The Relationship Between The Amino Acid Metabolism And The Developmental Potential Of Embryos

BackgroundSince the first test tube baby come into this world in 1978, in vitro fertilization (IVF) has become one of the most widely used assisted reproductive techniques. However, despite the widening of its application, low implantation rates and high multiple pregnancy rates remain to be two difficult issues, which are, at least in part, due to our inability to adequately assess the potential of individual embryo development. According to data from SART (the Society for Assisted Reproductive Technology), more then 40% conception derived of IVF cycles were multiple pregnancy. The high multiple pregnancy rates associated with IVF may encounter serious socio-economical and public health consequences. Numerous maternal and neonates’complications such as preterm births, low birth weight, pregnancy induced hypertension and post-partum hemorrhage are associated with multiple pregnancy. In recent years, the medical and financial complications associates with multiple pregnancies have now led a number of countries to impose legal restrictions on the number of embryos transferred in IVF cycles. The suggestion of the American Society for Reproductive Medicine (ASRM) is to transfer less than 2 embryos per cycle, in order to reduce the multiple pregnancy rates.In order to keep high pregnancy rate and meet the desire of the patients, multiple embryos transfer was historically performed by ART centers, accepting the related risk of multiple pregnancies. Yet, single embryo transfer (SET) is the best way to avoid multiple pregnancies. In Europe, many centers have tried to do SET in IVF cycles. Several centers in our country also have investigated the feasibility of single embryo transfer. If one good quality day3 embryo or one blastocyst was transferred, a relatively acceptable pregnancy rate and less multiple pregnancies would be achieved. But, because of the lack of effective method to evaluate the embryo development potential, SET is difficult to implement.Although morphology and cleavage rate remain the mainstay of embryo assessment, it could always be found that’good’embryos may fail, while some’bad’ quality embryos had the ability to implant. Thus a number of additional technologies for this application are under investigation. Blastocyst culture is also used as a selection method of good embryos. But only 30-40% cleavage stage embryos can develop into blastocyst. Some patients will have no opportunity of ET if there is no blastocyst formation which is the reason why most centers still prefer cleavage stage embryo transfer in fresh cycles.Embryonic metabolome was supposed to provide a rapid, non-invasive way to choose embryos, but till now none of embryonic metabolome parameters could be routinely used in clinical practice. To improve the understanding of embryo viability may help to identify the embryos that are most likely to result in a pregnancy and reduce the number of embryos to be transferred. This in turn may reduce the likelihood of multiple gestations while maintaining or even increasing implantation rates. Additional studies will be necessary to determine the value and limitations of the use of metabolomics in IVF practice. This is indeed a critical goal for reproductive medicine.Most recently the embryonic metabolome analysis include the measurement of glucose, pyruvate or amino acid levels in the embryo culture media, assessment of oxygen consumption of the embryos, human leukocyte antigen, platelet activating factor and leptin. Studies also suggest that amino acids are important regulatory factors in the process of embryo development. Amino acids are essential for embryonic well-being, and thought to be the building blocks for protein synthesis and key players in metabolic processes. Amino acids are involved in carbohydrate metabolism, osmotic pressure and the regulation of intracellular pH value etc. Amino acids are not only conducive to maintaining normal cell function, but also can promote the embryo development and differentiation. They can improve the embryos developmental potential after implantation.The knowledge of how these amino acid pathways functions allowed scientists to investigate the consumption and secretion of specific amino acids and their relation to overall embryo metabolism. In previous studies, an association was found between some amino acids in the culture media and the blastocyst development and clinical pregnancy rates. High-performance liquid chromatography (HPLC) was used to examine the changes in the concentration of amino acids secreted by individually cultured human embryos. However, the application of these techniques to the clinical practice could not been achieved for a variety of reasons. This technology requires complex equipment and dedicated staff, which would be cost prohibitive to most embryology laboratories, and results could not be got quickly enough to allow the information to be used clinically in the limited window of time acceptable for embryo transfer in the fresh cycle. Thus, the need remains for a validated technology that non-invasively predicts viability of embryos, through a rapid, on-site evaluation of multiple samples. Mass spectrum, (MS)-based metabolomics provides a more analytically sensitive alternative with the ability to detect metabolites at the level of micro molar concentration, typical of physiological concentrations. A recently developed technique, HPLC-MS could determine the concentrations of amino acids in embryo culture medium very quick, which provide insight into the phenotype of embryos with increasing reproductive potential.Objects1. To investigate the relationship between amino acids metabolism and the embryo quality by animal experiments.2. To investigate the relationship between amino acid metabolism