CN110343615B - Culture dish for sperm-egg in-vitro combined fertilization and use method - Google Patents
Culture dish for sperm-egg in-vitro combined fertilization and use method Download PDFInfo
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- CN110343615B CN110343615B CN201910795566.0A CN201910795566A CN110343615B CN 110343615 B CN110343615 B CN 110343615B CN 201910795566 A CN201910795566 A CN 201910795566A CN 110343615 B CN110343615 B CN 110343615B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/10—Petri dish
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/061—Sperm cells, spermatogonia
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
The invention discloses a culture dish for in-vitro combined fertilization of sperm and eggs and a use method thereof, and relates to the technical field of assisted reproduction, wherein the culture dish comprises a substrate and a first groove body, a protrusion is arranged in the first groove body, the top surface of the protrusion is a curved surface which radiates from the middle to the periphery and gradually descends in height, and the curved surface protrudes towards the bottom surface of the protrusion; the middle of the top surface of the protrusion is downwards recessed to form an ovum groove for accommodating an ovum, and the notch of the ovum groove is upwards opened; a plurality of blocking rings are arranged on the curved surface at intervals along the radial direction, the blocking rings and the first groove body are coaxially arranged, and the heights of the top ends of the blocking rings are lower than the heights of the groove openings of the ovum grooves; the baffle ring positioned at the outermost side and the inner wall of the first groove body enclose a sperm ring groove for adding sperms on the top surface of the protrusion. The invention can perform in vitro horizontal and vertical bidirectional optimization selection on sperms, effectively reduces the incidence rate of fertilization of multiple sperms of the ova, and improves the fertilization rate, the high-quality embryo formation rate and the clinical pregnancy rate of the ova.
Description
Technical Field
The application relates to the technical field of assisted reproduction, in particular to a culture dish for in vitro combined fertilization of sperm and egg and a use method.
Background
Along with the release of the national comprehensive 'two children' policy, the domestic couples conforming to the two children standard are about 9000 ten thousand pairs, wherein 6000 ten thousand women are over 35 years old, and along with the superposition influence of environmental pollution, great working and living pressure, age increase, bad life style and other factors, the present investigation report of the sterility of China published by China population association and national family planning Commission shows that the incidence of sterility of China is 12.5% -15%, and the number of patients is about 4000 ten thousand. For fertility purposes, the most effective approach is by assisted reproductive technologies (artificial reproductive technology, ART) typified by in vitro fertilization-embryo transfer (in vitro fertilization-embryo transfer, IVF-ET).
ART involves complex, multi-linked laboratory procedures, but among many steps, the handling of In Vitro Fertilization (IVF) is certainly the core of the overall technology. When clinical indications such as female salpingemphraxis, ovulation disorder, endometriosis, immunological infertility, male mild oligospermia, infertility caused by unknown reasons are met, a conventional in vitro fertilization technology (first-generation test tube infant technology) is applied, so that sperms and ova with certain concentration are incubated together and combined to form fertilized ova. However, in vitro fertilization modes adopted by most reproductive medicine centers are not very much two at present: one is to add the treated sperm to a four-well plate or petri dish droplet containing the ovum, and the other is to add the ovum to a sperm culture solution of a conditioned density. However, in either method, the sperm is in direct contact with the ovum, which results in sperm contacting the ovum having sperm or dead sperm with no forward motility in addition to normal sperm, while even normal sperm have uneven quality (some moving in a rapid forward direction and others moving in a slower curved line) which may result in sperm associated with the ovum being of poor quality at a relatively short distance. In addition, during conventional in vitro fertilization operation of various large reproductive medicine centers, the number of added sperms is generally 2000-20000 sperms/egg, which is greatly higher than the number of sperms (tens or even less) reaching the fertilization site and contacting the ovum during natural conception, and high-concentration sperms (including dead sperms) can generate metabolites and even toxins, which can adversely affect fertilization ending of the ovum, subsequent cleavage and development of the embryo, and finally result in reduction of clinical pregnancy rate and embryo planting rate.
Natural conception is a process in which sperm undergoes a long journey to find ova, and the process undergoes strict natural physiological selection, and is a process of eliminating the winner and the obsolete of most (tens of millions or even hundreds of millions) sperm. In vitro fertilization technology has produced the first example of the test tube infant Louise Brown worldwide since 1978, which has been over 40 years old, and while helping thousands of patients to solve the problem of fertility difficulties, the safety of the in vitro fertilization procedure itself is not well defined in the current community due to the lack of sufficiently large sample offspring safety studies. The influence of in vitro operation is reduced as much as possible, so that the sperm is optimized in the horizontal direction and the vertical direction in vitro, and the sperm is combined with the ovum for fertilization after weak retention is eliminated, which is clearly favorable for producing high-quality embryos and healthy offspring.
