Embryo Glue: Clinical Effectiveness - Research Proposal Example

Introduction EmbryoGlue is increasingly being used to improve the probability of embryo implantation after fertilisation. The product is especially popular where in vitro fertilisation (IVF) has been undertaken, since many cases that get IVF referral involve failure of the embryo to attach to the endometrium. Despite its growing popularity, various research studies have come up with differing opinions regarding the usefulness of the product in enhancing the rates of embryo attachment to the endometrium. For instance, Check et al. (2012) and Safari, Razi, Safari and Razi (2014) observed that the product did not attract significant improvement over the more common media in increasing the rate of embryo implantation among IVF recipients. On the other hand, Valojerdi, Karimian and Baghestani (2006) and Madani and Jahangiri (2012) encouraged use of embryo transfer mediums including EmbryoGlue, based on the positive results the product had returned in the two separate studies. Awford (2014) added his supporting voice by highlighting a case of a couple that had gotten a baby by the help of EmbryoGlue following numerous previous attempts at conception. Several factors are involved in the determination of how successful an implantation is. Two leading factors are the viscosity and toxicity of the media used (Madani and Jahangiri, 2012). Objective The manner in which viscosity affects embryo development remains a topic for discussion and further research. However, toxicity has negative impact on an embryo’s development. The study investigates the differences in viscosity levels between the ordinary media used for embryo development and a mixture of the said media and EmbryoGlue. For this reason, this study will focus on two main aspects of viscosity and toxicity: 1) Investigate how viscosities of the ordinary media and a mixture of the media and EmbryoGlue affect the development of the embryo. 2) Investigate how the toxicity levels within the two setups change over time. Consequently, the following hypotheses will guide the research object: H1: The use of EmbryoGlue significantly improves the rate of embryo development when compared to the ordinary media used for embryo development. H2: The levels of toxicity remain relatively equal for the two setups over time. Literature Review The most abundant compound in EmbryoGlue is hyaluronan (a polymer), which is also found in the fallopian tubes, uterus, and follicles. This compound is crucial for converting the endometrium into a blood-supply-rich layer (Khan, Ritcher, Blake and Yankov, 2007). The process of conversion is also accompanied by emergence of more active glands to enable the endometrium receive the embryo and nourish it further (Layyous, 2014). EmbryoGlue also contains the valuable nutritional chemicals amino acids and carbohydrates that increase the ability of the embryo to attach to the wall of the uterus. The product has greatly influenced IVF. Accordingly, subsequent IVF tests that follow a series of failures are accompanied by the introduction of the success-propelling EmbryoGlue, mostly in countries in which it is formally regulated (A. Englinde, personal communication, 16th February 2015). The use of EmbryoGlue also comes with the application of few more products that work on a supplementary basis with the product. One such is the luteal phase supporting intravaginal progesterone (Cyclogest 400, Coxph, UK) (Valojerdi et al, 2006). Hyperviscosity (too high) and too low viscosity levels are detrimental to the ability of the spermatozoon and the ovule to fuse successfully (IVF News Direct, 2008). During the embryo transfer phase, it is important to correctly time the duration it takes to transfer the embryo from the micro-droplets of EmbryoGlue since the process involves removal of the embryo from its optimal storage conditions outside the body. As such, long exposure to sub-optimal environment could easily kill the embryo (Halvaei, Khalili, Razi, Agha-Rahimi and Nottola, 2013; Hassani et al, 2013; Dhont, 2013). Normally, the perfect timing should be around twenty minutes, with a possible bias of ten minutes less or in addition. Testing the effectiveness of EmbryoGlue in enhancing embryo development is outlined in the methods section. Service evaluation of the performance of the product would involve examination of the rates of increment/ or lack of change in the numbers of viable pregnancies that are basically the result of its introduction. Generally, the improvement should be compared with the improvements witnessed for other products within the IVF. Nurture Fertility (2014) noted that the rate of improvement observed with the application of EmbryoGlue are substantially high within the field of clinical practice. As noted in the introduction, many studies that tried to evaluate the working success of EmbryoGlue returned failure results. This was mainly influenced by the shallow body of knowledge surrounding IVF when the product first came into use. However, this appears to have changed considerably over time, with present studies mainly returning success results, again as indicated. One improvement associated with the current success levels for the product is the thorough testing it is accorded at the manufacturing stage. Tests include ascertaining the concentration levels of each component (hyaluronan, amino acids and carbohydrates), and testing viscosity. Other tests carried out include pilot tests on animals to establish whether variations advised on the product over time have significant