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Embryo quality is crucial to the outcome of in vitro fertilization (IVF); however, the ability to precisely distinguish the embryos with higher reproductive potential from others is poor. Morphologic evaluation used to play an important role in assessing embryo quality, but it is somewhat subjective. The culture medium is the immediate environment...
Context in source publication
Context 1
... control sample (CM) incubated under the same conditions without an embryo was also collected and used for normalization. The morphological classification of the embryos in this experiment was described in detail in Table 3. The embryos with cell count ≥6 and class A or B are considered as morphologically high-scored embryos, otherwise, the embryos with cell count <6 and class A or B or cell count ≥6, but grade C, are considered as morphologically low-scored embryos. ...
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In this study, a rapid and simple method which combines drop coating deposition and Raman spectroscopy (DCDR) was developed to characterize the dry embryo culture media (ECM) droplet. We demonstrated that Raman spectra obtained from the droplet edge presented useful and characteristic signatures for protein and amino acids assessment. Using a diffe...
Citations
... As quality assessment protocols still require improvement, numerous novel methods of quality assessment have been proposed in recent years. One is the metabolic profiling of the embryos, which is achieved by the chemical analysis of spent culture media (Nagy et al., 2009;Zhao et al., 2013;Salmerón et al., 2021). Another technique, fluorescent lifetime imaging microscopy (FLIM) uses the differences in the exponential decay rate of the photon emission of autofluorescent coenzymes NAD(P)H and FAD 2+ and allows for quantification of their concentration and thus energy metabolism of the embryo (Ma et al., 2019;Venturas et al., 2022;Venturas et al., 2023). ...
Assisted Reproductive Technologies (ART) have revolutionized infertility treatment and animal breeding, but their success largely depends on selecting high-quality oocytes for fertilization and embryos for transfer. During preimplantation development, embryos undergo complex morphogenetic processes, such as compaction and cavitation, driven by cellular forces dependent on cytoskeletal dynamics and cell-cell interactions. These processes are pivotal in dictating an embryo’s capacity to implant and progress to full-term development. Hence, a comprehensive grasp of the biomechanical attributes characterizing healthy oocytes and embryos is essential for selecting those with higher developmental potential. Various noninvasive techniques have emerged as valuable tools for assessing biomechanical properties without disturbing the oocyte or embryo physiological state, including morphokinetics, analysis of cytoplasmic movement velocity, or quantification of cortical tension and elasticity using microaspiration. By shedding light on the cytoskeletal processes involved in chromosome segregation, cytokinesis, cellular trafficking, and cell adhesion, underlying oogenesis, and embryonic development, this review explores the significance of embryo biomechanics in ART and its potential implications for improving clinical IVF outcomes, offering valuable insights and research directions to enhance oocyte and embryo selection procedures.
... Some studies supported a spent embryo culture medium metabolomic approach. Zhao et al. used Raman spectroscopy to investigate metabolomic profiling of day 3 embryo culture media combined with morphology to successfully predict 6 out of 7 embryos implanting potential [16]. Bastu et al. who also used a Raman spectroscopy approach. ...
Background
Metabolites in spent embryo culture medium correlate with the embryo’s viability. However, there is no widely accepted method using metabolite dada to predict successful implantation. We sought to combine metabolomic profiling of spent embryo culture medium and clinical variables to create an implantation prediction model as an adjunct to morphological screening of day 3 embryos.
Methods
This investigation was a prospective, nested case-control study. Forty-two day 3 embryos from 34 patients were transferred, and the spent embryo culture medium was collected. Twenty-two embryos implanted successfully, and the others failed. Metabolites in the medium relevant to implantation were detected and measured by Liquid Chromatography-Mass Spectrometry. Clinical signatures relevant to embryo implantation were subjected to univariate analysis to select candidates for a prediction model. Multivariate logistical regression of the clinical and metabolomic candidates was used to construct a prediction model for embryo implantation potential.
