阅读说明：本技术 一种用压电破膜技术进行pgd/pgs胚胎活检的方法 (Method for carrying out PGD/PGS embryo biopsy by piezoelectric membrane rupture technology ) 是由 刘晓 刘璟青 王娟 于 2021-08-11 设计创作，主要内容包括：本发明公开了一种用压电破膜技术进行PGD/PGS胚胎活检的方法。本发明解决了发育中的胚胎穿过透明带分离捕获单个极体或卵裂球、孵出前的滋养层细胞的问题,避免了激光捕获辅助技术应用带来的各种实际问题,尽可能最大程度上保持了需要处理的胚胎发育的活性,这一技术是单个极体或卵裂球、孵出前的滋养层细胞分离的基础,也是后续各项针对上述获取细胞样品进行检测的前期基础,这一技术还可以根据不同需要处理胚胎情况进行各项参数调整并记录,方便了实际操作,提高了实验的工作效率,保证并提高了检测的成功率,最为重要的是这种方法极大地保护了需要处理的各种胚胎活性,提高了后续胚胎发育、着床受孕、活产等的成功率。(The invention discloses a method for carrying out PGD/PGS embryo biopsy by using a piezoelectric membrane rupture technology. The invention solves the problem that the developing embryo passes through the zona pellucida to separate and capture single polar body or blastomere and trophoblast cells before hatching, avoids various practical problems brought by the application of laser capture auxiliary technology, maintains the development activity of the embryo to be processed to the greatest extent, the technology is the basis of the separation of trophoblast cells before the single polar body or blastomere and the hatching, and is also the early basis of the detection of the obtained cell samples in the following items, the technology can adjust and record various parameters according to different embryo processing conditions, thereby facilitating practical operation, improving the working efficiency of experiments, ensuring and improving the success rate of detection.)

1. A method for PGD/PGS embryo biopsy by using a piezoelectric membrane rupture technology is characterized by comprising the following steps:

step 1, preparing a plurality of operation liquid drops and control liquid drops in the middle of a culture dish by using a liquid transfer device and a matched gun head;

step 2, moving the blastocyst to be processed into the operation liquid drop, and observing the structural form of the blastocyst to be processed in a focusing manner under a microscope;

step 3, dripping heavy oil into the micro-operation needle by using a liquid transfer device and a micro-sample loading needle, and pushing the heavy oil to the head of the micro-operation needle by using a connected oil pressure type micro-injection instrument after installing a micro-operation system;

step 4, operating the operation side micromanipulator, lowering the operation needle into the operation liquid drop to test the function of the piezoelectric membrane rupture device, and controlling the operation liquid drop to clean the head of the operation needle by using the oil pressure injection device until the pressure is balanced;

step 5, operating the micromanipulator (fixed side) on the other side, and adjusting the operating needle to fix the blastocyst to be processed;

step 6, operating the operation side micromanipulator to drive the micromanipulator needle to move to the edge of the fixed blastocyst, and contacting the operating needle with the transparent belt of the blastocyst;

step 7, starting piezoelectric vibration to drive the micro-operation needle to vibrate the contact part on the transparent belt, and observing the formation phenomenon of holes on the transparent belt of the blastocyst;

step 8, according to the actual situation under the microscope, adjusting various proper parameters of the piezoelectric film rupture instrument to achieve the purposes of completing the hole formation on the transparent belt and preserving the embryo to be processed;

9, if the punching result is satisfactory, stopping the work of the piezoelectric membrane rupturing instrument, and controlling the operation needle to suck out enough trophoblast cells through the holes on the transparent belt;

step 10, cutting off trophoblast cells and blastocysts in the needle port on the embryo fixing needle by using a biopsy needle through the matching of the operation needles on the two sides, and then spitting out the trophoblast cells in the biopsy needle port to complete the operation action of separation;

and step 11, moving the operating needles and the fixing needles on the two sides to perform the next operating action of processing the embryo.

2. The method for PGD/PGS embryo biopsy by piezoelectric rupture of membranes technology according to claim 1, wherein the pipette of step 1 is 100 μm pipette and the culture dish is 60mm dish.

