IVF Unica - Institute of Reproductive Medicine, oocytes, Oocyte Donation, Donated eggs, IVF, Brno, Czech republic
IVF Unica - Institute of Reproductive Medicine, oocytes, Oocyte Donation, Donated eggs, IVF, Brno, Czech republic
Starting the stimulation cycle
On the day of the stimulation cycle initiation, the couple receives a detailed schedule specifying the use of all prescribed medicaments. Upon the start, the methods of administration of individual medicaments and injections are explained.To enable proper embryo formation and subsequent transfer, the woman must use all the prescribed medicaments responsibly (hormonal pills, injections, nasal spray, etc.) according to the schedule. Consequently, the ovaries produce multiple follicles with maturing eggs. The time of follicle maturation and egg release = ovulation may be estimated in advance. This is very important since the oocytes must be collected shortly before an egg release via spontaneous ovulation would occur.
Follicular monitoring
The degree of oocyte maturation is assessed by ultrasound measurement of the follicular growth (i.e. follicular monitoring). A normally developing follicle grows by 2 mm a day. Along with hormone analyses, it is possible to estimate fairly exactly the optimum time for collection of mature oocytes, capable of fertilization.Follicular puncture and oocyte retrieval
The ultrasound-guided follicular puncture and aspiration of the follicular fluid with oocytes is performed vaginally in short complete anaesthesia. Follicles are pictured using a vaginal ultrasound probe and a needle is inserted in direction of the puncture line to follicle centre in order to evacuate the follicular liquid with the egg.During the puncture, oocyte presence in the collected follicular fluid is checked continuously and in case the oocyte is not detected, the follicle may be repeatedly flushed with medium to draw the egg. In some cases the egg is not retrieved from the follicle despite repeated flushing. After a basic control under the microscope, each egg is inserted in a labelled special culture plate and placed into an incubator.
Egg insemination – fertilization
The sperm cells collected from the husband/partner are treated separately and they are either added to the eggs several hours after the ovarian puncture and left in mutual contact (IVF), eventually selected spermatozoa are injected directly into the oocyte cytoplasm (ICSI) employing a microscope for micromanipulation.IVF
The basic therapeutic method is called in vitro fertilization (IVF). The popular expression is “artificial fertilization” or “test-tube fertilization”, because the connection of the sperm with the egg happens in a laboratory, outside of the woman’s body.During IVF, sperms are added to the retrieved eggs in a special plate and special media, but they have to penetrate the individual eggs by themselves. In case of men with normal sperm, the so-called normospermics, approx. 50% of retrieved eggs get fertilized in this way, i.e. even with normospermics, a quality sperm does not fertilize each egg.
The quality of sperm that penetrates into an egg during IVF is unknown. It remains a matter of coincidence.
Unfortunately, an abnormal spermatozoon may also penetrate the egg and fertilize it. An embryo may develop even from such an abnormal sperm and the woman may get pregnant with such abnormal embryo. However, in the fifth, seventh or ninth week of the pregnancy the embryo’s development stops and the woman experiences a missed abortion.
Only a sperm of normal morphology, i.e. with a head, neck and tail, and of progressive motility, should contain all the information necessary to produce an embryo “programmed all the way to the bassinet”, when combined with the information from a healthy egg.
Table 1 presents the basic values of a normal spermiogram according to the World Health Organization (WHO).
Table 1. Normal spermiogram
| number of sperms | more than 20 million per ml |
| total sperm motility | more than 50 % moving |
| progressive movement (forward) | more than 25 % moving progressively |
| sperm morphology (shape of head, neck, tail) | more than 30 % shaped normally |
Table 2 summarizes normal and basic pathological findings. Pathological conditions are labelled male (andrological) factor of sterility.
