Raman-based noninvasive metabolic profile evaluation of in vitro bovine embryos

Érika Cristina dos Santos; Herculano S. da Silva Martinho; Kelly Annes; Thais da Silva; Carlos Alexandre Soares; Roberta Ferreira Leite; Marcella Pecora Milazzotto

doi:10.1117/1.JBO.21.7.075002

6 July 2016 Raman-based noninvasive metabolic profile evaluation of in vitro bovine embryos

Érika Cristina dos Santos, Herculano S. da Silva Martinho, Kelly Annes, Thais da Silva, Carlos Alexandre Soares, Roberta Ferreira Leite, Marcella Pecora Milazzotto

Author Affiliations +

Journal of Biomedical Optics, Vol. 21, Issue 7, 075002 (July 2016). https://doi.org/10.1117/1.JBO.21.7.075002

Abstract

The timing of the first embryonic cell divisions may predict the ability of an embryo to establish pregnancy. Similarly, metabolic profiles may be markers of embryonic viability. However, in bovine, data about the metabolomics profile of these embryos are still not available. In the present work, we describe Raman-based metabolomic profiles of culture media of bovine embryos with different developmental kinetics (fast x slow) throughout the in vitro culture. The principal component analysis enabled us to classify embryos with different developmental kinetics since they presented specific spectroscopic profiles for each evaluated time point. We noticed that bands at 1076 cm−¹ (lipids), 1300 cm−¹ (Amide III), and 2719 cm−¹ (DNA nitrogen bases) gave the most relevant spectral features, enabling the separation between fast and slow groups. Bands at 1001 cm−¹ (phenylalanine) and 2892 cm−¹ (methylene group of the polymethylene chain) presented specific patterns related to embryonic stage and can be considered as biomarkers of embryonic development by Raman spectroscopy. The culture media analysis by Raman spectroscopy proved to be a simple and sensitive technique that can be applied with high efficiency to characterize the profiles of in vitro produced bovine embryos with different development kinetics and different stages of development.

1. Introduction

In recent years, in vitro production (IVP) became the technique of choice for bovine embryos production leading to new possibilities in the bovine genetics market.¹ However, despite the improvement of IVP process, the quality of the embryo and its ability to generate pregnancy are still lower than their in vivo counterpart.²

Methods to identify the better embryos according to their development potential and viability to establish pregnancy, mainly based on noninvasive techniques, are being developed as a strategy to increase the IVP efficiency.³ The assessment of embryonic kinetic patterns,⁴^,⁵—the levels of glucose, lactate, pyruvate,⁶ and amino acids in the culture media of IVP embryos,⁷^,⁸—the oxygen consumption by the embryo,⁹^,¹⁰ and secretome and metabolome patterns¹¹^–¹⁵ are some examples.

The metabolome is the systematic analysis of metabolites representing the functional phenotype of a cell and offers a unique opportunity to investigate the relationship between genotype and the resulting phenotype.¹⁶ The metabolites generated by the cell from the use of energetic precursor and compounds present in the medium are then secreted into the extracellular space, thereby altering the cellular microenvironment.³^,¹⁷ Thus, the analysis of the culture medium can, directly or indirectly, indicate events that are occurring in the cell, which can be correlated with the embryo viability and development potential.¹⁵^,¹⁸

Similarly, embryonic viability can be related with development kinetics, although the results, so far, are still controversial. Mice embryos⁸ and bovine embryos⁵ with fast development seem to be more viable. However, studies show that the methylation state, the expression of imprinted genes and genes associated with stress, as well as the metabolic profile of the in vitro mice embryos with slow to moderate development rates are more similar to in vivo embryos.¹⁹

Raman spectroscopy technique is a potential technique to determine the metabolic profile of IVP embryos. It is a noninvasive and nondestructive method, requiring a minimal previous preparation and small sample volume (few microliters), and it is also relatively cheaper when compared to other techniques.¹²^,¹⁵ In addition, it is a high-resolution method to detect molecular vibrations, which allow the identification of the molecular structure and its components conformation, providing characteristic spectral profiles from the inherent biochemical composition of the analyzed sample.²⁰

Considering the culture media analysis, Raman spectroscopy has been successfully described as a tool for the identification of the fertile potential of IVP human embryo.¹¹ The evaluation of the metabolic profile of human embryos culture media by Raman spectroscopy, combined with the application of bioinformatics tools, is a fast and noninvasive way of assessing the reproductive potential of embryos and pregnancy rates.¹² Also, different spectral profiles were obtained from embryos with good reproductive potential compared to embryos, which resulted in implantation failure.¹¹ Furthermore, Zhao et al.¹⁵ reported a relation between morphology and the relative concentrations of sodium pyruvate and phenylalanine in culture media by Raman spectroscopy, which can be used to predict the embryo reproductive potential with an accuracy of 85.7% for clinical pregnancy. However, it is important to notice that the evaluation of embryos culture media by Raman spectroscopy had not been reported to other species than human. Although human and bovine embryos present several aspects of the metabolism in common, bovine embryos present differences regarding kinetics of development, lipid content, among others, which hinder the use of Raman results from human embryos for this species.²¹ Besides that, there is still a lack of noninvasive methods to evaluate the viability of bovine blastocysts prior to embryo transfer.