However, in the process of implementing the technical solution in the embodiment of the present application, the present inventors have found that at least the following technical problems exist in the above prior art:
as sperm is not screened, the occurrence rate of multi-sperm fertilization in the sperm-egg in-vitro combination mode of the existing sperm and the ovum is higher, but the normal fertilization rate, the high-quality embryo formation rate and the clinical pregnancy rate of the ovum are lower.
Content of the application
According to the culture dish for sperm-egg in-vitro combined fertilization and the application method, the problems that sperm is not screened, the sperm-egg in-vitro combined mode of the existing sperm and the ovum is high in fertilization incidence rate, but the normal fertilization rate, the high-quality embryo formation rate and the clinical pregnancy rate of the ovum are low in the prior art are solved. According to the embodiment of the application, the volcano-shaped protrusion is arranged, the sperm ring groove is arranged at the bottom of the protrusion, the ovum groove is arranged at the top of the protrusion, sperms in the sperm ring groove are required to climb the slope to be combined with the ovum in the ovum groove, namely, the physical distance between the horizontal direction and the vertical direction is required to be travelled, so that the sperms combined with the ovum in the ovum groove are required to be screened through the horizontal movement and the vertical movement, the sperms are subjected to in vitro horizontal and vertical bidirectional optimization selection, dead sperm and weak sperm which are mingled are eliminated, the sperm concentration in the ovum groove is reduced, adverse effects on fertilization and incubation of the ovum caused by overlarge sperm concentration are avoided, the fertilization rate, the high-quality embryo formation rate and the clinical pregnancy rate of the ovum are improved while the fertilization rate of the ovum is effectively reduced, and the safety of filial generation is better ensured.
In order to solve the above problems, in a first aspect, an embodiment of the present application provides a culture dish for in vitro fertilization of sperm and eggs, where the culture dish includes a substrate and a plurality of first tanks, the first tanks are disposed on the substrate, and openings of the first tanks are opened upwards;
the first groove body is a circular groove, the central axis direction of the first groove body is taken as an axial direction, the diameter direction of the first groove body is taken as a radial direction, and a protrusion is coaxially arranged in the first groove body; the bottom surface of the protrusion is fixed on the inner bottom surface of the first groove body, the top surface of the protrusion is positioned in the first groove body, the top surface of the protrusion is a curved surface which radiates from the middle to the periphery and gradually descends in height, and the curved surface protrudes towards the bottom surface of the protrusion; the middle of the top surface of the protrusion is downwards recessed to form an ovum groove for accommodating an ovum, and the notch of the ovum groove is upwards opened;
a plurality of blocking rings are arranged on the curved surface at intervals along the radial direction, the blocking rings and the first groove body are coaxially arranged, the bottom ends of the blocking rings are fixed on the top surface of the protrusion, the top ends of the blocking rings vertically extend upwards, and the heights of the top ends of the blocking rings are lower than the heights of the grooves of the ovum grooves;
the barrier ring positioned at the outermost side and the inner wall of the first groove body enclose a sperm ring groove for adding sperms on the top surface of the protrusion.
Further, the height of the top end of each of the baffle rings is gradually increased from outside to inside along the radial direction.
Further, a second groove body is further arranged on the substrate, and an opening of the second groove body is opened upwards.
Further, the second groove body is a circular groove, and the number of the second groove bodies is one.
Furthermore, the protrusions below the ovum grooves are hollowed out, and the bottoms of the ovum grooves form transparent observation surfaces.
Further, the substrate is transparent.
Further, a liquid level scale mark is arranged in the inner wall of the first groove body, and the liquid level scale mark is positioned on the ovum groove.
Further, the ovum groove is a circular groove, and the ovum groove and the first groove body are coaxially arranged.
Further, the diameter of the egg groove is not smaller than 4mm.