Table 2. Spermiogram results and pregnancy potential
| perm condition | evaluation | fertilization method |
| normospermia | see Table 1 | spontaneous, natural, IVF |
| oligospermia | less than 20 mil. / ml | spontaneous, natural, IVF, ICSI |
| asthenospermia | limited motility | IVF, ICSI |
| teratospermia | more than 70 % pathological shape | ICSI |
| kryptospermia | less than 1 mil. / ml | ICSI |
| azoospermia | none capable of fertilization | MESA, TESE, ICSI, donor |
The cause of an ever growing number of men with insufficient sperm quality remains unknown. The sperm quality decrease is mostly represented by a disorder in production and development of spermatozoa in testes. Recent studies show that e.g. men with asthenoteratospermia have 80 % of sperms with defective DNA! Neither the exact cause nor effective treatment of this condition is known.
ICSI
If the infertility of a couple is influenced by the male factor, which is the case in approximately 60 % of couples, the IVF itself does not ensure success. Sperms either do not fertilize the eggs at all or eggs get penetrated by non-quality sperms.If case of the male infertility factor it is always more suitable to select a quality spermatozoon and inject it into the cytoplasm of the oocyte. This injection of a selected sperm to the oocyte cytoplasm is called ICSI - (intracytoplasmic sperm injection).
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When using ICSI both the oocyte and the spermatozoon are selected and connected. However, the combination of their genetic information cannot be influenced further. That remains a matter of coincidence. The ICSI principle itself, i.e. the selection of both the sperm and the egg, significantly increases the probability of a “favourable combination” and a perfect pregnancy, as opposed to IVF where even the quality of the connected cells is a matter of chance.
IMSI
Because medicine today cannot effectively cure non-quality sperm production, it searches for techniques of quality sperm selection. The most important criterion for sperm quality is their morphology, i.e. their appearance. Therefore, a microscope has been developed which can enlarge the sperm up to 6000 times. A normal sperm moves progressively forward and it would disappear fast from the field of vision of the microscope with such magnification. A special computer technique records a picture of the sperm and allows a more detailed examination of its morphology. This method is called IMSI (intracytoplasmic morphologically selected sperm injection). At present, there are not enough data available to prove that this more thorough selection of spermatozoa really leads to a higher number of children born.Extended culture
After 16 - 18 hours, an embryologist checks under a microscope if the insemination (oocyte fertilization) was successful, i.e. whether two so-called pro-nuclei formed in the egg. Until 1998, embryos were transferred following a two-day culture, in the stage of four cells per embryo, which is still considered a standard procedure. However, the stage of four cells can be reached by numerous potentially defective embryos and it is virtually impossible to distinguish a quality embryo at this early stage. Development of new cultivation media allowed for prolonged cultivation of the embryos in a laboratory (in vitro). Such extended culture allows for better assessment of the embryos. It is possible to set apart embryos whose development slows down or stops and select the embryo or embryos with adequate development for transfer. Their culture can be extended to up to 5 days following the oocyte collection via ovary puncture. The decision which day of in vitro culture is the most suitable for embryo transfer depends on the number and development stages of individual embryos which may vary in different couples.Number of embryos transferred
The transfer of two embryos is still considered optimal in our country. However, the partners must ready for the possibility of twin pregnancy. A transfer of one embryo brings about approximately 30% probability of conception. Transfer of two embryos increases such probability to 50 %, but it is necessary to take into account that more than one third of these cases lead to twin pregnancy. Three embryos are transferred only exceptionally in women older than 40 years. In Europe, a transfer of one embryo is becoming promoted more and more as multiple pregnancies are often accompanied with complications during pregnancy, premature labour, and immature newborns. To motivate the couples towards one embryo transfers, e.g. the health insurance system in Belgium covers six IVF cycles.Embryo transfer (ET)
Embryos are carefully introduced into the uterine cavity through a thin and flexible catheter. The procedure is painless and easy for the patient, similar to a regular gynaecological examination. The presence of the partner during the procedure is welcome. Following the transfer of the embryo(s), the patient remains in the horizontal position for a short time and may go home afterwards.The process of embryo implantation to the endometrium cannot be actively supported. One may only try not to disturb it by adverse acts such as smoking, rash body movements or excessive physical strain. However, it is not necessary to remain lying for more than 15 minutes following the embryo transfer.
The endometrium, into which the embryo is implanted, may be compared to a butter layer on a slice of bread on which poppy seeds are placed. The seeds will not fall out, even when the slice of bread is turned upside down! On the other hand, mental composure of the patient is very important.
