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.

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].

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.

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.

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.

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].

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.

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.

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.

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.