LBRC outside projects
Spectroscopy for morphological selection of embryo and oocyte
Soon after the report of the first successful pregnancy with in-vitro fertilization (IVF) and development of controlled ovarian stimulation in order to generate more than one embryo in any given cycle, it became apparent that morphology and cleavage rate of embryos correlate with their implantation potential. Thereafter, grading systems based on embryo cleavage rate and morphology were developed leading to significant improvements in implantation and pregnancy rates and reductions in multiple gestation rates. Today, among the treatment modalities offered to infertile couples, those utilizing IVF are associated with the highest success rates. Consequently, IVF use has been increasing steadily, from 64,681 cycles reported in 1996 in the US to 134,260 cycles in 2005. However, despite its widening application, IVF is currently associated with two important issues that derive, at least in part, from our inability to adequately assess the reproductive potential of individual embryos: (i) low implantation rates and (ii) high multiple pregnancy rates.
Approximately two of three IVF cycles fail to result in pregnancy, causing significant physical, emotional and financial distress for those undergoing infertility treatment. Even more striking is the failure of more than 8 of 10 transferred embryos to implant. Our inability to determine the embryos with highest reproductive potential is likely to contribute to the high rate of implantation and IVF cycle failures. In order to prevent failures, centers have historically chosen to perform simultaneous transfer of multiple embryos, accepting the related risk of multiple pregnancies. In the US, a mean number of 2.45 embryos were transferred in IVF cycles using fresh non-donor oocytes in 2006, resulting in a 34.3% live birth rate per transfer, of which 32% were multiple-infant live births. In total, while IVF treatment accounts for only 1% of all births in the US, 18% of multiple births result from IVF. Moreover, 51% of all IVF neonates are the products of multiple gestations, a frequency of 15- to 20-fold greater than with spontaneous conceptions.
The high multiple pregnancy rates associated with IVF have also significant public health consequences as the increased rate of preterm delivery in multiple-infant pregnancies compromises the survival of neonates and increases their risk of lifelong disability. Indeed, while multiple births constitute approximately 1/33 of all births in the US, they account for 1/8 of preterm births (37 weeks) and 1/4 of very low birth weight infants (2000 g). Largely due to the increased incidence of prematurity, cerebral palsy is increased 8-fold in twins and 47-fold in triplets, while infant deaths (birth to 1 year) are increased 6-fold in twins, and 17-fold in triplets and higher order gestation. Recently, it has been estimated that preterm births that result from IVF-related multiple pregnancies, account for $1 billion of cost to the society in the US yearly. These estimations do not include maternal complications associated with multiple gestations, including a 2- to 4-fold increase in pregnancy induced hypertension and post-partum hemorrhage.
The medical and financial complications associated with multiple pregnancies have now led a number of countries to impose legal restrictions on the number of embryos transferred in IVF cycles. Consequently, decreasing multiple gestations while maintaining or increasing overall pregnancy rates is an important goal of contemporary infertility treatment and an improvement over the current methods of embryo assessment would be beneficial for this purpose.
Morphology and cleavage rate remain the mainstay of embryo assessment. However, a number of additional technologies for this application are under investigation. These include the measurement of glucose, lactate, pyruvate or amino acid levels in the embryo culture media (ECM), assessment of oxygen consumption by the embryo, genomic and proteomic profiling, and most recently, analytical examination of the embryonic metabolome. As the number of assisted reproduction cycles increases worldwide, improvements in the ability to quickly and non-invasively identify the best embryos for transfer remain a critical goal for reproductive medicine.
The assessment of oocyte quality is rapidly becoming one of the major objectives of embryologists in human IVF. In fact, although classical IVF does not include any process of oocyte selection and only embryos (sometimes zygotes) undergo selection, the general trend toward limiting embryo "overproduction" and the rapidly improving results of oocyte cryostorage, have challenged embryologists to identify criteria to select, among all oocytes retrieved, the best oocytes to inseminate. Multiple methods of oocyte selection have been proposed. The study of oocyte morphology is very popular, being relatively quick and simple; however, it leads to identify more frequently "negative" than "positive" predictors of oocyte quality, and overall is not fully satisfactory.
Follicular fluid (FF) provides a very important microenvironment for the development of oocytes. FF is a product of both the transfer of blood plasma constituents that cross the blood follicular barrier and of the secretory activity of granulosa and theca cells. It is reasonable to think that some biochemical characteristics of the FF surrounding the oocyte may play a critical role in determining oocyte quality and the subsequent potential to achieve fertilization and embryo development. The analysis of FF components may also provide information on metabolic changes in blood serum, as the circulating biochemical milieu may be reflected in the composition of FF. FF is easily available as it is aspirated together with the oocyte at the time of oocytes collection, namely ovum pick-up (OPU).
- Embryo/oocyte selection using Raman spectroscopy on embryonic culture media (ECM) and follicular fluid (FF)
Metabolomics of the ECM/FF is the dynamic quantitative assessment of all low molecular weight substances that are present in ECM/FF at a given time. We will apply Raman spectroscopy to monitor metabolites, such as glucose, lactate and pyruvate. We will explore the relation between fertilization and maturation rate, pregnancy rate and take home baby rate with investigated ECM/FF by spectroscopy.
- Embryo/oocyte selection using confocal Raman and quantitative phase microscopy
The current techniques available for assessment of embryo/oocyte morphology are not very reliable. We will apply confocal Raman microscopy and quantitative phase microscopy to quantitatively assess embryo/oocyte morphology and chemical composition. We will explore the relation between fertilization and maturation rate, pregnancy rate and take home baby rate with investigate embryo/oocyte by microscopy.