Non-invasive metabolomic profiling of Day 3 embryo culture media using near-infrared spectroscopy to assess the development potential of embryos

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1 Acta Biochim Biophys Sin 2013, 45: ª The Author Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: /abbs/gmt115. Advance Access Publication 17 October 2013 Lab Note Non-invasive metabolomic profiling of Day 3 embryo culture media using near-infrared spectroscopy to assess the development potential of embryos Jing Fu 1, Juan Shao 1, Xiong Li 1, Yan Xu 1, Suying Liu 1, and Xiaoxi Sun 1,2 * 1 Shanghai Ji Ai Genetics & IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai , China 2 Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai , China *Correspondence address. Tel: þ ; Fax: þ ; steven3019@hotmail.com The success rate and multiple pregnancy rate are two important issues emerging in assisted reproductive technology cycles [1]. It is usually used to increase the number of embryos; however, it increases the risk of multiple pregnancies. Therefore, the key point to improve the success rate is to select the best-quality embryos [2 4]. Currently, noninvasive embryo selection emerges which is primarily based on morphological criteria, such as pronuclear morphology [5 7], appearance of much fragmentation [8 11], and multinucleation of blastomeres [12] and unequal-sized blastomeres [13 16] by light microscopy analysis. Although it is easy and convenient to obtain data by using these morphological criteria which are known to contribute to pregnancy outcomes, they have limited predictive value for ongoing pregnancy and the accuracy to select embryos of high reproductive potential almost depends on the experience of embryologist [17 19]. Therefore, new selection techniques are needed [20,21]. Evolving from genomics, transcriptomics and proteomics, metabolomics, an emerging omics science, is the systematic analysis of the inventory of metabolites, which represent the functional phenotype at cellular level [22]. In particular, metabolomics can be used to reveal the underlying change of disease and abnormal health conditions in a biological system. Metabolomics offers a unique opportunity to investigate the relationship between an organism s genotype and its resultant phenotype, as metabolome is the end product of gene expression and also marks a relationship between an organism s physiology and environmental conditions [23,24]. Spent culture media of embryos contain many metabolites, which are the ultimate products of cellular metabolism and are very diverse in their physical and chemical properties [25]. It can give the information of changes in embryo development process and in the gene expression of embryos, so it can be used as a predictor of embryo development potential. To investigate complex metabolic profiles of a biological system, non-selective but specific analytical technology is required. Many spectrometric and chromatographic techniques are excellent candidates and provide automated, high-throughput methodologies with informationrich profiles of target biological fluids. One of the optical spectroscope techniques to investigate the biological fluids is near-infrared (NIR) spectroscopy, which has bright future in the application in clinical setting because of its simple platform and inexpensive price [26]. To study metabolomic profiling by NIR spectroscopy, here, we measured embryo viability by ascertaining the changes in concentrations of key functional groups such as ROH, SH, C¼C, CH, OH, and NH, which correspond to albumin, lactate, pyruvate, glutamate, and glucose in culture media. In the present study, a total of 172 patients participating in the study were recruited from those undergoing double embryos transfer and single embryo transfer (SET) on Day 3 at Shanghai Ji-Ai Genetics and IVF Center (Shanghai, China) from April 2011 to December All of these selected patients were aged years, tube factor, male factor, and both factor for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) within the first or second cycle. Those with abnormal endometrial development [,6 mm in peak thickness or failure to develop trilaminar pattern before human chorionic gonadotropin (HCG) administration] were excluded. The study was approved by the ethics committee at Obstetrics and Gynecology Hospital, Fudan University (Shanghai, China) and the patients signed informed consent forms before participation. All of these patients were in the fresh cycles. The ovarian stimulation was performed by using Suprecur, a GnRH agonists (Aventis Pharma, Frankfurt, Germany), and Gonal-F, a recombinant follicle-stimulating hormone (Merck-Serono, Geneva, Switzerland). HCG (Profasi; Merck-Serono) was injected as long as more than three follicles of 17 mm were seen with ultrasound. Oocyte retrieval was performed by transvaginal Acta Biochim Biophys Sin (2013) Volume 45 Issue 12 Page 1074

