RU2613788C1 - Method for trophoblast cells biopsy at multiplicity of cells that do not participate in compaction at blastocyst stage for human embryos preimplantation genetic screening programmes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for trophoblast cells biopsy in human embryos preimplantation genetic screening programs, blastocysts pellucid zone is completely removed in the presence of multiple cells that do not participate in compaction. Trophoblast cells are freed, biopsy is performed not including the cells that are not involved in compaction into the material transferred to genetic diagnosis.

EFFECT: method allows to obtain biopsy material of good quality due to the complete removal of the pellucid zone.

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Description

Preimplantation genetic screening (ASG) is currently the most modern and most accurate method of breeding embryos in vitro to prevent the birth of children with congenital and hereditary diseases. ASG serves as an alternative to prenatal diagnosis and selective termination of early pregnancy in couples with a high risk of having children with congenital anomalies (Dahdouh E. et al., 2015). As a material for the genetic study of embryos, one or two blastomeres of the embryo can be used for 3 days of cultivation, or trophoblast cells of the blastocyst. Compared to a blastomere biopsy, trophoectoderm biopsy reduces the likelihood of selection for analysis of mosaic cells, which increases the sensitivity and specificity of ASG (Huang J. et al., 2015; Twisk M. et al., 2006). However, when culturing human embryos in in vitro fertilization programs, not all blastocysts obtained are suitable for biopsy and subsequent diagnosis. In some patients, during the formation of a compact morula, and subsequently blastocysts, not all blastomeres form an embryo. Some cells do not participate in compaction, but at the same time remain inside the pellicidal zone. Such cells (the so-called extracellular cells) can interfere with the formation of the blastocele and the subsequent exit of the embryo from the transparent membrane. The reasons why extracells are not involved in blastocyst formation are currently unknown.

, 2003). Поверхностные микроворсинки наблюдают лишь в некоторых базальных участках, основная их масса расположена на апикальном полюсе клеток, который проявляет повышенную лиганд-связывающую способность. Актиновые микрофиламенты концентрируются под апикальной поверхностью, микротрубочки и митохондрии выстраиваются параллельно базолатеральным мембранам, колонки эндоцитозных пузырьков располагаются между апикальным полюсом и базально-расположенными ядрами.It has been shown that during compaction, changes in the extra- and intracellular organization occur, including the polarization of blastomeres, the formation of intercellular contacts and the interaction of the cytoskeleton of neighboring blastomeres (Fleming et al., 2000., 2001), membrane transport systems, surface glycoproteins, antigens change. Prior to compaction, the blastomeres are spherical in shape and have a small number of intercellular contacts, microvilli are evenly distributed over the surface of the cells, and mitochondria in the cytoplasm are arranged in random order. In the early stages of compaction, cells acquire epithelial polarity, apical microvilli, and smoothed lateral surfaces. Changes in the location of microvilli are accompanied by a redistribution of the localization of mitochondria and cortical elements of the cytoskeleton (Veeck,

, 2003). Superficial microvilli are observed only in some basal areas, their bulk is located at the apical pole of the cells, which exhibits an increased ligand-binding ability. Actin microfilaments are concentrated under the apical surface, microtubules and mitochondria line up parallel to the basolateral membranes, columns of endocytotic vesicles are located between the apical pole and the basally located nuclei.

Apparently, the appearance of extracells indicates a violation of the above-described processes of compaction in individual blastomeres of an 8-cell embryo, which leads to their elimination from further development.

During a blastocyst biopsy, the embryologist takes no more than 5-8 trophoblast cells for analysis. Trophoblast cells are harvested after preliminary application of assisted hatching on day 4 after fertilization. According to European recommendations, it is advisable to conduct a biopsy of only those trophoblast cells that have left the pellucid zone so as not to damage the emerging embryo. If trophoblast cells do not exit the zone, prolonged cultivation is recommended. The appearance of extracells in the blastocyst significantly complicates the biopsy procedure.

It should also be remembered that extracells during trophoblast biopsy can lead to contamination of the biopsy material, and even lead to incorrect diagnosis, since these cells do not belong to the forming embryo. In the published foreign and domestic literature (due to the relative novelty of the ASG technique on trophoblast cells), there are no indications of a suboptimal quality blastocyst biopsy technique.

Currently, the world lacks uniform requirements and approved protocols for blastocyst biopsy, as well as a single standard for the implementation of ASG programs. This is due to various legislative acts in different countries (including differences in the ethical components of manipulations with human embryos). The guidelines of the European Association for Human Reproduction (ESHRE) describe the appearance of extracellules, but recommendations for suboptimal quality blastocyst biopsies with extracells are not given (ESHRE, 2010). The use of traditional biopsy techniques can lead to collapse and subsequent degeneration of the blastocyst.