alterations on previous response and whether the results are as desired (A. Englinde, personal communication, 16th February 2015). In order to test viscosity of the glue, tests of physiological concentrations in the fluids that comprise it are undertaken. Higher levels of viscosity are better for successful transfer of the embryo, but too high viscosity is again highly likely to negatively impact the implantation process. Generally, the time that the embryo spends in the glue affects the likelihood for success. Hyaluronan has a high cell to cell adhesion capacity. CD44, receptor for hyaluronan (HA) mediated motility (RHAMM), and the intercellular adhesion molecule 1 (ICAM-1) are the documented cell receptors for the compound (Aftoonian and Asgharnia, 2006; Fredler et al, 2007). ICAM-1 plays the role of a metabolic cell surface receptor (Reitinger et al, 2007). The role of CD44 is far more significant for hyaluronan’s role in EmbryoGlue, since it is the main receptor of HA on the surface of the cell. It mediates the process of cell interaction with HA, thereby helping kick-start various physiologic processes, including cell-to-cell and cell-substrate adhesion. This receptor, therefore, underlines the usefulness of HA in EmbryoGlue. The product’s viscosity will then be evaluated by evaluating the time it takes to flow from a designated point relative to a standard liquid, such as pure water. The present selling price per cycle is £150 (Nurture Fertility, 2014). The majority of equipment needed for the project are mainly hospital-based, including ultrasound devices, labotect catheter (Labotect, Germany), culture dish, among others (Valojerdi et al, 2006). Methods A comparative approach will be adopted, where embryos’ development will be observed under two distinct environments/ media – one involving assisted embryo development procedure using the traditional methods (applying media and observing the growth/ development of the embryo with a microscope). The parallel procedure will involve assisting embryo development in a medium comprising the traditional media and EmbryoGlue. The media will be tested for viscosity, after which the mixture comprising EmbryoGlue and the media will also be tested for the same. Specifically, the kinematic viscosity values for the products will be assessed. The Brookfield Digital Viscometer model DV-II has been selected for this purpose due to its ease of measurement and readings that do not require further conversion (Lubrizol, 2006). The device is also suitable for a wide range of viscous fluids, including water, glue, and oil (K. Michael, personal communication, 28th Feb. 2015). According to this source, the viscosity of the media-EmbryoGlue mixture is highly likely within the Brookfield Digital Viscometer. The procedure will involve immersing sufficient volumes of the media/ media and EmbryoGlue mixture into the Brookfield Digital Viscometer model DV-II, covering with a spindle when the device is level, and noting the subsequent readings expressed in poises. It is essential to adjust the temperature to that of the pig’s body for practical evaluation of closely related environmental conditions. The viscosity will be evaluated over time, calibrated at five minutes intervals for ease of data collection. Measurements will be taken for periods spanning to over 30 minutes. Subsequently, tests on the penetration of sperm on the selected eggs will be carried out. The sperms selected are mixed with the egg in a controlled environment that contains media and media/ EmbryoGlue mixture, and the number of sperms that penetrate the egg noted for each case. This helps to not only estimate the percentage of viable sperm cells out of the ones reserved, it also helps in estimating how likely a positive IVF outcome may be expected. The development process of the resultant pig embryo will be assessed using the embryoscope. Embryoscope is an IVF incubator that contains an in-built camera that collects automated time-lapse images of fertilized oocytes in an incubator from conception to transfer time. Using the device, the initial multiplication of the cuboidal epithelial cells in the newly formed pig embryo will be observed. Photos of the developing embryo will be taken after day 3 and day 5 to observe the differences in development of the embryos subjected to the two media. The inner cell masses will be assessed alongside the photo evaluation. Levels of toxicity will be assessed using the Microtox M500 (Shettlemore and Bundy, 2006). The different levels of viscosity of the product will be applied to the newly developing embryos and the development stages evaluated under embryoscope. This will give the needed data on how various levels of viscosity is aggregated against the success rates of implantation. The different viscosity levels will be recorded against observed success rates. This will basically require documenting materials for keeping records, particularly notebooks or a tablet. The general environment for embryo development is set to standards that allow maximal comparison of aspects of the pig’s womb. This includes temperature and concentration regulation for components of the media. The optimal temperature for embryo development/ incubation ranges between 15 °C (59 °F) to 20 °C (68 °F). Temperatures below and above this range are detrimental for pig embryo development. The embryo is forced to restructure when temperatures vary considerably, thereby leading to lowered chances of survival. Sodium chloride (NaCl) and glucose are the leading compounds in the media culture. The optimal