Extracorporeal fertilization (In Vitro fertilization)
IVF program consists of four steps. Each of them are very important in its own way, that’s why one must be very accurate carrying out all the doctor’s instructions.
Step I. Stimulation of superovulation with hormone preparations (10–30 days).
Step II. Puncture of the follicles of oocytes under the general anesthesia. Obtaining of sperm at the same time (1 day).
Step III. Sperm combines with the oocyte. Fertilization of the oocyte in vitro (1day). Cultivating of the embyos, monitoring of the embryos division (2–5 days).
Step IV. Transferring of the obtained embryos into the woman’s uterus cavity, the rest of embryos are frozen if it’s necessary (1 day).
5.1 Stimulation of superovulation. Only one oocyte usually ripens during the menstrual cycle. But in case of extracorporeal fertilization the risk theta oocyte isn’t fertilized can be too high. To increase chances of pregnancy after one procedure of extracorporeal fertilization, several mature cells must be obtained. That’s why Step I is stimulation of superovulation, when the woman is prescribed special medicines, which provide ripening of several follicles simultaneously. Before the beginning of stimulation the doctor together with the patient chooses the proper variant of the treatment and medicines, establish sequence of their taking that is called “protocol of stimulation”.
Follicle-stimulating hormone (FSH) is responsible for ripening of follicles, that’s why the medicines containing FSG are taken for stimulation of superovulation. Recombinant preparations are considered to be the most effective nowadays. They increase chances of pregnancy and reduce cost of treatment. Any chosen protocol of stimulation is followed by ultrasonic monitoring and hormonal analysis as well.
The risk of hyperstimulation of the ovaries
After the stimulation of superovulation many so-called yellow bodies are formed on the place of the follicles. Excessive hormones, produced by the yellow bodies cause the light form of Ovarian hyper stimulation syndrome in 10% of cases. The symptoms are: nausea, vomiting, abdominal pain, slight swelling of the belly. Some patients have weakness, low blood pressure, decreasing of urine amount. In cases of appearance of any of these symptoms the patient must see the doctor. If the hyperstimulation is in a light form the patient is recommended to drink more water and to avoid physical activity. In more serious cases the dropper with special medicines is prescribed. If the spasms and uterine bleeding are observed, the patient must be taken to the hospital immediately.
Discussions about the IVF influence on the development of malignant tumors haven’t stopped since the procedures of extracorporeal fertilization were started. Swedish scientists however have managed to refute all suspicions. In the result of large-scale examination they found out that the women who had decided to be undergone IVF procedures, risk of fall ill with one of the “female” kind of cancer was 25% less compared with women who had got pregnant by natural way.
5.2 Follicles puncture. The purpose of the puncture of the ovarian follicles is obtaining of oocytes. The doctor usually uses a thin needle, injecting it through the vagina under the ultrasonic monitoring. This method of puncturing is called transvaginal. The content of the follicle is aspirated with the syringe or a special aspirator. This procedure is very important so the patient should be very responsible to deal with it: she mustn’t drink and eat anything the day before the procedure, even after the midnight. As a rule the puncture is carried out in the morning under the general intravenous anesthesia.
Complications at the puncture of the follicles. Although the puncture provides minimal interfering, it is however the kind of operation, that can cause some complications. Injections of the puncture can cause slight vaginal bleeding and the application of the general anesthesia can cause wekness. This symptoms are quite natural and fade in several hours after the operation. In very exceptional cases introducing of the needle in the abdominal cavity can cause infection or internal bleeding. The patient with the risk of such complications are recommended to stay in the hospital.
5.3 Cultivation of the embryos and applying in vitro for fertilization of the oocyte. Collecting of sperm. After the puncture the oocytes are transferred to the laboratory for the detailed analyses. Mature oocytes are selected from immature ones and placed in a specially prepared solution. The spouse collects his sperm by masturbating, the collected sperm will be used for oocytes fertilization. This step is not difficult but may cause serious and explained stress. In cases of some problems the collecting of sperm can be done beforehand, and then the collected sperm will be sent to the laboratory to be preserved at the optimal temperature.