and blastocyst formation using human embryos medium analysis.3. To investigate the relationship between amino acid metabolism and the blastocyst quality.Methods1. Animal experimentsa) The ICR mice were sacrificed after controlled ovary stimulation, and two-cell embryo were collected and individually cultured in vitro.b) Deliver the cleavage stage mouse embryos into blastocyst culture medium on day 3 after embryo retrieval, and then the cleavage stage medium were collected. Concentrations of 30 kinds of amino acid in the medium were measured using HPLC-MS.c) Most cleavage stage mouse embryos developed into blastocyst, the blastocysts were double fluorescent stained and the number of ICM and TE cells was counted.d) Analysis the relationship between the amino acid concentrations in cleavage culture medium and ICM, TE cell numbers.e) Individual amino acid concentration alteration rate was calculated by the following method: Amino acid concentration of cleavage stage embryos medium was subtracted by that of blank medium, and then divided by that of blank medium. Amino acids with obvious alteration rate were selected for analysis. Stepwise regression method was used to analyze the relations between the concentration alteration rate and ICM and TE cell numbers of blastocyst.2. Human embryo experimentsa) The infertile women of 25 to 35 years old with bilateral fallopian tube obstruction in first treatment cycle were informed consent for measurement of their spent culture medium and considered for participation in the study.b) The embryos were individually cultured in 25ul droplets until day3.c) The remaining cleavage stage embryos were delivered into blastocyst culture medium and individually cultured after ET.d) The spent cleavage stage medium was collected, and amino acid concentrations were determined using HPLC-MS method.e) Subsequent blastocyst formations were observed and image of the blastocysts were obtained. Morphological parameters including the number of TE cells distributed along the circumference of zona pellucida and the area of ICM were collected.f) Concentrations of 30 kinds of amino acid were determined by HPLC-MS of the spent cleavage medium and blank medium.g) Individual amino acid concentration alteration rate was calculated by the following method: Amino acid concentration of cleavage stage embryos medium was subtracted by that of blank medium, and then divided by that of blank medium. Amino acids with obvious alteration rate were selected for the analysis of the next step.h) Using logistic regression method to analyze the relation between the amino acid concentration alteration rates and blastocyst formation.i) Using stepwise regression method to analyze the relations between the amino acid concentration alteration rates and ICM area and TE cell numbers of blastocysts.Results1. Animal experimentsa) Fifteen ICR mice were sacrificed after controlled ovarian hyper stimulation, a total of 503 2-cell embryos were collected. Every embryo was individually cultured in 20ul medium droplets. A total of 428 blastocysts were formed. Blastocysts formation rate was 85.1%.b) Double fluorescent staining was done in all hatched blastocysts. The images were collected under fluorescence microscope. The number of ICM and TE cells was countedc) The average number of TE cells was 83.69±18.96, which of ICM cells was 21.92±4.23 per blastocyst. Multiple stepwise regression analysis suggested that TE cell number was positively related to glutamine concentration alteration rate (P<0.05). No significant correlation was found between other amino acids concentration alteration rate and two parameters of blastocyst quality.2. Human embryo experimentsa) A total of 67 samples of spent cleavage embryos medium were collected from 14 patients, and amino acids concentration of which were determined. All of the 67 embryos were moved to blastocyst culture droplets separately on day3 after oocyte retrieval.b) Of the 67 embryos,29 expansion blastocysts formed after 2-3 days of culture. Blastocyst formation rate was 43.2%。c) The images of the blastocysts were saved and morphological parameters of the embryos were calculated. The average ICM area was 3637.79±3254.68 um2, and the average TE cell number along the perimeter of blastocysts was 7.8±2.6.d) Logistic regression analyze suggested that alteration rate of Asn has the most statistical significant influence on the formation rate of the blastocyst. The prediction of blastocyst formation was elevated from 56.7% to 70.1% if Asn alteration rate was considered.e) Stepwise regression analysis was used to reveal the relations between AA alteration rates and TE cell numbers, a positive statistical significant correlation was found between the number of TE cells and the alteration rate of Asn. Furthermore, the similar correlation was found between ICM area and the alteration rate of Ala.Conclusions1. HPLC-MS is a stable and reliable technique to determine amino acid concentration with a tiny volume of medium. Results can be got quickly enough to allow the information applied clinically in the limited window of time acceptable for embryo transfer in the fresh IVF cycle. Possibility exists for clinical application of HPLC-MS in IVF treatment.2. A correlation was found to be existed between glutamine alteration rate of mouse cleavage stage embryos and the consequent blastocyst quality.3. There was an association between alteration rate of specific amino acids and blastocyst formation of human embryos which might enhance the accuracy of the prediction of blastocyst formation.4. A relation exists between alteration rate of amino acids of human cleavage stage embryos and blastocyst morphological parameters, including TE cell number and ICM area, which suggested that there is a significant correlation between amino acid metabolism and quality of blastocysts.