embryo selection technic

embryo selection technic Paper details: Please worki on file "Embryo Selection Techniques revised 160627A" it has already corrected partly, and compare with the other file ( "Embryo Selection Techniques revised 160627") Add references on introduction part. Add a table that compare the succes rate of IVF based on morphological based seleection, morphokinetic, OMICS study and metabolic profiling. Add a figure or table that explain the differences IVF, ART, ICSI process. Change the references style form Harvard to vancouver system, using number. Article Review In-Vitrio Fertilization: Assessment of invasive and non-Invasive in-vitrio embryo transfer techniques for successful fertilization . Article history Received: Revised: Accepted: *Corresponding Author: Email: Abstract: Infertility in the United States is a common problem affecting over 6.1 million couples. The inability to conceive or carry a pregnancy to term has had many couples turning to assisted reproductive technology (ART), and most particularly, In-Vitrio Fertilization (IVF), which has been in existence for over 40 years, and has assisted in the birth of thousands of babies worldwide every year. Visual assessment of embryo morphology has been the primary tool for selecting embryo for IVF procedures. This is the most popular method and assesses several parameters at different developmental stages. The method provides valuable information about embryo quality as it grades embryos according to the morphology of their pronuclei after fertilization. Though morphological assessment is cost-efficient and easy to implement in a medical environment, it has a number of setbacks. Firstly, the method requires significant expertise as it is largely subjective. Additionally, even when the requisite expertise is available, the techniques lack accuracy due to lack of scientific standards. Notably, low quality embryos exhibits high developmental potential and can, unfortunately develop to term. However, despite the remarkable advance in the field of reproductive medicine and biology, the efficacy of IVF procedures remains relative low. The need for non-invasive and reliable embryo selection methods, which has largely been blamed for the low success rate of IVF, is important in ensuring that only embryos with the highest developmental potential are selected for transfer. In the present study, various methods of embryo selection that are currently in use will be assessed. These methods include metabolism, quality of the genetic material, OMICS technology, and the traditional embryonic morphology. The study explores novel procedure for embryonic assessment that are founded on advanced time-lapse imaging techniques, which have shown significant promise and are anticipated to lead to increased IVF efficiencies. Keywords: non-invasive in-vitro embryo transfer techniques, in-vitro fertilization Introduction Assisted reproduction technology (ART) is one of the fastest evolving scientific disciplines spurred by association of various researches both in biological and medical areas. This evolution led to the advent of human in vitro fertilization (IVF) over 40 years ago, and remains one of the major medicinal breakthroughs in recent world history. IVF has made the oocyte and preimplantation embryo possible and uniquely accessible, giving rise to the development of micromanipulation practices. IVF has become one of the primary medical procedures applied in clinics to overcome a range of female or male, or combined factor fertility medical conditions. The process starts with a hormonal therapy that stimulates the development of multiple follicles in the ovary. When follicular maturity is complete, the oocytes are inseminated under laboratory conditions. Once fertilized, the oocytes are then cultured into early embryos in a special medical equipment. It is noteworthy that the embryo is normally between 2 to 6 days. The clinician has to observe early embryo development and record the results during the in vitro culture period. Selected embryos are then implanted into a woman’s uterus and the patient kept under observation for approximately 12 weeks after the implantation. Normally, a successful implantation is defined as ultrasound-detected fetal cardiac activity during observation period. IVF success is predominantly determined by the embryo selection methodology. When selecting embryo from a cohort of available embryos, it is important that the most viable embryo is selected. Normally, if multiple embryos are selected, the probability of successful pregnancy outcome is heightened. However, the risk of multiple gestations is also increased. Multiple gestation is considered as one of the primary complications of assisted reproductive procedures, associated with increased costs of healthcare and poorer maternal and perinatal conditions. Consequently, there has been concerted