WO2015192352A1

WO2015192352A1 - Grade classification method for evaluating in-vitro fertilization treatment embryo according to cleavage behaviors

Info

Publication number: WO2015192352A1
Application number: PCT/CN2014/080289
Authority: WO
Inventors: 林戈; 卢光琇; 杨舒亭
Original assignee: 湖南光琇高新生命科技有限公司; 中南大学
Priority date: 2014-06-19
Filing date: 2014-06-19
Publication date: 2015-12-23
Also published as: CN104718298A

Abstract

Disclosed is a grade classification method for evaluating an in-vitro fertilization treatment embryo according to cleavage behaviors. In the present invention, in a case in which a human embryo cell is interfered and not damaged, data of dynamic observation on a human embryo is obtained by using a time-lapse imaging technology, and abnormal cleavage behaviors of the human cleavage-stage embryo of in-vitro fertilization are collected and described in detail and are divided into 7 types. The qualitative embryo grade classification method with little subjectivity is established according to the degree of severity of abnormal cleavage, the blastomere range affected by the abnormal cleavage behaviors and the occurrence period of the abnormal cleavage behaviors. The method can effectively screen out a cleavage-stage embryo with optimal blastocyst development potential.

Description

A classification method for evaluating in vitro fertilization treated embryos based on splitting behavior

The invention relates to the evaluation of embryos for in vitro fertilization treatment in the assisted reproductive technology and the selection of high quality embryos for transplantation, in particular, the embryos observed by the time difference imaging technique in the treatment are classified according to the abnormal pattern of the splitting behavior, and the embryos are screened. The method of transplantation.

Background technique

In vitro fertilization-embryo transfer (IVF-ET) has become a routine treatment for infertility. Traditional methods often rely on simultaneous transplantation of multiple embryos to increase pregnancy rate, but multiple embryo transfer leads to increased multiple pregnancy rates, increasing the risk of iatrogenic complications in mothers and fetuses. A feasible approach is to establish an effective embryo assessment. Methods High quality embryos were selected for transplantation during cleavage. The traditional cleavage stage static morphological embryo screening system uses the prokaryotic morphology, the number of embryo blastomeres on the third day, symmetry, and the number of fragments to evaluate and select embryos. However, this method has a single evaluation index, which is subjective and cannot accurately determine the shortcomings of embryo development potential. Time-lapse technology is an imaging technique for continuous exposure continuous shooting. It can acquire the morphology of each stage of embryonic development and the specific time of each event, and combine all recorded photos into dynamic video to achieve the embryo. Full monitoring of the development process. The morphological dynamics embryo evaluation screening method using time difference technique is to utilize the cleavage time during embryonic development (such as the time of fertilization to five-cell formation) and cleavage synchronization (such as from three-cell formation to four-cell formation). The time of formation) and other indicators to evaluate the embryo. However, this method has the following disadvantages: The morphokinetic index is influenced by various factors such as the culture system, the age of the patient, the maturity of the egg, the method of insemination, and the promotion scheme. Only a few indicators have been unanimously recognized, and there is some fluctuation in the scope of the same indicator. Therefore, morphodynamic indicators are not available in all laboratories.

Summary of the invention

SUMMARY OF THE INVENTION The present invention is directed to overcoming the deficiencies of the prior art and provides a hierarchical classification method for evaluating in vitro fertilization treated embryos based on splitting behavior.

In order to achieve the above object, the technical solution provided by the present invention is;

The grade classification method for evaluating the in vitro fertilization treatment embryo according to the splitting behavior is to transfer the embryo on the first day after fertilization into the embryo culture medium, and observe the embryonic eggs of the first to fifth days after fertilization under the time difference system camera of the incubator. In the case of cracking, set the camera shooting frequency to 5-15min/time; then classify the embryos according to the following steps: (1) Divide the embryo's splitting behavior into the following eight types:

1 normal division; 2 abnormal division; 3 unequal; 4 blastomere fragmentation; 5 large fragments; 6 abnormal cytoplasmic movement; 7 developmental block; 8 disordered sequence;

(2) According to the degree of influence of each splitting behavior on blastocyst formation, the above eight splitting behaviors of embryos are summarized into the following two categories:

Class I splitting behavior: normal division, abnormal cytoplasmic movement, unequal size, large debris on blastocyst formation less than 50%;

Class II division behavior: developmental block, divisional abnormality, disordered sequence, blastomere fragmentation on blastocyst formation degree of 50% or more;

