In vitro fertilization

About the technology of artificial insemination, the history of IVF and the problems of embryo development

Roman Shafei, Post-science

In vitro fertilization is a medical technology for overcoming infertility, in which fertilization of an egg is performed in artificial conditions outside the mother's body. The term itself is translated from Latin as "fertilization outside the body": extra – ‘outside', corpus – ‘body'.

Initially, in vitro fertilization was developed to overcome infertility in women with the absence of fallopian tubes. As a rule, the tubes are missing after surgical removal of an ectopic pregnancy. Before the era of IVF, a woman who survived two ectopic pregnancies, deprived of both fallopian tubes, had no chance to give birth to a child. It is through the tubes that the sperm swim to the eggs for fertilization. Organizing a meeting of sperm and egg "in vitro" seemed to be the solution to the problem. Now IVF is used for a variety of forms of infertility.

History of IVF

The first pregnancy carried out by in vitro fertilization was achieved in the UK in November 1977. As a result of this procedure, in 1978, the first person in the history of the Earth conceived "in vitro" was born – a girl, Louise Brown. Now Louise Brown is an adult woman with two children conceived naturally. The doctors who successfully performed IVF for the first time are recognized as the founders of artificial insemination technology – gynecologist Patrick Steptoe and embryologist Robert Edwards. The technology of in vitro (or artificial) fertilization always involves two specialists: a gynecologist and an embryologist. A gynecologist works with a woman, and an embryologist works with germ cells and embryos. Robert Edwards received the Nobel Prize in Physiology or Medicine in 2010 for the development of in vitro fertilization technology.

In the USSR, the birth of the first child after IVF occurred in 1986. The IVF procedure was performed in Moscow at the Center for Maternal and Child Health (now the Scientific Center of Obstetrics, Gynecology and Perinatology of the Ministry of Health of the Russian Federation) by gynecologist Elena A. Kalinina and embryologist Valentin Alekseevich Lukin. The first Soviet child "from a test tube" Elena Dontsova has a 30–year anniversary this year. Now she lives in Ukraine, is the mother of a 9-year-old son conceived naturally. Over the past 30 years, ECO technology has gone far from its origins, and yet it is still imperfect. According to the European Society of Human Embryology and Reproduction, only 33% of patients become pregnant after transferring an embryo obtained "in vitro" into the uterus. However, in nature, the probability of pregnancy is similar – more than 60% of human embryos die during the first 7 days of development. In such cases, there is no delay in menstruation, and the woman does not realize that there was an embryo in her uterus that died. In this sense, the IVF procedure is not at all inferior to natural conception.

Preparation of a woman

In vitro fertilization is preceded by hormonal preparation of the patient. In natural conditions, only one egg matures during a woman's menstrual cycle, but this is not enough for an effective IVF program. Therefore, with the help of hormonal drugs, the doctor stimulates the development of several eggs. The number of 6-8 eggs is considered optimal for IVF. It is impossible to see developing eggs in a woman's body. With the help of ultrasound, the doctor sees growing follicles – spherical formations in the ovaries, inside which eggs are contained. Mature follicles reach a diameter of about 2 cm, while the egg has a diameter of about 0.1 mm.

Selection of germ cells

On a certain day, eggs are taken – this procedure is usually carried out under general anesthesia. Using a syringe with a thin long needle, a gynecologist selects follicular fluid and gives it to an embryologist specialist to search for eggs. You can only see the eggs in the follicular fluid under a microscope. The found eggs are washed from the follicular fluid and placed in cups with a culture medium. The culture medium is a solution of many necessary physiological components: salts, sugars, amino acids, vitamins, proteins – about 40 substances in total. In such an environment, eggs, spermatozoa and embryos feel "at home". The cup with the medium in which the eggs are placed (and then the embryos) is kept in an incubator – a special cabinet, inside which the temperature is maintained at 37 ° C and the carbon dioxide level is 6%.

