CN107475078A - A kind of sperm screening based on microflow control technique and bionic device in vitro fertilization - Google Patents
A kind of sperm screening based on microflow control technique and bionic device in vitro fertilization Download PDFInfo
- Publication number
- CN107475078A CN107475078A CN201710944615.3A CN201710944615A CN107475078A CN 107475078 A CN107475078 A CN 107475078A CN 201710944615 A CN201710944615 A CN 201710944615A CN 107475078 A CN107475078 A CN 107475078A
- Authority
- CN
- China
- Prior art keywords
- sperm
- pipeline
- adding mouth
- pool
- outlet pond
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12M23/16—Microfluidic devices; Capillary tubes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Clinical Laboratory Science (AREA)
- Dispersion Chemistry (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of sperm screening based on microflow control technique and bionic device in vitro fertilization, including bionic device body, the bionic device body is by slide layer, silica gel pipe layers and dimethyl silicone polymer layer composition, central pool is provided with the middle part of the silica gel pipe layers, entrance pool is provided with the outside of the central pool, first outlet pond and second outlet pond, the central pool and entrance pool, first outlet pond and second outlet pond pass through the first pipeline respectively, second pipe and the 3rd pipeline communication, first pipeline, the first adding mouth is further respectively had on second pipe and the 3rd pipeline, second adding mouth and the 3rd adding mouth.This bionic device simulates natural conception process completely, avoids various operations to gamete and the potential for adverse effects of embryo, and can avoid the generation of polyspermy；The embryo quality under simulation natural conception state can be obtained, success rate in vitro fertilization can be greatly improved, the potential cause for causing infertility can be identified, so as to be pointed the direction for clinical treatment.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of micro fluidic device, more particularly to one kind is based on microflow control technique
Sperm screening with bionic device in vitro fertilization and should based on microflow control technique sperm screening with bionic device in vitro fertilization
Preparation method and method of work.
Background technology
The infertile incidence of disease is about 15%~20% in world wide, and has cumulative year after year trend.It is pre- according to WHO
Survey, in the near future, healthy reproduction problem will be that the third-largest disease of human health is threatened after cardiovascular and cerebrovascular disease and tumour
Disease.The increase of the infertile incidence of disease, and the relieving of national " two children " policy, have promoted the hair of auxiliary procreation technology significantly
Exhibition.First generation tube-test baby techniques --- IVF-ET Technique (IVF-ET) is since 1978 carry out, to many
Infertility Mr. and Mrs bring Gospel；Second generation tube-test baby techniques --- intracytoplasmic sperm injection (ICSI) technology and
The further abundant and perfect auxiliary procreation technology of third-generation technology --- PGD (PGD).Nevertheless,
It is many during auxiliary procreation technology to operate the damage that cause embryo, embryo's agensis or miscarriage are ultimately resulted in, this
It is the reason for causing tube-test baby techniques success rate to be difficult to more than 50%.These operations for having damage include：After sperm swim-up
Centrifugally operated, density gradient centrifugation operation and separating liquid in some compositions influence, wear ovum operation and culture environment and culture
Influence of liquid composition etc..And the influence that avoid these from operating, it is in vitro fertilization to simulate natural conception as far as possible.
The normal process of becoming pregnant is needed by three phases：The generation of sperm and maturation；Sexuality；Sperm by cervix,
Uterine cavity, fallopian tubal smoothly reach ampulla and met with ovum.Wherein, first and phase III and infertile relation the most
Closely.Spermatogenesis with it is ripe abnormal it is most common show as oligospermatism and asthénospermie, but these patients always have part
The normal sperm of vigor, the technology of the conventional high-quality sperm of separation in laboratory is upper reaches method and density-gradient centrifugation method, and these are grasped
High-quality sperm of opposing can cause to damage, and can reach Natural Selection vigor eupyrene sperm by micropore depth difference on micro-fluidic chip
Purpose；Running resistance of the sperm in female genital tract is heavy, as cervical mucus can to sperm motility rise screening effect, only
The stronger sperm of vigor could march on towards uterus through cervical mucus；Selective barrier is played in uterus and fallopian tube junction, can be with
Stop that weak sperm passes through；Oviductal Fluid is advantageous to capacitation, is prepared for next step acrosome reaction and sperm-egg fusion；And ovum is thin
The secretion of born of the same parents and cumulus cell has chemotaxis screening effect.Therefore in the case of natural conception, only with good vigor and
The sperm of chemotactic response ability could complete fertilization process, and microfluidic chip technology can screen such sperm, and in vitro
Simulate this natural conception process.
