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The IVF program - In Vitro Fertilization
 IVF is named as "program", as it is not reduced to unitary intervention, and provides preparation the woman and man, the operation itself, care of development of an ootid, introduction of an ootid in the uterus, control of pregnancy development. The in vitro fertilization since 80 years is the most effective way of treatment infertility, especially in cases, when the woman has obstruction of uterine tubes or at the man in a sperm the low contents of spermatozoon or down stroke of their mobility is observed. Also in cases, when the idiopathic reason infertility, is possible to use an artificial fertilization, if the other forms of medical treatment have not brought desirable results.
The indications for IVF
 1) Pathology of uterine tubes
 2) Endocrine infringements
 3) Endometriosis
 4) Set of symptoms of a polycystosis of ovaries
 5) Infertility of an obscure genesis
 6) Low contents in an ejaculate of active - mobile spermatozoon (asthenospermia)
Essence of in vitro fertilization (IVF)
 The essence of procedure of in vitro fertilization (IVF) consists of reception from the ovaries of woman of mature ootids, fertilization by husband spermatozoon (or at the request of both spouses by semen of the donor), cultivation turned out embryos in an incubator within 48-72 hours, and transfer embryo in a uterus of patient.
 The medical cycle of IVF lasts 15-30 days (depending on a kind of the protocol) and consists of 4 consecutive stages:
 1. Stimulation of ovulation – the use of medicinal preparations for stimulation of height both maturing follicles and ootids, contained in them, in ovaries the woman; the stimulation of an ovulation includes:
Laboratory researches - numerous definition of concentration of hormone oestradiolum in the blood of woman which enable to control optimally the process of maturing follicles;
Ultrasonic monitoring - ultrasonic research for controlling development follicles in ovaries and condition of endometrium (mucosa of a uterus)
 2. Puncture follicles (fence of the ripened ootids from follicles) insertion of a special needle in each follicle of an ovary of the woman with a suction follicle’s liquid containing an ootid. The procedure is carried out in hospital, under the ultrasonic control by a special needle through the wall of a vagina. In avoidance of discomfort, the puncture is carried out under an intravenous narcosis;
 3. Cultivation of embryo - in laboratory with the help of a microscope are searched the ootids which are fertilized by spermatozoon of a semen of the spouse in follicular liquid e which has been handed on the same day. The observation of development embrocated embryos is made in laboratory within 2-3 day;
 4. The transfer of embryos – premise of 1-3 embryo in a cavity of the uterus with the help of a special catheter (thin plastic straw), which is entered through the cervix of a uterus in a cavity.
IN VITRO FERTILIZATION

