RU2593740C1 - Method of controlling degree of zygosity of pregnancy in extracorporeal fertilisation and embryo transfer programs - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, particularly to obstetrics and gynaecology, and can be used for embryo transfer (ET) after extracorporeal fertilisation (EF). For this purpose, method includes forming 2 cohorts of oocytes, zygotes and embryos, originating from right and left ovary. In case of successful cultivation and development of at least 2 prime quality embryos (PQE) to obtain singleton pregnancy PQE is transferred from one cohort. To produce dizygotic pregnancy, method includes transfer of at least 2 PQE from different cohorts.

EFFECT: method enables to control onset of singleton or multi-zygotic (dizygotic) pregnancies in ET and EF programs.

1 cl, 1 tbl, 7 ex

Description

The invention relates to medicine, namely to obstetrics and gynecology, and can be used in the transfer of embryos (PE) after in vitro fertilization (IVF). The method allows you to regulate the onset of single or multiple (dizygotic) pregnancies in programs (IVF and PE).

Currently, the problem of unwanted multiple pregnancy (MP) in the implementation of the IVF and PE program remains unresolved. Iatrogenic MP accounts for 30-80% of multiple pregnancies, which is associated primarily with the use of modern methods of infertility treatment: hormonal stimulation of superovulation and IVF with the transfer of several good quality embryos.

The phenomenon of MP is still not fully understood. It is important to distinguish between monozygous and dizygotic (polysygotic) MP. In turn, dizygotic (or polysygotic) MP is associated with simultaneous ovulation (during one ovarian cycle), followed by fertilization of two or more eggs that matured in different follicles of one ovary (ovulatio uniovarialis), and with simultaneous ovulation, followed by fertilization of two or more eggs matured in different follicles in both ovaries (ovulatio biovarialis) [Makarov OV, Obstetrics. Clinical lectures. Publisher - M.: GEOTAR-Media, 2007. - 640 p.].

MP has medical, social, psychological, ethical and financial aspects. The medical aspects include, first of all, the risks of preterm birth and higher rates of perinatal mortality.

Thus, the decrease in MP is an urgent problem. Currently, the most common way to solve this problem in the field of assisted reproductive technologies is the method of selective transfer of a single embryo (Elective single-embryo transfer, eSET) [Thurin et al., 2004; Magli, 2007; Allen et al., 2006; De Neubourg et al., 2002; Ubaldi et al., 2015].

However, this method involves a number of additional circumstances.

Selective transfer of a single embryo, selected on the basis of a traditional morphological assessment of the quality of the transferred embryo, leads to a significant decrease in the effectiveness of IVF + PE programs, especially in the relative number of successful deliveries [Nazarenko T.A. Stimulation of ovarian function. - M .: Medpress - inform, 2008. - 272 p., 2008; Whitney J.B., Nugent N., Zozula S., Anderson R.E., Schiewe M.C. Single embryo transfer (SET) yields equivalent clinical pregnancy rates and higher implantation rates compared to dual embryo transfer (DET) of euploid blastocysts // Fertil. Steril. 2013 100 (3): S29].

Several approaches are known for improving the method of selective transfer of the single most complete embryo.

a) Invasive, including preimplantation genetic diagnosis of the polar body, one of the blastomeres, or blastocyst trophoblast cells to select the most healthy embryo [Harper JC1, Sengupta SB. Preimplantation genetic diagnosis: state of the art 2011 // Hum Genet. 2012; 131 (2): 175-86]. However, there is information that trauma to the shiny sheath of the embryo during ICSI, which is necessary in this case and repeated trauma during cell collection, can increase the number of monozygous MPs [Haimov-Kochman R1, Daum H, Lossos F, Aizenman E, Werner M, Yagel S , Laufer N, Simon A, Hurwitz A. Monozygotic multiple gestation after intracytoplasmic sperm injection and preimplantation genetic diagnosis // Fertil Steril. 2009 Dec; 92 (6): 2037. e11-7].

b) Non-invasive, including systems for continuous monitoring of embryo development (time-lapse embryo selection).

