What is infertility and how to deal with it
ECO: the long story of a short meeting
Dmitry Nikiforov, Elena Ryabchikova, Tatiana Ovsyannikova, Zhanna Popova
"First-hand Science" No. 2 (62), 2015
Published on the website "Elements"
Miracles do not happen, as our Professor Preobrazhensky proved.
Mikhail Bulgakov
Our life is a priceless gift. As well as the very ability to give life to a new person, to continue their kind. However, now there are about fifty million married couples living in the world, to whom nature has denied the opportunity to become parents. New reproductive technologies based on the latest achievements in genetics, molecular biology, immunology and other related sciences can "challenge" this verdict and enable thousands of people to experience the joy of fatherhood and motherhood.
The World Health Organization defines infertility as the inability of a couple of reproductive age to conceive and bear a fetus for a year or more of regular unprotected sexual relations. An absolutely reliable assessment of the level of infertility in individual countries and regions cannot be obtained for obvious reasons, but according to WHO, this figure is about 5% of the reproductive human population. Thus, today at least 3 million people should live in Russia alone, infertile according to objective medical indicators, i.e. due to anatomical, genetic, endocrine, immunological and other factors. These figures are confirmed by monitoring data conducted in 2011 by the joint efforts of Rosstat, the Ministry of Health of the Russian Federation, the UN Population Fund and a number of other organizations, according to which almost 5% of respondents who have ever been married reported that they or their partners were diagnosed with infertility (Sakevich, 2013).
There has not been a significant increase in the level of infertility in the world and in our country, but for each individual married couple it remains a tragedy. It is not surprising that the number of appeals to medical institutions on this occasion is increasing, which is due, on the one hand, to an increase in the level of education and well–being, and, on the other, to the rapid development of so-called assisted reproductive technologies (ART) designed to help infertile couples cope with the problem. However, the origin of life is a much more intimate and fundamental process than simply following instructions in a routine sequence of techniques, which makes artificial insemination a difficult task.
The simple mystery of life
Normally, each woman matures one, maximum two eggs in one month. Each egg is surrounded by a shell and a layer of cumulus cells feeding it, in the spaces between which there is an intercellular substance consisting mainly of hyaluronic acid. On the surface of each normal sperm there is a special cellular structure – the acrosome, which contains the enzyme hyaluronidase, which destroys hyaluronic acid.
As you know, only one sperm is needed to fertilize an egg, but in order for fertilization to become possible, hundreds of male germ cells must necessarily participate in the process. When the spermatozoa reach the egg, they work together to first dissolve the intercellular substance of the egg shell. A lot of spermatozoa empty their acrosomes containing hyaluronidase, diligently trying to drill through the protective layer of the egg with the help of translational movements of the tail.
As a result, there remains a very thin shell, which is broken through by one of the spermatozoa that happens to be at the right time and in the right place. This is the most successful sperm and fertilizes the egg. Simultaneously with the penetration of the sperm, a shell is formed in the egg, which prevents the penetration of other sperm. That's it, the fertilization process is complete. Then the development of the embryo begins, which in about seven days should attach to the surface of the uterine cavity of the mother.
Editing Nature
Male and female infertility is observed in approximately equal numbers of cases. In women, its causes are most often a violation of the ovulation process – the formation of mature eggs, obstruction of the fallopian tubes, as well as a disease such as endometriosis. The most common cause of male infertility is a violation of spermatogenesis: a decrease in concentration, activity, and a decrease in the quality of sperm. An illustrative fact: according to experts of the ART of Moscow, already now the vast majority of men in the Russian capital have deviations in sperm counts, and this trend is true for any large city. Moreover, a decrease in sperm quality relative to the minimum criteria established by WHO has been recorded in many regions of Russia, including those remote from megacities (Osadchuk et al., 2012).