In a second aspect, embodiments of the present application further provide a method for using a human sperm-egg in vitro-binding fertilization culture dish, comprising the steps of:
step 110: adding a fertilization culture solution into the first tank body, enabling the liquid level of the fertilization culture solution to be positioned at the liquid level scale mark, adding paraffin oil above the fertilization culture solution to maintain the osmotic pressure and the temperature stability of the fertilization culture solution, and finally placing the culture dish in an incubator for balancing;
step 120: placing the egg crown-cumulus compound in the second groove body for rinsing to obtain eggs, and then respectively placing the eggs in the egg grooves, wherein 1-10 eggs are placed in each egg groove;
step 130: placing the culture dish with the ovum back into an incubator for incubation, and then adding the semen sample into the semen groove;
step 140: the sperms positioned below and at the periphery move towards the ovum groove positioned above and in the middle, some dead sperms and sperms moving in situ are firstly retained in the ovum groove, while other sperms with poor activity are blocked by each blocking ring one by one, and only high-quality sperms with horizontal movement and vertical movement can be turned over each blocking ring one by one to reach the ovum groove and combined with the ovum for fertilization, so that sperms are preferred;
step 150: and (3) placing the culture dish added with the ovum and the sperm into an incubator for incubation, and completing the whole in-vitro combined fertilization process.
The above-mentioned one or more technical solutions in the embodiments of the present application at least have one or more of the following technical effects:
1. the sperm ring groove is arranged at the bottom of the protrusion, the sperm groove is arranged at the top of the protrusion, the sperm in the sperm ring groove is required to climb a slope to be combined with the ovum in the ovum groove, namely, the physical distance between the horizontal direction and the vertical direction is required to be travelled, so that the sperm which enters the ovum groove and is combined with the ovum is required to be screened through horizontal movement and vertical movement, the sperm is subjected to in vitro horizontal and vertical bidirectional optimization selection, the dead sperm and the weak sperm which are mingled are eliminated, the sperm concentration in the ovum groove is reduced, the adverse effect of the excessive sperm concentration on fertilization and incubation of the ovum is avoided, the fertilization rate, the high-quality embryo formation rate and the clinical pregnancy rate of the ovum are improved while the fertilization rate of the ovum is effectively reduced, and the safety of offspring is better ensured.
2. The height of the top end of each blocking ring is gradually increased from outside to inside along the radial direction, so that the optimization of sperms is carried out step by step, namely dead sperms and in-situ motile sperms are firstly retained in the sperm ring grooves, other sperms with insufficient vigor are blocked down one by one (forward and upward power is blocked by blocking plates with different distances and heights layer by layer), and only high-quality sperms with the capabilities of both horizontal swimming and vertical swimming can turn over the hillside on the upper surface of the protrusion to reach the egg grooves and are combined with eggs for fertilization, so that the effect of screening sperms is further played.
3. The top surface of the protrusion is a curved surface, the outer edge of the curved surface is connected with the inner wall of the first groove body, and the curved surface is in a certain radian to imitate the inner wall of the uterine cavity, so that the curved surface is as close to a sperm swimming path in a natural state as possible.
4. The baffle ring positioned at the outermost side can avoid artificial pushing of semen caused by artificial excessive boosting of the sample adding gun in the adding process, the circular sperm ring groove increases the area of the sperm sample adding area, and the same physical distance with the ovum groove can be kept no matter from which position of the sperm ring groove is added, so that the optimization of sperms is not influenced.
5. The second tank body is convenient for rinsing the cumulus compound of the egg crown so as to elute the adhered blood clots, impurities and residual follicular flushing fluid.
6. The protruding cavity of cuboid form is hollowed out to ovum groove below, and the chamber bottom of cavity with the tank bottom of ovum groove is all transparent, and the fretwork makes the ovum groove bottom surface forms a transparent slim observation surface, then can effectively avoid "volcano form" protruding too thick adverse effect that leads to the fact to ovum fertilization observation.
7. The arrangement of the liquid level scale marks can ensure that the culture solution can overflow the ovum groove, and can also play a role in verifying whether the volume of the added culture solution is quantitative or not.
8. The diameter of the egg grooves is not smaller than 4mm, and the number of eggs placed in each egg groove is preferably 4-6, so that the egg crown-cumulus compound is guaranteed to have enough space positions, the outer layers of the eggs are provided with radial egg dome granular cells during fertilization, adhesion and occupation of mutual space are easy to occur between the granular cells, and if the space is too small, the meeting and combination of sperms and the eggs are affected, so that fertilization fails.