3. The method for PGD/PGS embryo biopsy using piezoelectric rupture of membranes technique of claim 1, wherein step 4 is dropping 4 μm heavy oil.

4. The method for PGD/PGS embryo biopsy by piezoelectric rupture of membranes technique of claim 3, wherein the heavy oil is Sigma FC-770.

5. The method for PGD/PGS embryo biopsy with piezoelectric rupture of membranes technology of claim 1, wherein the parameters of the piezoelectric rupture of membranes instrument of step 8 are intensity 30, speed 20, and infinite number of times.

Technical Field

The invention relates to the technical field of smart cities, in particular to a method for carrying out PGD/PGS embryo biopsy by using a piezoelectric membrane rupture technology.

Background

At present, along with the development of modern society, the sterile and infertile population is more and more, various test-tube infant technologies are developed, wherein besides IVF-ET (in vitro fertilization) and ICSI (single sperm intracytoplasmic injection), related reproductive medicine technologies of various PGD/PGS (genetic diagnosis before embryo transplantation/genetic screening before embryo transplantation) for blocking various genetic defects are developed almost simultaneously, the methods need to obtain biological samples capable of carrying out subsequent detection and diagnosis in advance, including various polar bodies, blastomeres, trophoblasts and the like, then obtain different genetic information through subsequent FISH (in situ hybridization), PCR (polymerase chain reaction), sequencing technologies and the like, and then determine whether the detected embryo is continuously developed, so different technologies for obtaining the cells are correspondingly developed, and the current biopsy technology of various polar bodies, blastomeres and trophoblasts before hatching is realized through various laser beam rupture meters to generate adjustable laser beams on the periphery of the embryo The transparent upper part of the needle is punched or thinned to assist various subsequent biopsy needles to insert and sample along the corresponding parts on the treated transparent belt.

In the operation process, a complex and expensive laser membrane rupturing instrument is needed, the condition of each embryo zona pellucida to be processed is different, the generated laser beam cannot necessarily reach the optimal state, different parameter combinations such as the intensity, time, diameter and the like of the laser beam need to be debugged repeatedly, time and labor are wasted, particularly, the heat effect caused by the laser beam generated in the operation process can cause damage to the embryo to be processed to a certain extent in the process, the sample detection rate is reduced, more seriously, the heat damage caused by the laser operation can also influence the subsequent development of the embryo, in addition, the laser beam is often perpendicular to the edge of the embryo in the actual operation and goes through a hole in the tangential direction, a groove is directly formed on the surface of the embryo zona pellucida in the mode, the damage to the embryo is larger instead of an ideal hole oil, the characteristics are that the embryo development rate is directly reduced, the implantation rate, the conception rate and the live rate of the operated embryo are reduced, and how to avoid the damage caused by various assisted reproductive micromanipulations needing laser assistance technology becomes a great technical problem to be solved urgently.

Disclosure of Invention

1. Technical problem to be solved

The invention provides a PGD/PGS embryo biopsy method by piezoelectric film-breaking technology, which comprises the steps of punching or thinning transparent tapes on the embryo transparent tapes by a special piezoelectric film-breaking instrument driven by high-frequency mechanical vibration generated after electrification, stopping piezoelectric film-breaking vibration, and inserting needles along the holes or weak points on the processed transparent tapes by the micromanipulation needles to complete the subsequent various purposes of separating and capturing single polar bodies or blastomeres and trophoblast cells before hatching.

2. Technical scheme

In order to achieve the purpose, the invention provides the following technical scheme: a method for PGD/PGS embryo biopsy by piezoelectric rupture of membranes comprises the following steps:

step 1, preparing a plurality of operation liquid drops and control liquid drops in the middle of a culture dish by using a liquid transfer device and a matched gun head;

step 2, moving the blastocyst to be processed into the operation liquid drop, and observing the structural form of the blastocyst to be processed in a focusing manner under a microscope;

step 3, dripping heavy oil into the micro-operation needle by using a liquid transfer device and a micro-sample loading needle, and pushing the heavy oil to the head of the micro-operation needle by using a connected oil pressure type micro-injection instrument after installing a micro-operation system;