efforts from various fields across the world that are geared towards reducing the number of embryos transferred during IVF procedures. Indeed, embryo selection is the primary determinant of the success of IVF and the identification of an embryo that has the highest potential of reproduction is a major challenge for embryologist. There is an interesting question about how the performance of many novel embryo selection approaches performs in relation with the classical morphology-based embryo selection . In this regard, the present study explores the evolution of embryo selection methods such as metabolic profiling and OMICS technologies from the classical morphology based embryo selection. Recent studies have presented non-invasive metabolic profiling and omics technologies, method for assessing embryo’s reproductive potential. By using Raman spectroscopy combined with morphology for the metabolic embryo profiling, there are increased chances of success, a factor that has proved that metabolic profiling is an effective and non-invasive clinical tool for embryo selection. Studies have revealed that thorough metabolic profiling; the number of transplanted embryos could be significantly reduced. Indeed, this embryo selection method is expected to improve embryo implantation and pregnancy success rate leading to reduced medical costs, as well as reducing multiple infants outcomes from ART. In a recent publication, a metabolic profiling method that evaluates embryos by means of near infrared (NIR) spectroscopy on spent culture media of IVF/ICSI embryos has been shown to have the ability to assess embryo’s reproductive potential. Indeed, these and many more innovations are indicators of the huge potential of success for novel noninvasive embryo selection techniques. The study examines whether there has been significant improvement in IVF procedures’ success rate as researchers come up with more embryo selection methods. Method of Literature Selection The search criteria were designed to obtain information that was relevant to the research question using specific keywords and inclusion and exclusion criteria. The search criteria utilized various quality assessment methods to ensure valid data sources for the selected studies. Fig. 1. Method of Literature Selection PubMed electronic database was used as the primary source of research data. The search was conducted using keywords ‘embryo assessment’ and ‘embryo selection’ within the study’s title, abstract and the publications. Since the study’s core objective is to assess various embryo selection methods and the emerging technologies on embryo selection, the literature search covered the time interval between January 1, 2000 to January 31, 2016 to include most recent research findings in the study. After data review, eight of the selected articles did not show enough relevance and were excluded. The next level of selection included carefully reading abstracts for the remaining tables and 21 more papers did not meet eligibility and were eliminated. Lastly, the remaining 29 articles were eligible and complied with the requirements of the research question; they, therefore, were scrutinized. Nineteen articles were biased and so they were excluded. They were considered biased as they only provided information on morphologically biased embryo evaluation technique. Ultimately, ten publications were selected for this study as they met the eligibility criteria. Result of Literature Study Morphological Evaluation For the last two decades, the quality of embryos has been based on morphological assessments. The criteria that have been used over the years are the rate of cell division, pronuclear morphology, and progression to the blastocyst stage. The first cleaving embryos tend to implant at a higher rate. However, none of these criteria has been accurate in predicting the success of pregnancy, and this has been evident for the occurrence of situations in which poor quality embryos have been implanted and yielded multiple pregnancies, while in other cases there was no conception at all despite the implantation of quality embryos. To reduce cases of multiple pregnancies, a fine selection procedure has been developed to ensure that only one embryo is implanted. Embryologists have been using retrospective scoring methods such as pronuclear scoring system where embryo quality is assessed after fertilization and before cleavage to assess nucleoli size, number, and distribution. A strong correlation has been found to exist between the pronuclear scoring system and successful implantation. Morphological evaluation of the pre-implantation embryos’ viability has resulted in increased implantation and pregnancy rates. However, with the recent significant technological advancements, experts have developed more embryo selection technologies that either enhance or substitute the conservative morphology-based embryo