The foregoing description is only an overview of the technical solutions of the present application, and may be implemented according to the content of the specification in order to make the technical means of the present application more clearly understood, and in order to make the above-mentioned and other objects, features and advantages of the present application more clearly understood, the following detailed description of the present application will be given.
Drawings
FIG. 1 is a schematic diagram of a culture dish for in vitro fertilization of sperm and eggs according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a first tank of a culture dish for in vitro fertilization of sperm and eggs according to an embodiment of the present invention;
FIG. 3 is a top view of a culture dish for in vitro fertilization of sperm and eggs in accordance with an embodiment of the present invention;
fig. 4 is a bottom view of a culture dish for in vitro fertilization of sperm and eggs in accordance with an embodiment of the present invention.
Reference numerals illustrate: the first groove body 1, the protrusion 11, the inner wall 12 of the first groove body, the liquid level scale mark 121, the sperm ring groove 13, the top surface 14 of the protrusion, the blocking rings 111, 141 and 142, the ovum groove 151, the notch 15, the concave cavity 16, the observation surface 17, the base plate 2 and the second groove body 3.
Detailed Description
According to the culture dish for sperm-egg in-vitro combined fertilization and the application method, the problems that sperm is not screened, the sperm-egg in-vitro combined mode of the existing sperm and the ovum is high in fertilization incidence rate, but the normal fertilization rate, the high-quality embryo formation rate and the clinical pregnancy rate of the ovum are low in the prior art are solved.
In order to solve the technical problems, the technical scheme provided by the application has the following overall thought: through setting up volcano form protruding, will the sperm annular is in protruding bottom, will the ovum groove sets up protruding top, the sperm in the sperm annular is to need climbing with the ovum combination in the ovum groove, needs the horizontal direction promptly and the vertical physical distance that need walk around for get into the sperm that combines with the ovum in the ovum groove need pass through horizontal motion and vertical motion's screening, with the horizontal, vertical bidirectional optimization selection to the sperm, elimination the dead sperm of inclusion, weak sperm, and reduced the sperm concentration in the ovum groove, so as to avoid the too big adverse effect that causes to the ovum fertilization and incubation of sperm concentration, also improved fertilization rate, high-quality embryo formation rate, clinical pregnancy rate, better assurance filial security of ovum when effectively reducing the fertilization rate of ovum many sperm.
The following detailed description of the technical solutions of the present application will be made with reference to the accompanying drawings and specific embodiments, and it should be understood that the specific features of the embodiments and embodiments of the present application are detailed descriptions of the technical solutions of the present application, and not limiting the technical solutions of the present application, and the technical features of the embodiments and embodiments of the present application may be combined with each other without conflict.
Example 1
Fig. 1 is a schematic structural diagram of a culture dish for in vitro fertilization of sperm and eggs, as shown in fig. 1, wherein the culture dish for in vitro fertilization of sperm and eggs comprises a substrate 2 and a plurality of first groove bodies 1, the first groove bodies 1 are arranged on the substrate 2, and openings of the first groove bodies 1 are opened upwards.
As shown in fig. 2 and 3, the first groove body 1 is a circular groove, the central axis direction of the first groove body 1 is taken as an axial direction, the diameter direction of the first groove body 1 is taken as a radial direction, and a protrusion 11 is coaxially arranged in the first groove body 1; the bottom surface of the protrusion 11 is fixed on the inner bottom surface of the first groove body 1, the top surface 14 of the protrusion 11 is positioned in the first groove body 1, the top surface 14 of the protrusion 11 is a curved surface which radiates from the middle to the periphery and gradually descends in height, and the curved surface protrudes towards the bottom surface direction of the protrusion 11; the middle of the protruding top surface 14 is recessed downward to form an ovum groove 151 for holding an ovum, and the notch 15 of the ovum groove 151 is opened upward.
Specifically, the top surface 14 of the protrusion 11 is a curved surface, the outer edge of the curved surface is connected with the inner wall 12 of the first groove body 1, and the curved surface has a certain radian to imitate the inner wall of the uterine cavity, so that the curved surface is as close to the sperm swimming path in a natural state as possible.
The curved surface is provided with a plurality of blocking rings 111, 141 and 142 at intervals along the radial direction, the blocking rings 111, 141 and 142 and the first groove body 1 are coaxially arranged, the bottom ends of the blocking rings 111, 141 and 142 are fixed on the top surface 14 of the protrusion 11, the top ends of the blocking rings 111, 141 and 142 vertically extend upwards, and the heights of the top ends of the blocking rings 111, 141 and 142 are lower than the heights of the notch 15 of the ovum groove 151.
