupled with mass spectrometry (MS), Raman or near-infrared spectroscopy, nuclear magnetic resonance (NMR), and gas or liquid chromatography (GC or LC, respectively). MS is particularly appropriate due to the small sample sizes required and the complex nature of the ECMS [9,98]. Unlike non-optical spectroscopy methods like MS and NMR, which necessitate costly equipment and specialized personnel, optical spectroscopy methods such as near-infrared spectroscopy are more affordable and do not require sample preparation or separation [99]. Fluorescence lifetime imaging microscopy (FLIM) enables the measurement of a molecule’s fluorescence lifetime [100]. FLIM can be used to quantitatively assess the metabolic states of CCs, oocytes, and embryos by analyzing autofluorescent metabolites such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD+) [101-104]. Moreover, recent studies on CCs and embryos have demonstrated that FLIM can sensitively detect metabolic changes not only between samples from different patients but also within samples from a single individual. A study conducted in mice indicated minimal photodamage from FLIM measurements [105]; however, additional safety studies using biophysical models in human oocytes and embryos are warranted [106]. Given the variety of metabolic molecules identified in various studies, more extensive research is needed to identify metabolic biomarkers secreted into the embryo culture medium that could predict future fertility.

2) Proteomics assessment in the ECMS

2) Proteomics assessment in the ECMS

Proteomics represents a noninvasive method for embryo assessment and holds promise as a technology for identifying potential biomarkers for embryo selection in ART. Currently, the analysis of amino acids in spent culture media (SCM) is somewhat useful in evaluating embryo development [107]. However, despite recent advancements in proteomics technologies, our understanding of the proteome of mammalian preimplantation embryos is still limited [108]. The primary challenges include limited sample availability, low levels of protein expression, and insufficient sensitivity of proteomics platforms [109]. Various techniques are employed in proteomics research, such as two-dimensional gel electrophoresis, Western blotting, MS, and protein microarrays.

In a retrospective study utilizing protein microarrays, 120 antibody targets were employed to compare the conditioned media of implanted versus non-implanted blastocysts. The results showed an increase in the expression of soluble tumor necrosis factor receptor 1 and IL-10 in the conditioned media. Conversely, decreases were observed in the expression of macrophage-stimulating protein alpha, SCF, CXC chemokine ligand 13 (CXCL13), tumor necrosis factor-related apoptosis-inducing ligand receptor 3 (TRAILR3), and macrophage inflammatory protein 1 beta compared to the control medium [110].

Some findings indicate that the concentration of lipocalin-1 is elevated in the secretome of aneuploid embryos, suggesting its potential as a biomarker for noninvasive aneuploidy screening [111]. Additionally, the expression levels of human chorionic gonadotropin beta subunit [112] and IL-6 [113] in culture media are correlated with embryo quality and implantation rate, respectively. In degenerating blastocysts, heparin-binding epidermal growth factor-like growth factor precursor [114,115] is upregulated in the blastocyst media.

Huo et al. [107]. analyzed the amino acid concentrations in the embryo culture media of 98 patients who had undergone IVF embryo transfer treatment. The results of high-performance liquid chromatography (HPLC) revealed significant differences in the concentrations of serine, aspartate, histidine, and alanine between the culture media of embryos that resulted in pregnancy and those that failed to implant.

The identification of a reliable and reproducible proteomic/secretome signature presents a key challenge. Such a signature should be directly associated with embryo viability and competency, as well as with successful pregnancy and live birth outcomes. This task is particularly daunting due to the complexity, heterogeneity, and diversity of human embryos, along with the variety of culture media used and their associated protein contaminants. Additionally, the clinical application of proteomic methods is time-consuming. The processes of sample preparation, measurement, and data analysis are not currently feasible within the necessary time window for IVF [116].

3) Genomic assessment in ECMS: a new approach

3) Genomic assessment in ECMS: a new approach

Chromosomal screening through PGT necessitates an embryo biopsy and has inherent limitations. Nevertheless, recent research has identified the presence of cell-free DNA (cf-DNA) in the biological fluids of embryos cultured in vitro, such as blastocyst fluid and SCM [117-119]. This discovery has facilitated the development of ni-PGT in ART. Of these approaches, SCM analysis appears to be the most promising option [43,117-121]. The cf-DNA present in the ECMS can originate from both the ICM and the TE. However, a study utilizing DNA-specific fluorochromes has shown that the primary source of apoptotic cell DNA is the ICM [122].