The content of the invention
Goal of the invention：For overcome the deficiencies in the prior art, it is an object of the invention to provide one kind to be based on microflow control technique
Sperm screening with bionic device in vitro fertilization and should based on microflow control technique sperm screening with bionic device in vitro fertilization
Preparation method and method of work, this device simulates natural conception process completely, avoid centrifuge, wash in vitro, to wear ovum etc. each
Kind operation can avoid the generation of polyspermy to gamete and the potential for adverse effects of embryo.
Technical scheme：A kind of sperm screening and bionic device in vitro fertilization, including bionic device based on microflow control technique
Body, the bionic device body is by slide layer, silica gel pipe layers (SU-8 layer of silica gel) and dimethyl silicone polymer (PDMS) layer
Composition, the silica gel pipe layers middle part are provided with central pool, are provided with entrance pool on the outside of the central pool, first outlet pond and second goes out
Mouth pond, the central pool pass through the first pipeline, second pipe and the 3rd respectively with entrance pool, first outlet pond and second outlet pond
Pipeline communication, the first adding mouth, the second adding mouth and are further respectively had on first pipeline, second pipe and the 3rd pipeline
Three adding mouths.Sperm screening and bionic device in vitro fertilization, structure setting of the present invention based on microflow control technique are compact
Rationally, completely simulation natural conception process, avoid centrifuge, wash in vitro, wear ovum etc. it is various operate gamete and embryo are dived
Adversely affecting, and the generation of polyspermy can be avoided.It can be obtained using this invention device excellent under simulation natural conception state
Matter embryo, success rate in vitro fertilization can be greatly improved.Can be identified using this device causes the potential cause of infertility, such as essence
Son can not penetrate cervical mucus, capacitation and acrosome reaction obstacle etc. occurs, so as to be pointed the direction for clinical treatment.This invention device
The reproductive center of for example each medical institutions of human reproduction is applicable not only to, and is applied to animal breeding.
Further, above-mentioned sperm screening and bionic device in vitro fertilization, the central pool based on microflow control technique
Add sperm nutrient solution；Semen sample is added in the entrance pool；The first outlet pond and second outlet pond add ovarian follicle training
Nutrient solution and/or ovarian cumulus compound；The filling cervical mucus of first pipeline；Second pipe and the 3rd filling Oviductal Fluid of pipeline.
It is rational in infrastructure, can be bionical with high-quality.
Further, above-mentioned sperm screening and bionic device in vitro fertilization, the central pool based on microflow control technique
Radius 5mm, depth 0.1mm；The entrance pool radius is 2mm, depth 6mm；The first outlet pond and second outlet pond half
Footpath is 4mm, depth 6mm；First pipeline, second pipe and the 3rd pipeline long 10mm, it is wide 50 μm, it is high 20 μm.Structure is closed
Reason, it is easy to accomplish.
Further, the above-mentioned sperm screening based on microflow control technique and bionic device in vitro fertilization, first pipe
Road, second pipe and the 3rd pipeline are centered on central pool, respectively in 120 ° of settings.It is reasonable to set, it is easy to accomplish.
Further, the above-mentioned sperm screening based on microflow control technique is glued with bionic device in vitro fertilization, the uterine neck
Liquid is from male spouse, jenny or is artificial cervical mucus, and the artificial cervical mucus includes egg；The sperm
Nutrient solution includes commercial Earle ' s liquid, BWW liquid；The Oviductal Fluid includes commercial HTF liquid, MHTF liquid；The follicle culture liquid
Including commercial KSOM nutrient solutions, CZB nutrient solutions；The ovarian cumulus compound is derived from male with occasionally jenny.Various nutrient solutions
It is wide with ovarian cumulus compound source, using convenient.
Further, the above-mentioned sperm screening based on microflow control technique and bionic device in vitro fertilization, the sperm training
Nutrient solution, it is composed of the following components：NaCl 0.721g, MgSO47H2O 0.247g, fructose 1g, Na2HPO4 3.581g,
KH2PO4 0.136g, pure water 1000ml.Sperm nutrient solution component is reasonable, and the vigor of sperm can be effectively ensured, and effect is good.