In cases when sperm is obtained by surgical way from small seminal ducts or testicles, it will be sperm frozen at cryogenic temperature and after that – prepared for use.
Regardless of how the sperm has been collected it is examined and then is prepared for fertilization. Seminal fluid, decreasing fertilizing influence of sperm is removed, and spermatozoa are selected into a special test-tube (vitro) . the most active spermatozoa are selected as they can help to achieve good results. Then the most decisive process – process of fertilization – begins.
Process of the fertilization of the oocyte
The process of fertilization of the oocytes is going on by two methods: classic, that means fertilization in the test-tube (vitro) (FIV), and Intracytoplasmic sperm injection(ICSI). Depending on the causes of infertility the doctor choose the proper method of the oocyte fertilization.
Method FIV is applied when the cause of infertility is in the woman, while the quality of sperm is satisfied or a little distorted. Some time later after injection of the puncture the follicles of the oocytes are combined with the spermatozoa on the special substrate, that provides their vital activity. The substrate is sent for preservation that requires 37gr, and it must be preserved from 4-20 hours. And only one spermazoon from the great number of spermatozoa will reach and will get through the plasmatic membrane of the female oocyte and will manage to fertilize it.
The biologist detects through the microscope if fertilization has occurred or not in 18-20 hours. Successfully fertilized cell looks like the cell with two nuclei, that is often called pronucleus; one nucleus will be father’s nucleus and the other – mother’s. At this stage of forming the cell is called zygote. Zygote is not the embryo, because mother’s and father’s DNA won’t combined yet. After that the biologist selects two or three the most proper zygotes for transferring into the woman’s uterus. The rest of zygotes are frozen, as they may be used in the following attempts, it is necessary.
Different anomalies in extracorporeal fertilization sometimes occur and they prevent zygotes from forming to transfer them into the uterus and also to preserve at the cryogenic temperature for the future attempts.
5.4 Fertilization of the oocyte in vitro with the use of ISCI procedure. ICSI is intracytoplasmic injection of the spermatozoon directly into the oocyte, which is the new, but well elaborated procedure in extracorporeal fertilization, that has already gained confidence and it was carried out for the first time in 1992. Procedure ICSI was worked out for the treatment of male infertility.
Procedure ICSI is a procedure when a single spermatozoon is penetrated directly insite the oocyte with the use of the thin glass needle of micro size. The first stages of procedure ICSI occur as at the usual cycle of extracorporeal fertilization. The doctor prescribes injections of hormonal preparations to the woman, that are applied for stimulation of maturing of the follicles in ovaries. Spermatozoa, obtained from ejaculate or through biopsy of the testicle or of epididymis, are treated in a special method in necessary mediums. Spermatozoa can be obtained from the frozen sample of seminal fluid. Spermatozoa are placed in a special medium after that the biologist has indicated maturity of the oocytes. Spermatozoa with normal morphology are only selected, are got in immobilized condition, sucked into the tip of the very thin glass needle, and then is injected directly into inner part of the oocyte. At this time the oocyte is fixed with the special holding pipette.
This procedure demands the specialist’s high qualification and a special micromanipulator. This sequence of the biologist’s actions repeats for the each obtained oocyte. As the membrane of the oocyte is elastic enough the closing of the hole occurs very quickly, although about 1% of oocytes are damaged at the procedure ICSI. The oocytes are examined the next morning to make sure that fertilization has happened or that it has failed. The embyos are cultivated during the next 2-5 days, when their division and the following development are going on.
Risks of use of ICSI method.