Therefore, the present study aimed to identify the metabolomic profile of bovine embryos with different developmental kinetics throughout the in vitro culture (IVC) by the analysis of Raman spectroscopy from their culture media.

2. Materials and Methods

All procedures and protocols were performed in accordance with the Ethical Principles in Animal Research set forth by the Brazilian College of Animal Experimentation, with approval from “Ethic Committee in the Use of Animal” of Universidade Federal do ABC (protocol No. 008/2014).

2.1.

Experimental Design

Bovine oocytes were in vitro-matured (IVM) and in vitro-fertilized (IVF). Presumptive zygotes were cultured individually (IVC) in an adapted well of the well system²² and classified according to their number of cells at 22 h after the onset of IVC in fast (four cells) or slow (two cells) embryos, generating two groups that were evaluated at the following time points: 22 h past the beginning of culture (hpc)—cleavage stage, 96 hpc—period next to embryonic genome activation, and 168 hpc—blastocyst stage. Groups were named as fast (FCL) and slow (SCL) cleaved embryos at 22 hpc, fast (FMO) and slow (SMO) embryos with $> 16$ cells at 96 hpc, and fast (FBL) and slow (SBL) blastocysts at 168 hpc. The culture media from each embryo were collected and kept under $- 80 ° C$ for posterior analysis by Raman spectroscopy. A total of 150 embryos (25 embryos per group) were produced in vitro and Raman analysis was performed in pools of culture media from five embryos (five pools containing five embryos in each group). Raman spectra were collected from five biological replicates and two technical replicates. Figure 1 shows the schematic model of experimental design.

Fig. 1

Experimental design.

2.2.

In Vitro Production

Embryos were produced following standard protocols for IVM and IVF.²³ During the IVC, the zygotes were transferred individually into microdroplets of $20 μ l$ of a culture medium of potassium simplex optimization medium (KSOM) (MR-106-D Millipore^®) supplemented with 10% fetal calf serum (FCS), gentamicin, and nonessential amino acids. At 22 hpc, the KSOM medium was replaced by synthetic oviductal fluid (SOFaa) (supplemented with 5% FCS, essential and nonessential amino acids). Culture followed for 7 days at 38.5°C in 5% ${CO}_{2}$ and saturated humidity.

2.3.

Raman Spectroscopy

The Raman spectroscopy setup details and analysis were described elsewhere.²⁴ Briefly, the equipment used on this study was the Triple T64000 Raman Spectrometer (Horiba Jobin-Yvon S.A.S., France) with microanalysis option and CCD detector $1024 \times 256$ —OPEN-3LD/R with quantum response of $\sim 40 %$ . The excitation laser was 532 nm (Verdi G5, Coherent Inc., United States) focused on a spot with 5-mW power. Two Raman spectra were collected per droplet of culture media from embryos in three different stages of development. Each droplet ( $10 μ L$ ) was placed in a petri dish and covered with mineral oil (3.5 mL). The spectra were performed by using a plan achromatic $50 \times$ objective glass ( $0.20 mm / NA = 0.50$ ), the acquisition $number \times time of exposure$ was $3 \times 20 s$ , and confocal aperture in $6.5 μ m$ .

2.4.

Data Analysis

Experimental data were plotted using the Origin 8.0 software (OriginLab, Northampton, Massachusetts). Spectra were acquired by Labscap 5.0 software and preprocessed to remove spikes by an algorithm, which compares different spectra and subtracts those data points that seems to be aberrant. The baseline correction and normalization by area of the spectra was performed by Fityk 0.9.8 software.²⁵ Spectra were analyzed by principal component analysis (PCA), subroutines of the software Minitab 16 (Minitab Inc.). Vector normalization was made for the analysis done by PCA, and PCs were obtained from a covariance matrix. The following comparisons were made $FCL \times SCL$ , $FMO \times SMO$ , and $FBL \times SBL$ . For each comparison, analyses were performed in two spectral windows: 500 to $1800 {cm}^{- 1}$ and 1800 to $3200 {cm}^{- 1}$ . The spectral features enabling discrimination were found by visual inspection of the PC scores in the loading plot (LP).

3. Results

The spectra of the analyzed groups (culture media from FCL, SCL, FMO, SMO, FBL, and SBL) and pure culture media (SOFaa) are shown in Fig. 2. The corresponding vibrational bands assignment is presented on Table 1.²⁴

Fig. 2

Raman spectra: (a) 800 to $1600 {cm}^{- 1}$ region and (b) 2700 to $3100 {cm}^{- 1}$ region of the following groups: fast and slow cleaved embryos (FCL/SCL), fast and slow $> 16$ cells embryos (FMO/SMO), fast and slow blastocyst (FBL/SBL) and pure culture medium (SOFaa).