An example of this approach to solving the problem of the single most complete embryo intended for transfer is a method for determining changes in the cell population (US 8265357). This invention is a method and system for establishing changes in a cell population, as well as evaluating qualitative changes in embryos and selecting embryos for IVF by comparing successive images of embryos. The known method includes:

- measurement of the duration of the period of cell division — the duration of cell division from the beginning of the division of the cytoplasm to the transformation of the original cell into two daughter cells;

- measurement of the length of the period between divisions - the length of the resting period between cell divisions;

- measuring the duration of the cell cycle - the length of the period from the beginning of one division to the beginning of the next;

- measurement of intracellular mobility (excluding poor membrane mobility);

- measurement of the mobility of intracellular organelles;

- tracking the movement of cells within the embryo, etc.

Thus, the present invention is a method and system for tracking changes in cell populations, including within a developing embryo. In accordance with this method, the following characteristics are inherent in superior quality embryos:

- short period of cell division (less than 1 hour);

- the presence of only weak cellular movements in the interphase period, i.e. the cell center is stationary (shift less than 3 µm), cell membranes are mostly motionless (shift less than 3 µm);

- early onset of the first cell division (less than 25 hours after fertilization;

- a short period of cell movements in the interval between crushing (less than 3 hours);

- uniform distribution of cellular movement within the embryo throughout the observation period, i.e. the absence of inactive areas, zones, or volumes within the embryo, where cellular movements are not observed for more than 24 hours. The presence of such fixed sites may indicate the presence of dead or dying blastomeres and fragments that may impede further development.

The known method requires complex instrumentation and software.

c) Analysis of the molecular genetic profile of the follicular fluid of each oocyte and sperm on the day of transvaginal puncture (TBP) of the ovaries.

This includes a method for predicting pregnancy in an IVF program with selective PE by evaluating the molecular genetic profile of gametes using PCR-PB (RU 2550965). The aim of the known invention is to reduce the frequency of pregnancy MP, as well as the ability to predict the onset of pregnancy in the IVF program. The authors of this method solve the problem by selecting an embryo having a high potential for implantation with selective transfer of embryos. In the process of implementing the known invention, an IVF and PE program was conducted with stimulation of superovulation according to standard protocols. Fertilization was carried out using standard IVF methods or using ICSI (in the presence of subfertile abnormalities of the spermogram on TVP day from normal according to WHO criteria, 2010) in equal proportion (p = 0.833). Individual embryo cultivation was carried out in individual wells of a four-well plate in accordance with the number of the follicular fluid sample. The choice of the embryo was carried out on the basis of morphological evaluation. Selective transfer of one or two embryos was performed at the blastocyst stage. In this case, the transcriptome profile of male and female gametes was evaluated. A generally accepted morphological evaluation of the resulting embryos was performed. PE was made on the basis of a comprehensive morphological and molecular genetic assessment of gametes. In addition, the authors developed a mathematical model that can be used as an additional criterion for an objective assessment of gamete quality and allows classifying couples according to the outcome of IVF and PE depending on the combined data on the molecular genetic profile of follicular fluid and sperm on the day of ovarian TVP.

Despite the success of the selective selection of a single embryo in overcoming the MP problem, a common obstacle to the use of the above methods of additional preimplantation evaluation of gametes and embryos is their extreme complexity and high cost in terms of the volume of necessary research, the high cost of consumables, the required hardware and software. This makes them not always accessible, given the widespread use of IVF + PE at the moment in many perinatal IVF centers and laboratories with different levels of equipment and providing qualified personnel.

Another significant obstacle to the use of selective transfer of a single embryo is the socio-psychological attitude of a large number of patients undergoing infertility treatment using IVF + PE methods and overwhelmingly opposed to transferring a single embryo. Only less than 9% of patients agree to transfer one embryo, while 40% of patients refuse to transfer one embryo not for fear of a negative outcome of the program, but because they would like to have twins. This factor is important if we take into account the psychophysiological, as well as material costs of patients in the treatment process.

The approaches described above do not take into account the peculiarity of the IVF program, in which both ovaries are a priori stimulated and oocyte aspiration during TBP is carried out from both ovaries. It is this aspect that is central in the claimed method.

Closest to the claimed method according to the number of matching essential features selected method of IVF (RU 2190978). As follows from the description of the known method and the given clinical examples, the authors solve the problem of optimizing the selection of preovulatory oocytes for further cultivation and a qualitative assessment of the transferred embryos into the uterine cavity, taking into account the morphofunctional asymmetry of the female reproductive system.

The task of the authors of the prototype method is solved as follows.

- At the time of follicular puncture, the preovulatory oocytes and follicular fluid are separately aspirated from the right and left ovaries, the number of oocytes and the activity of proteolytic enzymes (acid peptide hydrolases) in the follicular fluid are determined.