Unused qualitative embryos could be frozen (cryoconserwation) for the further transplantations of embryo according to the contract signed with a married couple.
Information about IVF efficiency.
 The success of IVF depends on set of different factors: patient age, condition of a uterus, general condition of an organism, reason of infertility. But it is inconvenient to estimate the chances precisely. Besides the success of procedure IVF depends on skill of the doctors, equipment of clinic, selection of medicines for therapy.
At last, there are more - less objective factors influencing efficiency IVF.
They are the following ones:
The age of the woman: with age the quantity and quality of ootids is reduced. It is considered, that at 43-45 years the chances to become pregnant during one course IVF are 3 times less, than at the age of 35 years.
The reason of handicapping of an ootid implantation is defects of development and defects of uterus.
Thus, taking into account all set of the factors "for" and "against", the efficiency of the program IVF usually does not exceed 35 - 40%.
Treatment man's infertility (ICSI)
 One of the most essential improvements in treatment of man’s infertility was the reception of fertilization pathological samples of a sperm by using ICSI.
The word ICSI occurs from reduction of the first letters of English word collocation Intra Cytoplasm Sperm Injection that is translated literally as «the Introduction of a spermatozoon in cytoplasm of oocyte is a version IVF. The given procedure is carried out, as a rule, at the serious forms of man's infertility, when the fertilizing ability of a sperm is essentially reduced. Thus the procedure will be carried out, when one or several spermatozoa are placed inside of a mature ootid for the subsequent fertilization.
ICSI allows bypassing difficulties connected to the features of an environment of an ootid. At times fertilization does not occur, in spite of the fact that both the spermatozoon and ootid are normal. Because of any features of a structure of an outside membrane of an ootid the spermatozoon can not penetrate through it. The microinjection inside allows to bypass all the barriers, and after the injection of alive spermatozoon the frequency of a fertilization reaches 70-80 %.
ICSI is recommended, if at presence of a normal sperm the fertilization was not received even at one procedure of IVF, and is also recommended to the married couples, where at the man are impassable deferent canals and there are no chances of fertilization with the help of usual IVF. If in an ejaculate there are no spermatozoa in general, then for ICSI the spermatozoa are taken immediately from testicles (method refers to as TESA) or from their appendages (the method refers to as MESA). This is made simultaneously with a puncture of follicles at the woman.
In therapy of female and man's infertility the indication to realize the method of ICSI are the following conditions:
Azoospermia - complete absence of a semen as a consequence of serious disease of testicles, and also blockade or absence of deferent ducts
Oligozoospermia - down stroke of concentration of spermatozoon less than 2 million/ml;
Asthenozoospermia - less than 1 million of active - mobile spermatozoon in 1 ml of an ejaculate;
Teratozoospermia - less than 5 % of spermatozoon of a normal structure;
The varicocele is accompanied by formation of mass of the extended sinuous veins of a scrotum, similarly to a varicose phlebectasia of the bottom extremities;
The combined pathology of semen (is probable various combinations of changes in concentration, mobility and structure of spermatozoon, that considerably reduces fertilizing ability of a semen);
Presence of antispermal antibodies in an ejaculate (MAR-test more than 50 %), which interfere natural fertilization, even in case of normal concentration of spermatozoa, absence or unsatisfactory fertilization of ootids in the previous attempt of IVF.
The qualitative performance of ICSI procedure in our laboratory is promoted by the help of modern equipment. The procedure is carried out by the microscope of firm "Leica" (Germany) through micromanipulators of firm Narishige (Japan).
Due to presence of video equipment, we have an opportunity of video and computer record of all manipulations.

Microscope with micromanipulator
Engineering of realization ICSI
 The ICSI procedure is carried out on the same day of reception of ootids from the woman, or on the next day, if independent fertilization has not taken place. The micro fertilization for the woman represents the same procedure, as well as at IVF fertilization.
Before realization ICSI the cells of a radiate crownare which surround an ootid are leaved. A micromanipulation is carried out only on mature ootids at presence first polar body.
The reception of spermatozoon for ICSI can be carried out from an ejaculate or surgical methods (Testicular biopsy).
With the help of micro instruments under a microscope the spermatozoon of high quality gets out. He is located in a micro needle, with the help of which the puncture of a brilliant environment of an ootid is carried out, and then the spermatozoon is entered in cytoplasm of an ootid. A fertilization of all ootids, received at a puncture thus will be carried out.

Introduction of a spermatozoon inside of an ootid.
Outcome of ICSI
 The parameters of frequency of fertilization at ICSI can change from 20 up to 60 %. There is no guarantee, that any of ootids after realization of procedure of fertilizing. The disability of an ootid to a fertilization and division can be connected to difficulties of the procedure (damage of an ootid), and also with the quality of sex cells. So, for example, at a choice of spermatozoon for ICSI externally they can be of high quality (mobility and structure), but can contain chromosome anomaly. The quality of ootids also influences outcomes of realization ICSI and IVF program.
As a whole, the frequency of offensive of pregnancy after realization ICSI does not differ from those on the average at the usual program IVF and makes 30-35 %.
It is necessary to note genetic risk of the program ICSI at serious variants of man's infertility. Some genetic infringements at the men result in infringement of a spermatogenesis (process of formation of spermatozoon) and to infertility. The treatment by a method IVF with realization ICSI is suggested to such married couples. At realization of a micromanipulation the spermatozoon with genetic infringements can fertilize of an ootid. If there is a pregnancy by a fetus of a male, there is a high risk of transfer of such disease, and, hence, and infertility by right of succession to son. For bringing such risk to a minimum before the beginning of treatment the complex inspection of a married couple should be carried out including genetic consultation, especially of the man,.
Auxiliary Hatching
Processing of an environment of embryo (auxiliary hatching - zone drilling/assisted hatching)
 The processing of an environment of embryo is a new method, with the help of which it is possible to facilitate an implantation of embryonic a uterus.
This method consists of weakening an environment embryo that promotes stronger fastening her in a wall of a uterus.
The human ootid is surrounded with an extra cellular environment named transparent zone or zone pellucid. The functions of a brilliant environment are the following:
Fertilization (on her there are receptors of a recognition and linkage with spermatozoon)
Protective function (preservation of an ootid or embryo during passage on uterine tubes)
For the attachment of embryo under a mucosa of a uterus, it should be released from this environment. Density of a brilliant environment of an ootid can be changed after the influence of different factors; therefore arisen embryo could not independently be liberated from it, and accordingly can not take root into a cavity of the uterus.
The reasons of density change of a brilliant environment are the following ones: The age factor (possible endocrine changes at the women is older than 38 years);
Cultivation outside of an organism, that is in laboratory conditions (disadvantage of medium cultivation of some substances);
  