The main risk of use of ICSI method is a chance of transferring of defective genes to the patient’s posterity. To decrease and exclude this risk , a special genetic screening is carried out, that provides revealing different anomalies. The procedure ICSI is safe for the posterity. Infertile parents can become the cause of genetic diseases for their children, i.e. they have influence on their children’s health. Preimplantation genetic diagnosis provides revealing chromosomal and genetic defects in the embryo. This diagnostics is carried out at the stage of more that six blastomeres.
5.5Assisted hatching. Successful fertilization of the uterus depends on the implantation of the embryo in it. It depends on the external membrane and quality of the embryo. The embryo and the oocyte are protected with two layered membrane, which exists only at the early stage of the development. This membrane plays the important role in the process of fertilization. Due to the two-layered membrane the process of the fertilization of the oocyte occurs easier. Many researches register that the membrane gets denser that can prevent the embryo from attaching to the wall of the uterus, so to achieve the pregnancy it must be removed. This procedure is called hatching. Lower frequency of implantation depends on changing of density and thickness of the membrane of the embryos, obtained in the result of extracorporeal fertilization.
Laboratory embryos are often observed to excrete less ferments than it is necessary for the dissolving of the membrane. That’s why a new artificial model of the procedure of hatching, called assisted hatching has been worked out. Assisted hatching is cutting a small hole in the zona pellicula at the proper moment of the embryo’s development to make its fertilization easier. .The embryologist is the specialist who carries out this manipulation at the days of optimal condition of endometrium, which is called the implantation window. The embryologist’s synchronous and correct manipulations provide good chance for pregnancy.
5.6 The embyos development and their transfer. The embryos are transferred into the patient’s uterus for several days after collection of oocytes. At the second day the embryo is developed into 2-4 cells, and at the third day the embryo develops the stage of 6-8 cells. The transferring of the embryo into the uterus is painless and safe procedure. The embryos are placed into a thin flexible catheter. Opening the cervix of the uterus with the vaginal speculum the gynecologist carefully injects the catheter with the embryo into the cavity of the uterus.
Transfer of the embryo into the uterus is often carried out under the echography control to monitor the movement of the catheter in the uterus to be sure that the embyos are located in the right place. Several embyos are usually placed in the uterus at the same time. The result depends on the number of the embryos. The more embyos are placed the more babies are born. The number of embryos placed into the uterus are agreed with the couple and their doctor.
The blood sampling of the mother is done approximately twelve days after the transfer of the embryos to the uterus to make the test for pregnancy to detect the results of the procedure. If the test is positive the echography is planned about for weeks later after the transfer to control the process of the pregnancy. Generally speaking the pregnancy rate after the procedure ICSE doesn’t differ much from usual IVF. The average pregnancy rate for extracorporeal program is about 30-35%.
Possible risks at embyos transfer. Embryos transfer is rather simple, safe and painless procedure.While the catheter is being injected the patient should relax, mustn’t tighten up the muscles of the low part of the abdomen. The woman should be in the positive mood. After the transfer of the embryos, the patient should lie horizontally for some time.
How many embryos must be transferred into the uterus is one of the most difficult issues, which the doctors carrying out extracorporeal fertilization have to decide. Placement of multiple embryos carries the risk of multiple pregnancy, that may cause different hard aftercomings both for the fetus and for mother. This problem becomes more complicated because the embryo can divide that leads to the birth of enzygotic twins.
Embryos transfer is the last step in the cycle of extracorporeal fertilization, after which the doctor prescribes various preparations for keeping lutein phase, rising the chance of implantation. The doctor may prescribe additionally preparations of progesterone, estrogens or HCG hormone. The most difficult part of the IVF program is the period after transfer the embryos into the uterus, as every patient is tormented by tension and uncertainty guesing if pregnancy has occurred or not.
Pregnancy is detected with the use of test, that shows the presence of HCG hormone in the blood serum 10-14 days after the transfer. HCG hormone is only synthesized by the embryo cells. For all patients these 10-14 days seem the longest in their lives!