Future perspectives of non-invasive techniques for evaluating oocyte and embryo quality

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INTRODUCTION
According to the innovation conference of 2023, women's health plays a crucial role in shaping the future of humanity. 1Due to social, environmental and many other factors, infertility has become a key issue plaguing women's health.To combat this issue, assisted reproductive technology (ART) has emerged as a viable solution.ART encompasses various fertility treatments including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), with more than seven million children estimated to have been born through ART.However, the efficacy of ART remains relatively low.According to the statistics, among 100 oocytes collected, only 31 become high-quality embryos, and only 5 result in the birth of offspring. 2Due to the lack of objective parameters for evaluating oocyte or embryo quality, multiple embryos are transferred into the uterus to ensure an acceptable clinical pregnancy rate, resulting in a higher risk of multiple pregnancy from the IVF cycle.
There is no doubt that the goal of an IVF/ICSI cycle is to achieve one healthy baby per transfer.Selection of high-quality oocytes and/or embryos is the crucial initial step toward ensuring the success of an ART procedure.Oocyte quality, or oocyte developmental competence, refers to an oocyte's ability to undergo successful meiosis, fertilization, preimplantation embryo development, implantation, and ultimately result in the birth of a healthy offspring.Human oocytes and embryos are small in size (~ 120 µm).In addition, they are quite vulnerable to tiny changes in culture conditions, such as fluctuations in temperature, pH, osmolarity and toxicity.As a result, the assessment of oocytes and embryos is challenging.Clinically, the assessment involves both non-invasive morphological evaluation and invasive preimplantation genetic testing for aneuploidy screening (PGT-A).PGT-A is invasive in nature and requires additionalmanipulation and expense.In addition, consumption due to embryo biopsy, chromosome screening, and vitrification cannot be neglected.Therefore, more objective, accurate, quantitative, non-invasive, rapid, and repeatable evaluation methods are clinically demanded.
Recent developments in the technology of ultrastructure observation, autoluminescent detection, and microfluidic chips, among others, may provide a novel opportunity to overcome current limitations in the evaluation of oocytes and embryos.Here, we present various non-invasive techniques for evaluating oocyte and embryo quality (Figure 1), such as mechanical, optical, and cell metabolism secreted factor-based methods.Additionally, we introduce the use of automation and artificial intelligence (AI) approaches to increase the efficiency and precision in assessing oocyte and embryo quality, ultimately aiming to increase the efficiency of ART.

MECHANICAL-BASED METHODS
The mechanical properties of cells, such as elasticity, viscoelasticity, and shear modulus, play crucial roles in various cellular functions including cell growth, division, motility, and adhesion.Assessing the mechanical properties of oocytes may provide valuable insights into their quality and developmental potential for successful use in ART.
One commonly used method for assessing oocyte mechanics is analysis of the zona pellucida, which is the protein coating surrounding the oocyte plasma membrane.Techniques such as indentation force, micropipette aspiration, and atomic force microscopy are utilized to measure the hardness, elasticity, and viscosity of the zona pellucida.Typically, these non-invasive techniques involve the use of glass tubes of various sizes or probes to place pressure or suction on the zona pellucida.The resulting deformation of the zona pellucida is then analyzed to assess its mechanical properties.Several mechanical methods can also be conveniently appliedduring ICSI, facilitating the integration of these methods into clinical research.I Yanez et al., reported that the viscoelastic properties of embryos can be indicative of their viability for blastocyst development. 3Embryos with moderate levels of softness and hardness were identified as having higher chances of successful development.Excessive force applied via mechanical methods may cause deformation and damage to oocytes or embryos, so determining the appropriate amount of force is an important area for future research on mechanical methods.

OPTICAL-BASED METHODS
Optical-based methods, including absorption, refractive index, and autofluorescence, are used to measure various properties of oocyte development.A lab-on-a-chip (LOC) system combined with an optical sensor was previously developed to measure absorption and the refractive index at different stages of oocyte development. 4This system automates the capture, movement, and trapping of a single human oocyte using a microfluidic device.The amount of light absorbed and transmitted by the oocyte was measured using two illumination and collection optical fibers.Higher quality oocytes with a homogeneous cytoplasm had a lower absorbance ratio, in contrast, lower quality oocytes with heterogeneous cytoplasm and inclusions exhibited a higher absorbance ratio.
Optical-based methods are also utilized to assess the localization, shape, and refringence of the meiotic spindle using polarized light.The meiotic spindle plays a crucial role in aligning and separating chromosomes during meiosis, thus making it a key focus in the examination of oocyte quality.There is a correlation between meiotic spindle length and oocyte quality.Oocytes with a birefringent spindle, as observed under polarized light, tend to have better embryonic development than do those with a nonbirefringent spindle.Additionally, oocytes lacking a visible meiotic spindle under polarized light are linked to lower rates of fertilization and blastocyst formation.
The autoluminescent metabolic factors NADH and FAD are currently receiving increasing amounts of attention in relation to oocytes and embryos.These factors are directly linked to mitochondrial activity, and the fluorescence intensity of NADH and FAD has been used to quantify mitochondrial activity and metabolic changes in oocytes and embryos.To capture these autoluminescent metabolic cofactors, laser scanning confocal microscopy and fluorescence lifetime microscopy (FLIM) are commonly used.Confocal microscopy allows for 3D imaging of mitochondria in oocytes and embryos.FLIM has been successfully used to assess the metabolic processes of oocytes and provides information on the spatial distribution of metabolic cofactors in both the cytoplasm and mitochondria.Phototoxicity is a safety issue that must be considered in optical methods.High-intensity light irradiation may significantly affect the quality of oocytes and embryos, resulting in delayed embryo development or even failure to develop blastocysts.Theorectically, both confocal microscopy and FLIM will lead to higher phototoxicity.Using low-phototoxicity microscopy systems, such as two-photon excited fluorescence (TPEF) microscopy with a femtosecond laser and hyperspectral microscopy, is a potential solution.TPEF microscopy enables imaging at greater depths and reduces the risk of photodamage in out-offocus areas, while hyperspectral microscopy uses a broad spectrum of light to detect a variety of endogenous fluorophores and allows for a deeper understanding of the metabolism and content of oocytes and embryos.Hyperspectral microscopy has demonstrated its ability to differentiate between good and poor quality bovine embryos. 5Therefore, it is promising as a non-invasive approach for detecting metabolic changes associated with oocyte and embryo quality.