The outermost barrier ring 111 and the inner wall 12 of the first groove 1 enclose a sperm ring groove 13 for adding sperm on the top surface 14 of the protrusion 11.
Further, the height of the tip of each of the baffle rings 111, 141, 142 gradually increases from the outside to the inside in the radial direction.
Specifically, the height of the top ends of the blocking rings 111, 141, 142 gradually increases from outside to inside along the radial direction, so that the optimization of sperms is performed step by step, that is, dead sperm and in-situ motile sperms will be retained in the sperm ring groove 13 first, while other sperm with insufficient motility will block the blocking rings 111, 141, 142 one by one (forward and upward power is blocked by blocking plates with different distances and heights layer by layer), and only high-quality sperms with both horizontal swimming and vertical swimming capability can turn over the hillside on the upper surface of the protrusion 11 to reach the ovum groove 151 and perform combined fertilization with the ovum, thereby further playing the role of screening sperms.
Specifically, the radial direction is inward toward the axial direction, and the radial direction is outward away from the axial direction. The blocking ring 111 located at the outermost side can avoid semen from being pushed manually due to excessive manual pushing of the sample gun during the adding process, the circular sperm ring groove 13 increases the area of the sperm sample adding area, and the same physical distance as the ovum groove 151 can be maintained no matter from which position of the sperm ring groove 13 is added, so that the optimization of sperm is not affected.
Specifically, the protrusion 11 is similar to volcanic, the sperm groove 13 is arranged on a mountain foot, the ovum groove 151 is arranged on a volcanic mouth at the mountain top, the sperm is required to climb a slope to the ovum, so that a physical distance between the sperm and the ovum in the horizontal and vertical directions is formed, and the sperm is required to cross a barrier (i.e. barrier rings 111, 141, 142) on the slope of the fire (the top surface 14 of the protrusion 11), thereby providing conditions for natural screening of the sperm.
The sperm ring groove 13 is arranged at the bottom of the protrusion 11, the sperm groove 151 is arranged at the top of the protrusion 11, the sperm in the sperm ring groove 13 is required to climb a slope to be combined with the ovum in the ovum groove 151, namely, the physical distance between the horizontal direction and the vertical direction is required to be travelled, and the sperm need to pass through each baffle ring 111, 141 and 142 one by one, so that the sperm which enters the ovum groove 151 and is combined with the ovum is required to be screened through horizontal movement and vertical movement, the sperm is subjected to in vitro horizontal and vertical bidirectional optimization selection, the dead sperm and the weak sperm which are mingled are eliminated, the sperm concentration in the ovum groove 151 is reduced, the adverse influence on fertilization and incubation of the ovum caused by the excessive sperm concentration is avoided, the fertilization rate, the high-quality embryo formation rate and the clinical pregnancy rate of the ovum are improved, and the safety of offspring is better ensured.
Further, a second groove body 3 is further disposed on the substrate 2, and an opening of the second groove body 3 is opened upwards.
Specifically, the second tank body 3 facilitates rinsing of the cumulus compound to elute the adhered blood clot, impurities and residual follicular flushing liquid.
Further, the second groove body 3 is a circular groove, and the number of the second groove bodies 3 is one.
Further, the number of the first grooves 1 is three, and the first grooves 1 and the second grooves 3 are arranged in a matrix on the substrate 2.
Further, the protrusion 11 below the ovum groove 151 is hollowed out, the groove bottom 1511 of the ovum groove 151 forms a transparent observation surface 17, as shown in fig. 4, and the substrate 2 is transparent.
Specifically, the protrusion 11 below the ovum groove 151 is hollowed out to form a cuboid cavity 16, the cavity bottom 161 of the cavity 16 and the groove bottom 1511 of the ovum groove 151 are transparent, and the hollowed-out bottom surface of the ovum groove 151 forms a transparent thin observation surface 17, so that adverse effects on fertilized observation of the ovum caused by the excessive thickness of the volcano-shaped protrusion 11 can be effectively avoided.
Further, a liquid level scale line 121 is disposed on the inner wall 12 of the first tank body 1, and the liquid level scale line 121 is located above the ovum groove 151.
Specifically, the liquid level scale marks 121 are arranged to ensure that the culture solution can overflow the egg groove 151 and to verify whether the volume of the culture solution is quantitative.