selection. Concerning the above discussions, we shall focus on the most recent embryo assessment methods and evaluate whether they relate to the IVF effectiveness when compared with the conventional morphological systems (Desai, Szeptycki and Goldfarb, 2005). Listed in table 1 below, are the most recent approaches used in the selection of embryos and oocytes in the IVF laboratories. The first is referred to as Morphological evaluation. The method wholly depends on a light microscope for morphological evaluation (Dermer, 1971). However, most researchers work towards the establishment of additional parameters to enhance the extrapolative power of this model. During the early stages of cleavage, it is possible to obtain information on the oocyte –sperm interaction as it is provided in the pronuclear morphology (Yang et al., 2008). The second and third days involve the assessment of the number, proportion, and size of the blastomeres. During this period, the level of fragmentation, multinucleation, and cytoplasmic appearances are observed and their scores recorded (Craft and al-Shawaf, 1991). Table 1. Morphological Evaluation No. Stages Observation References 1. Early stage Oocyte –sperm interaction as it is provided in the pronuclear morphology. Yang et al., 2008 2. The second and third days number, proportion and size of the blastomeres, level of fragmentation, multinucleation, and cytoplasmic appearances Craft and al-Shawaf, 1991 Morphokinetics Technological advancements has led to the development of techniques of time-lapse imaging of early embryo development in the IVF laboratory (Gardner et al, 2001). This technique enables morphokinetic analysis by evaluating the change in embryo morphology over time by automatically acquiring images at predefined time points. Compared to static observations, morphokinetic analysis has proved to be a useful tool as it has the capability to explain the dynamic nature of embryo development (Keskintepe, 2012). Preliminary morphokinetic studies have shown that the potential of embryo development is correlated with accurate timing of specific events such as early cleavage, pronuclear formation, initiation of blastulation, and cell division intervals. In their study, Rubio et al (2003) showed that the implantation rate was significantly improved with morphokinetic evaluation (44.9%) compared with standard morphological evaluation (37.1%). In the same study, it was shown that there was significant decrease in pregnancy loss with the use of morphokinetic features for embryo selection (Jones, 2001). Omics technology Due to the limitations of traditional morphological methods, there has been a concerted effort in research both in medical and biological fields intended to develop alternative methods that are able to provide information that is more accurate. Advances in Omics technologies have greatly improved the examination of embryo molecular constitution and the interaction between the embryo and its external culture media. The interaction has enabled identification of potential nonmorphology markers of embryo viability. Omics research’ main objective is to identify how many copies of each different molecules that exist in a biological sample. The RNA, DNA, metabolite content, and protein constitutions of embryo culture media are examined by transcriptomics, genomics, metabolomics, and proteomics approaches. One of the omics’ selection approaches is preimplantation genetic screening (PGS), which originated from the need to improve IVF outcomes in special cases such as people presenting recurring implantation failure or miscarriages, mothers of advanced age, or cases of severe male factor infertility (Reddy, 2007). PGS has made it possible to end the transmission of hereditary diseases because of combination of molecular biology and blastomere biopsy techniques, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), and multiple displacement amplification (MDA) among others. It is noteworthy that unlike embryo morphological procedures, PGS involves screening for chromosomal and genetic disorders in the embryo prior to the transfer (Devreker, 2007). Although PGS was originally offered for vulnerable patients, its scope has been extended to patients with re-current IVF failures. However, PGS is an invasive embryo assessment approach and primarily tests for aneuploidy. A recent study showed that PGS with FISH is associated with lower pregnancy and live birth rates. Nevertheless, screening using more recent single nucleotide polymorphism array or genomic hybridization technologies has shown more promising results in improving IVF success rates. Polar body-based aneuploidy technique has been proved a less invasive method though the resulting implantation rate is not significantly different from transfers without aneuploidy screening. In their study in an RCT setting, Forman et al (2013) tested whether there