- Ovary is considered dominant, peptide-hydrolase activity, in the follicular fluid of which is 20 mg / ml for 1 hour or more, exceeds the corresponding indicator of another ovary.

- The selection of the best quality oocytes for further cultivation and the selection of transferred embryos are carried out taking into account the dominance of the ovary, the transfer of embryos into the uterine cavity is performed on the endometrial side, ipsilateral dominant ovary.

- Aspiration of oocytes and follicular fluid is performed 36 hours after the administration of an ovulatory dose of CG separately from the right and left ovaries.

- In order to obtain oocytes from ovarian follicles and follicular fluid, TVP is produced.

- The quality of oocytes obtained by puncture is evaluated by the level of activity of peptide hydrolases. Good oocyte quality is exhibited at a peptide-hydrolase activity level above 75 mg / ml / hour, average quality at a level of 40-74 mg / ml / hour, poor quality at a level below 40 mg / ml / hour.

- Transfer of embryos into the uterine cavity is carried out, as a rule, 48-50 hours after TBP. Embryos at the stage of 2-8 blastomeres.

However, the use of the prototype method does not solve the problem of preventing the occurrence of unwanted MP during the implementation of IVF and PE programs.

The claimed invention is aimed at solving the problem of regulating the onset of a single or multiple (dizygotic) pregnancy, as well as preventing the occurrence of unwanted MP during the implementation of IVF and PE programs.

Using in clinical practice the proposed method allows to achieve several technical (therapeutic) results:

- optimization of the choice of embryos with high potential for transfer into the uterine cavity;

- ensuring the outcome of the IVF and PE program in the form of a single or multiple pregnancy with a probability of about 93% due to the fact that all embryological stages of the program are carried out by forming two cohorts of embryos originating from different ovaries (ovarian cohorts) and their independent cultivation;

- the possibility of an informed choice: the development of a dizygotic or monozygous pregnancy, taking into account the wishes of the patient herself and her physiological abilities to carry a pregnancy MP due to the transfer of identified high-quality embryos (EVCs) from one or of 2 different formed cohorts of embryos;

- low cost and availability for use by many perinatal centers and IVF laboratories with different levels of equipment.

These technical (therapeutic) results in the implementation of the invention are achieved due to the fact that, as in the known method, they produce separate TBP of follicles and aspiration of preovulatory oocytes from the right and left ovaries.

A feature of the proposed method lies in the fact that they carry out the formation of 2 cohorts of oocytes, zygotes and embryos originating from various ovaries, by separately conducting each stage of IVF. To obtain a predominant result in the form of a single pregnancy, they carry out the transfer of the 1st, 2nd, rarely 3 EVCs from one cohort into the uterine cavity. To obtain a predominant result in the form of a dizygous pregnancy, 2, rarely 3, EVCs from both cohorts are transferred into the uterine cavity.

Disclosure of invention

A feature of the IVF program in accordance with the claimed methodology is that both ovaries are activated and oocyte collection during TBP is carried out from both ovaries, however, without mixing the cohorts aspirated from different ovaries.

The claimed invention is based on the use of separate cultivation of oocytes and fertilized zygotes belonging to different ovarian cohorts of oocytes, which allows a non-invasive and comprehensive approach to the choice of an embryo that has the maximum chance of implantation in the IVF and PE program from each cohort. The inventive method, in fact, is a partial reproduction of the natural mechanism of isolating the dominant follicle from the cohort of follicles that have overcome the meiosis block inside the ovary by extracorporeal selection of the leading EVC from the cohort of developing embryos.

The inventors have found that by the third day of development in vitro, embryos combined into ovarian cohorts based on their origin from a particular ovary are distributed according to the speed and quality of crushing. Each speed category is usually represented by a certain number of members. Among the embryos that make up the cohort, the isolation of “leaders” is observed. If these “leaders” contain 6-7-8 blastomeres (reflection of the optimal crushing rate, i.e., passing the third division of the crushing) of the same volume, with good turgor, light homogeneous cytoplasm (blastomere quality “a”) and less than 25% fragmentation, they are classified as embryos of the highest category (EVC) and their number is calculated. Qualitative assessment of cohort embryos, counting of EVCs and selection of leaders applying for transfer are carried out independently in both ovarian cohorts.