The zone pellucida changes associated by age. On the picture to the left - the view of normal ootid under electronic microscope,
to the right - ootid with age changes.
Cryoconservation (freezing embryos)
Therefore at presence of certain indications, by the embryologist are done(made) special notches on an environment of embryo with the purpose of auxiliary to liberate it - so-called auxiliary hatching. Auxiliary hatching can be carried out by three ways:
By chemical influence on a transparent zone by a solution of an acid or special enzyme, that dissolvent it.
By mechanical section of transparent zone with help of micro instruments.
Creation of micro apertures in a transparent zone with the help of laser.
In our laboratory is carried out auxiliary hatching by a mechanical way with the help of micro instruments.
The indications of Auxiliary Hatching realization
1. The woman is older than 38 years;
2. Previous unsuccessful attempts of an implantation;
3. Presence of embryos with bad morphological parameters;
4. Raised level of folliculostimulation hormone (FH) in the blood;
5. Transfer of defrozen embryos;
6. Smoking.
Before the beginning of IVF program the treating doctor discusses with a married couple an opportunity of realization of this manipulation. Thus the appropriate decision is made by the spouses in the special column of the agreement on treatment by a method IVF.
Cryoconservation of embryos

What is this?

Embryos deep freezing in a liquid nitrogen.

When will be carried out cryoconservation of embryos?
 Cryoconservation of embryos is carried out on “to a residual principle". In that case, when the realization of IVF program and transfer of embryos in a cavity of the uterus, at a married couple remain "superfluous" embryos of high quality, it is offered to couple to freeze it. The storage of frozen embryos is carried out in special marked plastic straws, placed in liquid nitrogen. Term of storage embryos is not circumscribed. In the literature there are data on offensive of pregnancy and labors after carry defrozen embryos stored 10 years.
  