The aim of the present invention is to develop a biopsy method of a human blastocyst in preimplantation genetic screening programs for multiple extracells inside the perivitelline space under the zone of the pellucide.

This problem is solved by using the trophoblast cell biopsy method for multiple blastocyst extracells in preimplantation genetic screening programs for human embryos, which is distinguished by the fact that such blastocysts remove the pellucid zone, release trophoblast cells, conduct biopsy and do not include extracells in the material transferred for genetic diagnosis.

The developed biopsy method allows to increase the number of blastocysts analyzed by comparative genomic hybridization by 24 pairs of chromosomes, to select more euploid embryos for transfer to the uterine cavity in infertility treatment programs using assisted reproductive technologies.

The advantages of the proposed blastocyst trophectoderm cell biopsy technique have been confirmed on 17 human blastocysts. All patients signed voluntary informed consent. Blastocysts of suboptimal quality were biopsied using the complete removal of the pellucide zone, the material was given for genetic diagnosis, the embryos were cryopreserved. After thawing, all embryos were of good quality and continued the expansion process.

Clinical examples

1. Patient A., 39 years old, came for treatment of infertility with IVF / ICSI / ASG and embryo transfer. A complete clinical examination was carried out. A history of four in vitro fertilization programs with the absence of good quality blastocysts for transfer. Pregnancy in the patient did not occur. Gynecological and endocrinological statuses without features. Husband, 43 years old, according to spermogram results - asthenozoospermia, secondary infertility. Spouse was stimulated with ovulation according to the modified short protocol with gonadotropin releasing hormone antagonists, transvaginal follicular puncture was performed, and 8 mature oocytes were obtained. Fertilization was carried out using the Pixie method (sperm selection for binding to hyaluronic acid). On day 3 of cultivation, fragmentation of all embryos was observed. On the 5th day of cultivation, two embryos - cavitating morula with many extracells. A trophoblast biopsy was not possible, as there was a high risk of contamination of the material with extracells. It was decided to completely remove the pellucide zone. The implementation of auxiliary hatching allowed to completely release the cells of the trophoblast and embryoblast and correctly conduct a biopsy. As a result, a genome-wide screening was performed, euploid embryos and genetically normal embryo transfer to the uterine cavity were selected. Pregnancy has come. Currently progressing.

2. Patient B., 30 years old, applied for the treatment of infertility using IVF / ICSI / ASG and embryo transfer. A complete clinical examination was carried out. Gynecological and endocrinological statuses without features. Karyotype of patient 46, XX, der (14; 21) (q10; q10), Robertson translocation between chromosomes 14 and 21. There is a healthy girl (the karyotype is unknown). A history of late termination of pregnancy due to fetal pathology (46, XX, der (14; 21) (q10; q10), +21). Pregnancy has come on its own. Husband, 33 years old, according to spermogram results - asthenozoospermia, secondary infertility. Spouse was stimulated with ovulation according to the modified short protocol with gonadotropin releasing hormone antagonists, transvaginal follicle puncture was performed, 3 mature oocytes were obtained. Fertilization was performed by the ICSI method. On the 5th day of cultivation, two embryos - a cavitating morula (with extracells with perivitellin space) and a good quality blastocyst. The third embryo stopped developing at the stage of 8 fragmentation blastomeres.

A good quality blastocyst trophoblast biopsy was performed using the classical method. On cavitating morula, a complete removal of the pellucide zone was carried out. After 4 hours, the expanded blastocyst was probiotic. As a result, a genome-wide screening was performed. Of the two embryos, 1 is euploid, 2 - heteroploid with multiple genetic pathologies. A genetically normal embryo was transferred to the uterine cavity. Pregnancy has not come. The couple is preparing for a re-program of IVF / ICSI / ASG.

Thus, the developed trophoblast cell biopsy technique is an effective method for working with suboptimal blastocysts, which allows trophoblast cell sampling, preimplantation genetic screening, and an increase in the effectiveness of infertility treatment programs using assisted reproductive technologies.

Bibliography

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Claims (1)

The trophoblast cell biopsy method in preimplantation genetic screening programs for human embryos, characterized in that the blastocysts in the presence of multiple cells that are not involved in compaction are completely removed from the pellucid zone, trophoblast cells are released, biopsy is performed and cells that are not involved in compaction are not included in the material transmitted to genetic diagnosis.