Table 1

Raman bands frequencies of the culture medium and their corresponding assignments.

Frequencies (cm−1)		Assignment
Experimental	Literature	Assignment
845	844²⁶	Proteins (v ( $C ─ C$ ))
891	865–942²⁷	Threonine ( $δ ({CO}_{2} H)$ )
952	955²⁸	v ( ${PO}_{4}$ )
1001	1001²⁸	Phenylalanine (Symmetric CC aromatic ring breathing)
1062	1063–1295²⁷	$Ɣ t$ ( ${CH}_{2}$ ) (Weak, couples with adjacent ${CH}_{2}$ groups)
1076	1082²⁰	Lipids ( $C ─ C$ or $C ─ O$ stretching mode)
1152	1156²⁹	$β$ -carotene ( $C ═ C$ stretch)
1300	1301²⁹	Amide III ( $δ$ ( $N ─ H$ )-30%, $α$ -helix, $ν$ ( $C ─ N$ )-40% & $δ ({CH}_{3})$ )
1346	1342²⁰	Collagen ( ${CH}_{3} {CH}_{2}$ wagging mode)
1442	1428–1471²⁹/1442³⁰	Lipid, protein ( $δ$ (CN) bending, $δ ({CH}_{3})$ ) / $β$ -carotene
1455	1451³¹	Lipids, proteins- Kerantin ( ${CH}_{2}$ bending)
2719	2727²⁹	T, A, G of DNA in bend overtone( ${CH}_{3}$ in phase deformation)
2892	2891²⁹	Poly methylene chain (v ( ${CH}_{2}$ , FR))
2957	2960³²	$υ (C ─ H)$

Note: Different vibration types: δ: deformation vibration; ρ: rocking vibration; Ɣt : twisting vibration; v: stretching vibration; δ: bending vibration; FR: Fermi resonance.

Figure 3 represents the results of PCA analysis of $FCL \times SCL$ [Figs. 3(a) and 3(b)], $FMO \times SMO$ [Figs. 3(c) and 3(d)], and $FBL \times SBL$ [Figs. 3(e) and 3(f)] in the 500 to $1800 {cm}^{- 1}$ and 1800 to $3200 {cm}^{- 1}$ ranges. Differences between fast and slow groups at different stages of development (CL, MO, and BL) in the two spectral windows evaluated (500 to $1800 {cm}^{- 1}$ and 1800 to $3200 {cm}^{- 1}$ ) were better observed by PCA analysis. Table 2 shows the percentage of contribution of each PC to the data variability.

Fig. 3

PCA of fast (FCL, FMO, FBL) and slow (SCL, SMO, SBL) groups: (a) PCA from 500 to $1800 {cm}^{- 1}$ region of CL groups; (b) PCA from 1800 to $3200 {cm}^{- 1}$ region of CL groups; (c) PCA from 500 to $1800 {cm}^{- 1}$ region of MO groups; (d) PCA from 1800 to $3200 {cm}^{- 1}$ region of MO groups; (e) PCA from 500 to $1800 {cm}^{- 1}$ region of BL groups; and (f) PCA from 1800 to $3200 {cm}^{- 1}$ region of BL groups.

Table 2

Percentage of variance of PC1, PC2 and PC3 in each spectral region for CL, MO, and BL groups.

Groups	PCs (%)			Raman shift (cm−1)
Groups	PC1	PC2	PC3	Raman shift (cm−1)
CL	94.8	4.8	0.4	500 to 1800
MO	99.8	1.2	0.1
BL	98.8	1.1	0.1
CL	99.7	0.2	0.1	1800 to 3200
MO	99.9	$< 0.01$	$< 0.01$
BL	99.9	$< 0.01$	$< 0.01$

Figure 4 presents the results of LP analysis of $FCL \times SCL$ [Figs. 4(a) and 4(b)], $FMO \times SMO$ [Figs 4(c) and 4(d)], and $FBL \times SBL$ [Figs. 4(e) and 4(f)] in the two spectral windows evaluated. In the first spectrum range measured the PC scores of the CL group [Fig. 4(a)], in particular, $1001 {cm}^{- 1}$ (phenylalanine), $1062 {cm}^{- 1}$ ( $─ {CH}_{2}$ ) and $1076 {cm}^{- 1}$ (lipids) bands, contributed to a greater extent in the separation of the fast and slow groups. In the second spectrum range measured [Fig. 4(b)], the region from 2700 to $2950 {cm}^{- 1}$ , especially, the $2719 {cm}^{- 1}$ (DNA nitrogenous bases T, A, and G) and $2892 {cm}^{- 1}$ (methylene group of polymethylene chain) had opposed profiles.