(3) According to step (1) and step (2), divide the embryo into six grades: A, B, C, D, E, F:

Grade A: first (on the first to two days after fertilization, from one cell to two cells), second (on the second to third days after fertilization, from two cells to four cells), third egg An embryo that undergoes normal division behavior in the first half to four days after fertilization (from four cells to eight cells);

Grade B: Embryos with at least one class I splitting behavior (abnormal cytoplasmic movement, unequal size, large fragments) in addition to normal splitting behavior in the first, second, and third cleavage;

Grade C: First, secondary cleavage occurs Class I splitting behavior, third cleavage occurs Class II splitting behavior (developmental arrest, abnormal division, disordered division, blastomere fragmentation);

Grade D: The first cleavage occurs in the class I splitting behavior, and in the second cleavage, one of the cells (partially) has a class II splitting behavior;

Grade E: The first cleavage occurred in the class I splitting behavior, and the second cleavage occurred in both cells (all) with class II splitting behavior;

Class F: The first cleavage occurs in the class II division of the embryo;

Among them, Class A embryos are the best choice for embryos for transplantation; if there is no Class A embryos, Class B embryos are selected for transplantation; if there are no Class A and Class B embryos, Class C embryos are selected for transplantation; E and F embryos are not used for transplantation.

In the above technical solution, the normal division refers to the division of the embryo: in one to five days after fertilization, one mitosis forms two sub-cells having a diameter difference of less than 1/5 and a dispersion of less than 10% of the original cell volume. Nuclear debris;

The abnormal division of the embryo refers to the division of the embryo: one to five days after fertilization, one mitosis forms three or more sub-cells with a diameter difference of 1/5 or less;

The unequal size means that the division of the embryo is: Within one to five days after fertilization, the diameters of the two daughter cells formed after the division differ by more than 1/5;

The cleavage of the blastomere means that the division of the embryo is: Within one to five days after fertilization, the daughter cells formed by the division are broken into 4-10 pieces of cell-free nuclear fragments with a diameter of 1/3 or less of the original cell diameter or more than 50% of the original cell volume produced by the cell-free nuclear fragment;

Large fragments refer to the division of the embryo as follows: Within one to five days after fertilization, two sub-cells with a diameter difference of 1/5 or less and 1-2 pieces with a diameter of 1/3 to 1/2 of the diameter of the daughter cells are produced during the division. Nuclear debris or scattered, cell-free nuclear debris that accounts for 10% to 50% of the original cell volume;

Abnormal cytoplasmic movement refers to the division of the embryo as follows: Within one to five days after fertilization, the rupture of the membranous sulcus in the cell membrane during the division but the cleavage of the blastomere does not occur or abnormal movement such as cytoplasmic distortion, rotation, etc.;

Developmental blockade refers to the division of the embryo: within one to five days after fertilization, the blastomere develops stagnation and does not continue to undergo mitosis to form the corresponding daughter cells;

Disruption of the order of division means that the division of the embryo is as follows: Within one to five days after fertilization, the order of division occurs in the order of L; under normal circumstances, A cells undergo mitosis to form two sub-cells of Al and A2, and A1 cells undergo mitosis to form Al l And A12 two daughter cells, A2 cells undergo mitosis to form two sub-cells A21 and A22, and so on; the order of division is disordered, A cells undergo a mitosis to form two sub-cells of Al and A2, and A1 cells undergo mitosis to form two sub-groups of Al and A12. Cells, when A2 cells have not yet split, Al l cells or A12 cells prematurely mitotically form two corresponding daughter cells, and then A2 cells undergo mitosis to form corresponding two daughter cells.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The data of dynamic observation of human embryos by time-lapse imaging technique were used to collect, describe and classify the abnormal behavior of splitting in in vitro fertilized human cleavage embryos for evaluation of in vitro fertilization embryos.

(2) classify the splitting behavior according to the severity of the influence of the splitting behavior on the embryonic development potential, and classify the splitting behavior with less than 50% influence on blastocyst formation into one class, defined as class I splitting behavior; The splitting behaviors with a degree of influence greater than or equal to 50% are classified into one class, defined as class II splitting behavior.

(3) Combining different cleavage periods and the extent of mitotic behavior in the embryo, a classification method for evaluating the in vitro fertilization treated embryos according to the division behavior was established.

