Hum. Reprod. Advance Access published online on April 18, 2008
Human Reproduction, doi:10.1093/humrep/den111
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Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection
1 Department of Obstetrics, Gynaecology and Reproductive Medicine, VU University Medical Center, Amsterdam, the Netherlands 2 Molecular Biometrics LLC, Chester, NJ, USA 3 McGill University, Department of Chemistry, Montreal, QC, Canada
4 Correspondence address. VU University Medical Center, department of Obstetrics, Gynaecology and Reproductive Medicine, PO box 7057, 1007 MB, Amsterdam, the Netherlands. Tel: +31-204443229; Fax: +31-204443267; E-mail: carlijn.vergouw{at}vumc.nl
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Abstract |
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BACKGROUND: The morphology of an embryo has a limited predictive value for assessing viability and ongoing pregnancy, therefore new selection tools are needed to maintain success rates with single-embryo transfer (SET). In this study, we investigated if metabolomic profiling of biomarkers of embryo culture medium by near-infrared (NIR) spectroscopy has a correlation with ongoing pregnancy in SET.
METHODS: A total of 333 patients scheduled for in vitro fertilization (IVF) with SET were included in the study. Embryos were selected for transfer by morphological criteria on Days 2 and 3 of in vitro culture, and left over culture media samples were analyzed by NIR spectroscopy.
RESULTS: The NIR spectral analysis produced unique metabolomic profiles that correlated to an embryo's reproductive potential. Resulting relative viability scores between positive and negative pregnancy outcomes were statistically significant (P < 0.03). A logistic regression of factors correlated to pregnancy outcomes showed that maternal age, percent fragmentation and relative viability scores all demonstrated a relationship. The extent of the correlation was determined by accuracy computation, where the accuracy of assessing viable embryos on Day 3 by metabolomic profiling was 53.6% and the accuracy of the morphological selection was 38.5%. In addition, the positive predictive value of metabolomic profiling was 0.365 and the negative predictive value was 0.830.
CONCLUSIONS: NIR metabolomic profiling of spent embryo culture media was able to distinguish viable embryos from non-viable embryos for reproduction.
Key words: non-invasive embryo selection/single-embryo transfer (SET)/near-infrared (NIR) spectroscopy/metabolomic profiling
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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One of the most important complications of in vitro fertilization (IVF) treatment is a high multiple pregnancy rate. Multiple gestations lead to a higher incidence of medical (both perinatal and neonatal) and socioeconomic complications (Lieberman, 1998
; Templeton, 2000; Gerris et al., 2004; Lukassen et al., 2004). Single-embryo transfer (SET) is an effective way to minimize the risks of multiple pregnancies post-IVF treatment (Gerris and van Royen, 2000; Pandian et al., 2005; van Montfoort et al., 2006). Given that a single embryo is transferred, the embryo selection process is crucial. However, general acceptance of SET is limited as there lacks an accurate method in assessing embryo reproductive viability (Brison et al., 2004). Currently, non-invasive embryo selection is primarily based on morphological criteria using light microscope analysis. Many studies have shown that an embryo's morphological appearance and developmental potential are biomarkers of its viability. Early cleavage (Lundin et al., 2001; Salumets et al., 2003; van Montfoort et al., 2004) and embryos that develop at a certain cleavage rate (Ziebe et al., 1997; van Royen et al., 1999; Thurin et al., 2005) were shown to be good prognostic factors. Pronuclear morphology (Tesarik and Greco, 1999; Scott et al., 2000; Gianeroli et al., 2007), the appearance of much fragmentation (Giorgetti et al., 1995; Ziebe et al., 1997; van Royen et al., 1999, 2001), multi nucleation of blastomeres (van Royen et al., 2003) and unequally sized blastomeres (Hardarson et al., 2001; Hnida et al., 2004; Holte et al., 2006; Scott et al., 2007) have also shown to be important. Although all of these factors are known to contribute to pregnancy results, they have a limited predictive value for ongoing pregnancy. Because of the limited predictive value, new selection tools are needed (Gardner et al., 2001; Brison et al., 2004). Additional methods which claim to be increasingly accurate, such as preimplantation genetic screening, are at a disadvantage in that one or two blastomere(s) is/are aspirated from an embryo. As an invasive measurement, this method provides little additional reproductive assessment value and shows no improvement in pregnancy rates (Staessen et al., 2007). One other study even reported lower pregnancy and live birth rates (Mastenbroek et al., 2007).