An important issue with ni-PGT is the potential detection of extra-embryonic DNA in the SCM. Practitioners of ni-PGT must understand the risks associated with time-dependent DNA degradation and contamination by maternal DNA. However, these risks can be significantly mitigated by carefully removing CCs and thoroughly washing the embryos in single-culture microdroplets. Additionally, employing intracytoplasmic sperm injection (ICSI) as the fertilization method can help prevent contamination with paternal DNA [43].

In a study by Shitara et al. [117], the concordance rate between PGT-A and outgrowth embryos was 43.8%, which is lower than previous reports have indicated. In the same study, the concordance rate for ni-PGT-A and outgrowth samples was found to be 56.3%, representing an improvement over the concordance rate for PGT-A samples alone.

Research has shown that the ratio of mitochondrial DNA (mtDNA) to genomic DNA (gDNA) in embryo culture medium correlates with embryonic development and can be used in place of biopsied embryo cells to predict the embryo quality and implantation rate [123]. Studies by Stigliani et al. [124] and Hashimoto et al. [125] have suggested that the mtDNA/gDNA ratio in embryo culture medium is associated with blastulation and may have predictive value. In contrast, Victor et al. [126] reported that mtDNA content does not correlate with ploidy or blastocyst viability. Regarding this potential noninvasive marker of pregnancy rate, Sayed et al. [127] measured the mtDNA/gDNA ratio using real-time PCR in the embryo culture media of patients with polycystic ovary syndrome (PCOS). They found that the mtDNA/gDNA ratio in the culture media of PCOS embryos was significantly lower than that in the control group [127]. In summary, the findings of various studies support the use of ni-PGT on cf-DNA in SCM. With the development of more effective methods, preimplantation screening could become simpler, potentially circumventing the adverse effects of invasive biopsy procedures on embryos.

4) Transcriptomics assessment in ECMS: a new approach

4) Transcriptomics assessment in ECMS: a new approach

MicroRNAs (miRNAs) are a class of single-stranded RNAs involved in the transcriptional or post-transcriptional regulation of gene expression. miRNAs are believed to be secreted by human embryos into embryonic culture media; thus, they can potentially serve as noninvasive biomarkers in assisted reproduction [128,129]. To measure the levels of these circulating miRNAs, RT-qPCR and droplet digital PCR (ddPCR) are reliable methods. In particular, ddPCR offers the ability to detect and quantify less abundant targets with greater precision and sensitivity than RT-qPCR [130-132]. After ddPCR is performed, the initial concentration of the target is estimated by counting the number of positive (containing the amplified target) and negative (no amplified target detected) reactions, applying Poisson statistical analysis.

Conversely, Rosenbluth et al. [133] observed that three miRNAs (miR-372, miR-191, and miR-645) were differentially expressed on day 5 in the SCM of embryos that resulted in successful pregnancies compared to those that did not. Fang et al. [134] suggested that the presence of hsa-miR-26b-5p and hsa-miR-21-5p in the culture media of cleavage-stage embryos could serve as potential biomarkers for reproductive outcomes. Additionally, Wang et al. [135] identified three miRNAs (hsa-miR-199a-5p, hsa-miR-483-5p, and hsa-miR-432-5p) that may act as biomarkers of embryo quality in IVF cycles. The analysis of miRNAs from SCM alone represents an initial step towards simplifying the process and developing a diagnostic assay to select reproductively competent embryos, aiming for affordability, reduced complexity, and shorter processing times.

5) Measurement of oxygen consumption in the ECMS

5) Measurement of oxygen consumption in the ECMS

An embryo’s oxygen consumption is a critical indicator of its overall metabolic activity and serves as a useful marker of embryo quality. As an embryo develops, changes in energy metabolism precede morphological alterations, and these shifts are mirrored by changes in mitochondrial activity.