The inventors also found that multiple pregnancy is directly related to the belonging of the transferred embryos to different cohorts, originating from oocytes from different ovaries. According to observations of the results of existing practice, the transfer of 2 EVCs taken from different ovarian cohorts led to the development of MP in 80% of cases, and the transfer of 2 EVCs from one cohort in 10% of cases.

The authors of the proposed method conducted a retrospective study of 64 patients in whom they received a clinical pregnancy after transferring at least 2 EVCs under the condition of separate cultivation of groups of oocytes, zygotes and embryos belonging to different ovarian cohorts. Patients were divided into 2 groups: patients with the transfer of EVCs from one of the ovarian cohorts (uniovarialis) were assigned to one group, patients of the other group were transferred to the EVCs originating from different ovarian cohorts (biovarialis).

The results of comparing the main indicators for these groups of patients are presented in the table, which shows the ratio of MP in two groups of patients.

As can be seen from the table, 92.3% (24 of 26) of multiple pregnancies that ended in childbirth developed as a result of TBP of oocytes from different ovaries, their separate cultivation and transfer of EVC from two different ovarian cohorts. This can be considered factual evidence that dizygous pregnancies are associated with PE from different ovarian cohorts.

In the process, an assessment was made of the sensitivity and specificity of the method used. The following results are obtained.

The sensitivity of the method = 24 * 100 / (24 + 2) = 92.3%.

The specificity of the method = 100 * 18/24 = 75%.

Diagnostic test accuracy = 100 * (24 + 18) / 50 = 84%.

The predictive value of a positive test result (PPR) of the test (positive predictive value) = 100 * 24 / (24 + 6) = 80%.

The predictive value of the negative predictive value (POR) of the test (negative predictive value) = 100 * 18 / (2 + 18) = 90% [Album A., Norell S. Introduction to modern epidemiology / Per. from English - Tallinn: Institute of Experiment. and clinic. Medicine (Estonia), Danish Cancer Society. 1996. 122 p.].

The method is as follows.

The preparation of patients is carried out in accordance with the standard protocol of hormonal stimulation of superovulation (to increase the number of eggs). Starting from TBP, the stages of the IVF + PE program are carried out in such a way that both cohorts of oocytes, zygotes and embryos originating from two different ovaries are located at each stage of processing and cultivation in separate media. Washing, fertilization and further cultivation are carried out in 2 separate containers (Petri dishes, and later in the nucleon wells), so that the cohorts of oocytes from different ovaries do not mix.

Thus provide a separate (separate) cultivation of both cohorts. On the third day of cultivation, an individual assessment is made of the embryos that make up different cohorts (of the reached development stage and its quality: crushing rate, that is, the passage through the third crushing division), of the same volume, with good turgor, light homogeneous cytoplasm (blastomere quality “a”) , fragmentation not exceeding 25%. They are classified as embryos of the highest category (EVC) (tri-pronuclear zygotes detected the day after fertilization are removed from the ovarian cohort).

In the case of successful cultivation, leading to the development of two or more EVCs by the third day of cultivation, PE is performed (returning in vivo one or two, rarely three embryos). In some cases, PE is produced on the 5th day.

If EVCs are formed in both ovarian cohorts, then with PE from both cohorts, the likelihood of MP and the possibility of a conscious choice arises: obtaining a dizygotic or monozygous pregnancy. The participants in the process make a decision taking into account the wishes of the patient herself, her previous experience and her physiological abilities to bear a multiple pregnancy.

Embryo transfer is carried out taking into account their belonging to a particular cohort.

Two (rarely three, by agreement with the patient) carry out the morphologically most promising EVCs. These include embryos having, by the beginning of the 3rd day of development, 6-7-8 blastomeres (i.e., having an optimal fragmentation rate). They have already crossed (6 and 7) or have completed (8) III division of crushing. The blastomeres are isometric, spherical (good turgor), with a light homogeneous cytoplasm, compactly collected in the early morula (in contrast to the so-called planar elongated ones), the fragmentation of which does not exceed 25%. At this stage, it is important to take into account the belonging of the embryos allocated for transfer to one cohort or two different cohorts.

A retrospective study conducted by the authors of the claimed invention showed that in 92.3% of cases (24 cases out of 26), a dizygous pregnancy develops subject to the transfer of an EVA from both cohorts. If a dizygotic MP is undesirable, only one cohort is taken for transfer of an EVC. The remaining EVCs from this cohort, as well as EVCs from another cohort, are subjected to cryopreservation, observing the same principle of non-mixing of embryos from different cohorts (information is recorded in the accompanying documentation). Thus, if the current IVF + PE attempt is unsuccessful and a decision is made to defrost the embryos for the so-called cryotransference, the ability to control pregnancy zygosity will be fully preserved.