step 4, operating the operation side micromanipulator, lowering the operation needle into the operation liquid drop to test the function of the piezoelectric membrane rupture device, and controlling the operation liquid drop to clean the head of the operation needle by using the oil pressure injection device until the pressure is balanced;

step 5, operating the micromanipulator (fixed side) on the other side, and adjusting the operating needle to fix the blastocyst to be processed;

step 6, operating the operation side micromanipulator to drive the micromanipulator needle to move to the edge of the fixed blastocyst, and contacting the operating needle with the transparent belt of the blastocyst;

step 7, starting piezoelectric vibration to drive the micro-operation needle to vibrate the contact part on the transparent belt, and observing the formation phenomenon of holes on the transparent belt of the blastocyst;

step 8, according to the actual situation under the microscope, adjusting various proper parameters of the piezoelectric film rupture instrument to achieve the purposes of completing the hole formation on the transparent belt and preserving the embryo to be processed;

9, if the punching result is satisfactory, stopping the work of the piezoelectric membrane rupturing instrument, and controlling the operation needle to suck out enough trophoblast cells through the holes on the transparent belt;

step 10, cutting off trophoblast cells and blastocysts in the needle port on the embryo fixing needle by using a biopsy needle through the matching of the operation needles on the two sides, and then spitting out the trophoblast cells in the biopsy needle port to complete the operation action of separation;

and step 11, moving the operating needles and the fixing needles on the two sides to perform the next operating action of processing the embryo.

The method for embryo biopsy, wherein the pipette in step 1 is a 100 μm pipette, and the culture dish is a 60mm culture dish.

The method for embryo biopsy, wherein step 4 comprises dripping 4 μm heavy oil.

The embryo biopsy method, wherein the heavy oil is Sigma FC-770.

The method for embryo biopsy, wherein the parameters of the piezoelectric film breaking instrument in the step 8 are intensity 30, speed 20 and infinite times.

3. Advantageous effects

In conclusion, the beneficial effects of the invention are as follows:

(1) the method for embryo biopsy solves the problems that the developing embryo passes through the zona pellucida to be separated and capture a single polar body or blastomere and trophoblast cells before hatching, avoids various practical problems caused by the application of a laser capture auxiliary technology, and keeps the development activity of the embryo to be processed to the greatest extent possible;

(2) the embryo biopsy method is the basis for separating trophoblast cells before single polar body or blastomere and hatching, and is also the early basis for detecting the obtained cell samples in the following steps;

(3) the embryo biopsy method provided by the invention can adjust and record various parameters according to different embryo processing conditions, thereby facilitating actual operation, improving the working efficiency of experiments, and ensuring and improving the success rate of detection;

(4) the embryo biopsy method greatly protects the activity of various embryos needing to be processed, and improves the success rate of subsequent embryo development, implantation and pregnancy, live birth and the like.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The basic configuration of the method of embryo biopsy of the present invention is as follows;

(5) micromanipulator x2 (for precision moving micromanipulator needle)

(6) Manual air pressure type microinjector x1 (for absorbing and releasing plant cells or conventional single cell samples)

(7) Manual oil pressure microinjector x1 (for sucking and releasing conventional single cell sample or single plant stem cell of plant)

(8) Inverted microscope full set x1 (for accurate observation of plant unicellular samples, various conventional inverted mirrors can be used)

(9) Piezoelectric membrane-breaking instrument x1 (for generating a suitable vibration under the cooperation of the above device and consumable material, transmitting to a connected micromanipulation needle, perforating or thinning a zona pellucida containing various embryos to be processed by the vibration of the micromanipulation needle, and then selectively moving and releasing various single cell samples such as single polar bodies or blastomeres containing intact nuclei required to be selected by the device)

(10)10uL pipette x1, with several tips (for adding heavy oil to the micromanipulation needle, generating piezoelectric vibration, and for preparing various micromanipulation drops below 10 uL)

(11)100uL pipette x1, with several tips (for the preparation of various 10-100uL micromanipulation droplets)

(12) Microscope adapter (microscope to microscope)

The required consumables are as follows;

(1) several 60mm cell culture dishes (embryo treatment and single cell separation droplets, and blank control droplets were prepared using their dish lids as carriers, and various related cell micromanipulations were performed.)