were improved neonatal and obstetric outcomes compared with transfer of non-tested embryos. By performing e-SET after aneuploidy screening of trophectoderm biopsy, the study revealed that the delivery rate were similar (69% vs 72% respectively). Other noninvasive methods of embryo selection are based on analysis of embryo culture media. For example, proteomics technologies are being used to profile the secretome that includes protein synthesized and secreted by the embryo into the surrounding culture media (Lafrance, 2004). Though preliminary studies have shown that secretome profile and altered gene expression are positively correlated with embryo viability, there is a need to demonstrate the clinical benefits from proteomic and transcriptomic analysis of granulosa cells in randomized controlled trials (RCTS). Metabolomics-based embryo selection methods profile changes in metabolite levels in culture media associated with amino acid and carbohydrate metabolism turnover as pointers of normal preimplantation embryo development (Simlara, 2011). Various studies have identified potential biomarker metabolites including glucose, pyruvate, lactate, asparagine, and glutamate. Particularly, pyruvate uptake on the first five days has be shown to be associated with blastocyst and embryo development. Additionally, higher levels of lactate production between the third and fifth days and glucose uptake on the fourth day have been shown to be correlated with blastocyst development (Bromer, 2008). Amino acids turnover in the culture media has been shown to play an important role in preimplantation embryo development. It is noteworthy that this is the most commonly studied noninvasive embryo selection method. In a study conducted by Vergouw et al (2008), it was shown that live birth rate after NIR spectroscopy was slightly different from live birth rate after embryo selection by morphology alone (31.7% vs 28.9% respectively). However, the study established that metabolomics improved implantation rate for day 5 transfers (46.8% vs 28.9%). Metabolite Profiling Transcriptomics technique is important in assessing oocytes and embryos. This method requires the clinicians to identify particular gene markers by evaluating the gene expression levels in granulosa and cumulus cells. The gene markers define the embryos’ eligibility. Similarly, proteomics and metabolomics are noninvasive models for embryo’s assessment and selection. Unlike most approaches, these methods assess the interaction between the embryo and its culture media. Embryos that show positive interaction in their culture media are considered eligible for transfer. However, metabolomics and proteomics are not commonly used due to the intense technology they require. Lack of rapid technology and bench-top instruments inhibits the efficacy of the mentioned approaches in the IVF laboratories. The metabolic process begins at the pronuclear stage during the embryo’s development; during the compacting of the embryo, ATP synthesis from lactate and carboxylic acid pyruvate is the source of energy. This trend of energy synthesis continues until glucose becomes the primary metabolite, which is a normal occurence at the blastocyst stage. During development, the embryo absorbs certain substances from its surrounding environment and excretes some other by-products. Ultra-microfluorescence is then used to observe this environmental change. Other separation techniques are also used to determine the substances that have been released or consumed by the spent media; the information obtained is then used to evaluate the cellular activities and potential development of the embryo during the culture period. The table represents an analysis of other researchers regarding the use of metabolites to determine the embryo viability. Hardy et al (1989), in their experiment conducted concerning the uptake of glucose and pyruvate by an embryo and blastocyst within a span of six days showed increased pyruvate uptake on the first four days indicating embryo development. Up to the fourth day, glucose concentration remained unchanged and on the fifth day, both glucose and pyruvate uptake increased showing blastocyst development. On the other hand, Turner et al (1994), focused on a two days experiment that analyzed pyruvate uptake to test pregnancy. According to the authors, embryos with good morphology had high chances of implantation if they showed an intermediate pyruvate uptake. Conaghan et al (1993) also presented an implantation test for the second and third days by evaluating pyruvate uptake; according to them, decreased pyruvate uptake was a sign of implantation. Gott et al (1990) reflected on pyruvate, glucose, and lactate uptake for 3 to 6 days to test embryo’s development. According to them, increased pyruvate uptake on days 2-4 indicated embryo development. Up to day four, glucose concentration remained