The present invention also provides a kind of sperm screening based on microflow control technique and the preparation method of bionic device in vitro fertilization,
Comprise the following steps：
(1) the film type mask plate of silica gel pipe layers is made；
(2) light transmission features of film are utilized, the silica gel pipe layers are obtained by photoresist developing photoetching；
(3) dimethyl silicone polymer prepolymer is mixed with curing agent by 10: 1, be sufficiently stirred, inhaled bubble with vavuum pump
Go out, then slowly pour in silica gel pipe layers, vacuum outgas, 1.5~2h curing moldings in 72 DEG C of baking ovens, obtain the poly- diformazan
Radical siloxane layer；
(4) after the dimethyl silicone polymer of solidification is cut, with card punch, centre is beaten above central pool and entrance pool
Diameter 4mm hole, centre beats diameter 8mm hole above first outlet pond and second outlet pond, in the first adding mouth, second
Diameter 2mm hole is beaten at adding mouth and the 3rd adding mouth；
(5) after being cleaned with ultrasonic cleaning machine, cleaned with deionized water, dried up with nitrogen；
(6) dimethyl silicone polymer layer is bonded with slide layer with plasma generator, obtains and described be based on microflow control technique
Sperm screening with bionic device in vitro fertilization.
Sperm screening and the preparation method of bionic device in vitro fertilization, method of the present invention based on microflow control technique
Rationally, application is convenient, suitable large-scale industrial production, and production cost is low.
The present invention also provides a kind of sperm screening based on microflow control technique and the method for work of bionic device in vitro fertilization,
Comprise the following steps：
(1) after using the pipeline of deionized water rinsing first, second pipe and the 3rd pipeline, dried in 60 DEG C of baking ovens；
(2) use sample injector from the filling cervical mucus of the first adding mouth into the first pipeline, filling total amount is 10nl；
(3) managed respectively with sample injector from the second adding mouth and the 3rd filling Oviductal Fluid of adding mouth to second pipe and the 3rd
In road, filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool；
(5) add follicle culture liquid in first outlet pond and second outlet pond and ovarian cumulus compound, follicle culture liquid add
It is 350 μ l to enter amount；
(6) the μ l of semen liquefaction sample 100 are added in entrance pool, bionic device surface is covered with diaphragm seal, that is, covers
Central pool, entrance pool, first outlet pond, second outlet pond, the first adding mouth, the second adding mouth and the 3rd adding mouth, then will
Bionic device body is horizontally placed in 37 DEG C of wet box；
(7) observed per 10min under inverted microscope once, monitor sperm running situation；Sperm reaches first outlet pond
Continue 2~3d of culture with second outlet pond and with after ovum fertilization, taking out embryonated egg, obtain fertilization embryo.
Sperm screening and the method for work of bionic device in vitro fertilization, method of the present invention based on microflow control technique
Rationally, using that conveniently, can obtain fertilization embryo in vitro, the fertilization radicle of acquisition is developed according to male with occasionally female uterus
Fertilization embryonic implantation is entered male in good time and matched somebody with somebody occasionally in female uterus by situation.
Further, above-mentioned sperm screening and the method for work of bionic device in vitro fertilization based on microflow control technique,
Comprise the following steps：
(1) after using the pipeline of deionized water rinsing first, second pipe and the 3rd pipeline, dried in 60 DEG C of baking ovens；
(2) use sample injector from the filling cervical mucus of the first adding mouth into the first pipeline, filling total amount is 10nl；
(3) managed respectively with sample injector from the second adding mouth and the 3rd filling Oviductal Fluid of adding mouth to second pipe and the 3rd
In road, filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool；
(5) sperm nutrient solution is added in first outlet pond and second outlet pond, addition is 350 μ l；
(6) 100 μ l liquefied semen samples are added in entrance pool, bionic device surface is covered with diaphragm seal, that is, seals
Lid central pool, entrance pool, first outlet pond, second outlet pond, the first adding mouth, the second adding mouth and the 3rd adding mouth, then
Bionic device body is horizontally placed in 37 DEG C of wet box；
(7) sperm cultures of the 5 μ l containing sperm is taken after 50min-120min from first outlet pond and/or second outlet pond
Liquid, and carry out sperm count.
The application of this bionic device is convenient, and sperm can be screened, and obtains the sperm that form is normal, vigor is good.
