Maternal and embryonic factors greatly determine the development of the embryo. If it occurs outside the womb (in vitro), embryos produce growth factors that encourage their development. There is proof that insulin-like growth factor II (IGF-II) promotes growth in early development stages of embryos and creates a leeway in which genes manipulate development before implantation. In this study, a recombinant form of growth will be added onto culture media in which human embryo will be grown. This will be compared with a control media in which no growth factor was added. Culturing of the embryo will take 168 days. The number of inner cell mass and trophectoderm will be counted to asses the effect of the factor with respect to mitotic division. The number of embryo that shall have reached the blastocysts stage will also be counted.
- To determine the effect of insulin-like growth factor II on human preimplantation embryo development invitro.
- To determine the effect the growth factor on cell multiplication.
- To monitor the impact this growth factor on the development of the embryo.
The recognition of factors responsible for growth in embryos of mammals or available in the female reproductive system has lately developed fundamental concern and is widely employed in several realistic appliances Heyner, 1993. Flourishing implantation necessitate proper maturity of the preimplantation embryo leading to a devise blastocyst capable of embedding into the uterine wall. Substantial data point out that certain growth factor produced by the endothelium work on the embryo to take care of this process (Sharkey et al.1996).
Fertilization in vitro has been used to come up with possible, moveable embryos though little is known concerning the impact of such systems on the resulting embryo. Cloning technology has been reported to result to abnormally large offsprings. The technology of fertilizing an egg outside the mother’s body as well as tissue culturing in the same environment has been employed to produce possible, manageable embryos. Nevertheless, more research is needed in this field to comprehend issues concerning the likely outcome on maturity of the resulting young organism. Resulting of extraordinarily big children has been linked with the techniques of genetic copying (Kruip and den 1997). To add onto this, research has indicated that in vitro media growing of embryos (Walker et al. 1996), embryo relocation (Wilmut and Sales 1981), or administration of progesterone hormone to females after producing an ovum (Kleemann et al.1994) lead to bigger embryos, fetuses, or offspring than usual.
It has been noted that in-vitro fertilization (IVF) in man, embryos are more often than not relocated to the uterus on second or third day of their growth, following-on a 13 % rate of implantation. Over two million children have been given birth to following in vitro fertilization (IVF) procedures. On 25 July 1978, the first “test tube” baby, called Louise Brown, was born after successive experiments. These studies had been done for other animals like the rabbits and mice but it’s evident that similar studies can be applied to human beings. Basically an ovum is fused with sperm in vitro then relocated to the uterus for implantation thereby setting up a successful pregnancy. This technology has now turned out to be conventional, converting the healing of barrenness in both women and men, and bringing about the reward of children to innumerable couples (Gardner and Lane 1998).
In spite of the achievement of IVF, anxiety in relation to the procedures still lingers in peoples minds. Even though the accomplishment of any given artificial reproductive technologies (ART) has been made better last twenty five years, flourishing results for whichever couple is not guaranteed (Schoolcraft et al. 2001). Embryos may be unsuccessful to embed, and there also be chances of frightening effect of numerous pregnancies, though this can be conquered by decreasing the quantity of embryos relocated at one given time. Lastly, there are concerns and anxiety that IVF babies are at larger threat than normally conceived babies for abnormalities in development (Fleming et al. 2004).
The inherent feature of the oocyte facing fertilization largely dictates whether a zygote has the likelihood to progress all the way through pregnancy (Lonergan et al. 2003). Generally, human ova necessary for IVF are obtained from a set of eggs obtained by endoscopy following successive stimulation of the ovarian even though, not all the ova are of equal value. Assortment of embryos for relocation following mitotic division singly or doubly more often than not is based on their form and structural manifestation, and in accordance to criterion that most embryologists are familiar with as illogical and inexact (Ebner et al. 2003). For that reason, the embryologists tend to grow the embryos in various media for a number of days prior to transfer (Gardner and Lane 2003). Since embryos that split and get to the blastocysts stage fastest are expected to have development ability (Lonergan et al. 2003).
Even though the quality of the oocyte is vital for conquering IVF, the culturing media settings where the zygote develops and later on divides also determine the potential of the oocyte (Lonergan et al. 2003). Earlier research has established that in vitro culture may result into epigenetic alteration in the embryonic genome (Mann et al. 2004) as well as manipulating gene appearance as a whole (Lonergan et al. 2003). All these may lead to unfavorable effect on the development of the embryo, the latest research have indicated the effect of the “large offspring syndrome” commonly distinguished in ruminant species following in vitro culturing of embryos in unfavorable conditions.