concentration for NaCl is 85 Mm NaCl for optimal synthesis of amino acids. Studies have shown that variation below and above this point leads to sub-optimal synthesis of the amino acids. Glucose concentration levels are irrelevant. Variation of glucose concentrations does not affect embryo development. The points to consider when choosing the number of embryos to use is the likelihood to survive. The researcher chose to use twenty embryos, in the hope that ten will provide viable results for analysis. This assumption dwells on a 50% survival prediction for the embryos. Equipment Embryoscope Brookfield Digital Viscometer model DV-II Ultrasound devices Labotect catheter (Labotect, Germany) Culture dish Analysis The independent samples t-test will be used to evaluate whether there is observable, significant differences between the rate of embryo development for corresponding levels of viscosity of EmbryoGlue. The rate of development will be assessed for embryos within the two setups and the growth rates compared. The rate of development will be treated as the dependent variable. The viscosity levels will be treated as the independent variable. Similarly, the toxicity levels will be assessed against time for the two media setups. Table 1. Timetable of follow-up of activities for the research. Dates Action February – 10th March Proposal writing 15th March – 15th April Attending ethical committee and sample identification 16th April – 15th May Pilot study (data collection and questionnaire modification) 1st June – 10th June Conduct experiments and collect data 15th June – 30th June Data analysis 1st July – 31st July Compile report Table 2. Budgetary forecasts for the research exercise. Item Number Cost Writing/ recording material £20.00 Experimentation material £200.00 Travel £300.00 Assistants’ incentives £500.00 Miscellaneous £300.00 Total £1320.00 The contingency plan would be the introduction of an alternative less time-consuming project, which would be duly discussed with the supervisor. Table 3. Step-wise timeframe table. Step Time Sperm assay concentration measurement 4 hours Embryo cleaning 2 hours Transfer to culture media 15 minutes Exploration of embryo development in culture 5 days References Aflatoonian, A. and Asgharnia, M. 2006. Factors affecting the successful embryo transfer. Iranian Journal of Reproductive Medicine. 4(2): 45-50. Awford, J. 2014. Miracle baby Ethan survived because doctors glued him into his mother’s womb. Daily Mail. Retrieved from http://www.dailymail.co.uk/news/article-2798980/miracle-baby-ethan-survived-doctors-glued-mother-s-womb.html. Check, J. H., Summers-Chase, D., Yuan, W., Swenson, K., Horwath, D. and Press, M. 2012. “Embryo glue” does not seem to improve chances of subsequent pregnancy in refractory in vitro fertilization cases. Clinical and Experimental Obstetrics & Gynaecology. 39(1): 11-12. Dhont, M. 2013. Evidence-based reproductive medicine: A critical appraisal. Facts, Views & Vision in ObGyn. 5(3): 233-240. Fredler, S., Schachter, M., Strassburger, D., Esther, K., El, R. R. and Raziel, A. 2007. A randomized clinical trial comparing recombinant hyaluroran/ recombinant albumin versus human tubal fluid for cleavage stage embryo transfer in patients with multiple IVF-embryo transfer failure. Oxford Journals: Human Reproduction. 22(9): 2444-2448. Halvaei, I., Khalili, M. A., Razi, M. H., Agha-Rahimi, A. and Nottola, S. A. 2013. Impact of different embryo loading techniques on pregnancy rates in in vitro fertilization/ embryo transfer cycles. Journal of Human Reproductive Sciences. 6(1): 65-69. Hassani, F., Eftekhari-Yazdi, P., Karimian, L., Valojerdi, M. R., Movaghar, B., Fazel, M…and Johansson, L. 2013. The effects of ISM1 medium on embryo quality and outcomes of IVF/ICSI cycles. International Journal of Fertility & Sterility. 7(2): 108-115. IVF News Direct 2008. Seminal hyperviscosity may affect the positive outcome of IVF and embryo transfer. Retrieved from http://www.ivfnewsdirect.com/?p=94. Khan, N., Richter, K. S., Blake, B. J. and Yankov, V. I. 2007. Case-matched comparison of intramuscular versus vaginal progesterone for luteal phase support after in vitro fertilization and embryo transfer. Fertility & Sterility. 87(4): S13. Layyous, N. 2014. Embryo glue. Retrieved from http://www.layyous.com/en/assisted-reproduction/embryo-glue/2-150. Lubrizol 2006. Viscosity – Brookfield. Ohio: Lubrizol Advanced Material, Inc. Madani, T. and Jahangiri, N. 2012. Increasing pregnancy by improving embryo transfer techniques. Advances in Embryo Transfer. Wu, B. (Ed.). Zagreb: Intech. Nurture Fertility 2014. EmbryoGlue – Improves pregnancy rates by 19%. Retrieved from http://www.nurture.ac.uk/news/embryoglue-improves-pregnancy-rates-by-19. Reitinger, S., Laschober, G. T., Fehrer, C., Greiderer, B. and Lepperdinger, G. 2007. Mouse testicular hyaluronidase-like proteins SPAM1 and HYAL5 but not HYALP1 degrade hyaluronan. Biochemical Journal. 401(1): 79-85. Safari, S., Razi, M. H., Safari, S. and Razi, Y. 2014. Routine use of EmbryoGlue as embryo transfer medium does not improve the ART outcomes. Archives of Gynaecology and Obstetrics. 291(2): 433-437. Shettlemore, M. G. and Bundy, K. 2006. Toxicity measurement of orthopaedic implant alloy degradation products using a bioluminescent bacterial assay. Journal of Biomedical Materials Research. 45(4): 395-403. Valojerdi, M. R., Karimian, L. and Baghestani, A. R. 2006. Efficacy of a human embryo transfer medium: A prospective, randomized clinical trial study. Journal of Assisted Reproduction and Genetics. 23(5): 207-212. Read More