Fig. 4

LPs of the CL, MO, and BL groups, emphasizing the spectra regions and/or specific bands that presented a major contribution to separate fast and slow embryos. (a) CL at the spectral window 1000 to $1300 {cm}^{- 1}$ ; (b) CL at the spectral window 2700 to $3000 {cm}^{- 1}$ ; (c) MO at the spectral window 1000 to $1300 {cm}^{- 1}$ ; (d) MO at the spectral window 2700 to $3000 {cm}^{- 1}$ ; (e) BL at the spectral window 1000 to $1300 {cm}^{- 1}$ ; and (f) BL at the spectral window 2700 to $3000 {cm}^{- 1}$ . The x axis represents Raman shift ( ${cm}^{- 1}$ ).

The first range evaluated of the MO group [Fig. 4(c)] presented a major contribution to separate the fast and slow groups in the $1001 {cm}^{- 1}$ peak (phenylalanine). In the second spectral range evaluated [Fig. 4(d)], $2892 {cm}^{- 1}$ (methylene group of polymethylene chain) and $2957 {cm}^{- 1}$ ( $─ CH$ ) peaks appear to have a greater contribution to the separation of fast and slow groups.

Finally, for the first range evaluated of the BL group [Fig. 4(e)], the greater contribution to separate the fast and slow groups was observed in the $1001 {cm}^{- 1}$ (phenylalanine), $1076 {cm}^{- 1}$ (lipids), and $1300 {cm}^{- 1}$ (amide III) peaks. Regarding the second range evaluated [Fig. 4(f)], the $2892 {cm}^{- 1}$ (methylene group of polymethylene chain) peak showed a major contribution in the groups separation.

4. Discussion

Embryo selection, based on cleavage rate and morphology, has been developed in recent years aiming the improvement in embryo implantation and pregnancy rates. Although the morphological evaluation is fast and noninvasive, it can be considered a subjective and imprecise method.⁷^,¹³^,¹⁴ Recent studies suggest that the evaluation of metabolome by the culture media analysis can identify embryos with higher implantation potential, since during the IVP, the embryos consume and produce metabolites in the culture media that reflect their cellular activity and development potential.¹⁵^,¹⁸^,²⁰ Similarly, embryo viability has been associated with the development kinetics of embryos.⁵^,⁸ Therefore, the Raman spectroscopy technique, for culture media analysis, was used in this study to discriminate metabolic profiles of bovine IVP embryos with different development kinetics.

PCA demonstrated that the culture medium of fast and slow developing embryos presents different spectroscopic profiles in the whole spectrum range analyzed (500 to $3200 {cm}^{- 1}$ ) at all stages evaluated (Fig. 3). Indeed, in humans, the analysis of culture media by Raman spectroscopy has been suggested as a high sensitive method to select embryos with higher viability. In this study, the metabolomic profiling of embryonic development was associated with implantation rates in IVF, predicting delivery or failed implantation with 80.5% accuracy.¹²

By LP analysis, we identified the bands that contributed to the discrimination among fast and slow groups. In the CL group, the DNA nitrogenous bases (T, A, G) seem to strongly contribute to the separation of these groups. There is, indeed, a correlation between the presence of free DNA in culture media and embryo quality.³³ Genomic DNA (gDNA) and mitochondrial DNA (mtDNA) are detectable in human embryos secretome at the cleavage stage and a high proportion of mtDNA/gDNA in the secretome is associated with poor embryo quality and a high degree of fragmentation. Moreover, the assessment of the DNAmt/gDNA ratio in the day 3 of the embryo secretome in combination with the morphological classification has the potential to improve the success in the identification of viable embryos with higher development.³³^,³⁴ Thus, further studies quantifying the DNA found in the culture media and the evaluation of mtDNA/gDNA ratio may provide important information on the relationship between development kinetics and embryo viability in the bovine species.

Furthermore, the results show that lipids greatly contribute to the separation of the CL and BL embryos from fast and slow groups. The relationship between lipid metabolism and embryo quality has been described in bovines and humans.³⁵^–³⁷ Changes in the embryo lipid profile can be correlated with biosynthetic activity, membrane structure, and functional specialization of the embryos. These modifications can also be used to monitor the metabolic state during embryonic development and to evaluate the impact of the in vitro conditions on the embryo.³⁸ In our study, differences in lipid profile of culture medium of embryos at the stage of CL and BL coupled to differences in amino acid profile, such as phenylalanine, may represent blastomere metabolic changes related to, for example, the ability to use different energy substrates and consequently differences from the excess accumulation of this metabolism. Embryos with the capacity to develop to the blastocyst stage had a lower amino acid turnover than those that developmentally arrested.³⁹^,⁴⁰ The highest consumption of amino acids that is also directed to the TCA cycle may promote an increase in acetyl-CoA synthesis, a precursor of lipid metabolism.⁴¹ As a result of different preferred pathways, the fast and slow embryos of CL and BL groups could accumulate different energy precursors, one directing the exceeding glucose to glycogen synthesis while the other accumulating lipids.