2 ultrasound h after HCG injection. Oocytes were fertilized using either conventional IVF or ICSI and incubated in fertilization media (Vitrolife, Kungsbacka, Sweden). Normal fertilization was assessed and confirmed by the presence of two pronuclei and second polar body at h after insemination. The embryos were washed and cultured individually in cleavage media (Vitrolife) for 48 h before being transferred. Volume of each of culture droplet was 30 ml. Routine examination of embryo quality included measuring the number and uniformity of blastomeres, and the degree of fragmentation. Embryo morphology was scored as follows [12,27]: grade A, 7 cells, no fragments, and equal blastomeres, which scored 4; grade B, 7 cells,,20% fragmentation, equal or unequal blastomeres, which scored 3; grade C, 6 cells, or equal or unequal blastomeres, 20% 50% fragments, which scored 2; grade D, equal or unequal blastomeres,.50% fragments, which scored 1. Pregnancy outcomes were recorded for each patient at 8 10 weeks gestational age. Positive pregnancy was defined as fetal cardiac activity at that time. On Day 3, two embryos of good quality were selected by using morphology score system for transfer and the spent culture media were tested by using NIR spectroscopy after removing the transferred embryos. Analysis for individual embryo culture media samples was performed as described previously [28 30]. A total of ml of IVF culture media sample was then slowly injected by using an Eppendorf pipette into an individual sample cell designed to fit in the NIR instrument (ANTARISII; Thermo Fisher Scientific Inc., New York, USA). Sample cells were then placed in a sample chamber maintained at 23.58C and NIR transmittance spectra was measured using 512 element Table 1. Comparison of patients general conditions between the two groups InGaAs array NIR spectrometer with an operating wavelength of nm. The analysis time for each sample was 1 min. The measurement was repeated with corresponding control medium for each sample to account for any influence of variations in culturing conditions. A final spectral profile was then generated for each embryo by analyzing the embryo culture medium and control medium. Quantification of sample properties from NIR spectra consisted of variables determination in selected wavelength domains and genetic algorithm optimization. Experiment was carried out according to previous report [31]. Briefly, to objectively select wavelength regions, a preprocessing method based on Haar wavelets was used. The combination of wavelength regions, which most parsimoniously estimated implantation outcomes, was determined by using inverse least-squares regression ( partial least square-discriminant analysis) and genetic algorithm optimization. The following formula was used: Y ¼ b 0 þ b 1 X 1 þ b 2 X 2 þ...þ b n X n where Y was the dependent variable (pregnancy outcome of 0 or 1), X 1, X 2... and X n were independent variables (i.e. integrated wavelength region), and b 0, b 1...and b n were the coefficients. Data were analyzed by using Matlab software (version 7.1; MathWorks Inc., New Mexico, USA) and SPSS (version 18.0; SPSS Inc., Chicago, USA). P, 0.05 was considered significant for all comparisons. The baseline characteristics of pregnant and non-pregnant patients were listed in Table 1. A total of 339 embryos were transferred to 172 patients. There were no significant Item Pregnancy group Non-pregnancy group P Patients number No. of embryo transfer Age NS Infertility duration NS Cycles NS Infertility indication Tube factor (%) Male factor (%) Both factor (%) Proportion of IVF (%) Proportion of ICSI (%) Peak E2 value (pg/ml) NS Endometrial thickness (mm) NS Cell number NS Morphology score NS Viability index P, 0.01 NS, no significant difference. Acta Biochim Biophys Sin (2013) Volume 45 Issue 12 Page 1075