Recent studies have presented non-invasive metabolomic profiling methods of assessing an embryo's reproductive potential. It is thought that the physiology and viability of the preimplantation embryo is affected by the environment to which the embryo is exposed in vitro (Gardner and Lane, 2005). Suboptimal culture conditions induce cellular stress and lead to metabolic transformation. As metabolic transformation is associated with a change in viability, embryo metabolism assessment may be used to determine embryo viability before transfer (Gardner and Sakkas, 2003). The intrinsic quality of the embryo can also lead to differences in metabolism.
In previous studies, viability differences were found in embryo metabolism of pyruvate (Conaghan et al., 1993; Gardner et al., 2001), glucose (Gardner et al., 2001) and amino acid turnover (Houghton et al., 2002; Brison et al., 2004; Stokes et al., 2007). Although these studies have solidified the increasing potential of metabolomic profiling, they are of limited use in routine IVF laboratories. The techniques can be very expensive and often require complex instrumentation and specific skilled staff. In addition, receiving results of these tests are not within the time frame required to make an embryo selection. So the need for a rapid, accurate and non-invasive tool to predict embryo viability in SET in IVF remains.
In a recent publication, a method has been introduced that evaluates, by means of near-infrared (NIR) spectroscopy, the metabolomic profiles of spent culture media of IVF/ICSI embryos showing its ability to assess an embryo's reproductive potential (Seli et al., 2007). However, this study was conducted using medium samples of multiple transferred embryos, whereas this method may be of particular value in the selection process with SET. Techniques such as proteomics and metabolomics are able to provide important clinical information concerning the modification of an embryo's in vitro environment. In relation to metabolomics by NIR spectroscopy, this information is measured biologically by ascertaining the concentrations of key functional groups such as ROH, -SH, C=C, -CH, -OH and -NH groups. The possible constituents of the culture media that could represent these changes include albumin, lactate, pyruvate, glutamate and glucose. In this pilot study, we retrospectively investigated if metabolomic profiling of spent culture media by NIR spectroscopy, correlates with ongoing pregnancy when the transferred embryos were selected by conventional morphological selection criteria.
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Materials and Methods |
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Patient population
The ethic committee of the VU University medical center gave approval for this study. From July 2006—April 2007, embryos from patients of 333 IVF or ICSI cycles (304, Day 3; 29, Day 2) with SET were included. Pregnancy outcomes were recorded for each patient at 12 weeks gestational age. Positive pregnancy was defined as fetal cardiac activity (FCA) at this time.
Stimulation protocol
Stimulation protocols were performed as previously described (Roseboom et al., 1995; Goverde et al., 2000). Briefly, patients under the age of 38 years or with previous good response in a IVF or ICSI treatment underwent controlled ovarian hyperstimulation with a ‘long’ protocol with GnRH agonist [Decapeptyl (Ferring, Copenhagen, Denmark)] and gonadotropins [Gonal F (Serono, Geneva, Switzerland), Puregon (Organon, Oss, the Netherlands) or Menopur (Ferring)]. In women older than 38 years or with a previous poor response a short GnRH agonist protocol was applied.
Ovarian response was monitored by vaginal ultrasonography and serum estradiol determinations. Human chorionic gonadotrophine [Pregnyl (Organon)] 10 000 IU s.c. was administered, when there was at least one follicle 
18 mm and three or more follicles 16 mm. Ultrasonographic directed oocyte retrieval was performed 36 h later.
Laboratory procedure
Oocyte insemination was initiated 
40 h after HCG injection using standard IVF and/or ICSI procedures. At 16–18 h after insemination, the fertilization was scored. Embryos were cultured individually in 25 µl pre-equilibrated medium drops (human tubal fluid, Lonza, Belgium with 10% protein solution Sanquin, the Netherlands) and alongside, embryo-free media drops were incubated as controls. Morphological assessment was assigned for each embryo by combining the number of blastomeres and the percentage fragmentation of an embryo. Embryos were selected by their morphological appearance and developmental potential prior to transfer. The embryo with the highest number of blastomeres and the least fragmentation was transferred. If only one embryo was available, the transfer was performed on Day 2 after oocyte retrieval, irrespective of morphology. After transfer, the spent media drop, in which the transferred embryo was cultured, and a control media drop were immediately frozen (–196°C). Samples were shipped to Molecular Biometrics (Montreal, Canada) and stored at –80°C until analyzed by NIR spectroscopy.
