Article
Non-invasive viability assessment of day-4 frozen–thawed human embryos using near infrared spectroscopy

https://doi.org/10.1016/j.rbmo.2011.08.015Get rights and content

Abstract

This study investigated if metabolomic profiling of culture media using near infrared (NIR) spectroscopy was related to live-birth rates after single-embryo transfer of frozen–thawed embryos. Analysis of culture media of frozen–thawed embryos was performed by NIR spectroscopy. A viability score was calculated using a predictive multivariate algorithm of fresh day-5 embryos with known pregnancy outcomes. This algorithm generated with fresh day-5 embryos could help to identify the live-birth group from the no live-birth group. Multivariable regression models that tested the predictive ability of the viability score for live birth showed an odds ratio in the crude analysis of 1.50 (P = 0.008), after adjustment for embryo morphology, 1.44 (P = 0.022), and after adjustment for all variables, 1.71 (P = 0.005); based on a 0.1 step increase in viability scores. In conclusion, higher viability scores resulted in higher live-birth rates. An algorithm generated from fresh embryos might be used to predict viability of frozen–thawed embryos. Frozen–thawed embryos have different metabolic activity which is related to implantation potential. Therefore, this method might be useful to select the best embryo for transfer within a group of embryos with similar morphology.

In frozen–thawed embryo transfer (FET) cycles, usually more than one embryo is transferred. However, elective single embryo transfer (SET) might be effective in FET cycles when a good-quality embryo is selected. Viability assessment of frozen–thawed embryos is usually performed by morphological assessment. Although very helpful, morphological assessment remains subjective and can be unreliable in predicting embryo viability. New parameters to predict embryo viability, including non-invasive metabolomic profiling, have recently been studied. Metabolomics is the study of small-molecule metabolite byproducts left behind from cellular processes. By measuring byproducts of the embryonic metabolism in spent embryo culture media, a snapshot of the physiology of an embryo is obtained, which translates to viability. In this study, we investigated if metabolomic profiling by near infrared (NIR) spectroscopy was related to live-birth rates after SET of frozen–thawed embryos. Analysis of spent culture media of frozen–thawed embryos was performed by NIR spectroscopy and a viability score was calculated. The mean viability score from embryos with known implantation potential was significantly higher than the mean viability score of embryos which failed to implant: i.e. higher viability scores resulted in higher live-birth rates. Individual embryos showed a positive relationship between increased viability scores and increased live-birth rates. In other words, frozen–thawed embryos (of the same morphological grade) have different metabolic activity which is related to implantation potential. This indicates that the use of morphological and metabolomic criteria can both help with the decision of which embryo to transfer after thawing.

Introduction

Although elective single-embryo transfer (SET) is frequently applied in fresh cycles, it is generally assumed that the implantation potential of cryopreserved embryos is lower than that of fresh embryos (Edgar et al., 2000, Wang et al., 2001). Therefore, more than one embryo is usually transferred in frozen–thawed embryo transfer (FET) cycles. Several studies report higher clinical pregnancy rates, implantation rates and/or delivery rates after double-embryo transfer compared with SET in FET cycles, but double-embryo transfer is also associated with elevated multiple pregnancy rates (Edgar et al., 2007, Hydén-Granskog et al., 2005, Tang et al., 2006). However, some reports suggest that elective SET might be a feasible option in FET cycles when a good-quality embryo is selected for transfer (Hydén-Granskog et al., 2005, Tang et al., 2006). It is therefore of great importance to define parameters which are related to embryo viability post thawing (Edgar et al., 2007).