Execution examples

Examples of embryo transfer from two different ovarian cohorts

Example 1

Patient S., 42 years old. I contacted the GUBZ-MO "MOPTS" for conducting IVF programs. Conducted clinical and laboratory examinations. In preparation for the IVF program, the polyps were removed from the patient. Due to the presence of a pronounced male factor, IVF was used in IVF. In TVP, 6 and 4 oocytes are aspirated from the first and second ovaries, respectively. On the 5th day of development, 3 embryos were transferred: 2 early AA blastula from the first cohort and bl2AA from the second. The onset of pregnancy ended in the birth of twins.

Example 2

Patient D., 33 years old. I contacted the GUBZ-MO "MOPTS" for conducting IVF programs. Conducted clinical and laboratory examinations. In TVP, 4 and 7 oocytes are aspirated from both ovaries, respectively. On the 3rd day of development, 2 embryos were transferred: 8A from the first cohort and 8A from the second. By the 5th day of development, two good-quality blastocysts were formed in the second cohort that were cryopreserved. The onset of pregnancy ended in the birth of twins.

Example 3

Patient I., 30 years old. I contacted the GUBZ-MO "MOPTS" for conducting IVF programs. Conducted clinical and laboratory examinations. In TVP, 6 and 4 oocytes were aspirated from the ovaries, 3 embryos were transferred on day 5: 2 early AA blastula from the first cohort and bl2AA from the second. The onset of pregnancy ended in the birth of twins.

Example 4

Patient K., 24 years old. I contacted the GUBZ-MO "MOPTS" for conducting IVF programs. Conducted clinical and laboratory examinations. 6 and 6 oocytes were aspirated from the ovaries, 2 embryos were transferred on day 3: 8A and 7A, respectively. The onset of pregnancy ended in the birth of twins.

Examples of embryo transfer from one ovarian cohort

Example 5

Patient M., 32 years old. I contacted the GUBZ-MO "MOPTS" for conducting IVF programs. Conducted clinical and laboratory examinations. 7 and 6 oocytes were aspirated from the ovaries, 3 embryos were transferred from the first cohort (8a, 8a, 6a) on day 3; no transfer of embryos was made from another ovarian cohort. On day 5, a good quality blastocyst was formed in it, which was cryopreserved. The onset of pregnancy ended in the birth of one child.

Example 6

Patient G., 33 years old. I contacted the GUBZ-MO "MOPTS" for conducting IVF programs. Conducted clinical and laboratory examinations. Of both ovaries, 9 and 5 oocytes are aspirated. From the first cohort, on the 3rd day of development, 3 embryos were transferred (8c, 8c, 8c), from another ovarian cohort, there was no transfer and by the 5th day of development 2 good blastocysts were formed in it, which were cryopreserved. The onset of pregnancy ended in the birth of one child.

Example 7

Patient I., 30 years old. I contacted the GUBZ-MO "MOPTS" for conducting IVF programs. Conducted clinical and laboratory examinations. From ovaries, 8 and 3 oocytes are aspirated. The ICSI procedure was performed with oocytes from the first ovarian cohort. On the 3rd day of development, 2 embryos were transferred from it (8a, 8a). In the second ovarian cohort, traditional IVF was performed, but fertilization did not occur. The onset of pregnancy ended in the birth of one child.

Thus, the claimed method of regulating the degree of pregnancy zygosity in IVF and PE programs has high specificity and sensitivity and helps to prevent the development of unwanted MP during the IVF program.

Claims (1)

A method for regulating the degree of pregnancy zygosity in in vitro fertilization (IVF) and embryo transfer (PE) programs, including separate aspiration of preovulatory oocytes from the right and left ovaries, fertilization, cultivation and transfer of embryos into the uterine cavity, characterized in that they form two cohorts of oocytes, zygotes and embryos originating from different ovaries by separately conducting each stage of IVF, if you successfully cultivate and develop at least two embryos, Of the highest quality (EVC), in order to obtain a predominant result in the form of a single pregnancy, the transfer of EVCs is carried out from one cohort; in order to obtain a preferential result in the form of a dysygous pregnancy, at least two EVCs from different cohorts are transferred.