LP analysis also evidenced that the amide III bands contributed to a greater extent to the separation of the fast and slow groups at BL stage. The analysis of the amide III vibrational mode (1230 to $1300 {cm}^{- 1}$ ), denominated by the nitrogen–hydrogen bonds vibration and by the carbon–nitrogen stretching vibration, can be used for conformational analysis of proteins. Amide III analysis can also be used for quantitative analysis of proteins secondary structure, using the ratio of $α$ -helices and $β$ -sheets bands at 1235 and $1275 {cm}^{- 1}$ , respectively.⁴²^,⁴³ Based on this information, it can be suggested that there is a correlation between the embryo development kinetics and its ability to promote changes in the structure of essential proteins. Interestingly, the changes in this spectral region were only evident after the activation of the embryonic genome and hence after mRNA synthesis by the embryo.⁴⁴ Therefore, these changes may result from failures in gene expression or genomic and epigenetic alterations occurred in the early stages of development.¹⁹

It is interesting to note that the LP analysis showed specific profiles in the vibration bands of phenylalanine and the methylene group of polymethylene chain, which greatly contributed to the separation of the fast and slow groups in all evaluated stages. Phenylalanine is an essential amino acid that is not produced by the organism but obtained through the diet⁴⁵ or in the case of IVP embryo, by supplementation of the culture medium. This amino acid has an important role in the preimplantation embryo development, especially in protein synthesis, energy supplement, and pH adjustment of the medium.¹⁸ The relationship between phenylalanine concentration in the culture medium and the embryonic viability has been described in other species. These concentrations are used as biomarkers of clinical pregnancy in humans¹⁵ and blastocyst formation in pigs.⁴ Furthermore, in vivo bovine blastocysts showed higher rates of phenylalanine, valine, and threonine when compared to in vitro blastocysts.⁴⁶ Thus, the fact that phenylalanine is related to embryonic viability and also with development kinetics, as showed in the results of this study, suggests that viability has indeed an important relation with kinetics.

Finally, there is no data in the literature that relate the methylene group of the polymethylene chain (methylene with ${CH}_{2}$ bond) with embryonic development. However, studies indicate that the analysis of the intensity proportion of ${CH}_{3} / {CH}_{2}$ bands can be related to molecules conformational changes.¹⁵ Analysis of different types of brain malignant tumor cells showed an increase in the spectrum bandwidth that included the peaks at 2935 and $2880 {cm}^{- 1}$ , which correspond to the groups methyl ( ${CH}_{3}$ bond) and methylene ( ${CH}_{2}$ bond), respectively. The decrease in intensity of the ${CH}_{3} / {CH}_{2}$ bands ratio, when compared to normal or benign cell types, also suggests changes in molecular conformation of malignant cells.⁴⁶ The similarity between embryonic and tumor cells regarding plasticity, high adaptability in the cell growing tissue, morphological correlations, among others⁴⁷^,⁴⁸ could indicate that the molecular conformation disorders evidenced by the ${CH}_{3} / {CH}_{2}$ bands ratio could also be valid for the identification of the embryo metabolic status.

Therefore, we suggest that the phenylalanine and methylene group of the polymethylene chain bands, which present opposing profiles at all stages of embryonic development, can be considered Raman spectroscopy biomarkers of bovine embryos development kinetics. Table 3 shows a summary of the biological function of the important bands for fast and slow embryos described on this study.

Table 3

Raman bands assignments in fast and slow embryo and their biological function or interpretation.

Frequencies (cm−1)	Assignment	Biological function/interpretation
2719	DNA nitrogenous bases	Identification of embryos with high potential for development³³^,³⁴
1076	Lipids	Embryo quality³⁶^,³⁸
1300	Amide III	Conformational analysis of proteins⁴²^,⁴³
1001	Phenylalanine	Embryo viability⁴^,¹⁵
2892	Polymethylene chain	Modifications to the molecular conformation¹⁵^,²⁹

5. Conclusion

The use of Raman spectroscopy for the analysis of IVP bovine embryos culture media has the potential to be applied as a diagnostic method for noninvasive evaluation and characterization of embryos. Some metabolites already described as biomarkers of embryonic viability appear to contribute in major proportion to the separation of the groups with different development kinetics at the stages evaluated, suggesting a relationship between kinetics and viability of bovine IVP embryos.

Acknowledgments

The authors would like to thank the Brazilian agencies FAPESP (Grant No. 2012/10351-2), CAPES, and UFABC for financial support; and Multiuser Central Facilities of UFABC for the experimental support.

References

1.

J. H. M. Viana et al., “Use of in vitro fertilization technique in the last decade and its effect on Brazilian embryo industry and animal production,” Acta Sci. Vet., 38 (2), 661 –674 (2010). Google Scholar

2.

L. G. B. Siqueira et al., “Factores que afectan a la fecundación in vitro en bovinos,” Spermova, 2 (1), 10 –12 (2012). Google Scholar

3.

M. Muñoz et al., “Prediction of pregnancy viability in bovine in vitro-produced embryos and recipient plasma with Fourier transform infrared spectroscopy,” J. Dairy Sci., 97 (9), 5497 –5507 (2014). http://dx.doi.org/10.3168/jds.2014-8067 Google Scholar

4.