Further, above-mentioned sperm screening and the method for work of bionic device in vitro fertilization based on microflow control technique,
The semen sample in entrance pool is suctioned out after step (7), 100 new μ l semen samples is rejoined, repeats the above steps, can be with
Reach the purpose of enrichment motile sperm.
Further, above-mentioned sperm screening and the method for work of bionic device in vitro fertilization based on microflow control technique,
Comprise the following steps：
(1) after using the pipeline of deionized water rinsing first, second pipe and the 3rd pipeline, dried in 60 DEG C of baking ovens；
(2) use sample injector from the filling cervical mucus of the first adding mouth into the first pipeline, filling total amount is 10nl；
(3) managed respectively with sample injector from the second adding mouth and the 3rd filling Oviductal Fluid of adding mouth to second pipe and the 3rd
In road, filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool；
(5) Oviductal Fluid is added in first outlet pond and second outlet pond, addition is 350 μ l, the Oviductal Fluid
In contain an ovarian cumulus compound；
(6) 100 μ l liquefied semen samples are added in entrance pool, bionic device surface is covered with diaphragm seal, that is, seals
Lid central pool, entrance pool, first outlet pond, second outlet pond, the first adding mouth, the second adding mouth and the 3rd adding mouth, then
Bionic device body is horizontally placed at 37 DEG C, cultivated in 5%CO2 incubators；
(7) observe in first outlet pond and/or second outlet pond whether there is sperm with inverted microscope after 1h, seen after 4~6h
Examine whether there is second polar body discharge or female-male pronucleus formed；If second polar body discharge or female-male pronucleus formed, prompt sperm with
Ovum has been fertilized to form embryonated egg, can now suction out embryonated egg, and transplanting is supplied after continuing culture；If discharged without second polar body
Or female-male pronucleus is formed, then continue to observe 2h, until sperm and Folliculogenesis embryonated egg.
This bionic device method is reasonable, can apply to sperm in vitro fertilization-embryonic implantation (IVF-ET), compared to facing at present
The conventional IVF-ET of bed, pregnancy rate can increase by 15%~20%, and polyspermy rate and abortion ratio can significantly reduce.
Above-mentioned technical proposal can be seen that compared with prior art, and the present invention has the advantages that：
(1) apparatus of the present invention simulate natural conception process completely, avoid during existing auxiliary procreation technology in vitro from
The heart, wash, wear the various operations such as ovum to gamete and the potential for adverse effects of embryo, and the generation of polyspermy can be avoided.
(2) apparatus of the present invention can obtain the embryo quality under simulation natural conception state, can greatly improve in vitro fertilization
Success rate.
(3) apparatus of the present invention can identify cause infertility potential cause, as sperm can not penetrate cervical mucus, capacitation and
Generation acrosome reaction obstacle etc., so as to be pointed the direction for clinical treatment.
(4) apparatus of the present invention are applied widely, and this device can be used in human reproduction and animal breeding.
Brief description of the drawings
Fig. 1 is the structural representation of silica gel pipe layers of the present invention；
Fig. 2 is the sperm screening of the present invention based on microflow control technique and the structure of bionic device section view in vitro fertilization
Schematic diagram；
In figure：100 bionic device bodies, 1 central pool, 2 entrance pools, 3 first outlet ponds, 4 second outlet ponds, 5 first pipes
Road, 6 second pipes, 7 the 3rd pipelines, 8 first adding mouths, 9 second adding mouths, 10 the 3rd adding mouths.
Embodiment
Below in conjunction with the accompanying drawings and specific embodiment, the present invention is furture elucidated.
Embodiment 1
The screening of the sperm based on microflow control technique as shown in Figure 1-2 and bionic device in vitro fertilization, including bionic device
Body 100, the bionic device body 100 is by slide layer, silica gel pipe layers (SU-8 layer of silica gel) and dimethyl silicone polymer
(PDMS) layer is formed, and central pool 1 is provided with the middle part of the silica gel pipe layers, and the outside of central pool 1 goes out provided with entrance pool 2, first
Mouth pond 3 and second outlet pond 4, the central pool 1 pass through first respectively with entrance pool 2, first outlet pond 3 and second outlet pond 4
Pipeline 5, second pipe 6 connect with the 3rd pipeline 7, are further respectively had on first pipeline 5, the pipeline 7 of second pipe 6 and the 3rd
First adding mouth 8, the second adding mouth 9 and the 3rd adding mouth 10.