CELL METABOLISM SECRETED FACTOR-BASED METHODS
Cell metabolism secreted factor-based methods involve the examination of follicular fluid and culture media from oocytes and embryos.Follicular fluid, which is obtained during oocyte retrieval, consists of secretory products from granulosa and theca cells, as well as components of blood plasma that cross the blood follicle barrier.Follicular fluid is in close contact with the maturing oocyte and may represent healthy or unhealthy developmental conditions.Techniques such as high-pressure liquid chromatography, mass spectrometry, and RNA sequencing have been employed to identify markers of oocyte quality in follicular fluid.Substances such as hormones, fatty acids, and small molecule metabolites have been identified as potential markers of oocyte quality.The quality of granulosa cells and/or cumulus cells can also provide insights into oocyte quality.GDF9, which is synthesized by oocytes and involved in the expansion of cumulus cells during final follicle development, has been identified as an essential marker of oocyte quality. 6Since the follicular fluid of each oocyte is relatively independent, it is important to focus solely on the associations between oocytes and their own individual follicular fluid.
The balance between the products of intracellular metabolism in oocytes and the culture media is constantly changing.Therefore, by using targeted and untargeted metabolomic approaches, it is possible to analyze metabolites in culture media to assess the health, developmental capacity, and selective advantage of oocytes and embryos in a non-invasive manner.However, there are various external factors, such as contamination from nonoocyte cells, limited culture time, and human error during manual manipulation, that can complicate the non-invasive assessment of oocytes.Currently, metabolic markers for oocyte quality are still under exploration. 7To discover predictive biomarkers for normal and abnormal metabolism, microfluidics platforms with automated, high-coverage, and high-throughput targeted metabolic assessments in real time will be necessary.

AUTOMATION AND AI METHODS
With the advantage of time-lapse embryo culture technology, consecutive images have been automatically captured in recent years.However, no universal algorithm has been developed for the selection of viable embryos for transfer, indicating that current morphological parameters may not be enough, and clinical factors, such as female age, ovarian reserve, infertility etiology, and ovarian stimulation protocol, should be considered.AI techniques have gained attention for their applications in medical imaging diagnostics by incorporating deep learning, computer vision image processing techniques, and clinical data.It has been applied in prediction, diagnosis, information management, data collection, and clinical practice. 8Moreover, AI can supplement existing conventional morphological indicators used clinically and integrate new indicators in the future to improve the accuracy of quality screening.Recently, a prediction model for ovarian reserve or polycystic ovary syndrome has been reported. 9,10It is promising to develop an AI model for oocyte and embryo quality assessment based on a large amount of clinical and laboratory data for model training.

IMPROVING OOCYTE QUALITY METHODS
In addition to selecting high-quality oocytes and embryos, improving oocyte quality is crucial for the success of ART procedures.Clinically, doctors typically rely on different ovarian stimulation protocols to enhance oocyte quality.Mitochondria have garnered increased attention due to their significant role in regulating oocyte metabolism and epigenetics.Antioxidants such as coenzyme Q10 are orally administered or added to culture media during IVF/ICSI treatment to improve oocyte quality.The development of AI and cell metabolism secreted factor-based methods may also benefit the enhancement of oocyte quality.This approach may benefit the development of a more personalized ovarian stimulation protocol for specific patients.In addition, small molecule substances discovered through secreted factor methods may be orally consumed or added to culture media to improve oocyte quality.

CONCLUSION
In the field of ART, selecting high-quality oocytes and embryos is a crucial step.The exploration of new non-invasive methods for evaluating oocytes and embryos is clinically demanded.The future of non-invasive assessment of oocytes and embryos lies in physics, microscopy, incorporating genomics, microfluidics, AI, and other biomedical technologies into the study of oocyte and embryo biology.However, there are still challenges to overcome, particularly in terms of technology and clinical applications.To address these challenges, it is important to encourage interdisciplinary research and collaboration between medicine and industry.Doing this will expand our understanding of unexplored ultrastructural fields and change traditional methods to develop more reliable techniques for the assessment of oocyte and embryo

Figure 1 .
Figure 1.Non-invasive assessments for selection of high-quality oocytes and embryos.