RU2625777C1 - Method of determination in vitro of perspective embryones for the following implantation in the uterine in the examination of the extracorporal fertilization procedure (efp) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: to determine in vitro promising embryos for subsequent implantation into the uterus during the procedure of in vitro fertilization (IVF) by means of the time-lapse video recording in 2-cell embryos, the sequence and orientation of the planes of division (horizontal/equatorial E, vertical/meridional-M) of each blastomer. It is taken into account that such planes can be: successive equatorial (EE), successive meridional (MM), meridional, then equatorial (ME), equatorial, then meridional (EM). And if the sequences of divisions in these planes correspond to the variants (EM) or (MM), then such embryos are considered promising for subsequent implantation into the uterus during the IVF procedure.

EFFECT: early detection and transfer of the most promising embryos for implantation, which increases the frequency of implantation and pregnancy.

2 dwg, 2 tbl, 2 ex

Description

The invention relates to medicine, namely to gynecology and reproductologists, embryology, and can be used in in vitro fertilization (IVF) and embryo transfer (PE) programs to predict their outcomes.

Modern knowledge and experience gained over the past two decades in the treatment of infertility with the rapid progress of assisted reproductive technologies (ART) have made significant progress in this direction. This became possible due to the introduction of improved superovulation inducers into the IVF program, which allows to increase the number of oocytes, as well as as a result of improving the quality of culture media.

At the same time, reproductologists are dissatisfied with the results of in vitro fertilization programs: the frequency of pregnancy after the use of IVF and PE, even in the best clinics in the world, does not exceed 30% per embryo transfer and 10-15% per stimulated cycle (V.I. Kulakov, B.V. Leonov, 1998).

Apparently, these measures allowed approaching a certain threshold of effectiveness, and therefore, the search for new ways leading to an improvement in the effectiveness of IVF and PE programs is very relevant.

One of the ways to further increase the effectiveness of IVF programs may be to develop methods of objective control over the selection of the most complete and promising embryos for further implantation in the uterus.

Early detection of the most promising embryos for implantation is crucial, since the transfer of early (crushing) embryos to the mother's body increases the frequency of implantation and pregnancy.

It is known that 24-36 hours after fertilization, the first division of the zygote occurs and from that moment the fertilized oocyte becomes a 2-cell embryo. The cells of the embryo at this stage are called blastomeres.

At this stage, one can assess the quality of the embryo by the degree of deformation, vacuolization, fragmentation (the volume of the embryo occupied by nuclear-free fragments of the cytoplasm), the more there are, the lower the potential of this embryo for implantation and further development is considered. In addition to these data, the shape and relative sizes of the blastomeres are evaluated.

The most common classification of crushing embryos by quality is a - ab - b, where a is the best, b is the worst.

On the 2nd day of development, the human embryo consists of 2, 3 or 4 blastomeres.

After another day, the embryo normally already consists of 6-8 blastomeres, but 4 blastomeres are allowed, if on the 2nd day the embryo was 2-cell. Before the 8-cell stage, all cells of the human embryo are totipotent, i.e. each of them can give rise to a whole organism.

On the 4th day of development, the human embryo already consists, as a rule, of 8-16 cells, the morula stage begins. It is at this stage in vivo that the embryo enters from the fallopian tube into the uterine cavity. Morules also differ in the degree of compaction of blastomeres (C1 - C2 - C3 - C4).

By the end of 4 days of development, a cavity gradually forms inside the morula - the cavitation process begins.

From the moment the cavity inside the morula reaches 50% of its volume, the embryo is called a blastocyst. The larger the blastocyst cavity and the better developed internal cell mass and trophoblast, the greater its potential for implantation.

At this stage, one can also assess the quality of the embryo by the degree of deformation, vacuolization, fragmentation. In addition, the shape and relative sizes of the blastomeres are evaluated.

The most generally accepted classification of crushing embryos on the 5th day of development according to quality is A - B - C, where A is the best, C is the worst. A two-letter designation is used - one for trophoblast, the other for embryoblast.

Subsequently, the embryo begins to increase in size. At the same time, blastomeres continue to divide.

When the blastocyst cavity reaches a significant size, the glossy shell that has become thinner due to stretching breaks and the process of hatching (hatching) the embryo from the shiny shell begins.

Only after the end of this process is the blastocyst able to implant (attach) to the uterine endometrium. Implantation occurs, as a rule, on the 6-7th day of embryo development, considering the day of fertilization to be zero.

Sometimes it is impossible to establish sharp dividing lines between one or another variant of embryo development. Therefore, the above classification is relative, approximate.