Wherein, the central pool 1 adds sperm nutrient solution；Semen sample is added in the entrance pool 2；The first outlet
Pond 3 and second outlet pond 4 add follicle culture liquid and/or ovarian cumulus compound；First pipeline, the 5 filling cervical mucus；Second
6 and the 3rd pipeline of pipeline, 7 filling Oviductal Fluid.
As shown in appendix 1, central pool 1 the radius 5mm, depth 0.1mm；The radius of entrance pool 2 is 2mm, and depth is
6mm；The first outlet pond 3 and second outlet pond 4 radius is 4mm, depth 6mm；First pipeline 5, the and of second pipe 6
3rd pipeline 7 long 10mm, it is wide 50 μm, it is high 20 μm.Also, first pipeline 5, the pipeline 7 of second pipe 6 and the 3rd are with central pool
Centered on 1, respectively in 120 ° of settings.It is reasonable to set, it is easy to accomplish.
1 different little Chi of subordinate list and pipe layers volume parameter
Embodiment 2
In the screening of the sperm based on microflow control technique of embodiment 1 and bionic device in vitro fertilization, the liquid in each little Chi
Source is as follows：
Semen sample can derive from the mankind or other animals；
The cervical mucus is from male spouse, jenny or is artificial cervical mucus, the artificial cervical mucus
Including egg；The sperm nutrient solution includes commercial Earle ' s liquid, BWW liquid；The Oviductal Fluid includes HTF liquid, MHTF
Liquid；The follicle culture liquid includes commercial KSOM nutrient solutions, CZB nutrient solutions；The ovarian cumulus compound is derived from male with occasionally female
Property animal, from ampulla of uterine tube obtain.
Embodiment 3
Artificial cervical mucus, egg can be taken to be diluted with physiological saline 1: 2~1: 5.
The sperm nutrient solution can be prepared voluntarily, composed of the following components：NaCl 0.721g, MgSO47H2O
0.247g, fructose 1g, Na2HPO4 3.581g, KH2PO4 0.136g, pure water 1000ml.
Embodiment 4
Sperm screening based on microflow control technique and the preparation method of bionic device in vitro fertilization, bag in a kind of embodiment 1
Include following steps：
(1) with reference to the accompanying drawings 1, accompanying drawing 2 and the content of subordinate list 1 draw design drawing, and with specific software such as AutoCAD design chips
Pipeline and raised structures domain, and change into printable film format file；Silica gel pipe layers are made according to design drawing
Film type mask plate；
(2) light transmission features of film are utilized, the silica gel pipe layers (silicon substrate SU-8 moulds are obtained by photoresist developing photoetching
Tool, SU-8 thickness can be adjusted)；
(3) dimethyl silicone polymer prepolymer is mixed with curing agent by 10: 1, be sufficiently stirred, inhaled bubble with vavuum pump
Go out, then slowly pour in silica gel pipe layers, vacuum outgas, 1.5~2h curing moldings in 72 DEG C of baking ovens, obtain the poly- diformazan
Radical siloxane layer；
(4) after the dimethyl silicone polymer of solidification is cut, with card punch above central pool 1 and entrance pool 2 centre
Diameter 4mm hole is beaten, centre beats diameter 8mm hole above first outlet pond 3 and second outlet pond 4, in the first adding mouth
8th, diameter 2mm hole is beaten at the second adding mouth 9 and the 3rd adding mouth 10；
(5) after being cleaned with ultrasonic cleaning machine, cleaned with deionized water, dried up with nitrogen；
(6) dimethyl silicone polymer layer is bonded with slide layer with plasma generator, obtains and described be based on microflow control technique
Sperm screening with bionic device in vitro fertilization.