unchanged, which meant there were very minimal metabolic activities and no embryo development. On day 5, both glucose and pyruvate uptake increased, which showed blastocyst development. And lastly, increased lactate uptake on day 3 to 5 indicated blastocyst development.All the above researchers used ultra-microfluorescence to observe this environmental changes. They also used other separation techniques to determine the substances that had been released or consumed by the spent media. Houghton et al (2002) presented an analysis of 18 amino acids, which included glutamine, alanine, Arginine, Methionine, and asparagine to test blastocyst development. According to them, reduced glutamine, methionine, and arginine uptake indicated blastocyst development; while reduced alanine and asparagine production showed blastocyst development. They used the HPLC techniques for separation. They too presented an analysis of 18 Amino acids, including alanine, serine, and glycine during the compacting eight cells; as well determined blastocyst development. The researchers found decreased serine uptake indicated blastocyst development, whereas reduced glycine and alanine uptake showed blastocyst development. Lastly, Briston and Sell (1991) presented a test for live birth and pregnancy during the second day, with a primary focus on leucine, glycine, glutamate and asparagine. According to Briston et al (2004), increased asparagine uptake, reduced glycine and leucine uptake indicated pregnancy and live birth, while according to Sell et al. (Sell et al, 2008), increased glutamate uptake showed improved pregnancy and live birth rates. Notably, lack of rapid technology and bench-top instruments inhibited the efficacy of the techniques used by the above researchers. Positive implantation outcome was a sign of a successful IVF process; implantation involves an embryo and a woman. However, implantation depends on a well-functioning endometrium and the level of protein as well as its competence. To enhance the implantation outcomes, scientists have managed to manipulate the endometrium embryo interaction; hence, some factors that prevent female infertility and implantation among women can be prevented. Table 2 gives an idea about the evaluation of preimplantation embryo metabolism as an indicator of reproductive viability. Table 2. Study of Examination Stage, Analyzed Metabolite, Assessed Outcome, Technology and the Findings Examination stage Analyzed metabolite Assessed outcome Technology Findings Refference Days 1 to 6 Glucose and pyruvate Development of embryo and blastocyst Ultra-microfluorescence assay Increased pyruvate uptake on the first four days indicating embryo development Up to day four glucose concentration remained unchanged On day 5, both glucose and pyruvate uptake increased showing blastocyst development Hardy et al, 1989 Day 2 Pyruvate Pregnancy Ultra-microfluorescence assay Embryos with good morphology had high chances of implantation if they showed an intermediate pyruvate uptake Turner et al., 1994 Day 2 to day 3 Pyruvate Implantation Ultra-microfluorescence assay Decrease in pyruvate uptake Conaghan et al.,1993 Day 32 to day 6 Pyruvate, lactate, and glucose Embryo development Ultra-microfluorescence assay Increased pyruvate uptake on days 2-4 indicating embryo development Up to day four glucose concentration remained unchanged On day 5, both glucose and pyruvate uptake increased showing blastocyst development Increased lactate uptake on day 3 to 5 indicating blastocyst development. Gott et al, 1990 Days 4 to day 6 Pyruvate and glucose Development and quality of blastocyst Ultra-microfluorescence assay High glucose and pyruvate and glucose uptake on day 4 indicating blastocyst development Gardner et al., 2001 Day 2- 3 18 amino acids which include glutamine, alanine, Arginine, Methionine, asparagine. Development of blastocyst High-performance liquid chromatography (HPLC) Reduced glutamine, methionine and arginine uptake indicating blastocyst development Reduced alanine and asparagine production showing blastocyst development Houghton et al., 2002 Compacting eight cells 18 Amino acids including, alanine, serine, glycine Development of blastocyst High-performance liquid chromatography (HPLC) Decreased serine uptake indicating blastocyst development, reduced glycine and alanine uptake showing blastocyst development Houghton et al., 2002 Day 2 Leucine, glycine, and asparagine Live birth, pregnancy High-performance liquid chromatography (HPLC) Increased Asparagine uptake and reduced glycine and leucine uptake which indicated pregnancy and live birth Brison et al., 2004 Day 2 Leucine, glutamate Glycine, and asparagine Pregnancy, live birth High-performance liquid chromatography (HPLC) Increased glutamate, pregnancy, and live birth Sell et al, 2008 Conclusion Since the invention of IVF, embryo selection has attracted immense interest and has been an active