The prior art method for determining the prospects of embryos by changing the light transmission of the active zones of the embryo associated with the spontaneous division of individual blastomeres [1].

Known methods are those in which promising embryos are selected based on the time of division [2], expression by the embryo of one or more miRNAs [3], based on measurement of the expression level of 45 genes, whereby the rate of subsequent implantation is predicted [7], based on the number and size of blastomeres on the second or third day, as well as by the percentage of fragmentation and the presence of multinucleation at the stage of 4 or 8 cells [8].

The closest (prototype) to the proposed method, we consider a method for determining in vitro promising embryos for subsequent implantation in the uterus during the in vitro fertilization (IVF) procedure, based on determining the spatial distribution of blastomeres (J Cell Sci. 2005 Feb 1; 118 (Pt 3) : 505-15. Epub 2005 Jan 18., Down regulation of Par3 and aPKC function directs cells towards the ICM in the preimplantation mouse embryo., Plusa B1, Frankenberg S, Chalmers A, Hadjantonakis AK, Moore CA, Papalopulu N, Papaioannou VE , Glover DM, Zernicka-Goetz M.).

However, in this source of information there is no information about the possibility of choosing promising embryos according to the planar sequence of the second division of blastomere fragmentation at the 2-cell embryo stage. In addition, this method was carried out on mice. We offer a method for determining in vitro promising embryos for subsequent implantation in the uterus during the in vitro fertilization (IVF) procedure in humans.

Further, the solution is illustrated by reference to the drawings, which show the following:

In FIG. 1. The formation of the universal tetrahedral form of 4-cell embryos as a result of successive equatorial (A), meridional (B) and meridional and equatorial (C) divisions in the second fragmentation cycle is shown. The equatorial and meridional division variant (not shown) differs from the ME variant only in the division sequence. The time after intracellular sperm injection (h: min) is indicated in the image field. An asterisk indicates the second polar body.

In FIG. Figure 2 shows the development paths of individual nuclei (gray lines), grouped in accordance with the order and orientation of the fission planes in the second fragmentation cycle: successive equatorial and meridional (A and B), meridional and equatorial (C) and equatorial and meridional (D) fissions blastomeres of 2-cell embryos. Solid contrast lines with markers are averaged development paths. The ellipses in FIG. B and D indicate the grouping of the trajectories of individual embryos in accordance with the development rate: 1 - high, 2 - low, 3 - intermediate. Dotted contrast lines are the average development paths for each of the groups that differ in the pace of development. The horizontal axis is the number of blastomeres in the embryo (the order of appearance of the blastomeres); vertical axis - post-ICSI time., hours

In table 1, the same superscript numbers indicate statistically significant differences in mean values (Van der Waerden test, P <0.05).

1) B - stage of blastocyst; M - stage of morula; the numbers indicate the gradation of embryos at the blastocyst stage; in parentheses indicate the number of embryos that have reached this stage.

2) The mean times between successive divisions of the blastomeres differ from each other in the same way (and with the same significance) as the duration of the periods of divisions of the blastomeres.

In table 2, the designations are the same as in table 1.

The method is as follows.

Ethical standards and legal restrictions allow the use of only non-invasive methods for researching conceptuses, i.e. only microscopic observations (excluding interventions if necessary, genetic analysis of the embryo). In this regard, time-lapse video recordings of the development of human embryos are analyzed (for example, using a Primo Vision microscope placed in a Thermo thermostat; other commercially available devices (EmbryoScope, Eeva, etc.) can be used) obtained using the standard cultivation protocol.

Analysis of a time-lapse video of the development of human embryos under standard (generally accepted) in vitro cultivation conditions. In particular, the determination of fission variants of blastomeres of 2-cell embryos. To do this, in successive frames determine: 1) the position of the second guide body, i.e. animal pole of the zygote, 2) the orientation of the plane of the first division of the fragmentation, which is normally assumed to be meridionally directed, i.e. dissecting the zygote from the animal to the vegetative pole; 3) determine the crushing plane of each of the two blastomeres: horizontal (equatorial - E) or vertical (meridional - M); 4) determine the sequence of the second crushing divisions in these planes, which can be: successive equatorial (EE), successive meridional (MM), meridional then equatorial (ME), equatorial then meridional (EM) (Fig. 1).

If the sequence of the second crushing divisions in these planes corresponds to (EM) or (MM), then such embryos are considered promising for subsequent implantation into the uterus during the IVF procedure.