Embodiment 5
A kind of sperm screening and the method for work of bionic device in vitro fertilization based on microflow control technique, including following step
Suddenly：
(1) with after the first pipeline of deionized water rinsing 5, the pipeline 7 of second pipe 6 and the 3rd, dried in 60 DEG C of baking ovens；
(2) with single pushing-type sample injector (available commercial) from 8 filling cervical mucus of the first adding mouth into the first pipeline 5,
Filling total amount is 10nl；
(3) respectively with single pushing-type sample injector from the second adding mouth 9 and 10 filling Oviductal Fluid of the 3rd adding mouth to the second pipe
In the pipeline 7 of road 6 and the 3rd, filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool 1；
(5) follicle culture liquid and ovarian cumulus compound, follicle culture liquid are added in first outlet pond 3 and second outlet pond 4
Addition is 350 μ l；
(6) the μ l of semen liquefaction sample 100 are added in entrance pool 2, bionic device surface is covered with diaphragm seal, that is, covers
Central pool 1, entrance pool 2, first outlet pond 3, second outlet pond 4, the first adding mouth 8, the second adding mouth 9 and the 3rd adding mouth
10, then bionic device body 100 is horizontally placed in 37 DEG C of wet box；
(7) observed per 10min under inverted microscope once, monitor sperm running situation；Sperm reaches first outlet pond 3
Continue 2~3d of culture with second outlet pond 4 and with after ovum fertilization, taking out embryonated egg, obtain fertilization embryo.
Fertilization embryonic implantation is entered into male in good time with occasionally female uterus developmental state according to male to move with occasionally female
Thing intrauterine.
Embodiment 6
The above-mentioned screening of the sperm based on microflow control technique is used for the work side of sperm screening with bionic device in vitro fertilization
Method, comprise the following steps：
(1) with after the first pipeline of deionized water rinsing 5, the pipeline 7 of second pipe 6 and the 3rd, dried in 60 DEG C of baking ovens；
(2) use sample injector from 8 filling cervical mucus of the first adding mouth into the first pipeline 5, filling total amount is 10nl；
(3) respectively with sample injector from the second adding mouth 9 and 10 filling Oviductal Fluid of the 3rd adding mouth to second pipe 6 and the
In three pipelines 7, filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool 1；
(5) Oviductal Fluid is added in first outlet pond 3 and second outlet pond 4；
(6) 100 μ l liquefied semen samples are added in entrance pool 2, bionic device surface is covered with diaphragm seal, that is, seals
Lid central pool 1, entrance pool 2, first outlet pond 3, second outlet pond 4, the first adding mouth 8, the second adding mouth 9 and the 3rd adding mouth
10, then bionic device body 100 is horizontally placed in 37 DEG C of wet box；
(7) after 30min in the sperm nutrient solution of central pool visible motile sperm, as time went on, motile sperm by
More than cumulative and enter the microchannel of the liquid containing HTF, movable essence can be seen after about 50min in first outlet pond 3 and second outlet pond 4
Son, take the HTF liquid containing sperm in 5 μ l outlet baths after 2h and carry out sperm count, if sperm sum up to 50000, can be direct
The sperm row screened with this is (IVF) in vitro fertilization, the sperm of this quantity enough with 1 oocyte fertilization.
By the screening of the present apparatus, sperm motility is more than 90%, and normal morphology sperm percentage also significantly improves, remote high
In 4%.If the number of motile sperm obtained is less, the semen sample in entrance pool can be suctioned out, rejoin 100 new μ l
Semen sample, so as to reach the purpose of enrichment motile sperm.
Embodiment 7
The above-mentioned screening of the sperm based on microflow control technique is applied to sperm in vitro fertilization-embryo with bionic device in vitro fertilization
The method of work of (IVF-ET) is transplanted, is comprised the following steps：
(1) with after the first pipeline of deionized water rinsing 5, the pipeline 7 of second pipe 6 and the 3rd, dried in 60 DEG C of baking ovens；
(2) use sample injector from 8 filling cervical mucus of the first adding mouth into the first pipeline 5, filling total amount is 10nl；
(3) respectively with sample injector from the second adding mouth 9 and 10 filling Oviductal Fluid (HTF) of the 3rd adding mouth to second pipe
6 and the 3rd in pipeline 7, and filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool 1；
(5) Oviductal Fluid is added in first outlet pond 3 and second outlet pond 4, addition is 350 μ l, the fallopian tubal
Contain an ovarian cumulus compound in liquid；
(6) 100 μ l liquefied semen samples are added in entrance pool 2, bionic device surface is covered with diaphragm seal, that is, seals
Lid central pool 1, entrance pool 2, first outlet pond 3, second outlet pond 4, the first adding mouth 8, the second adding mouth 9 and the 3rd adding mouth
10, bionic device body 100 is then horizontally placed at 37 DEG C, cultivated in 5%CO2 incubators；
(7) observe sperm is whether there is in first outlet pond (3) and/or second outlet pond (4) with inverted microscope after 1h, 4~
Observation whether there is second polar body discharge after 6h or female-male pronucleus is formed；If second polar body discharge or female-male pronucleus are formed, prompt
Sperm has been fertilized to form embryonated egg with ovum, can now suction out embryonated egg, and transplanting is supplied after continuing culture；If without the second pole
Body is discharged or female-male pronucleus is formed, then continues to observe 2h, until sperm and Folliculogenesis embryonated egg.Using this device compared to mesh
Preceding routine clinical IVF-ET, pregnancy rate can increase by 15%~20%, and polyspermy rate and abortion ratio can significantly reduce.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member for, under the premise without departing from the principles of the invention, to central pool 1, entrance pool 2, first outlet pond 3, second outlet pond 4,
First pipeline 5, the pipeline 7 of second pipe 6 and the 3rd, the first adding mouth 8, the second adding mouth 9 and the 3rd adding mouth 10 shape and
Size can be improved, and these improvement are regarded as protection scope of the present invention.