Further, the ovum groove 151 is a circular groove, and the ovum groove 151 is coaxially disposed with the first groove body 1.
Further, the diameter of the ovum groove 151 is not smaller than 4mm.
Specifically, the diameter of the ovum groove 151 is not smaller than 4mm, and the number of the ovum placed in each ovum groove 151 is preferably 4-6, so as to ensure that the ovum crown-cumulus compound has enough space, and the outer layers of the ovum during fertilization are all provided with radial ovum cumulus granule cells, so that adhesion and occupation of mutual space are easy to occur between the granule cells, and if the space is too small, the meeting and combination of sperms and the ovum are affected, thereby causing fertilization failure.
Example two
Based on the same inventive concept as the culture dish for in vitro combined fertilization of sperm and egg in the previous embodiment, the invention also provides a use method of the culture dish for in vitro combined fertilization of sperm and egg, which comprises the following steps:
step 110: adding a fertilization culture solution into the first tank body 1, enabling the liquid level of the fertilization culture solution to be positioned at the liquid level scale mark 121, adding paraffin oil above the fertilization culture solution to maintain the osmotic pressure and the temperature stability of the fertilization culture solution, and finally placing the culture dish in an incubator for balancing;
further, the step 110 specifically includes: adding a fertilization culture solution into the first tank body 1, enabling the liquid level of the fertilization culture solution to be located at the liquid level scale mark 121, adding a layer of culture paraffin oil which can just completely cover the liquid level of the fertilization culture solution above the fertilization culture solution so as to maintain the osmotic pressure and the temperature stability of the fertilization culture solution, and finally placing the culture dish into an incubator for balancing for 6-24 hours, wherein the setting environment in the incubator is as follows: the temperature was 37 ℃, the relative humidity was 100%, and the CO2 concentration was 6%.
Step 120: placing the egg crown-cumulus compound in the second groove body 3 for rinsing to obtain eggs, and then placing the eggs in the egg grooves 151 respectively, wherein 1-10 eggs are placed in each egg groove 151;
the step 120 specifically includes: placing the egg crown-cumulus compound (the eggs with granular cells on the periphery) obtained during the egg taking operation in the second groove body 3 for rinsing to elute the adhered blood clots, impurities and residual follicle flushing liquid, rinsing to obtain eggs, then respectively placing the eggs in the egg grooves 151, and placing 4-6 eggs in each egg groove 151;
step 130: placing the culture dish with the ovum back into an incubator for incubation, and then adding the semen sample into the semen groove;
the step 130 specifically includes: placing the culture dish with the ovum back into an incubator for incubation for 3-4 hours, wherein the setting environment in the incubator is as follows: the culture dish was removed from the incubator at 37℃with a relative humidity of 100% and a CO2 concentration of 6%, and a sperm sample of a certain concentration volume was sucked by a sample gun, gently added to the sperm cell 13, and uniformly added along the sperm cell 13.
Specifically, the manipulation is gentle when semen specimen is added to avoid artificial boosting of sperm.
Step 140: the sperm located below and at the periphery will swim to the egg groove 151 located above and in the middle, some dead sperm and sperm moving in situ will stay in the sperm groove 151 first, while other less viable sperm (curve slow swimming sperm) will be blocked by each blocking ring 111, 141, 142 one by one, only high quality sperm (straight fast swimming sperm) with both horizontal swimming and vertical swimming capability will flip one by one through each blocking ring 111, 141, 142 to the egg groove 151 and combine fertilization with an egg, thus sperm is preferred;
step 150: and (3) placing the culture dish added with the ovum and the sperm into an incubator for incubation, and completing the whole in-vitro combined fertilization process.
The step 150 specifically includes: placing the culture dish added with the ovum and the sperm into the incubator for incubation for 16-18 hours, wherein the environment in the incubator is as follows: the temperature is constant at 37 ℃, the relative humidity is 100%, the concentration of CO2 is constant at 6%, and the whole in vitro combined fertilization process is completed.
The various modifications and embodiments of the culture dish for in vitro fertilization of sperm and egg in the first embodiment of fig. 1 to 3 are equally applicable to the method for using the culture dish for in vitro fertilization of sperm and egg in the present embodiment, and by the detailed description of the culture dish for in vitro fertilization of sperm and egg in the foregoing, those skilled in the art will clearly know the method for using the culture dish for in vitro fertilization of sperm and egg in the present embodiment, so that the description will not be described in detail herein.