EE, MM, ME, and EM variants of the second division are found in 10.3, 19.1, 27.9, and 42.6% of embryos, respectively, which developed without structural abnormalities (N = 68). The frequency of occurrence of EE, MM, ME, and EM variants in embryos with structural defects is 30.3, 15.2, 24.2, and 30.3%, respectively (N = 33). The frequency of EE variants of division in abnormal embryos has a clear tendency to exceed (almost three times) that in normal embryos (x2 = 4.30, P = 0.038; P with Yeits correction is 0.072). Thus, EE embryos are more prone to organic developmental disabilities to a greater extent than embryos with other variants of the second cleavage division.

Accordingly, embryos with the EE variant of the second division of fragmentation should be considered the least promising for transfer and should at least be the subject of special attention of the embryologist.

It is well known that, in general, implantation-successful blastocysts are characterized by a high rate of fragmentation [23]. However, from the practice of embryo cultivation, it follows that morphologically (and genetically) perfect, rapidly fragmenting embryos do not always form a perfect blastocyst; and vice versa - embryos crushed at a moderate pace often turn into a textbook ideal blastocyst. The dynamics of the development of human embryos with different variants of the second division of the fragmentation differs significantly both in the parameters of the fragmentation cycles and the duration of the blastomere cycles (Fig. 2).

EE embryos are characterized by a smoothed “wavy” temporal development trajectory. In MM-, ME-, and EM-embryos, a “step-like” trajectory is more pronounced (Fig. 2). The averaged time trajectories (see Fig. 2) differ from each other (paired Wilcoxon test; the significance of the differences (P) between EE- and MM-, EE- and EM-, MM- and ME-, MM- and EM-options are 0.001 ; the significance of the differences between EE and EM is 0.002; between ME and EM variants is 0.023).

Differences in the duration of crushing cycles become apparent in the third cycle and are exacerbated in the 4th crushing cycle. The longest cycles are in EE-, the least - in MM embryos. EM and ME embryos occupy an intermediate position. The lengthening of complete crushing cycles is mainly associated with lengthening of periods of cell divisions. Accordingly, the division rate is maximal in MM and minimal in EE embryos (Table 1). MM embryos reach the stage of 16 cells after 80.5 ± 4.85 h (mean and standard deviation); EM embryos after 87.7 ± 9.47 h, and ME and EE embryos after 94.8 ± 11.29 and 98.1 ± 5.05 h (differences in mean values are statistically significant, except for those for EE and ME groups (P = 0.197 ); Van der Waerden criterion).

Thus, in accordance with a decrease in the rate of crushing, embryos with different variants of the second crushing division can be ranked as follows: MM, EM, ME, EE.

However, the frequencies of transformation of embryos with different variants of the second division of crushing into implantation-competent blastocysts do not differ.

The reason for the latter lies in the variability of developmental trajectories in ME- and EM-embryos, which is more noticeable than in EE- and MM-embryos (see Fig. 2; compare the corresponding values of standard deviations presented in Table 1).

ME- and EM-embryos can be divided into three groups in accordance with the similarity of the trajectories (Fig. 2, C, D). The averaged development trajectories of both ME- and EM-embryos from different groups significantly differ from each other (P = 0.0007 for all variants of comparisons, paired Wilcoxon test). Embryos of the second group are characterized by longer cycle times. Crushing cycles in embryos of the third groups can be comparable or exceed in duration the corresponding cycles of embryos from the first groups, and in some cases - cycles of embryos from the second groups (see details in Table 2).

ME embryos of the first, second and third groups reach the 16-cell developmental stage after 85.1 ± 4.28 (mean ± standard deviation), 110.5 ± 6.10 and 94.6 ± 2.57 hours; EM embryos after 74.9 ± 3.10, 102.1 ± 5.20 and 88.8 ± 3.05 h, respectively. The differences between these indicators are statistically significant (Van der Waerden test, P = 0.001, 0.024 and 0.001). At the same time, the averaged development paths of ME and EM embryos of the first, second, and third groups do not differ (paired Wilcoxon test; P = 0.256, 0.158, and 0.112, respectively).

By the end of the registration period, embryos from different groups reached different stages of development (Table 2). The first groups unite the embryos that formed developed blastocysts (grades 4 and 5). The second groups include slowly developing embryos that have reached the morula stage or begin to cavitate the blastocyst. The third groups are heterogeneous, since at the end of the observations, the embryos that make them up show the entire spectrum of blastocyst gradations, although the majority of blastocysts of the 2nd and 3rd gradations (Table 2).

A comparison of alternative distributions (the number of embryos that reached grades 5 and 4 with the number of earlier embryos at the end of the registration period) demonstrates the clear dominance of advanced in the development of both ME and EM embryos in group 1 and, accordingly, the dominance of lagging embryos in group 2 (two-sided exact Fisher test, P = 0.021 and 0.001). The same comparison for groups 1 and 3 showed a significant difference in alternative distributions in EM embryos (P = 0.019), but similarity in ME embryos (P = 0.277).