Results
The levels of 13 metabolites were significantly different between the successful and failed groups, among which five were most relevant and interpretable selected by Least Absolute Shrinkage and Selection Operator regression analysis. None of the clinical variables significantly affected day 3 embryo implantation. The most relevant and interpretable set of metabolites was used to construct a prediction model for day 3 embryo implantation potential with an accuracy of 0.88.
Conclusions
Day 3 embryos’implantation potential could be noninvasively predicted by the spent embryo culture medium’s metabolites measured by LC-MS. This approach may become a useful adjunct to morphological evaluation of day 3 embryos.
... Later studies using RS on embryo spent media have further expanded our understanding of compositional changes that may be related to embryo viability. In 2013, Zhao et al. (2013) (Brinster, 1965). In a similar study in 2016, the team of Parlatan et al. (2016) specifically examined the amino acid composition of spent media and observed the most pronounced differences in the band ratios correlating to glycine, glutamine and proline. ...
Background:
DNA damage is a hazard that affects all cells of the body. DNA-damage repair (DDR) mechanisms are in place to repair damage and restore cellular function, as are other damage-induced processes such as apoptosis, autophagy and senescence. The resilience of germ cells and embryos in response to DNA damage is less well studied compared with other cell types. Given that recent studies have described links between embryonic handling techniques and an increased likelihood of disease in post-natal life, an update is needed to summarize the sources of DNA damage in embryos and their capacity to repair it. In addition, numerous recent publications have detailed novel techniques for detecting and repairing DNA damage in embryos. This information is of interest to medical or scientific personnel who wish to obtain undamaged embryos for use in offspring generation by ART.
Objective and rationale:
This review aims to thoroughly discuss sources of DNA damage in male and female gametes and preimplantation embryos. Special consideration is given to current knowledge and limits in DNA damage detection and screening strategies. Finally, obstacles and future perspectives in clinical diagnosis and treatment (repair) of DNA damaged embryos are discussed.
Search methods:
Using PubMed and Google Scholar until May 2021, a comprehensive search for peer-reviewed original English-language articles was carried out using keywords relevant to the topic with no limits placed on time. Keywords included 'DNA damage repair', 'gametes', 'sperm', 'oocyte', 'zygote', 'blastocyst' and 'embryo'. References from retrieved articles were also used to obtain additional articles. Literature on the sources and consequences of DNA damage on germ cells and embryos was also searched. Additional papers cited by primary references were included. Results from our own studies were included where relevant.
Outcomes:
DNA damage in gametes and embryos can differ greatly based on the source and severity. This damage affects the development of the embryo and can lead to long-term health effects on offspring. DDR mechanisms can repair damage to a certain extent, but the factors that play a role in this process are numerous and altogether not well characterized. In this review, we describe the multifactorial origin of DNA damage in male and female gametes and in the embryo, and suggest screening strategies for the selection of healthy gametes and embryos. Furthermore, possible therapeutic solutions to decrease the frequency of DNA damaged gametes and embryos and eventually to repair DNA and increase mitochondrial quality in embryos before their implantation is discussed.
Wider implications:
Understanding DNA damage in gametes and embryos is essential for the improvement of techniques that could enhance embryo implantation and pregnancy success. While our knowledge about DNA damage factors and regulatory mechanisms in cells has advanced greatly, the number of feasible practical techniques to avoid or repair damaged embryos remains scarce. Our intention is therefore to focus on strategies to obtain embryos with as little DNA damage as possible, which will impact reproductive biology research with particular significance for reproductive clinicians and embryologists.
... All patients underwent IVF treatment with routine pituitary suppression protocol, as previously described [17]. In brief, patients were subcutaneously injected with gonadotropin-releasing hormone agonist (GnRH-a; Decapeptyl or Diphereline; Ispen, Paris, France) in the midluteal phase for at least 14 days. ...