For artificial insemination, sperm cannot be used "as it is" (experts call it native sperm): seminal fluid will spoil the environment and destroy the eggs. It is necessary to isolate mobile spermatozoa from the sperm (in the sperm of any healthy man, most of the spermatozoa are immobile) and transfer them to a culture medium, having cleaned them of seminal fluid. For this, embryologists use multiple centrifugation with gradual replacement of media. The resulting suspension of sperm in the medium can be used for artificial insemination.

IVF methods

Actually in vitro fertilization is carried out by one of two methods. The first method is called "in vitro insemination" and consists in the following: a suspension of spermatozoa is added to a cup with eggs. Sperm swim independently and fertilize eggs. But it is not possible to see the penetration process under a microscope: hundreds or even thousands of spermatozoa cling to the egg, they loosen the egg shells, producing a large amount of "garbage". The process takes about two hours – it is impossible to observe all this time in a microscope: the eggs must be in an incubator.

If the sperm is very bad and the spermatozoa are unable to swim and fertilize the eggs on their own, then a second method is used, which is called ICSI (from the English ICSI – intracytoplasmic sperm injection – the introduction of a sperm into the cytoplasm). With this method, the most "beautiful" sperm is selected under a microscope, caught with a glass needle and injected into the egg – thus fertilized. The ICSI method gives more stable results, so it gradually replaces the in vitro insemination method from IVF practice.

IVF method, sperm injection into the egg (wikipedia.org )

The results of fertilization are judged the next day – only then it will be possible to say whether fertilization was successful. A fertilized egg is called a zygote – this is a single-celled stage of human development. Two nuclei should form in the zygote – male and female (the so-called pronuclei). The male nucleus contains the chromosomes of the father, the female contains the chromosomes of the mother. The nuclei in the zygote appear 12-15 hours after the penetration of the sperm into the egg and remain visible for about 8 hours. After the disappearance of the pronuclei, the chromosomes of the father and mother are combined into a single "metaphase plate", and from that moment on, in all the nuclei of the future cells of the embryo (and then of the human), the chromosomes of the father and mother will be together. In this short period (about 8 hours, most of which falls at night), when the nuclei of the zygote are visible, it is necessary to examine the embryos in order to fix that fertilization was successful. If there are no pronuclei in the zygote or three pronuclei are visible, then such an embryo cannot be transplanted to the mother.

Embryo development

Over the next five days, the embryos continue to be cultured in an incubator, monitoring their development daily. Development is very fast, every day brings something new. On the second day after fertilization, the embryo consists of four cells, its size is still about 0.1 mm (the same as the size of the egg). On the third day, the number of cells increases to eight, but the embryo does not grow – just the cells become smaller. On the fourth day, the embryo already consists of 10-20 cells, but even now its size is the same – about 0.1 mm, and the cells have become even smaller. Due to the constant division of the embryo into all small cells, this stage of development is called "embryo fragmentation". The embryo has no time to grow, it does not waste time on this. His task now is to create a cell mass, from which morphogenesis – the formation of a body shape – will begin in the following days. While these 10-20 cells are a shapeless mass, they are all the same in size, appearance, biochemical characteristics and other parameters, but tomorrow, on the fifth day, the process of "modeling" the body of the future person will begin from this homogeneous mass of cells. On the fifth day, the number of cells increases to 50-80, the embryo forms a kind of ball from them – the so-called blastocyst. This ball is already slightly larger in size – 0.15 mm, and it has inside the so–called embryoblast - a special group of cells from which the body of the future newborn will be formed. As a rule, at the blastocyst stage, the embryo is transferred to the uterus of a woman in the hope that it will be able to attach to the uterine wall (experts call this process implantation). If we leave the embryo to be cultivated further, then on the sixth day we will see how the blastocyst hatches from its protein shell (this shell got to the blastocyst from the egg). For about another day, the blastocyst can remain without a shiny shell in the cup in an incubator, but without finding the uterine wall, it dies.

