Fertilization (fertilization) can be defined as the male and female reproductive cells oocyte and sperm, which combine to form the single-cell zygote, which will form the embryo with the subsequent cell divisions. Therefore, in order for fertilization to occur, the sperm must reach the oocyte. This process, "in vitro" environment (in the womb of the woman), sexual intercourse during the ejaculation of men spilled into the vagina of the transition from the cervix and the uterus in the transition from the passage of the uterus in the uterus and the uterus, the ovule to reach the oocyte in the tubular and waiting to connect the genetic structure within the sperm head (DNA) containing nucleus and many biomolecules involved in fertilization.
The first priority for in vitro fertilization is that a sufficient number of motile spermatozoa (~ 2 million) can reach the uterus. Secondly, the sperm must be in maturity at the part of the head called acrosome, located in the anterior part of the head, that connects the oocyte surrounding the cumulus cell layer to the zona pellucida layer, which is the outer protective sheath of the oocyte, and ultimately has the receptors and enzyme content necessary to reach the oocyte. Various negativities in this process (short of sperm number, tail defects affecting mobility, acrosomal defects) may prevent the occurrence of fertilization.
The first priority in IVF treatments is to skip the steps that may prevent the sperm from reaching the egg and to allow the sperm to be brought directly to the oocyte. For this purpose, IUI (Intra Uterine Insemination), IVF (In vitro Fertilization) or ICSI (Intra Cytoplasmic Sperm Injection) methods are used in infertile couples.
IUI ProcessThe aim of the IUI procedure is to overcome the possible cervical problems that prevent the sperm from reaching the uterus and to move the motile sperm cells directly into the uterus. Subsequent fertilization depends on the structural features of sperm and oocytes.
In cases where IUI cannot be achieved by IUI procedure or due to pre-defined negativity due to gametes, fertilization methods are applied in laboratory. For this purpose, IVF or ICSI is still preferred for infertility indication.
In the IVF procedure, the sperm cells obtained by the pretreatment procedures are maintained in the same culture medium as the oocytes obtained by the OPU (Oocyte Pick-up) process which is performed after KOH (Controlled Ovarian Hyperstimulation) treatment to increase the number of oocytes. In order to apply this method, the number and quality of the sperm should be sufficient (more than 2 million / ml and no significant morphological defects). Fertilization rates with IVF application are 50-60%.
ICSI MethodAlthough ICSI method is a preferred method especially in pairs with low sperm count, it is widely used because of the higher fertilization success (> 70%) compared to IVF method. In this method, the prepared sperm cells are injected directly into the oocyte under the microscope, with the help of specific micropipettes, using the preferred alternative selection methods depending on their morphological characteristics, mobility style and sperm structure. In this way, other possible factors that may prevent sperm from reaching the oocyte are overcome.
The ICSI method allows the sperm to reach directly into the oocyte; however, this does not mean that every oocyte will be fertilized. Achieving fertilization requires the sperm to function properly at the molecular level, beyond the physical reach of the oocyte.
The primary priority for the fertilization of oocytes is that some factors in the sperm cytoplasm can activate the oocyte. The oocyte is a cell that is pending at the stage of meiosis, defined as metaphase 2, and requires a warning by the sperm to continue the division. At this point, the most prominent factor identified so far is a biomolecule called phospholipase-C. Phospholipase-C is the initiator of a series of processes in the oocyte cytoplasm, which ultimately leads to an increase in intracellular calcium level and ongoing fluctuations. The meiosis of this oocyte, which receives this signal, continues and eventually the fertilized cell called zygote occurs.
The success rate in the ICSI method, which allows to overcome many obstacles to fertilization, is around 70-80 percent; however, in some cases (3-5%) no fertilization may occur. The underlying causes of fertilization failure are complex and depend on many factors, such as cycle specific parameters, oocyte count and quality, sperm count and quality. Ultimately, the main reason for failure of fertilization is that oocyte activation could not be achieved. At this point, the defects of the sperm attract the head.
Fertilization FailureIn order for normal fertilization to occur, the sperm must have sufficient nuclear maturity and there should be no defect in the packaging of the genetic material. During the sperm ripening process, in order to preserve the genetic structure against the environmental negative effects during sperm production, it is necessary to pass the tightly packed DNA to the looser packaging form. Sperm morphological defect, which is defined as globozoospermia, is the most striking anomaly in this regard. In addition to the absence of acrosome in the sperm with this defect, the DNA remains tightly packaged, which significantly affects the fertilization success (0-20%). In addition, the oocyte activating factors in the sperm are inadequate and they affect the fertilization success in the organelles defects called centriol, which constitute microtubule structures that govern the fertilization process.
One of the factors that affect the fertilization success is the inability of the oocyte cytoplasm to have sufficient maturity (cytoplasmic maturation). The maturity of oocyte is assessed in two parts as nuclear and cytoplasmic. Although the observation of the cell called polar body in the shingles indicates that the oocyte has reached nuclear maturity, an oocyte in this structure may not be sufficient in terms of the amount of organelles (especially mitochondria), which are defined as cytoplasmic maturation, which will play an important role in the fertilization process. In this case, fertilization may not occur even if there is no problem with sperm.
Alternative MethodsThere are alternative methods in order to overcome the failure of fertilization in the laboratory. The common point of these methods, defined as AOA (Assisted Oocyte Activation), is the artificial induction of intracellular calcium increase, which is the primary priority for the oocyte fertilization. The methods used for this purpose are divided into three categories as mechanical, electrical and chemical activation:
In the mechanical activation method, the oocyte cytoplasm is aimed to increase the likelihood of inoculation of calcium in the oocytes by micropipette injection several times. In addition, calcium may be introduced into the oocyte externally during microinjection. According to other methods, the chance of success is lower.
In the electrical activation method, low voltage electrical signals are given to the oocytes which have been microinjected by the use of this method and culture containers. It is aimed to expand the channels on the cell membrane (pore) and increase the entry of extracellular calcium into the cell.
In the chemical activation method, the oocytes are kept in various chemicals and it is aimed to expand the channels in the cell membrane as in the electric activation. Examples of such chemicals are ethanol, calcium ionophore A213187, ionomycin, puromycin, strontium chloride, phorbol ester and thimerosal.
Although oocyte activation has been shown to be particularly successful in sperm-induced fertilization failures (fertilization rates of up to 100 percent in globozoospermia cases), it may not be possible to achieve the same success in oocyte-related conditions. In this case, the most useful approach is to increase the probability of maturation of oocytes obtained by manipulation in the KOH process (elongation of time or testing of different protocols), in the case of failure of oocyte cytoplasmic maturation in cases with insemination failure in previous trials, prolongation of waiting time until microinjection after OPU.