How the program of transfer defrozen embryos is carried out?
 In case of absence of pregnancy after the IVF program, the transfer of defrozen embryos can be carried out already in the following cycle, which is with offensive of a menses. Thus the procedure of a stimulation of multiple heights of follicles is carried out, and the medicinal preparations for preparation of a mucosa of a uterus for transfer of embryo are nominated. These preparations contain natural female hormones, which in natural conditions cause changes mucosa of a uterus in the second phase of a menstrual cycle and prepare it for a possible implantation of embryo in case of a fertilization. Approximately in the middle of a menstrual cycle the mucosa of a uterus reaches the optimum characteristics (thickness and frame). The defreezing of embryos and transfer them in a cavity of the uterus will be carried out. Because during the cryoconservation of embryos their environment is condensed, before transfer of embryos should be carried out auxiliary hatching – notching of an environment embryo.
Conditions for embryos conservation realization
 Cryoconservation will be carried out only by embryos of high quality and at the certain stage of development. Embryos of bad quality are not subject to freezing, otherwise they will be destructed. The stage of embryo development also plays a role in the forecast cryoconservation. The embryos at a stage of zygotes two four, eight cells and blastocyste are subject to freezing.
The presence of filled agreement for cryoconservation of embryos.
Possible outcomes during the realization of the cryoconservation program and defreezing of embryos.
It is necessary to remember, that embryos after cryoconservation and the defreezings can be unsuitable for transfer owing to their degradation. In this case, the transfer of such embryos will not be carried out.
By preparation of a mucosa of a uterus will be carried out the hormonal analysis which is a marker of a condition of a mucosa. At deviations of hormonal parameters from norm, the transfer of embryos will not be carried out, as the mucosa of a uterus will be unprepared. In this case, the realization of the repeated program to preparation of endometrium at once in the following cycle is possible.
We do cryoconservation of embryos only when it is necessary for the patients, and they have agreed to storage of the germs. But in a result to have such "insurance" in case of failure is very favorable. Approximately 60 % of spouses have such opportunity.
First, kept embryos allow the spouses essentially to spare agents in the second attempt (medical cycle), spent on a medicine -stimulator.
Secondly, the woman is relieved of insertion of these preparations - daily "injections", and also the risk of hyper stimulation and unpleasant sensations at reception of the cells.
The frequency of offensive of pregnancy after the transfer of defrozen embryo makes 10-15 %.
How long can be stored cryo embryos?
 Frozen embryos are stored in liquid nitrogen at temperature -196? in special vessels. At an adequate regimen of vessel content term of storage of embryos can be estimated by years.
Whether use frozen embryos influences in the future at child?
 The survival rate of embryos at thawing after cryoconservation makes from 50 % up to 85 %, and developing of defrozen embryos by nothing differ from what are received in a standard cycle of IVF: they also successfully "get accustomed" and give normal pregnancies. Thus the risk of occurrence of any complications and pathologies does not exceed those at realization of a standard cycle of IVF.
What will be with cryo embryos, if the spouses become pregnant from the first attempt?
 The spouses carefully concern to such frozen children, they leave such embryos for planning birth of the following child. But all the same everyone of a steam independently defines terms of storage of embryos.
Cryoconservation of a sperm
 The conditions of our clinic allow making freezing of spermatozoa for their subsequent use in the programs of treatment of infertility with the help of methods of auxiliary reproductive technology. Freezing of a sperm is carried out in liquid nitrogen, with the help of the special equipment. The semen can be stored in the frozen condition for several years. As optimum term of storage of a sperm it is necessary to consider 3 years, as on expiration of this period the fertilizing ability of spermatozoon is worsened.
The basic indications for cryoconservation of a sperm
 1. Trades connected to high risk for reproductive health: work in conditions of contact with organic and inorganic substances, high and low temperatures ionizing radiation, extreme kinds of works (testers, military men during military actions).
 2. This procedure can be used by those of the patients, who plan treatment of any diseases by medicines, which action can have an effect on quality of a sperm - chemic- and radial therapy, hormonal treatment, bilateral vasectomy etc.
 3. Also is made cryoconservation of a sperm of the donors. It is not only allowed to frame bank of a sperm, but also to carry out diagnostic check behind the donors. A donor sperm with 2 negative results on infections transmitted by a sexual way, executed with an interval 3 months could be used only during the 6 months after its delivery, after reception at the donor.
 4. Conservation of a sperm as a biological material for diagnostic and medical procedures: at performance of a biopsy of a testicle, fence of a sperm from an epididymis. Also of this method could be used, for the spouse, for example who of specificity of his work, can not in time get in clinic for delivery of a semen necessary for realization of treatment by a method of IVF or intra uterus insemination.
For preservation is used the a sperm received after 3-4 days of sexual continence checked up on quality presence antispermal of antibodies and sterility.
In a case of cryoconservation of a sperm the agreement on realization of the given method is filled.
Possible outcomes at cryoconservation and defreezing of a sperm
 Mobility and fertilizing ability of spermatozoa after cryoconservation decrease on the average on 30-70 of %. Such wide variability of spermatozoa survival rate testifies about various cryoresistance (fastness to cryoconservation). And cryoresistance in turn, mainly, is defined by the function of plasmatic membranes: By their plasticity, condition of enzyme systems, ability to maintain deformative strains especially arising at such phase transitions as ice - water.

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