P. J. Booth, T. J. Watson and H. J. Leese, “Prediction of porcine blastocyst formation using morphological, kinetic, and amino acid depletion and appearance criteria determined during the early cleavage of in vitro-produced embryos,” Biol. Reprod., 77 765 –779 (2007). http://dx.doi.org/10.1095/biolreprod.107.062802 Google Scholar

5.

P. Holm, P. J. Booth and D. H. Callesen, “Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media,” Reproduction, 123 553 –565 (2002). http://dx.doi.org/10.1530/rep.0.1230553 Google Scholar

6.

D. K. Gardner et al., “Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential,” Fertil. Steril., 76 1175 –1180 (2001). http://dx.doi.org/10.1016/S0015-0282(01)02888-6 Google Scholar

7.

D. R. Brison et al., “Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover,” Human Reprod., 19 2319 –2324 (2004). http://dx.doi.org/10.1093/humrep/deh409 Google Scholar

8.

Y. S. Lee, G. A. Thouas and D. K. Gardner, “Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage,” Human Reprod., 30 (3), 543 –552 (2015). http://dx.doi.org/10.1093/humrep/deu334 Google Scholar

9.

A. S. Lopes, T. Greve and H. Callesen, “Quantification of embryo quality by respirometry,” Theriogenology, 67 21 –31 (2007). http://dx.doi.org/10.1016/j.theriogenology.2006.09.026 THGNBO 0093-691X Google Scholar

10.

J. G. Thompson et al., “Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos,” Reproduction, 106 (2), 299 –306 (1996). http://dx.doi.org/10.1530/jrf.0.1060299 Google Scholar

11.

E. Seli et al., “Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization,” Fertil. Steril., 88 (5), 1350 –1357 (2007). http://dx.doi.org/10.1016/j.fertnstert.2007.07.1390 Google Scholar

12.

R. T. Scott et al., “Non-invasive metabolomics profiling of human embryo culture media using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study,” Fertil. Steril., 90 77 –83 (2008). http://dx.doi.org/10.1016/j.fertnstert.2007.11.058 Google Scholar

13.

S. M. Pudakalakatti et al., “NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential,” NMR Biomed., 26 20 –27 (2013). http://dx.doi.org/10.1002/nbm.v26.1 Google Scholar

14.

C. G. Vergouw et al., “Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection,” Human Reprod., 23 (7), 1499 –1504 (2008). http://dx.doi.org/10.1093/humrep/den111 Google Scholar

15.

Q. Zhao et al., “Noninvasive metabolomic profiling of human embryo culture media using a simple spectroscopy adjunct to morphology for embryo assessment in in vitro fertilization (IVF),” Int. J. Mol. Sci., 14 (4), 6556 –6570 (2013). http://dx.doi.org/10.3390/ijms14046556 1422-0067 Google Scholar

16.

L. Botros, D. Sakkas and E. Seli, “Metabolomics and its application for non-invasive embryo assessment in IVF,” Mol. Human Reprod., 14 679 –690 (2008). http://dx.doi.org/10.1093/molehr/gan066 Google Scholar

17.

N. L. Themaat and Z. P. Nagy, “A review of the promises and pitfalls of oocyte and embryo metabolomics,” Placenta, 32 (3), S257 –S263 (2011). http://dx.doi.org/10.1016/j.placenta.2011.05.011 PLACDF 0143-4004 Google Scholar

18.

A. G. Shen et al., “Accurate and noninvasive embryos screening during in vitro fertilization (IVF) assisted by Raman analysis of embryos culture medium,” Laser Phys. Lett., 9 322 –328 (2012). http://dx.doi.org/10.1002/lapl.v9.4 1612-2011 Google Scholar

19.

B. A. Market Velker, M. M. Denomme and M. R. Mann, “Loss of genomic imprinting in mouse embryos with fast rates of preimplantation development in culture,” Biol. Reprod., 86 (143), 1 –16 (2012). http://dx.doi.org/10.1095/biolreprod.111.096602 Google Scholar

20.

Z. Huang et al., “Rapid and nondestructive method for evaluation of embryo culture media using drop coating deposition Raman spectroscopy,” J. Biomed. Opt., 18 (12), 127003 (2013). http://dx.doi.org/10.1117/1.JBO.18.12.127003 JBOPFO 1083-3668 Google Scholar

21.

Y. J. Ménézo and F. Hérubel, “Mouse and bovine models for human IVF,” Reprod. Biomed. Online, 4 (2), 170 –175 (2002). http://dx.doi.org/10.1016/S1472-6483(10)61936-0 Google Scholar

22.

G. Vajta et al., “New method for culture of zona-included or zona-free embryos: the well of the well (WOW) system,” Mol. Reprod. Dev., 55 256 –264 (2000). http://dx.doi.org/10.1002/(ISSN)1098-2795 Google Scholar

23.