The period of development of MM embryos to the 16-cell stage is intermediate between the corresponding indicators for ME and EM embryos from the first groups, significantly shorter than the first (Van der Waerden test, P = 0.017), but longer than the second (P = 0.001) indicator . The average fragmentation path of MM embryos differs from those for ME and EM embryos from the first groups (paired Wilcoxon test, P = 0.001 and 0.000, respectively).

The alternative distribution (advanced against developmental lags) in the MM group does not differ from similar distributions in the first groups of ME and EM embryos (Fisher's exact test, P = 0.590 and 0.148), but differs from distributions in the second groups (P = 0.015 for both comparisons).

The analysis presented above suggests that embryos with EM- and MM-variants of the second cleavage division are more successful (approximately twice) in the formation of a developed blastocyst, in comparison with embryos with ME- and EE-cleavage variants. In other words, the differentiation of the variants of the second division of crushing allows one to identify a priori embryos that are most promising for the formation of implantation-competent blastocysts.

Based on our studies, the technical result of the invention consists in the earliest and most accurate determination of the highest quality, and, accordingly, the most promising for the formation of implant-competent blastocysts for subsequent implantation into the uterus under the IVF protocol due to the fact that embryos with a sequence of second divisions crushing in planes: the equatorial then the meridional (EM) or sequential meridional (MM) are least prone to organic disturbances, and therefore, they will allow you to get the desired pregnancy and subsequently lead to the birth of a healthy baby.

Example 1

Patient Mr., was admitted to the Center for Human Reproduction in order to conduct her IVF and PE programs. The patient complains of infertility for 11 years. From the anamnesis it is known that the patient in 1989 underwent surgery for PCOS (no effect), and in 1998, laparoscopy for adhesions of the pelvic organs 3-4 degrees, bilateral salpingitis. Despite the scope of the operation - the separation of adhesions of the pelvic organs and the abdominal cavity, salpingo-ovariolysis, it was not possible to restore the patency of the fallopian tubes.

An additional examination by an endocrinologist revealed: hypothalamic syndrome; hypofunction of the ovaries; galactorrhea. Correction of endocrine disorders.

At the examination stage, before the IVF program, ureaplasmosis was detected, anti-inflammatory treatment was carried out (with effect).

Against the background of stimulation of superovulation (Humegon 75ED + 75ED 12 ampoules, clomiphencitrate 100 mg per day for 5 days), the time of administration of CG was determined depending on whether the leading follicle reached a diameter of at least 18 mm or 2-3 follicles of at least 17 mm. The ovulatory dose of Pretil was 10 thousand units.

In an ultrasound study, at the time of follicular puncture, 5 leading follicles were registered in the left ovary and 2 in the right. The thickness of the endometrium was 9 mm.

Oocytes and follicular fluid are aspirated 36 hours after the administration of an ovulatory dose of CG separately from the right and left ovaries. In order to obtain oocytes and follicular fluid from ovarian follicles, transvaginal puncture is performed using a vaginal puncture probe with a frequency of 5 MHz of the Aloka SSD-500 ultrasound apparatus.

The patient underwent follicular puncture and aspiration of preovulatory oocytes and follicular fluid. With follicular puncture from the left ovary, 3 oocytes were obtained, from the right ovary 1.

The obtained preovulatory oocytes from the right and left ovaries are cultured in special thermostats at a temperature of 37 ° C with a mixture of 5% CO ₂ in air in a culture medium Menezo B2.

In a follicular fluid aspirated at the time of puncture of the right and left ovaries, the activity of proteolytic enzymes (acid peptide-hydrolases) is determined by the increase in acid-soluble folin-positive compounds. The substrate for determining the activity of enzymes is lyophilized hemoglobin.

The follicular fluid is poured into 2 test tubes of 1 ml each (control and experimental) and 0.5 ml of a 1% solution of lyophilized commercial hemoglobin prepared in acetate buffer (0.2 M CH ₃ COONA and 0.2 M CH ₃ COOH are added to them). ) pH 4.2. After that, 1.5 ml of a 10% TCA solution is poured into the control sample, and the experimental sample is placed in a thermostat at 37 ° C for 1 hour, and 1.5 ml of 10% TCA are added at the end of the incubation. Then the samples are centrifuged for 10 minutes at 3000 rpm. 1.5 ml of the centrifugate is transferred to the test and control tubes, where 0.16 ml of 10% NaOH solution is added, after which 0.4 ml of the content is selected, in which the concentration of acid-soluble products (tyrosine) of hemoglobin proteolytic hydrolysis is determined. To the neutralized centrifugate, 2 ml of the working reagent solution are added to each tube, which is a mixture of 0.25 L of a 2% solution of sodium carbonate in a 0.1 M solution of sodium hydroxide and 0.01 L of a 0.5% solution of copper sulfate in 1% sodium citrate solution (this mixture is prepared before analysis).