Analysis of culture media samples by NIR spectroscopy
Spectral regions (ROH, -SH, C=C, -CH, -OH and -NH groups) of metabolomic profiles that discriminated between embryos with positive and negative pregnancy outcomes (as determined by FCA) were determined by a genetic algorithm search method. These spectral regions were quantified using inverse least-squares regression and leave-one-out cross validation, resulting in a relative ‘embryo viability score’ as a measure of an embryo's reproductive potential. For further details see Seli et al. (2007) and supplementary data: NIR spectroscopy details.
Statistical analysis
Subsequent to the relative viability score estimation, Student's t-test results were used to separately determine trends within the pregnancy outcome groups. Alpha error of <0.05 was considered significant for all comparisons. Values of accuracy, positive and negative predictive values (PPV and NPV) were also computed using,
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A logistic regression analysis was done to model the probability of pregnancy given maternal age, percent fragmentation and number of blastomeres of the embryo, type of infertility treatment (IVF or ICSI), endometrium thickness, duration and indication of infertility and the relative viability scores.
A Pearson correlation coefficient was also calculated to establish the association between the relative viability score and the morphology of an embryo.
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Results |
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The overall ongoing pregnancy rate after SET on Day 2 was 17.2% (5/29) and 29.3% (89/304) after Day 3 SET. There were no multiple pregnancies. NIR spectral analysis of discarded culture media samples collected from 333 SET cycles produced unique metabolomic profiles. The mathematical model (Supplementary data, Equation 1) produced relative embryo viability scores that correlated to an embryo's reproductive potential as determined by pregnancy outcomes. The mean relative viability scores of negative and positive FCA groupings after Day 2 SET were 0.2295 (±0.0985) and 0.2814 (±0.0224), respectively (P < 0.03). The mean relative viability scores of negative FCA and positive FCA groupings after Day 3 SET were 0.2767 (±0.1115) and 0.3076 (±0.0974), respectively (P < 0.02).
Table I shows values of accuracy, positive predictive values and the negative predictive values of metabolomic profiling and morphology assessment after Day 2 and Day 3 SET. The accuracies of metabolomic profiling and morphology in assessing embryo reproductive potential after Day 3 SET were 53.6 and 38.5%, respectively. The positive predictive values of the metabolomic profiling and morphology after Day 3 SET were 0.365 and 0.313 respectively. The negative predictive values of metabolomic profiling and morphology after Day 3 SET were 0.830 and 0.833, respectively. The accuracies of metabolomic profiling and morphology after Day 2 SET were 69.0 and 27.6%, respectively. The positive predictive values of metabolomic profiling and morphology after Day 2 SET were 0.333 and 0.100, respectively. The negative predictive values of metabolomic profiling and morphology after Day 2 SET were 0.941 and 0.667, respectively.
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Logistic regression analysis (Table II) of factors contributing to FCA revealed that after day 3 SET, maternal age, percent fragmentation of the embryo and the relative viability score were positively associated with ongoing pregnancy. Number of blastomeres, ICSI or IVF treatment, endometrium thickness, duration and indication of infertility were not associated with ongoing pregnancy and were excluded from the model. Logistic regression analysis of factors contributing to FCA for Day 2 SET was denied because of the small sample size.
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Figure 1 shows the relation between the relative viability score and number of blastomeres with a Pearson correlation coefficient of –0.013. The null hypothesis of no association is accepted (P = 0.826). Figure 2 shows the relation between the relative viability score and percent fragmentation with a Pearson correlation coefficient of 0.024. The null hypothesis of no association is also accepted (P = 0.682).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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Through this pilot study, we have demonstrated that metabolomic profiling of spent culture media by NIR spectroscopy is capable of accurately assessing the reproductive potential of a cleavage stage embryo, by correlating a relative viability score to ongoing pregnancy. Furthermore, it was suggested that the modification of culture media by oxidative modification may be an indicator of reproductive potential, and is independent of morphological selection criteria.
NIR spectroscopy is advantageous in the application of metabolomic profiling as it only requires small sample volumes (7–10 µl), is non-invasive and non-toxic to the embryo. It can be easily incorporated into a clinical setting as it is a compact, bench-top instrument that is rapid (analysis time per sample is <1 min) and simple to use. Even further, it offers objective selection criteria in comparison to morphological selection methods, where the morphology of an embryo is judged by a variation of embryologists and technicians with subjective opinions.