A number of research findings indicate that most IVF babies are born with low birth weights this raises an alarm for it is not known whether the children will reveal compensatory growth during adolescence and abnormally high blood pressure distinguished in logically conceived babies of low delivery weight (Fleming et al. 2004).
Fetal growth can be controlled by receptors which are two types: Type I and Type II, IGFBPs that is binding proteins as well as the insulin-like growth factors which is of two types also; IGF I and IGF II. (Guidice 1994). Early on studies recognized that receptors fastened to insulin and related growth factors (IGFs) are well demonstrated by experiments prior to implantation of embryos of animals like the mouse (Heyner, 1993 ). Using RT-PCR techniques, studies on this technology have been long-established at molecular level. Adding onto this, electron microscope of high resolution have revealed that insulin is contained in hollow mass of cells in the mouse embryo. Studies on immunology have also detected insulin in internal cell mass as well as the trophectoderm. Related studies which involve labbling IGF-I with gold have given away to discovery that this ligand is present in blastocysts of the mouse. Generally, all blastocysts articulate receptors that attach IGF-I onto exterior of the trophectoderm specifically the basolateral cells, the probability of 0.3 demonstrate receptors positioned at the apex (Heyner, 1993).However IGF-II is more of insulin like and its level in the blood is three times that of IGF-I. Although IGF-II has been considered as a predominant somatomedin during the pre-natal period, both area expressed in the human embryo and are involved in regulating foetal development and growth(Heyner,1993). Both IGF-I and IGF-II do not act independently, they have to coordinate with other growth factors in the body for proper fetal development (Blondin et al.2000).
Statistical data behind the fundamental role played by growth hormones throughout the stages of early embryonic development have been mounting up steadily (O’Neill 1998). Various types of cells from monkeys’ epithelial cells as well as human endometrial cells have proved to improve in-vitro embroyo development (Desai et al.2000). Liberation of little quantities of growth encouraging factors by body cell single layers could possibly be one of the ways by which co-culture cells manipulate embryonic development (Yeung et al.1996). Earlier studies in relation to co- cultures indicate that considerably elevated cell slicing rate of about 92% was achieve in the co-culture experimental set in contrast with the embryo not in co-culture set which had a rate of 83%. An elevated proportion of morphologically better embryos were noted in the experimental group than in the control group (Magli et al. 1995; Kervancioglu et al.1997)
Earlier research on mutant mice distinctly show augmented levels of IGF-II (Leighton et al.1995) demonstrating body weights that are 130% ± 5 than that of usual mice. Mice indicating reduced levels of Type-I receptor (Liu et al.1993) IGF-I (Liu et al.1993) and IGF-II (DeChiara et al.1990) reveal lesser body weights that than that of normal controls by a margin of 45% to 60%. There is evidence that adjustment in the phrase of IGF family members could be accountable for the diverse growth characteristics observed in fetuses and offspring initiating from embryos produced using in vitro experiments.
There are several existing culture media that permit embryos to grow to blastocysts stage at a speed equivalent with that taking place inside the womb (Summers and Biggers 2003), suggesting that embryos will be liberated from epigenetic marks which come as a result of the strain of in vitro culture. The constituents of most media are similar to those in the female reproductive system. (Gardner and Lane 1998). Studies carried out by (Sjöblom et al. 2005), describe a medium entirely lacking protein, which permitted more 95% plus of murine double-cell embryos from the fallopian tube of normally bred mice to proceed to blastocysts. It is not yet known whether the hollow ball of cells are of the same quality to those naturally conceived and budding in vivo. Some up to date studies on mouse embryos have been supportive of optimizing a culture medium with respect to its capability to encourage embryo growth (Ecker et al. 2004) or excluding some components like fetal bovine serum (Fernández-Gonzalez et al. 2004) may shun specific developmental and behavioral penalties that may occur after giving birth accredited to the preceding in vitro embryo culture. However, these studies also mean that negligible variation from most favorable practice can result to difficult to detect, unintentional penalties on the consequential pups.
This study will be carried out in three parts. The first section involves obtaining research permit from relevant authorities and taking ethical consideration into account, the subjects will need to be talked to. The second section will involve identifying subjects of study and sampling the oocytes. The last portion of the study involves laboratory experiments that entail culturing the embryo, adding insulin-like growth factor II, and counting cells as well as embryo following mitotic division.