As a rule, two embryos are transferred to the uterus of a woman. These are the best embryos selected according to different criteria. But even the transfer of two embryos only leads to pregnancy in a third of cases. Why does pregnancy not occur when transferring embryos into the uterus? As mentioned above, a similar pregnancy rate is also shown for natural conditions of conception. About 60% of all pregnancies recorded using a blood test for chorionic gonadotropin (this hormone releases an embryo into the mother's blood) stop before menstruation is delayed. Such a pregnancy can be recorded only by a blood test for chorionic gonadotropin. Without conducting such an analysis, a woman cannot guess about an aborted pregnancy. These data mean that in natural conditions about 60% of embryos that have reached the blastocyst stage cannot perform implantation and die. Why is it so arranged in nature? There is no exact answer. I see the following picture: it is known that many errors occur during the formation of germ cells, most of the human germ cells are unsuitable for normal embryo formation. Spermatozoa during the passage of the female genital tract undergo a very strict selection. Out of several tens of millions of sperm entering the female vagina during sexual intercourse, only a few hundred swim to the place of fertilization – the fallopian tube. This is one in a hundred thousand. In the female body, there are special barriers that spermatozoa need to overcome – first of all, "istmus" and "cervix". Eggs are selected after fertilization – only a third of fertilized eggs are able to develop into a blastocyst that can be implanted into the uterus, two-thirds of eggs cannot form a normal embryo and stop developing.

IVF problems

Unfortunately, it is not always possible to determine its "potential for development" by the appearance of the embryo, which is largely determined by the genetic normality of the embryo. Currently, there are methods of molecular biology that allow us to determine the "healthy genetics" of the embryo. This is the so-called preimplantation genetic diagnosis, or PGD. To carry out this procedure, one of its cells is taken from the embryo (as shown, taking part of the material does not harm the embryo) and DNA analysis is performed. Based on the results of such an analysis, it is possible to determine whether the number of chromosomes in the cells of the embryo is normal, to identify certain mutations. Unfortunately, due to the technical complexity and high cost, this procedure is not carried out in all medical centers and not every married couple.

If more than two embryos of good quality were obtained in the IVF program, then some of them that were not selected for transfer to the uterus can be frozen for storage. In the future, they can be thawed and transferred to the uterine cavity to achieve pregnancy. This happens if the pregnancy has not occurred or if the patient has already given birth to a child and wants to give birth to a second, third, fourth. Embryo freezing is a well-established procedure today. The survival rate of embryos after thawing is about 96%. Frozen storage is carried out in liquid nitrogen at a temperature of -196 ° C. Embryos are sealed in miniature plastic tubes and immersed in liquid nitrogen poured into giant thermos flasks – the so-called Dewar vessels. Theoretically, in this state, embryos can remain viable for many hundreds, and maybe even thousands of years. But the achievement of pregnancy after the transfer of a thawed embryo into the uterus, which had been lying in liquid nitrogen for about 20 years (USA), has been reliably shown. An embryo cannot be immersed in liquid nitrogen just like that – it is detrimental to any living cell. It is necessary to saturate the embryo cells with so–called cryoprotectors - substances that prevent the formation of ice crystals. Currently, ethylene glycol, glycerin and dimethyl sulfoxide are usually used as cryoprotectors.

According to the Russian Association of Human Reproduction (RARCH), in Russia in 2013, about 65 thousand IVF procedures were performed, the results of which were achieved slightly less than 20 thousand pregnancies, of which 13.5 thousand ended in childbirth. There are more than 200 medical centers operating on the territory of Russia that carry out the IVF procedure. Detailed statistics can be found on the RARCH website.

About the author:
Roman Shafei is a Candidate of Biological Sciences, a leading researcher at the Department of Embryology of the Faculty of Biology of Moscow State University. 

Portal "Eternal youth" http://vechnayamolodost.ru  28.03.2016