The above-mentioned one or more technical solutions in the embodiments of the present application at least have one or more of the following technical effects:
1. the sperm ring groove 13 is arranged at the bottom of the protrusion 11, the sperm groove 151 is arranged at the top of the protrusion 11, the sperm in the sperm ring groove 13 is required to climb a slope to be combined with the ovum in the ovum groove 151, namely, the physical distance between the horizontal direction and the vertical direction is required to be travelled, so that the sperm which enters the ovum groove 151 and is combined with the ovum is required to be screened through horizontal movement and vertical movement, the in vitro horizontal and vertical bidirectional optimization selection of the sperm is carried out, the dead sperm and the weak sperm which are mingled are eliminated, the sperm concentration in the ovum groove 151 is reduced, the adverse influence of the excessive sperm concentration on the fertilization and incubation of the ovum is avoided, the fertilization rate, the high-quality embryo formation rate and the clinical pregnancy rate of the ovum are improved while the fertilization rate of the multi-sperm is effectively reduced, and the safety of offspring is better ensured.
2. The height of the top ends of the blocking rings 111, 141, 142 gradually increases from outside to inside along the radial direction, so that the optimization of sperms is gradually performed, namely dead sperms and in-situ motile sperms are firstly retained in the sperm ring groove 13, other sperms with insufficient motility are blocked down the blocking rings 111, 141, 142 one by one (forward and upward power is blocked by blocking plates with different distances and heights layer by layer), and only high-quality sperms with both horizontal swimming and vertical swimming capabilities can turn over the hillside on the upper surface of the protrusion 11 to reach the egg groove 151 and combine with eggs for fertilization, thereby further playing the role of screening sperms.
3. The top surface 14 of the protrusion 11 is a curved surface, the outer edge of the curved surface is connected with the inner wall 12 of the first groove body 1, and the curved surface has a certain radian to imitate the inner wall of the uterine cavity, so that the curved surface is as close to the sperm swimming path in a natural state as possible.
4. The blocking ring 111 located at the outermost side can avoid semen from being pushed manually due to excessive manual pushing of the sample gun during the adding process, the circular sperm ring groove 13 increases the area of the sperm sample adding area, and the same physical distance as the ovum groove 151 can be maintained no matter from which position of the sperm ring groove 13 is added, so that the optimization of sperm is not affected.
5. The second tank body 3 is convenient for rinsing the cumulus compound to elute the adhered blood clots, impurities and residual follicular flushing liquid.
6. The protrusion 11 below the egg groove 151 is hollowed out to form a cuboid-shaped concave cavity 16, the cavity bottom 161 of the concave cavity 16 and the groove bottom 1511 of the egg groove 151 are transparent, and the hollowed-out bottom surface of the egg groove 151 forms a transparent thin observation surface 17, so that adverse effects on fertilized observation of eggs caused by excessively thick volcano-shaped protrusions can be effectively avoided.
7. The liquid level scale marks 121 are arranged to ensure that the culture solution can overflow the ovum groove 151 and to verify whether the volume of the culture solution is quantitative.
8. The diameter of the ovum groove 151 is not smaller than 4mm, and the number of the ovum placed in each ovum groove 151 is preferably 4-6, so as to ensure that the cumulus compound has enough space, and the outer layers of the ovum during fertilization are all provided with radial cumulus granule cells, so that adhesion and occupation of mutual space are easy to occur between the granule cells, and if the space is too small, the meeting and combination of sperms and the ovum are affected, and fertilization fails.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and equivalents are intended to be encompassed in the scope of the claims of the present invention.
Claims (8)
1. The culture dish for the in vitro combined fertilization of the sperm and the eggs is characterized by comprising a base plate and a plurality of first groove bodies, wherein the first groove bodies are arranged on the base plate, and the openings of the first groove bodies are upwards opened;
the first groove body is a circular groove, the central axis direction of the first groove body is taken as an axial direction, the diameter direction of the first groove body is taken as a radial direction, and a protrusion is coaxially arranged in the first groove body; the bottom surface of the protrusion is fixed on the inner bottom surface of the first groove body, the top surface of the protrusion is positioned in the first groove body, the top surface of the protrusion is a curved surface which radiates from the middle to the periphery and gradually descends in height, and the curved surface protrudes towards the bottom surface of the protrusion; the middle of the top surface of the protrusion is downwards recessed to form an ovum groove for accommodating an ovum, and the notch of the ovum groove is upwards opened;
a plurality of blocking rings are arranged on the curved surface at intervals along the radial direction, the blocking rings and the first groove body are coaxially arranged, the bottom ends of the blocking rings are fixed on the top surface of the protrusion, the top ends of the blocking rings vertically extend upwards, and the heights of the top ends of the blocking rings are lower than the heights of the grooves of the ovum grooves;
the barrier ring positioned at the outermost side and the inner wall of the first groove body enclose a sperm ring groove for adding sperms on the top surface of the protrusion;
the height of the top end of each blocking ring is gradually increased from outside to inside along the radial direction;
the substrate is also provided with a second groove body, and the opening of the second groove body is opened upwards.