In summary, the present invention aims to dynamically observe human embryos using time-lapse imaging techniques without disturbing human embryonic cells or destroying human embryonic cells, and to perform abnormal behaviors of human cleavage embryos in vitro fertilization. Collections and detailed descriptions were made and divided into 7 types. According to the severity of abnormal division, the range of blastomeres involved in abnormal division behavior and the occurrence period of abnormal division behavior, a qualitative and subjective small classification method of embryos was established, which can effectively screen out the best blastocysts. Developmental potential cleavage stage embryo.

detailed description

Example 1 The method is applied to the clinical evaluation of embryos undergoing in vitro fertilization treatment, and the embryos are observed in an in vitro culture process using a time difference imaging system conventionally built in an incubator. Set the shooting frequency to 5 min/time and the exposure time to 70 ms/time. On the first day after fertilization, the embryos to be selected were transferred to a 3x3/4x4W0W culture dish prepared with G1. 5 embryo culture medium. The embryo-containing W0W culture dish was placed under the time difference system camera, and the embryo was evaluated on the fifth day after fertilization using the cleavage behavioral embryo classification model (Fig. 1).

Among the 345 normal fertilized embryos, 96 of the first, second and third cleavage had normal division behavior, and were rated as Class A embryos; at least one of the first, second and third cleavage occurred except for normal division behavior. There were 102 class I splitting behaviors, which were rated as B-class embryos; the first and second cleavage occurred in class I splitting behavior, and the third cleavage occurred in class II splitting behavior, 37 of which were rated as C-class embryos. The first cleavage occurred class I splitting behavior, and in the second cleavage, one of the cells (partially) had 39 class II splitting behavior, which was rated as D-class embryo; the first cleavage occurred class I splitting behavior. In the second cleavage, 19 of the two types of cells (all) had class II division behavior, and were rated as E-class embryos; 52 of the first cleavage class II division behavior, rated as F-class Embryo. A, B, C, D, E, F embryo blastocyst formation rate (blastocyst formation is an indicator for evaluating embryonic development potential. Formation of blastocysts on the fifth day after fertilization indicates good embryo development potential, no blastocyst indicates embryo The developmental potential difference was 94.8%, 85.3%, 48.6%, 51.3%, 47.4%, 21. 2%, with a decreasing trend (P<0.05), which was statistically significant. ), can distinguish the developmental potential of the embryo.

Example 2

The method was applied to the selection of high quality embryos for clinical transplantation in patients undergoing in vitro fertilization treatment, and the embryos were observed by time difference technique in vitro culture. Embryos were evaluated and selected on the fifth day after fertilization using the cleavage behavioral embryo classification model (Fig. 1). The enrolled patients were randomly divided into a split behavioral group and a conventional morphological group. The environment of the two incubators was 37 °C, 6% C0 ₂ .

The splitting behavior group uses the method described in the present invention to set a shooting frequency of 5 min/time and an exposure time of 70 ms/time using a time difference observation system built in an incubator which is conventionally used in clinical practice. On the first day after fertilization, the embryos to be selected were transferred to a 3x3/4x4W0W culture dish prepared with G1. 5 embryo culture medium. The embryo-containing W0W culture dish was placed under the time difference system camera and photographed continuously until the transplantation. Embryos were evaluated and selected on the day of transplantation using the cleavage behavioral embryo classification method (Fig. 1).

The embryos were routinely cultured in the conventional morphological group. On the first day after fertilization, the embryos to be selected were transferred to a Petri dish prepared with G1. 5 culture medium. Embryos were evaluated and selected using conventional morphological methods on the day of transplantation.

A total of 20 patients were enrolled in the split behavioral group, 1 was abolished, and 19 were involved in the experimental analysis. There were 194 normal fertilized embryos in 19 people, with an average of 10. 2 normal fertilized embryos per person. Among the 194 normal fertilized embryos, 54 of the first, second and third cleavage had normal division behavior, and were rated as grade A embryos; the first, second and third cleavage were There were 57 cases of class I splitting behavior except for normal splitting behavior, which was rated as B-class embryo; the first and second cleavage occurred in class I splitting behavior, and the third cleavage occurred in class II splitting behavior. 33, were rated as C-class embryos; the first cleavage occurred class I splitting behavior, and the second cleavage occurred when one of the cells (partially) had 22 class II splitting behavior, which was rated as D-class embryo; There was a class I splitting behavior in one cleavage, and 10 of the two types of cell division (all) in the second cleavage were classified as E-class embryos; the first cleavage occurred in class II division. There are 18, rated as F-class embryos. Each patient chooses 2-3 optimal embryos for transplantation, and the embryos are selected in order of A, B, C, D, E, and F. In the absence of A-class embryos, B-class embryos are considered. analogy. Of the 39 embryos selected for transplantation, 29 were Class A embryos, 9 were Class B embryos, and 1 was Class C embryos. Of the 19 patients who participated in the experimental analysis, 2-3 embryos transplanted from 11 were grade A embryos, 6 were transplanted with at least 1 grade A embryo, and 2 were not transplanted with grade A embryos. Transplanted grade B or C. Embryo.