The mixed contents are kept at room temperature (18-25 ° C) for 10 minutes, after which 0.2 folin-Ciocalteu solution is poured and again, after thorough mixing, the samples are kept at room temperature (18-25 ° C) for 20 minutes.

The measurement of the optical test sample is carried out against the control sample in a cuvette with a layer thickness of 1 cm at a wavelength of 750 nm on a spectrophotometer or photoelectric colorimeter.

The sperm is subjected to flotation and centrifugation, after which its fertility is assessed and oocytes are inseminated.

3 embryos were obtained.

By time-lapse recording in embryos at the stage of crushing of 2 cells, the sequence of the second crushing divisions was determined. The sequence of the second cleavage divisions in the planes of two embryos corresponded (EE), one embryo (EM).

One embryo was transferred with a sequence of second cleavage divisions (EM) at the stage of 8 blastomeres using a special disposable catheter for difficult delivery by COOK (Australia) through the cervical canal into the uterine cavity.

The result of the IVF and PE program was pregnancy, confirmed by ultrasound and hormonal studies.

Example 2

Patient K. entered the Center for Human Reproduction with the aim of conducting an IVF and PE program. The patient complained of infertility for 19 years. From the anamnesis it is known that the patient in 1988 underwent a laparoscopic operation that did not reveal pathology from the fallopian tubes. Considering the data of laparoscopy and the ovarian hypofunction revealed during the examination (hyperestrogenism), a negative PCT, a patient after preliminary hormonal correction, 5 attempts of IISM were made (without effect).

The lack of effect of the therapy required further in-depth examination. With repeated laparoscopy in 1998, the diagnosis was established: grade 2 endometriosis. Coagulation of endometrioid heterotopies was carried out, Danoval treatment was carried out for 1/2 year. In the absence of the effect of the therapy, the patient is recommended a program of IVF and PE.

Superovulation was stimulated by 0.1 mg Dekapeptil from the 2nd to the 10th day of the menstrual cycle, Humegon 75 U + 75 U 24 ampoules from the 2nd to 7th day of MC. Upon reaching maturity by follicles on day 10, MC was assigned Pretil 10,000 units.

In an ultrasound study, at the time of follicular puncture, 4 leading follicles were registered in the left ovary and 3 in the right. The thickness of the endometrium was 11 mm.

The patient underwent follicular puncture and aspiration of preovulatory oocytes and follicular fluid. With follicular puncture from the left ovary, 3 oocytes were obtained, from the right ovary 1.

2 embryos were obtained.

By time-lapse video recording of embryos at the stage of crushing of 2 cells, the crushing plane and the sequence of the second crushing divisions in these planes were determined. The sequence of the second crushing divisions in one embryo corresponded (ME), the second embryo (EE).

Two embryos were transferred at the stage of 8 blastomeres using a special disposable catheter for difficult delivery of COOK (Australia) through the cervical canal into the uterine cavity.

The result of the described IVF and PE attempt was the absence of the fact of pregnancy in this patient.

We conducted a study of 100 women (100%) who underwent an IVF protocol. Of these, 25 women underwent PE with a second fragmentation division (EE) and 25 women underwent PE with a second fragmentation (ME) sequence. As a result, 2 women recorded the fact of pregnancy.

Women who underwent PE with sequences of the second division of crushing (EM) - 25 people and (MM) - 25 people, pregnancy occurred in 11 cases.

Thus, the study conducted by us indicates the possibility of identifying the most promising embryos in vitro already at the stage of a 2-cell embryo.

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Claims (1)

A method for determining in vitro promising embryos for subsequent implantation in the uterus during the in vitro fertilization (IVF) procedure, characterized in that by time-lapse recording in 2-cell embryos, the sequence and orientation of the division planes are determined (horizontal / equatorial - E, vertical / meridional - M ) of each blastomere, which can be: successive equatorial (EE), successive meridional (MM), meridional, then equatorial (ME), equatorial th, then meridional (EM) and if the sequence of divisions in these planes correspond to the embodiments (EM) or (MM), these embryos are considered promising for subsequent implantation in the uterus during the IVF procedure.

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