This is how human spermatozoa look before selection for artificial insemination: a – normal; b – with deviations (the phenomenon of agglutination – the heads stick together)
In recent years, the number of young patients seeking help from ART specialists has also increased markedly. Unfortunately, in the future, the problem of infertile marriage will only get younger, which is primarily due to the lifestyle of the younger generation. The desire to receive all the benefits of modern life inevitably affects the health of young boys and girls, which subsequently affects their reproductive system. Improper diet, alcohol and smoking, sedentary lifestyle, urbanization and the associated deterioration of the environmental situation – all these negative factors cannot but affect the health, including the reproductive system of potential moms and dads.
What methods are there in the medical arsenal to overcome infertility? ART specialists today are able to obtain sperm and eggs from an infertile couple, achieve fertilization, cultivate embryos, choose the best of them and transfer them for implantation into the uterine cavity of the expectant mother. At the same time, both germ cells and embryos can be frozen, and in this state they can be stored for years before subsequent defrosting. Moreover, after fertilization of the egg, it is possible to do a biopsy of the embryo's cellular material and conduct a so-called preimplantation genetic diagnosis (PGD), which will give information about the presence or absence of certain gene abnormalities.
The simplest ART method in terms of implementation is "intrauterine insemination", which does not require the extraction of eggs from the body of the expectant mother. All other methods involve fertilization of eggs outside the woman's body.
Such in vitro fertilization (IVF) can be carried out in two fundamentally different ways: by mixing isolated eggs with a suspension of sperm ("classical IVF", IVF, In Vitro Fertilization) or by injecting a single sperm into the cytoplasm of the egg (ICSI, ICSI, Intra Cellular Sperm Injection).
The path to victory
With intrauterine insemination, the sperm of the husband or donor is injected into the woman's uterine cavity. Normally, with natural conception, spermatozoa, before reaching the egg, overcome a long way: first through the aggressive (with high acidity) environment of the vagina, then the cervical canal filled with mucus (cervical canal), along the surface of the uterine cavity and, finally, through the uterine (fallopian) tubes – the organ through which the egg transported from the ovary. On the way to the cherished goal, many of the many sperm cells fail to cope with the task, and only the most selected male cells in the literal sense of the word reach the fallopian tubes. And here the question arises: how to artificially carry out the same selection of spermatozoa so that only the best participate in fertilization?
For this purpose, a system of sperm purification from seminal plasma (the non-cellular part of sperm formed from prostate secretions) and a sperm selection system based on the principle of centrifugation in a density gradient were developed. The most mature and suitable for fertilization spermatozoa have a heavy, compact and dense cell nucleus. Therefore, the heaviest and, therefore, the best of them will be as a result of centrifugation at the bottom.
Such spermatozoa are selected, re-purified, and as a result, a suspension of the highest quality spermatozoa available to the patient is obtained, which can be used in any of the ART methods, including for intrauterine insemination. If this suspension is collected into a catheter and brought directly into the uterine cavity, then it is possible to overcome such infertility factors as the cervical canal impassable for sperm, incompatibility of sperm with cervical mucus. In addition, special cleaning has an activating effect on spermatozoa and with mild factors of male infertility can also solve the problem.
The essence of the sacrament
As mentioned above, there are only two different methods of fertilization outside the body of the expectant mother, who is not capable of conceiving naturally. If the patient does not have fallopian tubes, and the migration of an egg from the ovary to the uterus is impossible, then the only way to realize the reproductive function is classical IVF. In classical IVF, eggs, along with their natural environment – feeding cells, are mixed with a suspension of treated sperm. At the same time, the process of sperm penetration into the egg itself occurs in a sense in a natural way.
If a married couple has problems with the quality of sperm or eggs, then this serves as an indication for ICSI. In this case, the eggs are pre-purified from the surrounding feeding cells with the help of the hyaluronidase enzyme, which makes it possible to select mature and high-quality oocytes, which is impossible during fertilization by classical IVF. The ICSI procedure itself is carried out in an extremely short time, since the presence of eggs outside the incubator adversely affects their ability to fertilize and further proper development of embryos.