These methods should possess features such as high sensitivity, non-invasiveness, a low detection limit, compatibility with other types of measurements (such as temperature, pH, chemical, and impedance), and reproducibility [136]. Previous methods, such as fluorescence and spectrophotometry, were not suitable for clinical use due to their low sensitivity and invasiveness [137]. Instead, newer oxygen measurement techniques, such as optical methods (fluorescence and phosphorescence), electrochemical methods (amperometric and potentiometric), magnetic resonance imaging (MRI), and electron paramagnetic resonance imaging (EPRI), have been employed. The pO₂ optical imaging method enables three-dimensional imaging of cells using oxygen sensor particles [138]. However, it has disadvantages, including issues with photostability, phototoxicity, and photobleaching, which complicate its clinical application [139]. MRI and EPRI imaging methods are also based on a noninvasive three-dimensional sensor, but they require a contrast agent and a magnetic resonance device [140,141].

Recent advances in techniques for measuring embryo oxygen consumption have led to the introduction of electrochemical methods suitable for clinical use. These include the use of self-referencing microelectrodes [142] and Clark-type electrochemical oxygen sensors, as well as scanning electrochemical microscopy [143,144]. This approach offers a distinct advantage over other methods by allowing the measurement of chemical species concentrations without the need for pretreatment of biological samples or optical probes. It also enables the simultaneous detection of multiple chemical constituents, such as neurotransmitters [145,146] and metabolic markers [147,148]. Kurosawa et al. [137] developed a novel device that employs a chip-sensing embryo respiration monitoring system, which automates the measurement of oxygen consumption using electrochemical methods. Tedjo et al. [136] utilized an electrochemical imaging system equipped with an integrated microelectrode array to measure the oxygen consumption of bovine oocytes. This system was found to enable imaging at low oxygen concentrations in vitro.

6) Measurement of oxidative stress in the ECMS

6) Measurement of oxidative stress in the ECMS

The production of reactive oxygen species (ROS) is a factor that influences infertility and embryo survival in ART. Physiological levels of ROS in culture media may result from internal factors, such as embryo metabolism, or external factors, such as the environment containing the embryo [149]. Internal sources of ROS can arise from events like sperm-mediated oocyte activation and the activation of the embryonic genome. The oxidative status can be used to predict the competency of early embryos. Thermochemiluminescence (TCL) is a technique for assessing ROS in embryo culture medium that is highly sensitive to sample oxidation [150]. Recently, Alegre et al. [151] employed the TCL method to measure ROS in embryonic culture medium prior to transfer, in conjunction with time-lapse evaluation for embryo selection. Their findings challenge the widespread view that oxidative stress is solely a negative factor. Instead, their research showed that high-quality embryos exhibit a more extensive oxidative metabolism and actually impose an “oxidative charge” on their environment.

Another system for measuring oxidative stress incorporates electrochemical technology to calculate the static oxidative-reduction potential (ORP) in millivolts (mV) [150]. Sallam et al. [152] demonstrated that the ORP levels in the culture medium of fertilized oocytes were lower than those of unfertilized oocytes. Similarly, the ORP levels measured in the culture medium of embryos that resulted in pregnancy were lower than those of embryos that did not lead to pregnancy.

However, the current conventional techniques for analyzing metabolites involved in embryonic oxidative processes are not yet suitable for clinical application. Further studies are needed to assess the impact of ROS produced in culture medium relative to existing embryo selection methods. These findings should be validated through large-scale, prospective randomized studies that employ various techniques to measure oxidative stress and its relationship with embryo implantation rates.

8. AI for embryo selection

8. AI for embryo selection

AI models may have the potential to identify embryos with the highest likelihood of resulting in pregnancy [153]. Machine learning can standardize a wide range of clinical data, mitigating potential biases and reducing variability among observers. This technology enables a more objective assessment of embryos compared to manual human evaluation [154]. AI models must be interpretable, facilitating embryo assessment based on biologically meaningful parameters [155]. The parameters used for the embryo selection process vary among AI models. The database used in one study categorized four distinct characteristics: fertilization method (IVF or ICSI), incubation period (e.g., 5 days), embryo quality (e.g., blastocyst, hatched blastocysts, fresh, or frozen), and outcome (for example, positive implantation, fetal heart rate, or live birth) [156]. Additionally, factors such as patient demographics, oocyte donation, culture medium, image quality criteria, and other clinical settings may be pertinent for characterizing a specific population of embryos. The selection of input features for the model can greatly impact its capacity to predict pregnancy probabilities (Figure 3). For instance, some models utilize a still image of the embryo, while others employ a time-lapse video of the embryo’s development. Curchoe et al. [157] scrutinized the relevance of AI study results in terms of clinical practice and identified four issues: unbalanced data sets, small sample sizes, limited performance measures, and non-generalizable settings. The overarching aim of these methods is to rank embryos based on their implantation potential and/or to predict the actual probability of pregnancy for each embryo. In recent years, numerous studies have focused on the commercialization and methodological advancements of AI in this field [158].