research field. Omics technologies, which are less invasive than the traditional morphological technique, have particularly been studied on a global scale. As the study has revealed, research carried over the last few decades provides significant repertoire of possible improved embryo selection techniques. Notably, some techniques such as PGS, pronuclear morphology and preimplantation genetic diagnosis predominantly focus on the nuclear component of the embryo looking for embryo with higher success potential in terms of chromosomal . Other techniques focus on the cytoplasm component , examining the quality of embryo metabolism. By combining these varying approaches, IVF success rate will rise as a result of better embryo selection methods. The study allows the increase in knowledge on embryo selection, and the field of assisted reproduction in general, thereby helping in designing new selection tests or diagnostic methods that are aimed at improving the success rates in ARTs. As shown in the study, there are several ongoing randomized controlled experiments performed at various cell biopsy stages that will provide crucial data on which type of embryo selection proves useful in ART setup. Though medical practitioners and researchers have not yet had a consensus on the best embryo selection method, it is clear that ongoing studies on new embryo assessment technologies will enable identification of new markers of the potential of embryos. It is important to note that these techniques are opening widening the spectrum of reproductive technology and medicine. Information obtained from this study and other similar studies will change how current IVF procedures are performed as they have proved to be better tools foe examining differences among human gametes and embryos, and follicles. Additionally, the study allows a systematic inquisition on modern non-invasive methods that have reported positive results due to their precision compared to morphological technique. Notably, the time lapse imaging technique has reported highest levels of accuracy, and it complements the morphological method by reducing chances of failure of implantation caused by exposing the embryo to conditions outside the incubator. Results from the other modern method including metabolic profiling, omics tecnology, whether they had lower pregnancy rates and significant implantation failure or not. From the results, one can judge that morphokinetics is the best model due to its high precision. On the integration of the time-lapse technique with classical morphology assessment, there was no significant difference between the time-lapse technique and the morphology evaluation. Although morphokinetics provides high-quality results, the traditional methods are continually being used because they provide an observational assessment of the embryo cells. Also, the morphology-based method is used worldwide due to the lack of consensus on the benefits of the novel embryo transfer techniques. Acknowledgement Special thanks to Dr. Santoso Gunardi, MS, PA (K) from the Department of Anatomy, Faculty of Medicine, who always gave support and insightful suggestions. Special thanks too to Dr. dr. Ninik Mujihartini, MS for explaining the metabolite examination regarding the IVF mentioned in this manuscript. Funding Information Private funding of both authors supported this research. Author’s Contributions This article review was written and revised based on the ideas of Ria Margiana. Silvia Werdhy Lestari reviewed, revised, and proofread the paper and did paper content control. Ethics There is no ethical issues that might arises from this article review. References Intracytoplasmic Sperm Injection (ICSI). Fertility and Sterility 82 (2004): 205. Web. Allahbadia, GautamNandkishore. "Endometriosis, Oocytes, and In Vitro Fertilization". IVF Lite 2.3 (2015): 73. Web. Boone, W. R. "Overview of the College of American Pathologists (CAP) Inspection Process for Assisted Reproductive Technology (ART) Laboratories". Journal of Reproductive Biotechnology and Fertility 2.1 (2011): 41-45. Web. Brison DR, Leese HJ. Energy metabolism in late preimplantation rat embryos. J Reprod Fertil.1991;93(1):245–251. [PubMed] Bromer JG, Seli E. Assessment of embryo viability in assisted reproductive technologies: shortcomings of current approaches and the emerging role of metabolomics. Curr Opin Obstet Gynecol.2008; 20:234–241. [PubMed] Campagnoli, C. et al. "In Vitro Fertilization and Embryo Transfer". Experientia 41.12 (1985): 1491-1494. Web. Carvalho, Luiz, and Stacey A. Missmer. "IVF Treatment in Patients with Endometriosis: A Challenger Approach". Journal of Fertilization: In vitro 02.01 (2012): n. pag. Web. Conaghan J, Hardy K, Handyside AH, Winston RM, Leese HJ. Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology. J Assist Reprod Genet. 