2. A culture dish for in vitro fertilization of sperm and eggs as claimed in claim 1 wherein said second well is a circular well and the number of said second wells is one.
3. The culture dish for in vitro fertilization of sperm and eggs according to claim 1, wherein the protrusions below the egg grooves are hollowed out, and the bottoms of the egg grooves form transparent observation surfaces.
4. A culture dish for in vitro fertilization of sperm-egg as described in claim 3 wherein said substrate is transparent.
5. The culture dish for in vitro fertilization of sperm and eggs according to claim 1, wherein liquid level graduation marks are arranged in the inner wall of the first groove body and are positioned above the ovum groove.
6. A culture dish for in vitro fertilization of sperm-egg as described in claim 1 wherein said egg well is a circular well and wherein said egg well is coaxially disposed with said first well.
7. A culture dish for in vitro fertilization of sperm-egg as described in claim 6 wherein said egg well has a diameter of no less than 4mm.
8. A method of using a sperm-egg in vitro fertilization culture dish as described in any one of claims 1 to 7, comprising the steps of:
step 110: adding a fertilization culture solution into the first tank body, enabling the liquid level of the fertilization culture solution to be positioned at the liquid level scale mark, adding paraffin oil above the fertilization culture solution to maintain the osmotic pressure and the temperature stability of the fertilization culture solution, and finally placing the culture dish in an incubator for balancing;
step 120: placing the egg crown-cumulus compound in the second groove body for rinsing to obtain eggs, and then respectively placing the eggs in the egg grooves, wherein 1-10 eggs are placed in each egg groove;
step 130: placing the culture dish with the ovum back into an incubator for incubation, and then adding the semen sample into the semen groove;
step 140: the sperms positioned below and at the periphery move towards the ovum groove positioned above and in the middle, some dead sperms and sperms moving in situ are firstly retained in the ovum groove, while other sperms with poor activity are blocked by each blocking ring one by one, and only high-quality sperms with horizontal movement and vertical movement can be turned over each blocking ring one by one to reach the ovum groove and combined with the ovum for fertilization, so that sperms are preferred;
step 150: and (3) placing the culture dish added with the ovum and the sperm into an incubator for incubation, and completing the whole in-vitro combined fertilization process.
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| JP2010094045A (en) * | 2008-10-14 | 2010-04-30 | Sumitomo Bakelite Co Ltd | Multi-well plate |
| CN103421683A (en) * | 2012-05-25 | 2013-12-04 | 曾勇 | Sperm competition fertilization dish and application method thereof |
| CN107593582A (en) * | 2017-08-24 | 2018-01-19 | 寿县临淮畜牧养殖有限公司 | A kind of breeding method for improving Anhui White Goats reproductive capacity |
| CN109468226A (en) * | 2018-11-16 | 2019-03-15 | 浙江大学 | Simulate the device in vitro fertilization and application method in sperm travelling path under natural mode |
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| US7618770B2 (en) * | 2005-07-29 | 2009-11-17 | Xy, Inc. | Methods and apparatus for reducing protein content in sperm cell extenders |
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| JP2010094045A (en) * | 2008-10-14 | 2010-04-30 | Sumitomo Bakelite Co Ltd | Multi-well plate |
| CN103421683A (en) * | 2012-05-25 | 2013-12-04 | 曾勇 | Sperm competition fertilization dish and application method thereof |
| CN107593582A (en) * | 2017-08-24 | 2018-01-19 | 寿县临淮畜牧养殖有限公司 | A kind of breeding method for improving Anhui White Goats reproductive capacity |
| CN109468226A (en) * | 2018-11-16 | 2019-03-15 | 浙江大学 | Simulate the device in vitro fertilization and application method in sperm travelling path under natural mode |
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