Ovaries will be obtained from willing women within the reproductive age that will be sampled randomly. The sperm collected should be put in a specially prepared sperm buffer that is specifically formulated in order to keep the pH at the right level (Magli et al. 1995). The sperm buffer is made in a way to sustain the optimum pH whenever the solution is exposed air. The buffer is usually used for preparing of samples of the semen and the solutions of these semen samples. This is then washed and processed outside the incubator. The sperm medium is the same as the Sperm Buffer only that the buffer is usually made of the right pH of the solution is usually maintained while in the incubator. This medium is vital for the ultimate resuspension of the sperms for use in IVF because the fertilization takes place inside the incubator (Magli et al.1995).
Embryo Culture Equipment: Laminar Flow Hood
The preparation of all media and solutions to be used in IVF occurs inside this particular covering, which blows air away in the direction of the embryologist. The air is sieved and the outpouring of air prevents any contaminants from blowing inside thus contaminating the solutions and embryo dishes being made. Samples of semen preparations used in IVF takes place in the sterile environment as well (Gardner and Lane 2003).
The Preparation Incubator
All dishware and solutions used for an IVF treatment are kept in the incubator until they are needed for use. As Gardner and Lane (2003) put it: “The incubator is sterile inside, is at 37°C, has a carbon dioxide concentration of 6.0%, and the environment is fully humidified to prevent any evaporation. All solutions and dishes to be used for treatment are equilibrated in this incubator for a minimum of 4 hours before use.”
Embryo Culture Incubator
The egg and embryo are incubated in this chamber throughout their life-time in IVF laboratory. The incubator is usually provided with the correct levels of oxygen (O2) and carbon dioxide (CO2) for the purpose of ensuring that the egg and the embryos are provided with optimum environment at all times. 37°C is the temperature maintained in the incubator to provide the favorable conditions. The incubator temperature and pressure have to be carefully monitored throughout and modern incubators is usually attached to a phone alarm system which will ring out to the embryologist during off hours incase an unsuitable or emergency condition arise (Gardner and Lane 2003).
Every time the embryos and the eggs are exposed outside the incubator for whatever reasons, they always have to be in the IVF Chamber. The chamber itself is usually created and modified for the reason of giving optimum surroundings for eggs and embryos, even when handled outside of the incubator (Gardner and Lane 2003).
After finishing washing at retrieval, the eggs are usually placed in a fertilization medium, that contains a an assortment of salt, sugar, amino-acids, proteins among other nutritious elements. This are basically used to enable the egg and sperm in IVF to grow throughout the progression of fertilization, which is ICSI and IVF. Fertilization medium and all additional culture media that follow are usually buffered with the correct mechanism for the purpose of regulating the pH to the right level of the solutions found inside the embryo incubator (Gardner and Lane 2003). Alternatively, the Cumulus-oocyte complexes will be collected from these ovaries and put in shock absorbing artificial human tubal fluid medium preferably HEPES-HTF (Quinn et al. 1985) and grown in media of average settings such as TCM-199 using tailored human tubal fluid medium (mod-HTF) where maturity as well as fertilization will take place. The entire chemical reagents of medium will be of tissue culture rating from recognized companies like Sigma Chemical Co. (St. Louis, MO). All the invitro culture media will contain mg/ml of gentamicin. (Blondin et al.2000). The full-grown oocytes will be fused with male gametes (semen) from male counterparts whose fertility is confirmed to produce embryos in vivo. All eggs that usually go through ordinary fertilization are subsequently positioned into cleavage medium. This is normally formulated exclusively to sustain the development requirements of the beginning cleavage stage embryo (Gardner and Lane 2003).
After sometime about twenty hours after insemination, zygotes will be washed for about six times in modified Tyrode’s-lactate Hepes and grown in groups of 20 zygotes in 1 ml of TCM-199 + 10% serum in vitro-produced with serum ie IVPS or one ml of TCM199 + 1% BSA in vitro-produced with serum restraint IVPSR. Three days after insemination exercise, IVPSR embryos will be relocated into newly prepared TCM-199 + 10% serum while IVPS embryos will have spanking new medium reinstated. Five days later, fresh TCM-199 + 10% serum will be substituted in both set ups. On the seventh day after insemination, the cells normally hollow-like considered to be at blastocyst-stage embryos will be collected and allocated a morphological value ranking following counting after staining (Blondin et al.2000). Mammalian embryos show signs of significant plasticity and will usually create blastocysts under a broad variety of culture situations, although most probably at some adaptive cost to their postgestational growth program.
Multiplication of cells will be determined by counting based on visualization. The cells will then be stained by using 4 µg/ml Hoechst dye (33342 bisbenzimide; Sigma) so that the cell nuclei is clearly visible. Embryos are put in the solution for approximately 50 minutes then they are made wet mounts. The tainted nuclei will be observed using mercury lamp UV illumination to determine the number of inner cell mass and trophectoderm (Stojanov 1999).
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