C. A. Soares et al., “Photobiological effect of low-level laser irradiation in bovine embryo production system,” J. Biomed. Opt., 19 (3), 035006 (2014). http://dx.doi.org/10.1117/1.JBO.19.3.035006 JBOPFO 1083-3668 Google Scholar

24.

E. C. Santos et al., “Rapid and noninvasive technique to assess the metabolomics profile of bovine embryos produced in vitro by Raman spectroscopy,” Biomed. Opt. Express, 6 (8), 2830 –2839 (2015). http://dx.doi.org/10.1364/BOE.6.002830 BOEICL 2156-7085 Google Scholar

25.

M. Wojdyr, “Fityk: a general-purpose peak fitting program,” J. Appl. Crystallogr., 43 1126 –1128 (2010). http://dx.doi.org/10.1107/S0021889810030499 Google Scholar

26.

M. S. Bergholt et al., “In vivo diagnosis of gastric cancer using Raman endoscopy and ant colony optimization techniques,” Int. J. Cancer, 128 (11), 2673 –2680 (2011). http://dx.doi.org/10.1002/ijc.v128.11 Google Scholar

27.

A. Barth and C. Zscherp, “What vibrations tell us about proteins,” Q. Rev. Biophys., 35 (4), 369 –430 (2002). http://dx.doi.org/10.1017/S0033583502003815 QURBAW 0033-5835 Google Scholar

28.

S. Cinta Pinzaru, A. Falamas and C. A. Dehelean, “Molecular conformation changes along the malignancy revealed by optical nanosensors,” J. Cell. Mol. Med., 17 (2), 277 –286 (2013). http://dx.doi.org/10.1111/jcmm.12006 Google Scholar

29.

Y. Zhou et al., “Human brain cancer studied by resonance Raman spectroscopy,” J. Biomed. Opt., 17 (11), 116021 (2012). http://dx.doi.org/10.1117/1.JBO.17.11.116021 JBOPFO 1083-3668 Google Scholar

30.

O. Samek et al., “Raman microspectroscopy of individual algal cells: sensing unsaturation of storage lipids in vivo,” Sensors, 10 (9), 8635 –8651 (2010). http://dx.doi.org/10.3390/s100908635 SNSRES 0746-9462 Google Scholar

31.

L. Xue et al., “Diagnosis of pathological minor salivary glands in primary Sjogren’s syndrome by using Raman spectroscopy,” Lasers Med. Sci., 29 (2), 723 –728 (2014). http://dx.doi.org/10.1007/s10103-013-1398-y Google Scholar

32.

A. Moshaverinia et al., “Modification of conventional glass-ionomer cements with N-vinylpyrrolidone containing polyacids, nano-hydroxy and fluoroapatite to improve mechanical properties,” Dent. Mater., 24 (10), 1381 –1390 (2008). http://dx.doi.org/10.1016/j.dental.2008.03.008 Google Scholar

33.

S. Stigliani et al., “Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation,” Human Reprod., 28 2652 –2660 (2013). http://dx.doi.org/10.1093/humrep/det314 Google Scholar

34.

S. Stigliani et al., “Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome,” Mol. Human Reprod., 20 (12), 1238 –1246 (2014). http://dx.doi.org/10.1093/molehr/gau086 Google Scholar

35.

E. M. Ferguson and H. J. Leese, “Triglyceride content of bovine oocytes and early embryos,” Reproduction, 116 373 –378 (1999). http://dx.doi.org/10.1530/jrf.0.1160373 Google Scholar

36.

M. L. Sutton-Mcdowall et al., “Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos,” Theriogenology, 77 (8), 1632 –1641 (2012). http://dx.doi.org/10.1016/j.theriogenology.2011.12.008 THGNBO 0093-691X Google Scholar

37.

P. Haggarty et al., “Fatty acid metabolism in human preimplantation embryos,” Human Reprod., 21 (3), 766 –773 (2006). http://dx.doi.org/10.1093/humrep/dei385 Google Scholar

38.

C. R. Ferreira et al., “Developmental phases of individual mouse preimplantation embryos characterized by lipid signatures using desorption electrospray ionization mass spectrometry,” Anal. Bioanal.Chem., 404 (10), 2915 –2926 (2012). http://dx.doi.org/10.1007/s00216-012-6426-4 ABCNBP 1618-2642 Google Scholar

39.

H. J. Leese, “Metabolism of the preimplantation embryo: 40 years on,” Reproduction, 143 (4), 417 –427 (2012). http://dx.doi.org/10.1530/REP-11-0484 Google Scholar

40.

C. G. Baumann et al., “The quiet embryo hypothesis: molecular characteristics favoring viability,” Mol. Reprod. Dev., 74 1345 –1353 (2007). http://dx.doi.org/10.1002/(ISSN)1098-2795 MREDEE 1098-2795 Google Scholar

41.

R. F. Kletzien, P. K. Harris and L. A. Foellmi, “Glucose-6-phosphate dehydrogenase: a “housekeeping” enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant stress,” FASEB J., 8 174 –181 (1994). Google Scholar

42.