With the ICSI method, the egg is fertilized by injecting a single sperm into it. With the help of a micromanipulator, the capillary pierces the egg shell, and the sperm is injected into the cytoplasm. The process is visualized using a digital camera that transmits an image to a computer screen.
But where do the eggs necessary for any IVF technique come from? They are obtained by puncture (puncture) of the ovarian follicles of the patient – this operation is most often performed under general anesthesia and ultrasound control. To increase the effectiveness of in vitro fertilization, they cause the growth of several follicles at once, which allows you to get several eggs at once, and each additional embryo increases the chances of pregnancy.
To stimulate the growth of follicles, hormonal preparations of the human pituitary gland are used, which are necessary for the normal maturation of follicles and eggs – follicle-stimulating (FSH) and luteinizing (LH), as well as human chorionic gonadotropin (hCG), which is secreted by the placenta after successful implantation of an egg into the uterus and plays an important role in the development of pregnancy.
Today, thanks to the achievements of modern genetic engineering, a culture of Chinese hamster ovarian cells is used to obtain these hormones. Thanks to the use of such recombinant technologies, the target product is produced in large quantities and is much easier to clean than a natural hormone. The availability of hormones has significantly increased the effectiveness of IVF cycles.
After stimulating the growth of follicles and surgically obtaining eggs, one of the most important moments of IVF comes – fertilization. After a certain period of time, the embryologist scans the cells with a microscope and determines whether fertilization has occurred. Note that the very expression "test tube baby" appeared precisely because after fertilization of the eggs, the resulting embryos are in a culture dish for some time. Although, to be precise, IVF specialists do not use test tubes, but laboratory glass cups (Petri dishes). So, strictly speaking, children appear "out of cups".
The embryos obtained by one method or another are then cultured for a maximum of 5-6 days in incubators at a temperature of 36.9–37.0 ° C in special media (Lane, Gardner, 2007).
Human embryos on the 1st day of development (1): 1a – with two pronuclei, evidence of proper fertilization; 1b – an abnormal embryo with three pronuclei, not suitable for transfer to the uterus. The yellow arrow points to the pronucleus.
After artificial insemination, the egg begins to split, and the embryo goes through the first stages of its development "in vitro".: Day 2 is the beginning of egg crushing (2), day 3 is the formation of cell mass (3), day 4 is the growth of cell mass (4). Embryos with abnormal development are rejected after microscopic examination. Embryo transfer into the uterine cavity is usually carried out on the 5th day of its "life" after genetic diagnosis
How to diagnose an embryo?
Embryo transfer into the uterine cavity is usually carried out on the fifth day of its "life", after genetic diagnosis. The need for this procedure is due to the fact that about 10-15% of pregnancies obtained with IVF do not end in childbirth due to fading fetal development or miscarriages due to genetic abnormalities. It should be added that this figure is not so great: it is believed that in Russia every fifth ordinary pregnancy does not end with childbirth, while stopping the development of the embryo often occurs at a period of no more than two weeks, so a woman may generally remain unaware that she was pregnant. But since, unlike the natural method of fertilization, the IVF procedure is time-consuming and costly, including emotionally, they try to exclude failure in advance.
Determining the sex of an embryo on the territory of the Russian Federation is prohibited by law, but such a diagnosis can be carried out simultaneously with the identification of genetic anomalies. And in the USA, on the contrary, it is the determination of the sex of the unborn child that is the most popular among all genetic analyses. Therefore, in this country, much more embryos are subjected to genetic diagnosis than in other countries.
To determine the genetic status of an embryo, a biopsy of its cellular material is performed – tissue in the case of an embryo or a polar egg cell. Modern technical capabilities allow such a procedure to be carried out on the third, fourth or fifth day after fertilization.
This is what a human embryo looks like on the 5th day after classical IVF. A thick non-cellular membrane is visible around the embryo and spermatozoa stuck to it that have not penetrated inside.