Our results indicate that embryos that implant and result in pregnancy affect their in vitro environment differently than embryos who fail to implant. This is in agreement with previous studies in which differences were found in embryo metabolism of glucose (Gardner et al., 2001), pyruvate (Conaghan et al., 1993; Gardner et al., 2001) and amino acid turnover (Houghton et al., 2002; Brison et al., 2004; Stokes et al., 2007). In contrast, NIR spectroscopy analyzes the changes in several organic functional groups (ROH, -SH, C=C, -CH, -OH and -NH groups) associated with these individual metabolites, and is therefore an indirect assay of culture media biomarkers. These organic functional groups are sensitive to reactive oxygen species, and the variation between viable and non-viable embryos may be a result of oxidative modification.
Metabolomic profiling by NIR spectroscopy might give a new dimension to embryo selection, in addition to morphological criteria. It is well known that selection based on morphology has its limitations, although most pregnancies are from high grade embryos (Conaghan et al., 1993; Gardner et al., 2001; Brison et al., 2004). In the current study, although >85% of the embryos were of good quality, the ongoing pregnancy rate after Day 3 SET was 29.3%. Even though there are other factors that influence pregnancy outcomes, apparently not all morphological good-quality embryos will implant. In comparing the selection accuracy of both metabolomic profiling and morphology, metabolomic profiling by NIR spectroscopy is 15% higher than the accuracy of morphology after Day 3 SET and even 40% higher in the Day 2 SET group, when transfer was performed irrespective of morphology. In practice, these data suggest that metabolomic profiling might be used in conjunction with morphological criteria to enhance the embryo selection procedure. While these outcomes are quite encouraging, it is important to note that the Day 2 SETs were all transferred because only one embryo was available, whereas only the morphologically best embryo was transferred on Day 3.
Our data confirm the recent results by Seli et al. (2007) in a clinical IVF setting, but with a larger number of cases and using single cultured and transferred embryos. Seli et al's data are very promising but with the SET data we can measure differences in metabolism between individual embryos and make one on one comparisons between pregnancy, metabolomic profiles and morphology data. In addition, we used much lower volumes (i.e. 4–5-fold) and a replaceable sample cell providing a closer representation of use in a clinical IVF setting. Most importantly, from a spectroscopy standpoint, this is the first time long wavelength NIR has been used. The long wavelength NIR allows the volume decrease.
The logistic regression of factors contributing to FCA with the Day 3 SET samples showed that maternal age, fragmentation of the embryo and the relative viability score were all factors contributing to FCA. Maternal age (Roseboom et al., 1995; Goverde et al., 2000; Strandell et al., 2000) and fragmentation (Giorgetti et al., 1995; Ziebe et al., 1997; van Royen et al., 1999, 2001) are known to contribute to FCA, but the relative viability score derived from metabolomic profiling by NIR spectroscopy provides a new parameter of embryo quality. This further indicates the usefulness of the relative viability score when selecting between embryos of equal morphological quality.
The Pearson correlation coefficients between the relative viability score and number of blastomeres; and between the relative viability score and fragmentation, are both close to 0, indicating no linear correlation between these variables (Altman, 1991). Considering that >85% of the transferred embryos were of good quality (<20% fragmentation, 4 blastomeres on Day 2 or 6–9 blastomeres on Day 3), this is a strong indication that we are looking at new aspects of an embryo's intrinsic quality. This is in agreement with an earlier study in which it was stated that morphology alone cannot discriminate between sibling embryos (Gardner et al., 2001).
The resulting data showed a large inter-patient or inter-embryo variability of the relative viability score. So far it is unknown what the cause of this variability is and if comparable variability will occur when we analyze individual embryos of the cohort of one patient. However, in general, a low relative viability score was highly predictive of poor pregnancy outcomes, so the data suggest that metabolomic profiling can be used in conjunction with morphology for embryo selection to distinguish between morphologically equal embryos.
Additional studies are required to provide further insight on the clinical value of metabolomic profiling by NIR spectroscopy, and the improvements in IVF embryo selection when morphology and metabolomic profiling are combined as an adjunct technique of assessing reproductive potential. Currently, prospective randomized trials applying the relative viability score on top of the morphology to select an embryo for SET are underway.
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This study was partially funded by Molecular Biometrics LLC, New Jersey, USA.
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We would like to thank all members of the IVF team from the VU University Medical Center in Amsterdam, especially the laboratory technicians and embryologists, for their assistance.
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Submitted on November 13, 2007; resubmitted on January 31, 2008; accepted on March 13, 2008.
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