Background: To evaluate both the impact of hepatitis B virus (HBV)-DNA copies in women with HBV infection on the ovarian reserve function and outcomes of in vitro fertilization (IVF). Methods: We conducted a retrospective study on a total of 9927 couples undergoing their first IVF cycle. After filtering, 1570 couples (546 HBV-seropositive women and 1024 HBV-seronegative women whose partners were HBV-seronegative) failed to meet inclusion criteria. According to the HBV-DNA titers in serum, the HBV-seropositive group was divided into three groups: DNA-high copy group (n = 139), DNA-low copy group (n = 241), and DNA-negative group (n = 166). All patients underwent controlled ovarian hyperstimulation using the long downregulation protocol followed by IVF. Results: Compared with the HBV-negative group, HBV-positive women with high DNA copy exhibited lower antral follicle count (AFC) (11.9 ± 4.3 vs 13.3 ± 3.2), lower number of oocyte retrieved (9.2 ± 5.7 vs 13.1 ± 6.1), larger proportion of AFC
... For instance, phenylalanine is one of the amino acids that inhibit hamster 1-cell embryo development in vitro and is harmful for blastocyst formation in pigs (McKiernan et al., 1995;Booth et al., 2007). Moreover, the concentrations of phenylalanine in embryo culture media are used as biomarkers for clinical pregnancy in humans (Zhao et al., 2013). Our results also indicate that the greater vibration at 1,004 cm −1 of phenylalanine in FF is a warning sign for oocyte development in PCOS, and are in line with the previous views that blastocyst development is modulated by amino acids, and the ideal environment for embryonic development needs the supplement of suitable amino acids (Gardner, 1998). ...
Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in reproductive women where abnormal folliculogenesis is considered as a common characteristic. Our aim is to evaluate the potential of follicular fluid (FF) Raman spectra to predict embryo development and pregnancy outcome, so as to prioritize the best promising embryo for implantation, reducing both physiological and economical burdens of PCOS patients. In addition, the altered metabolic profiles will be identified to explore the aetiology and pathobiology of PCOS. In this study, follicular fluid samples obtained from 150 PCOS and 150 non-PCOS women were measured with Raman spectroscopy. Individual Raman spectrum was analyzed to find biologic components contributing to the occurrence of PCOS. More importantly, the Raman spectra of follicular fluid from the 150 PCOS patients were analyzed via machine-learning algorithms to evaluate their predictive value for oocyte development potential and clinical pregnancy. Mean-centered Raman spectra and principal component analysis (PCA) showed global differences in the footprints of follicular fluid between PCOS and non-PCOS women. Two Raman zones (993–1,165 cm ⁻¹ and 1,439–1,678 cm ⁻¹ ) were identified for describing the largest variances between the two groups, with the former higher and the latter lower in PCOS FF. The tentative assignments of corresponding Raman bands included phenylalanine and β -carotene. Moreover, it was found that FF, in which oocytes would develop into high-quality blastocysts and obtain high clinical pregnancy rate, were detected with lower quantification of the integration at 993–1,165 cm ⁻¹ and higher quantification of the integration at 1,439–1,678 cm ⁻¹ in PCOS. In addition, based on Raman spectra of PCOS FF, the machine-learning algorithms via the fully connected artificial neural network (ANN) achieved the overall accuracies of 90 and 74% in correctly assigning oocyte developmental potential and clinical pregnancy, respectively. The study suggests that the PCOS displays unique metabolic profiles in follicular fluid which could be detected by Raman spectroscopy. Specific bands in Raman spectra have the biomarker potential to predict the embryo development and pregnancy outcome for PCOS patients. Importantly, these data may provide some valuable biochemical information and metabolic signatures that will help us to understand the abnormal follicular development in PCOS.