Until recently, research of embryonic factors that influence the outcome of FET cycles has mainly focused on embryo survival rate and post-thawing morphology and kinetics. Important embryonic factors influencing implantation in FET cycles are the developmental stage before cryopreservation (Edgar et al., 2000, Karlström et al., 1997, Salumets et al., 2003), the occurrence of cryopreservation-related blastomere loss (Burns et al., 1999, Edgar et al., 2000, El-Toukhy et al., 2003, Pal et al., 2004, Salumets et al., 2006, Tang et al., 2006, Van den Abbeel et al., 1997) and the resumption of cell division during post-thaw embryo culture (Guerif et al., 2002, Salumets et al., 2006, Van der Elst et al., 1997, Ziebe et al., 1998). Although very helpful, morphological assessment remains subjective and can be unreliable in predicting an embryo’s viability (Brison et al., 2004, Sturmey et al., 2009, Vergouw et al., 2008). Therefore, there is a strong need for other objective markers to aid the prediction of embryo viability. Conaghan et al. (1993) and Gardner et al. (2001) showed that differences in embryo metabolism of glucose and/or pyruvate are related to embryo viability. Others showed an association between amino-acid turnover and developmental or implantation potential using high-performance liquid chromatography in fresh (Brison et al., 2004, Houghton et al., 2002) and frozen–thawed embryos (Stokes et al., 2007). These studies claim that embryos with a low or quiet amino-acid turnover have the highest developmental potential. Seli et al. (2008) used proton nuclear magnetic resonance and found that embryos that resulted in clinical pregnancy and delivery had higher glutamate concentrations in the culture media.

Relatively new is the development of metabolomic profiling of spent embryo culture media by near infrared (NIR) and Raman spectroscopy. These technologies measure modifications of the chemical composition of culture media made by embryos or oocytes and generate a value reflective of the implantation potential of an embryo (Ahlström et al., 2011, Nagy et al., 2008, Scott et al., 2007, Seli et al., 2007, Vergouw et al., 2008) or nuclear maturity status and quality of oocytes (Nagy et al., 2009). Using this technology, distinct differences were found in culture-media composition of embryos with proven reproductive potential compared with embryos that did not implant (Ahlström et al., 2011, Hardarson et al., 2008, Nagy et al., 2008, Nagy et al., 2009, Scott et al., 2007, Seli et al., 2007, Seli et al., 2010, Vergouw et al., 2008). These differences were seen regardless of day of transfer and were independent of embryo morphology (Hardarson et al., 2008, Seli et al., 2010, Vergouw et al., 2008). Nagy et al. (2009) showed significant differences in NIR profiles of mature and immature oocytes. They also demonstrated that oocytes that resulted in implanted embryos had higher viability indices than oocytes that resulted in embryos that did not implant. Furthermore, using Fourier transform infrared spectroscopy, differences in metabolomic footprints were found between viable and non-viable embryos (Brison et al., 2007).

Previous studies using metabolomic profiling of spent culture media by NIR and Raman spectroscopy have all focused on fresh-embryo transfer cycles. There are no data available of metabolomic profiles analysed by NIR spectroscopy of frozen–thawed embryos. This study analysed if metabolomic profiling of spent culture media by NIR spectroscopy could also serve as a marker of embryo viability in frozen–thawed embryo transfer cycles.

Section snippets

Patients

From November 2007 to January 2009, spent embryo culture media was collected from 127 frozen–thawed single-embryo transfers of 115 patients. The outcomes were retrospectively reviewed. The study had ethical approval of the Institutional Review Board (IRB reference non-WMO).

Fresh-cycle protocol

Patients under the age of 38 years or with previous good response in an IVF or intracytoplasmic sperm injection (ICSI) treatment were treated as previously described (Goverde et al., 2000, Roseboom et al., 1995). In women

Results

The baseline characteristics of patients and embryos are presented in Table 1. A total of 127 embryos were transferred: 67 were compaction-stage embryos and 60 had blastocoelic cavities at the time of transfer. Of the 127 SET, 67 were performed when more than one embryo of sufficient quality was available (elective SET) and 60 when only one embryo was available for transfer (non-elective SET). A total of 120 embryos showed resumption of mitosis 20–24 h post thawing.

The live-birth rate per embryo

Discussion

This study analysed spent culture media of frozen–thawed embryos with known implantation potential after SET and related the results to live birth. When using a previously established predictive multivariate algorithm developed from fresh day-5 embryos, the data showed a positive relationship between increasing viability scores and live-birth outcomes: i.e. a higher viability score indicated a higher viability of frozen–thawed embryos. Other studies in which the same viability assessment method

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    Carlijn Vergouw completed her Master of Science degree in 2001 at Wageningen University, The Netherlands. She is now a clinical embryologist and IVF laboratory director at the Division of Reproductive Medicine, Department Obstetrics and Gynaecology of VU University Medical Center in Amsterdam, The Netherlands. Her thesis focuses on non-invasive embryo selection.

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