(2) Micro loader micro loading needles (for adding heavy oil for generating piezoelectric vibration into micro-operation needle tube)

(3) A plurality of micromanipulation needles/biopsy needles (for receiving vibration generated by the following piezoelectric membrane rupture device and transmitting the vibration to various embryo cell micromanipulation needles to cause vibration action, so as to cause the formation of holes at corresponding parts on the embryo transparent contacted with the micromanipulation needles or directly thin the parts on the transparent belt contacted with the micromanipulation needle openings, and then, the micromanipulation needles/biopsy needles can be matched with the equipment to select, move and release various cell samples needing genetic identification, such as various single polar bodies/blastomeres containing complete cell nuclei, trophoblasts and the like which are required to be selected.)

(4) Embryo fixing needle (for fixing embryo in micromanipulation)

The required reagents are as follows;

(5) the embryo operating fluid is a plurality

(6) Sigma FC-770 heavy oil 200uL (micro loading needle matched with 10uL pipette is added into micro-operation needle tube to generate controllable high-frequency micro vibration with proper intensity)

A method for PGD/PGS embryo biopsy by piezoelectric rupture of membranes comprises the following steps:

step 1, preparing a plurality of operation and control liquid drops in the middle of a 60mm culture dish by using a 100uL pipettor and a matched gun head;

step 2, moving the blastocyst to be processed into the operation liquid drop, and observing the structural form of the blastocyst to be processed in a focusing manner under a microscope;

step 3, filling about 5uL of heavy oil into the micro-operation needle by using a 10uL pipette and a micro-sample loading needle, and pushing the heavy oil to the head of the micro-operation needle by using a connected oil pressure type micro-injection instrument after a micro-operation system is installed;

step 4, operating the operation side micromanipulator, lowering the operation needle into the operation liquid drop to test the function of the piezoelectric membrane rupture device, controlling the operation liquid drop by using an oil pressure injection device to clean the head of the operation needle, and finally, preparing a formal experiment after pressure is balanced;

step 5, operating the micromanipulator (fixed side) on the other side, and adjusting the operating needle to fix the blastocyst to be processed;

step 6, operating the operation side micromanipulator to drive the micromanipulator needle to move to the edge of the fixed blastocyst, and contacting the operating needle with the transparent belt of the blastocyst;

step 4, starting piezoelectric vibration to drive the micro-operation needle to vibrate the contact part on the transparent band, wherein the hole formation phenomenon on the transparent band of the processed blastocyst can be seen under a microscope (in actual operation, once a hole is formed due to higher pressure in the blastocyst cavity, the expanded blastocyst cavity can be rapidly shrunk, and whether the hole is formed can also be judged);

step 8, according to the actual situation under the mirror, adjusting various appropriate parameters of the piezoelectric film rupture instrument, including strength, speed, frequency and the like (the experiment sets parameters, the strength is 30, the speed is 20 times/, the frequency is infinite, namely, the pedal is stepped on to generate vibration, and the pedal is released to stop vibration after the effect is achieved), so as to achieve the purposes of completing the hole forming on the transparent belt and storing the embryo to be processed, and if the hole forming result is satisfied, a whole set of parameter combination can be stored through a program recording function;

step 9, stopping the work of the piezoelectric membrane rupture instrument, and directly using the operation side operation instrument to control the operation needle to suck out enough trophoblast cells through the broken hole on the transparent band;

step 10, cutting off trophoblast cells and blastocysts in the needle port on the embryo fixing needle by using a biopsy needle through the matching of the operation needles on the two sides, and then spitting out the trophoblast cells in the biopsy needle port to complete the operation action of separation;

and 11, moving the operating needles and the fixed needles on the two sides, performing the next operation action of processing the embryo, and after separating the cell sample, performing various subsequent processing, such as FISH (in situ hybridization), PCR (polymerase chain reaction) detection, various single cell sequencing and other detection operations.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.