Cryopreservation – Freezing of embryos
Upon ovary stimulation numerous oocytes may mature. Subsequent fertilization with sperm results in multiple embryos. As a matter of fact, not all embryos are of comparable quality and some do not develop further. However, the regularly developing embryos bring hope for a successful pregnancy. One or two of them are selected and transferred into the uterus. The remaining embryos with optimistic development may be saved for subsequent pregnancy by cryopreservation, i.e. frozen.Cryopreservation represents a sophisticated process, whereby the embryos are frozen to the very low temperature of minus 196 degrees of Celsius. At this temperature, all biological processes stop in the cells. The embryos may be preserved in such state for many years. After thawing, the biological activities re-start. Healthy children born from such frozen and thawed embryos prove that cryopreservation is an important process of reproductive medicine.
Unfortunately, a perfect “restart” of all biological development activities cannot be guaranteed in all cells; thus, the probability of becoming pregnant following a cryo embryo transfer is lower when compared to “fresh” embryo transfer. However, it would be inaccurate to directly compare CET and ET.
Cryo Embryo Transfer – CET
CET does not represent an infertility treatment cycle, even though it is sometimes erroneously considered as such. CET is not included among the cycles covered by the health insurance. CET cannot be compared with ET, i.e. transfer of fresh embryos from the regular IVF stimulation cycle!CET is merely a continuation of the previous IVF cycle. It represents another transfer of embryos from an already finished treatment. The transfer of frozen and subsequently thawed embryos gives the woman a chance to attempt pregnancy several times without repeating the risks connected with the initial ovary stimulation, such as ovarian hyperstimulation syndrome, bleeding from ovaries after puncture, and infection.
CET does not pose any risks. CET is less stressful and does not interfere with the woman’s hormonal system. No stimulation, anaesthesia or puncture is involved. Everything is ready; the embryos are stored in the Cryobank. It is also incomparably less demanding from the financial point of view.
The embryologists thaw the embryos one or two days prior to the transfer and cultivate them further. Correctly developing embryos are then chosen for the transfer. This allows answering the question: “Has the biological development potential of the embryo remained preserved?” Of course, this is regardless of the fact how the embryo is “programmed inside”. Similarly to the “fresh” embryo monitoring, the development after thawing does not ensure perfect development of the embryo until birth. The fact that not all thawed embryos will develop further needs to be taken into account and multiple embryos should be thawed when available. The in vitro culture will show the more capable ones. If the embryo does not develop after thawing, CET does not take place and the respective portion of the fee is reimbursed.
Therefore, the previous paragraphs should help answer the frequently asked question: “Shall we consider potential embryo freezing?” It is indeed advisable.
Medicaments used following the transfer
The endometrium is supported with the yellow body (corpus luteum) hormone (e.g. Progesterone, Utrogestan, Duphaston, Agolutin, Crinone), and this treatment should continue in all cases until the pregnancy test. When the test is positive, it is suitable to continue using the medicaments. Medicament usage after the transfer is explained in detail to each patient.Pregnancy test
Pregnancy is accompanied by the production of pregnancy hormone hCG, which is otherwise not present in the body. Pregnancy may be diagnosed from urine no earlier than 14 days after embryo transfer. Pregnancy tests promise positive reaction already at 25 or 50 units of hCG. Some tests are not as sensitive as stated by their manufacturers, or the amount of hCG in urine may be lower. The result thus may be false negative. If the pregnancy test is negative, it is advisable to perform a blood screen and determine the values of pregnancy hormone hCG in the serum.The pregnancy test must always be performed, regardless of vaginal bleeding!
The embryo may hatch outside of the uterus, e.g. in the fallopian tube, and such extrauterine pregnancy may threaten the woman’s life. In these cases it is necessary for the patient to follow closely the instructions and advice of the physician. If the value of hCG exceeds 1000 units and ultrasound does not show a pregnancy in the uterus, extrauterine pregnancy is highly probable, which frequently requires surgery – in most cases via laparoscopy.
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