3 differences in the age, duration of infertility, treatment cycles, infertility cause, the proportion of IVF/ICSI, and the endometrial thickness between these two groups. However, the mean viability index was significantly different between these two groups, although the morphology scores were nearly the same ( vs , P. 0.05). Figure 1 showed the relationship between the implantation outcome and viability index. It seemed that the indices of all Figure 1 Relationship between the implantation outcome and viability indices, determined in blinded analysis of 223 embryos from 114 patients. One hundred and seventy-three embryos were from 88 patients on the left stand for 0% implantation. The viability indices of 47 embryos were above 0 (27%) and those of 126 embryos were below 0 (73%). Fifty embryos were from 26 patients on the right stand for 100% implantation. Figure 2 ROC curves of composite score (yellow), viability index (green), and morphology score (blue) for Day 3 embryo culture medium samples. The optimal cutoff value was AUC ROC for composite score was 0.93, AUC ROC for viability score was AUC ROC for morphological grade was P ¼ 0.004, composite score vs. morphology score. successful implantation embryos were above 0, and most of the failed implantation embryos (73%) had indices below 0. Figure 2 showed morphology grading, metabolomic viability scores, and composite scores of Day 3 embryo culture media samples by using receiver operating characteristic curve (ROC) analysis. The composite score was a combination of morphology score and viability index. The area under the ROC curve (AUC ROC ) of viability score was significantly higher than that of morphology score (0.96 vs. 0.53; P ¼ 0.004; Fig. 2), while the AUC ROC of composite score was not significantly different from that of viability score (0.93 vs. 0.96; P. 0.05; Fig. 2). The best threshold was In addition, the positive-predictive value of metabolomic profiling was and the negative-predictive value was Viability index or the combination of viability index and morphological grading was more accurate in predicting outcome than morphological grading alone. NIR spectroscopy is a rapid and non-invasive technique in analyzing spent embryo media to choose the best embryo for implantation. In the present study, each testing sample needed only 10 ml of media and the machine is small and easy to operate. These advantages make it valuable in science research and clinical application, which improves elective SET in a widespread use quickly. Generally, morphology scores were used to predict the development potential of embryos. However, the limitations of this technique have arisen more and more significantly. On one hand, there is no accurate morphological score system which is regarded as the best way to choose embryos; on the other hand, morphological score system is based on scientists subjective judgment. Some researchers have reported that the pregnancy rate of good-quality embryos, which are chosen by morphological score system, is 49%, while our result showed that the pregnancy rate was only 48.8%, even with the goodquality rate of 93.3%. Therefore, the morphology score was not the sensitive observation index of embryo functions. The embryos that have high morphology scores could also have some genetic and epigenetic defects which embarrass the implantation, while the metabolomics offer a unique opportunity to investigate the relationships between an organism s genotype and its resulting phenotype as the metabolome is the end product of gene expression, and also marks a relationship between an organism s physiology and environmental conditions [23,24]. Hence, that may explain why the metabolomic profiling of spent embryo culture media was more accurate than morphological grading. In conclusion, the metabolomic profiling data from early cleavage embryos showed similar findings to earlier work using this method with Day 5 embryos [16,26,32 37] and oocytes [35]: higher viability index results in higher pregnancy outcomes. Embryos (of the same morphological grade) have different metabolic activity which is related to implantation potential, indicating that the uses of Acta Biochim Biophys Sin (2013) Volume 45 Issue 12 Page 1076

4 morphological and metabolomic criteria both can help to decide which embryo to transfer. New techniques in assessing embryo quality may improve pregnancy and delivery rates per embryo transfer and therefore encourage the implementation of SET [38]. Metabolomic profiling by using NIR spectroscopy is a rapid, objective, and non-invasive embryo assessment technique which may provide extra information about the implantation potential of Day 3 embryos during transfer, which might reduce the number of embryos transferred in fresh cycles and therefore reduce the incidence of multiple pregnancies. Acknowledgements The authors thank Professor Yiping Du and Wenting Ma, who are from East China University of Science and Technology, for their help in the collection and analysis of NIR spectroscopy data. Funding This work was supported by the program from Science and Technology Commission of Shanghai Municipality (No ). References 1 Wright V, Cheng L, Jeng G and Macaluso M. 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