A total of 20 patients were enrolled in the conventional morphological group, 1 was abolished, and 19 were involved in the experimental analysis.

Clinical outcomes were evaluated four weeks after embryo transfer. Compared with the conventional morphological embryo selection method, the split-behavior embryo grade assessment method can improve the embryo implantation rate (69.2% vs. 42.1%, in each group of 20 cases (with one person canceling the transplant). P < 0.05, statistically significant) and clinical pregnancy rate (78.9% vs. 57.9%, Ρ > 0.05), no statistically significant. Baseline characteristics and clinical outcomes of the two groups of participants are shown in Table 1.

Table 1 Baseline characteristics and clinical outcome parameters of the two groups of participants. Scheduling behavior group (n=20) Conventional morphology group (η=20) Depreciation female party age (years) 28.4±3· 5 28.5±3· 9 0.883 Female BMI (kg/m ² ) 21.4±2· 3 21.1±1· 8 0.678 Infertility years (years) 4.3±2· 9 3.8±2· 1 0.524

The number of Mil eggs (pieces) 15.2±4· 2 12.3±4·3 0.047 Number of transplanted embryos 2.1±0· 2 2.0±0 0.324 Number of high-quality embryos 2.1±0· 2 1.9±0· 3 0.086 Basic FSH (IU/L) 6.5±1· 5 6.6±1· 5 0.834 Basic LH (IU/L) 4.7±1·8 4.7±1· 9 0.943 Base E ₂ (pg/mL) 41.7±13· 9 38.6±11· 5 0.458 Cancellation rate (%) 5.0 (1/20) 5.0 (1/20) 1.000 Clinical pregnancy rate (%) 78.9 (15/19) 57.9 (11/19) 0.163 Implantation rate (%) 69.2 (27/39) 42.1 (16/38) 0.017 Multiple pregnancy rate (%) 73.3 (11/15) 45.5 (5/11) 0.149 Abortion rate (%) 0 (0/15) 9.1 (1/11) 0.234

Claims

Rights request

1. A hierarchical classification method for evaluating embryos treated with in vitro fertilization based on division behavior. The method is characterized in that the method is to move the embryos on the first day after fertilization into the embryo culture medium and observe them under the time difference system camera of the incubator. Embryonic cleavage status from the first to fifth days after fertilization; then classify the embryos according to the following steps:

(1) The embryonic division behavior is divided into the following eight types:

①Normal division; ②Abnormal division; ③Unequal size; ④Blastomere fragmentation; ⑤Large fragments; ⑥Abnormal cytoplasmic movement; ⑦Development arrest; ⑧Disordered division order;

(2) The above eight types of division behaviors of embryos are summarized into the following two categories:

Category I division behavior: normal division, abnormal cytoplasmic movement, unequal size, and large fragments affect blastocyst formation by less than 50%;

Category II cleavage behavior: developmental arrest, abnormal cleavage, disordered cleavage sequence, and blastomere fragmentation that affect blastocyst formation by greater than or equal to 50%;

(3) According to steps (1) and (2), the embryos are divided into six levels: A, B, C, D, E, and F: Level A: The first, second, and third cleavages all occur. Embryos with normal dividing behavior;

Level B: Embryos with at least one type I cleavage behavior other than normal cleavage behavior in the first, second and third cleavage;

Grade C: embryos with type I cleavage behavior in the first and second cleavage, and type II cleavage behavior in the third cleavage;

Grade D: Embryos with type I cleavage behavior in the first cleavage and one cell with type II cleavage behavior in the second cleavage;

Grade E: Embryos with type I cleavage behavior in the first cleavage and type II cleavage behavior in both cells during the second cleavage;

Grade F: Embryos with type II cleavage behavior in the first cleavage;

Among them, grade A embryos are the optimal embryos for transplantation; if there are no grade A embryos, grade B embryos are selected for transplantation; if grade A and grade B embryos are not available, grade C embryos are selected for transplantation; D. Grade E and F embryos are not used for transplantation.