Clinical management of practical models

The primary objective of ART research should be to develop and validate practical models that utilize clinical data to predict the live birth rate of singletons following IVF with embryo selection. Accurate and long-term follow-up studies should be conducted regarding the health of babies born through embryo selection strategies. Embryos chosen based on current models may potentially possess certain undesirable traits, such as an increased risk of cancer or mental disorders. While investigating these outcomes may place a burden on families, further clinical trials are warranted. These trials should be limited in scope until we can confidently assert the safety and effectiveness of these technologies for the long-term health of children conceived via IVF [155]. As embryo selection technologies gain popularity, researchers are investigating whether these methods can improve pregnancy and live birth rates beyond what is achievable with morphology-based embryo selection alone. Independent research teams should critically assess and validate the safety and effectiveness of current models. Additionally, these technologies are associated with concerns around cultural, religious, or personal goals and values/beliefs regarding their use. Some patients may opt for IVF with PGT-A solely for sex selection purposes. Conversely, persistent worries exist about subjecting women to invasive procedures that are not medically necessary, the potential risks to children born through ART, and the possibility of discrimination against these children or their siblings [159]. Physicians face a challenging task in adhering to ethical principles when deciding whether to offer IVF with PGT-A to such patients. It is therefore essential to view embryo selection technologies as an adjunct to IVF that should be considered on a case-by-case basis. This approach will assist patients in determining whether they are likely to benefit from the technology. The decision should incorporate various factors, including the patient’s age, ovarian reserve, and outcomes from previous IVF cycles, as well as laboratory protocols.

Future perspectives and conclusion

Various embryo selection technologies have emerged within the past decade. These technologies are either used in combination with morphology or intended to replace conventional morphological evaluation [160]. Some of the most promising areas of research for noninvasive procedures include cf-DNA analysis, microscopy techniques coupled with AI, and omics analysis of blastocyst media. High-throughput proteomics, metabolomics, genomics, and transcriptomics technologies are valuable tools for noninvasive embryo analysis [45]. Embryo selection using morphokinetic markers has been shown to improve success rates compared with outcomes obtained through standard morphology-based embryo selection [160]. It is important to consider the practicality of current procedures in an IVF clinical environment. Implementing new low-profile, time-lapse microscopy combined with AI or bench-top matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS models could be relatively straightforward. However, techniques involving LC-MS/GS-MS or HPLC may be less practical for routine embryo screening [45]. Currently, the most common invasive test is PGT, which is used to analyze the chromosomal constitution of blastomeres or TE. The most critical aspect of such studies is the potential impact of biopsy and manipulation on subsequent embryo development [44]. To ensure test accuracy and potentially improve overall live birth outcomes with PGT-A, the issue of embryonic mosaicism must be addressed. Several studies support the application of different technologies by developing automated annotation software for morphokinetic analysis [161,162]. With successes in both high-throughput bioinformatic tools and clinically useful hardware, accurate selection of the best embryo for transfer is now accessible. A combination of these technologies will likely transform our understanding of embryo physiology and enhance our ability to select viable embryos for transfer in ART cycles. In this way, a nuanced view of embryo function related to its development, quality, and potential outcomes can be compiled. However, much work is still required to transition pilot research to the clinical setting.

Notes

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Author contributions

Conceptualization: ZB, ZZ. Methodology: ZB, AA. Formal analysis: ZB, MK. Data curation: ZB, AA, HT. Project administration: ZB, FA. Visualization: NS. Software: ZB. Validation: ZB. Investigation: ZB. Writing-original draft: ZB, AA, HT. Writing-review & editing: ZB, MG, FA. Approval of final manuscript: ZB, AA, MK, HT, MG, NS, ZZ, FA.

Acknowledgments

The authors would like to express their gratitude to the Shahid Akbarabadi Clinical Research Development Unit (ShACRDU) at the Iran University of Medical Sciences in Tehran, Iran, for its support throughout the study.

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