... For instance, phenylalanine is one of the amino acids that inhibit hamster 1-cell embryo development in vitro and is harmful for blastocyst formation in pigs [35,36]. Moreover, the concentrations of phenylalanine in embryo culture media are used as biomarkers for clinical pregnancy in humans [37]. Our results also indicate that the greater vibration at 1004 cm − 1 of phenylalanine in FF is a warning sign for oocyte development in PCOS, and are in line with the previous views that blastocyst development is modulated by amino acids, and the ideal environment for embryonic development needs the supplement of suitable amino acids [38]. ...
Background: Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in reproductive women where abnormal folliculogenesis is considered as a common characteristic. Our aim is to evaluate the potential of follicular fluid (FF) Raman spectra to predict oocyte development and pregnancy outcome, so as to prioritize the best promising oocyte for implantation, reducing both physiological and economical burdens of PCOS patients. In addition, the altered metabolic profiles will be identified to explore the aetiology and pathobiology of PCOS.
Methods: In this study, follicular fluid samples obtained from 150 PCOS and 150 non-PCOS women were measured with Raman spectroscopy. Individual Raman spectrum was analyzed to find biologic components contributing to the occurrence of PCOS. More importantly, the Raman spectra of follicular fluid from the 150 PCOS patients were analyzed via machine-learning algorithms to evaluate their predictive value for oocyte development potential and clinical pregnancy.
Results: Mean-centered Raman spectra and principal component analysis showed global differences in the footprints of follicular fluid between PCOS and non-PCOS women. Two Raman zones (993-1,165 cm-1 and 1,439-1,678cm⁻¹) were identified for describing the largest variances between the two groups, with the former higher and the latter lower in PCOS FF. The tentative assignments of corresponding Raman bands included phenylalanine and β -carotene. Moreover, it was found that FF, in which oocytes would develop into high-quality blastocysts and obtain high clinical pregnancy rate, were detected with lower quantification of the integration at 993-1,165 cm⁻¹ and higher quantification of the integration at 1,439-1,678 cm⁻¹ in PCOS. In addition, based on Raman spectra of PCOS FF, the machine-learning algorithms via the fully connected artificial neural network (ANN) achieved the overall accuracies of 90% and 74% in correctly assigning oocyte developmental potential and clinical pregnancy, respectively.
Conclusions: The study suggests that the PCOS displays unique metabolic profiles in follicular fluid which could be detected by Raman spectroscopy. Specific bands in Raman spectra have the biomarker potential to predict the oocyte development and pregnancy outcome for PCOS patients. Importantly, these data may provide some valuable biochemical information and metabolic signatures that will help us to understand the abnormal follicular development in PCOS.
... Twelve metabolites were identified and analyzed by 1H-NMR (proton-NMR), identifying that the increase in formate to glycine ratio and the decrease in citrate to alanine ratio was indicative of intrauterine pregnancy [28]. Metabolomic profiling by Raman spectroscopy identified sodium pyruvate and phenylalanine levels to be associated with embryo implantation potential [62]. The principal component and discriminative analysis were applied to amino acid spectral data obtained by HPLC. ...
There have been over 8 million babies born through in vitro fertilization (IVF) and this number continues to grow. There is a global trend to perform elective single embryo transfers, avoiding risks associated with multiple pregnancies. It is therefore important to understand where current research of noninvasive testing for embryos stands, and what are the most promising techniques currently used. Furthermore, it is important to identify the potential to translate research and development into clinically applicable methods that ultimately improve live birth and reduce time to pregnancy. The current focus in the field of human reproductive medicine is to develop a more rapid, quantitative, and noninvasive test. Some of the most promising fields of research for noninvasive assays
comprise cell-free DNA analysis, microscopy techniques coupled with artificial intelligence (AI) and omics analysis of the spent blastocyst media. High-throughput proteomics and metabolomics technologies are valuable tools for noninvasive embryo analysis. The biggest advantages of such technology are that it can differentiate between the embryos that appear morphologically identical and has the potential to identify the ploidy status noninvasively prior to transfer in a fresh cycle or before vitrification for a later frozen embryo transfer.