The most informative and reliable results are obtained by a biopsy of the cell mass of a five-day-old embryo. The selection of material during this period of development significantly reduces the risk of obtaining incorrect data due to the possible phenomenon of embryo mosaicism, when different cells carry a different set of genes.
The fact is that on the third day of development, an embryo can take only one or two cells out of a dozen available for analysis and not detect any genetic abnormalities in them. However, if at least one of the remaining cells contains a DNA mutation or a chromosomal anomaly, then the embryo will develop with deviations in the future. On the fifth day of embryo development, the number of its cells increases significantly, and the cell mass is combined, so at this time it is possible to take a relatively large area of tissue for analysis, which will give more accurate information about the genetic status of the embryo.
Genetic analysis of embryo cell material can be carried out using several methods: fluorescent in situ hybridization using fluorescent labeled DNA probes; comparative genomic hybridization based on comparison with control DNA, as well as sequencing, i.e. determination of the primary structure of the DNA sequence.
Full-genome genetic research of embryos is now widely used in American IVF practice and is becoming increasingly widespread in European countries. The most promising is comparative genomic hybridization, which makes it possible to diagnose aneuploidy (an incorrect number of chromosomes) and microstructural chromosomal abnormalities in all chromosomes at once (Glinkina et al., 2014). When genetic abnormalities are detected among the embryos obtained, a selection is carried out, and only those of them that will give rise to a normal pregnancy and lead to the birth of a healthy child are transferred to the mother's body.
Today, there are already more than four million people in the world who were conceived using in vitro fertilization, and millions of women have known the happiness of motherhood, which nature or circumstances denied them. Dozens of discoveries are made annually in the field of secondary reproductive technologies, thanks to which the effectiveness of IVF techniques is constantly increasing. Nevertheless, experts hope that in the future ART will not become a universal practice, but will remain a saving way out for a few. To do this, each of us already needs to take care of our own health, the health of our children and loved ones.
The publication uses photos of the Central Research Institute of the IHBFM SB RAS. All photos were taken on phase contrast microscopes, which allow you to observe the cells without damaging them.
References:
1. Glinkina Zh. I., Makhmudova G. M., Kibanov M. V. Preimplantation genetic diagnosis by comparative genomic hybridization – a new approach to the prevention of the birth of children with congenital and hereditary pathology in the framework of the ART program // Reproduction problems. 2014. № 4. C. 68–72.
2. Dedov I. I., Makarova N. P., Vityazeva I. I., Bogolyubov S. V. Morphological structures of the sperm affecting the effectiveness of fertilization by ICSI method // Problems of reproduction. 2010. № 3. C. 64–67.
3. Osadchuk L. V., Kleshchev M. A., Gutorova N. V. and others. Hormonal profile and sperm quality in men of Eastern Siberia // Bulletin of the Russian Academy of Medical Sciences. 2012. No. 3. C. 50-55.
4. Lane M., Gardner D. K. Embryo culture medium: which is the best? // Best Pract. Res. Clin. Obstet Gynaecol. 2007. V. 21. № 1. P. 83–100.
About the authors
Nikiforov Dmitry Vladimirovich – Engineer of the Laboratory of Reproduction Problems of the Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk). Laureate of the Russian President's Award for the Support of Talented Youth (2008).
Ryabchikova Elena Ivanovna – Doctor of Biological Sciences, Professor, Head of the Microscopic Research Group of the Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk), Associate Professor of the Department of Biomedical Physics of the Faculty of Physics of NSU, Associate Professor of the Department of Molecular Biology of the Faculty of Natural Sciences of NSU.
Ovsyannikova Tatiana Viktorovna – Doctor of Medical Sciences, Head of the Laboratory of Reproduction Problems at the Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk).
Popova Zhanna Yurievna is a reproductologist at the Center for Reproductive Health of the Center for New Medical Technologies (Novosibirsk).
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23.05.2016