M. Gniadecka et al., “Diagnosis of basal cell carcinoma by Raman spectroscopy,” J. Raman Spectrosc., 28 125 –129 (1997). http://dx.doi.org/10.1002/(ISSN)1097-4555 Google Scholar

43.

G. Rusciano et al., “Raman spectroscopy of Xenopus laevis oocytes,” Methods, 51 27 –36 (2010). http://dx.doi.org/10.1016/j.ymeth.2009.12.009 MTHDE9 1046-2023 Google Scholar

44.

Q. T. Wang et al., “A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo,” Dev. Cell, 6 (1), 133 –144 (2004). http://dx.doi.org/10.1016/S1534-5807(03)00404-0 Google Scholar

45.

D. Voet and J. G. Voet, Bioquímica, 3rd ed.Artmed, Porto Alegre (2006). Google Scholar

46.

R. G. Sturmey et al., “Amino acid metabolism of bovine blastocysts: a biomarker of sex and viability,” Mol. Reprod. Dev., 77 285 –296 (2010). http://dx.doi.org/10.1002/mrd.21145 MREDEE 1098-2795 Google Scholar

47.

R. E. Brown and M. F. Mcguire, “Oncogenesis recapitulates embryogenesis via the hypoxia pathway: morphoproteomics and biomedical analytics provide proof of concept and therapeutic options,” Ann. Clin. Lab. Sci., 42 (3), 243 –57 (2012). Google Scholar

48.

S. Koljenovic et al., “Discriminating vital tumor from necrotic tissue in human glioblastoma tissue samples by Raman spectroscopy,” Lab. Invest., 82 (10), 1265 –1277 (2002). http://dx.doi.org/10.1097/01.LAB.0000032545.96931.B8 LAINAW 0023-6837 Google Scholar

Biography

Érika Cristina dos Santos received her MSc in biotechnoscience at the Universidade Federal do ABC/Brazil. She is a member of the laboratory of Cellular and Molecular Biology of the Universidade Federal do ABC and has experience in the following topics: in vitro production of bovine embryos, metabolomics, Raman spectroscopy, and MALDI-TOF-MS.

Herculano Martinho received his PhD degree in physics from Universidade Estadual de Campinas/ Brazil. He is a professor at Universidade Federal do ABC. He is the head of the Multiuser Central Facilities and has experience in the following topics: low-level laser therapy, Raman-based optical biopsy, and dynamical properties of macromolecules.

Kelly Annes received her MSc in biotechnoscience at the Universidade Federal do ABC/Brazil. She is a member of the Laboratory of Cellular and Molecular Biology of the Universidade Federal do ABC and has experience in the following topics: in vitro production of bovine embryos, molecular biology techniques, lipidomics, and MALDI-TOF-MS.

Thais da Silva received her MSc in biotechnoscience at the Universidade Federal do ABC/Brazil. She was a member of the Laboratory of Cellular and Molecular Biology of the Universidade Federal do ABC and has experience in the following topics: in vitro production of bovine embryos and molecular biology techniques.

Carlos Alexandre Soares received his MSc in biotechnoscience at the Universidade Federal do ABC/Brazil. He was a member of the Laboratory of Cellular and Molecular Biology of the Universidade Federal do ABC and has experience in the following topics: computer programming, in vitro production of bovine embryos, molecular biology techniques, and biomodulation with low-level laser.

Roberta Ferreira Leite received her BS in veterinary medicine and her MS degree in biotechnoscience at the Universidade Federal do ABC/Brazil. She is a member of the Laboratory of Cellular and Molecular Biology of the Universidade Federal do ABC and has experience in the following topics: animal genetics, bioinformatics, in vitro production of bovine embryos, and molecular biology techniques.

Marcella Pecora Milazzotto received her PhD in biotechnology from University of Sao Paulo/Brazil. She is now a professor at Universidade Federal do ABC where she is responsible for the Laboratory of Cellular and Molecular Biology. She has experience in the following topics: in vitro fertilization, gametes, and embryos metabolism.

Citation Download Citation

Érika Cristina dos Santos, Herculano S. da Silva Martinho, Kelly Annes, Thais da Silva, Carlos Alexandre Soares, Roberta Ferreira Leite, and Marcella Pecora Milazzotto "Raman-based noninvasive metabolic profile evaluation of in vitro bovine embryos," Journal of Biomedical Optics 21(7), 075002 (6 July 2016). https://doi.org/10.1117/1.JBO.21.7.075002

Published: 6 July 2016

Access the abstract

JOURNAL ARTICLE
7 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY

GET CITATION

CITATIONS

Cited by 11 scholarly publications.

Explore citations on Lens.org

RIGHTS & PERMISSIONS

Get copyright permission Get copyright permission on Copyright Marketplace

KEYWORDS

Raman spectroscopy

In vitro testing

Principal component analysis

Molybdenum

Proteins

Biological research

Source mask optimization

1.

Introduction

2.

Materials and Methods