1993;10(1):21–30. Craft, I, and T al-Shawaf. "Limiting The Number of Oocytes and Embryos Transferred in GIFT and IVF.". BMJ 303.6795 (1991): 185-185. Web. De Vos, A. "Intracytoplasmic Sperm Injection (ICSI)". Human Reproduction 15.suppl 4 (2000): 59-64. Web. Dermer, Gerald B. "A Method for The Visualization of Pulmonary Surfactant in The Light Microscope". Arch Intern Med 127.3 (1971): 415. Web. Desai, N., J. Szeptycki, and J. Goldfarb. "Application of Vitrification Technology in the IVF Lab: Preliminary Experience with Human Cleavage Stage Embryos, Blastocysts and Oocytes Vitrified on Cryoloops". Fertility and Sterility 84 (2005): S180-S181. Web. Devreker F. Uptake and release of metabolites in human preimplantation embryos. In: Cohen J, Elder K, editors. Human Preimplantation Embryo Selection. London, UK: Taylor and Francis; 2007; 325–336. Forman, E. J., Hong, K. H., Ferry, K. M., Tao, X., Levy, B., Treff, N. R., et al. "In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial." Fertility and Sterility. 2013; 100:100-107. [PubMed] Fullick, Ann. In Vitro Fertilization. Oxford: Heinemann Library, 2002. Print. Gashi, Astrit M. "The Impact of Assisted Reproductive Technology (Art) to the Increasing Incidence of the High Order Multiple Pregnancies". Gynecology & Obstetrics 05.07 (2015): n. pag. Web. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertility and Sterility. 2001; 76:1175–1180. [PubMed] Ginsburg, Elizabeth S, and Catherine Racowsky. In Vitro Fertilization. New York: Springer, 2012. Print. Gott AL, Hardy K, Winston RM, Leese HJ. Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos. Hum Reprod. 1990;5(1):104–108.[PubMed] Hardy K, Hooper MA, Handyside AH, Rutherford AJ, Winston RM, Leese HJ. Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos.Hum Reprod. 1989;4(2):188–191. [PubMed] Houghton FD, Hawkhead JA, Humpherson PG, et al. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod. 2002;17(4):999–1005. [PubMed] Huzair, Farah, and Alexander Borda-Rodriguez. "Challenges for the Application and Development of Omics Health Technologies in Developing Countries". Drug Dev. Res. 73.7 (2012): 447-451. Web. Jones GM, Trounson AO, Vella PJ, Thouas GA, Lolatgis N, Wood C. Glucose metabolism of human morula and blastocyst-stage embryos and its relationship to viability after transfer. Reprod Biomed Online. 2001;3:124–132. [PubMed] Johnston, Robert et al. "Demographic and Obstetric Outcomes of Pregnancies Conceived by Assisted Reproductive Technology (ART) Compared to Non-ART Pregnancies". JBRA Assisted Reproduction 19.1 (2015): n. pag. Web. Keskintepe, Levent. "Human Embryo Culture Dilemma Continues: "Back to Nature" or "Let the Embryo Choose". Journal of Fertilization: In vitro 02.02 (2012): n. pag. Web. KM, Yenkie, and Diwekar UM. "Modeling and Prediction of Outcome for the Superovulation Stage in In-Vitro Fertilization(IVF)". J IVF Reprod Med Genet 02.02 (2014): n. pag. Web. KRUSE, ANTHON. "An In Vivo/Vitro Embryo Culture Technique". Hereditas 77.2 (2009): 219-224. Web. Lafrance D, Lands LC, Burns DH. In vivo lactate measurement in human tissue by near-infrared diffuse reflectance spectroscopy. Vib Spectrosc. 2004; 36:195–202. Lambert, R. D. "Safety Issues in Assisted Reproductive Technology: Aetiology of Health Problems in Singleton ART Babies". Human Reproduction 18.10 (2003): 1987-1991. Web. Li, Ye. "Advanced MR Imaging Technologies in Fetuses". OMICS J Radiology 01.04 (2013): n. pag. Web. Reddy UM, Wapner RJ, Rebar RW, Tasca RJ. Infertility, assisted reproductive technology, and adverse pregnancy outcomes. Obstet Gynecol. 2007; 109:967–977. [PubMed] Rubio, C., Simon, C., Vidal, F., Rodrigo, L., Pehlivan, T., Remohõ, J., et al. "Chromosomal abnormalities and embryo development in recurrent miscarriage couples." Human Reproduction. 2003; 182-188. [PubMed] Seli E, Botros L, Sakkas D, Burns DH. Noninvasive metabolomic profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril. 2008;90(6):2183–2189. [PubMed] Shirazi, Abbas. "Mammalian Oocyte Cryopreservation". J IVF Reprod Med Genet 02.02 (2012): n. pag. Web. Simlara SL, Rottemberg V, Raine-Fenning N, Bhattacharya S, Zamora J, Coomarasamy A. Association between the number of eggs and live birth in IVF treatment: an analysis of 400 135 treatment cycles. Hum Reprod. 2011;26(7):1768–1774. [PubMed] Turner K, Martin KL, Woodward BJ, Lenton EA, Leese HJ. Comparison of pyruvate uptake by embryos derived from conception and non-conception natural cycles. Hum Reprod. 1994;9(12):2362–2366.[PubMed] Vergouw CG, Botros LL, Roos P, Lens JW, Schats R, Hompes PGA, Burns DH, Lambalk CB. Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection. Hum Reprod. 2008;23:1499–1504. [PubMed] Wood, Carl, and Robyn Riley. IVF, In Vitro Fertilization. Melbourne: Hill of Content, 1992. Print. Yang, X.-Y. et al. "The Timing of First Cleavage of Different Cloned and IVF Embryos with Oocytes Recovered by Ovum Pickup". Journal of Zoology 276.3 (2008): 299-305. Web.