... To study the complex metabolic profiles of a biological system, effective analytical methods are introduced in the following. In the recent studies, spectroscopic methods such as near-infrared (NIR) spectroscopy [15,16], Raman [17,18] and Nuclear Magnetic Resonance (NMR) [19,20] have been employed. ...
... Some recent studies have suggested that embryo quality and viability is correlated with the changes in chemical composition of the culture medium (Vergouw et al., 2011(Vergouw et al., , 2012Muñoz et al., 2013;Munoz and Uyar, 2014;Nadal-Desbarats et al., 2013;Li et al., 2015;Nagy et al., 2008). The culture medium is the direct microenvironment of an in vitro cultured embryo (Zhao et al., 2013;Shen et al., 2012). The embryonic development requires the uptake of certain substances from the surrounding culture medium and discharge of metabolites in culture media. ...
... Several technologies have been used to evaluate culture media, such as near-infrared spectroscopy (NIR) (Seli et al., 2007), Fourier transform infrared spectroscopy (FT-IR) (Muñoz and Uyar, 2014), Raman spectroscopy (Zhao et al., 2013) and nuclear magnetic resonance (NMR) (Nadal-Desbarats et al., 2013). At present, Raman spectroscopy has been considered as a very promising tool to detect the structure information of biomolecules in aqueous solution and embryo culture media due to its high sensitivity to detect tiny biochemical and molecular variations in tissues or biological samples, and the weak Raman signal of water molecules (Wu et al., 2011). ...
... By measuring metabolites in the culture media, metabolic footprints have been used to reveal the phenotype of mutations of yeast genes (10). Studies have shown that changes in the composition of culture media correspond to differences in embryo quality and reproductive potential (11)(12)(13). In these attempts, correlations between pyruvate uptake (14), glucose uptake (15) or concentration of certain amino acids (11), and embryo viability were observed. ...
... In the present study, we applied Raman spectroscopy to detect changes in metabolic footprints caused by chromosomal abnormalities in embryo culture media. This novel technique can potentially avoid transfer of aneuploid embryos and benefit patients from expensive and laborious assessment of aneuploidy risk (2,6,7,9,(11)(12)(13)17). Furthermore, by applying machine-learning algorithms to build classification models based on Raman spectra, Raman spectroscopy shows potential for establishing a diagnostic platform providing rapid, noninvasive, and affordable cytogenetic assessment of embryo health. ...
Objective
To develop and validate Raman metabolic footprint analysis to determine chromosome euploidy and aneuploidy in embryos fertilized in vitro.
Design
Retrospective study.
Setting
Academic hospital.
Patient(s)
Unselected assisted reproductive technology population.
Intervention(s)
To establish the analysis protocol, spent embryo culture medium samples with known genetic outcomes from 87 human embryos were collected and measured with the use of Raman spectroscopy. Individual Raman spectra were analyzed to find biologic components contributing to either euploidy or aneuploidy. To validate the protocol via machine-learning algorithms, additional 1,107 Raman spectra from 123 embryo culture media (61 euploidy and 62 aneuploidy) were analyzed.
Main Outcome Measure(s)
Raman-based footprint profiling of spent culture media and preimplantation genetic testing for aneuploidy (PGT-A).
Result(s)
Mean-centered Raman spectra and principal component analysis showed differences in the footprints of euploid and aneuploid embryos growing in culture medium. Significant differences in Raman bands associated with small RNAs and lipids were also observed. Stacking classification based on k-nearest-neighbor, random forests, and extreme-gradient-boosting algorithms achieved an overall accuracy of 95.9% in correctly assigning either euploidy or aneuploidy based on Raman spectra, which was validated by PGT-A sequencing results.
Conclusion(s)
This study suggests that chromosomal abnormalities in embryos should lead to changes of metabolic footprints in embryo growth medium that can be detected by Raman spectroscopy. The ploidy status of embryos was analyzed by means of Raman-based footprint profiling of spent culture media and was consistent with PGT-A testing performed by next-generation sequencing.