Claims (10)
1. a kind of sperm screening based on microflow control technique and bionic device in vitro fertilization, it is characterised in that：Including bionic device
Body (100), the bionic device body (100) is made up of slide layer, silica gel pipe layers and dimethyl silicone polymer layer, described
Be provided with central pool (1) in the middle part of silica gel pipe layers, provided with entrance pool (2), first outlet pond (3) and the on the outside of the central pool (1)
Two outlet baths (4), the central pool (1) pass through first respectively with entrance pool (2), first outlet pond (3) and second outlet pond (4)
Pipeline (5), second pipe (6) connect with the 3rd pipeline (7), first pipeline (5), second pipe (6) and the 3rd pipeline (7)
On further respectively have the first adding mouth (8), the second adding mouth (9) and the 3rd adding mouth (10).
2. the sperm screening according to claim 1 based on microflow control technique exists with bionic device in vitro fertilization, its feature
In：The central pool (1) adds sperm nutrient solution；Semen sample is added in the entrance pool (2)；The first outlet pond (3)
Follicle culture liquid and/or ovarian cumulus compound are added with second outlet pond (4)；The filling cervical mucus of first pipeline (5)；Second
Pipeline (6) and the 3rd pipeline (7) filling Oviductal Fluid.
3. sperm screening and bionic device in vitro fertilization, its feature according to claim 1 or 2 based on microflow control technique
It is：Central pool (1) the radius 5mm, depth 0.1mm；Entrance pool (2) radius is 2mm, depth 6mm；Described first
Outlet bath (3) and second outlet pond (4) radius are 4mm, depth 6mm；First pipeline (5), second pipe (6) and the 3rd
Pipeline (7) long 10mm, it is wide 50 μm, it is high 20 μm.
4. the sperm screening according to claim 2 based on microflow control technique exists with bionic device in vitro fertilization, its feature
In：The cervical mucus is from male spouse, jenny or is artificial cervical mucus, and the artificial cervical mucus includes chicken
Egg white；The sperm nutrient solution includes commercial Earle ' s liquid, BWW liquid；The Oviductal Fluid includes commercial HTF liquid, MHTF liquid；
The follicle culture liquid includes commercial KSOM nutrient solutions, CZB nutrient solutions；It is dynamic with occasionally female that the ovarian cumulus compound is derived from male
Thing.
5. the sperm screening according to claim 2 based on microflow control technique exists with bionic device in vitro fertilization, its feature
In：The sperm nutrient solution, it is composed of the following components：NaCl 0.721g, MgSO47H2O 0.247g, fructose 1g,
Na2HPO4 3.581g, KH2PO4 0.136g, pure water 1000ml.
6. the screening of the sperm based on microflow control technique and bionic device in vitro fertilization according to claim any one of 1-5
Preparation method, it is characterised in that：Comprise the following steps：
(1) the film type mask plate of silica gel pipe layers is made；
(2) the silica gel pipe layers are obtained by photoresist developing photoetching；
(3) dimethyl silicone polymer prepolymer is mixed with curing agent, be sufficiently stirred, bubble is suctioned out with vavuum pump, then poured
In silica gel pipe layers, vacuum outgas, 1.5~2h curing moldings in 72 DEG C of baking ovens, the dimethyl silicone polymer layer is obtained；
(4) after the dimethyl silicone polymer of solidification is cut, with card punch in central pool (1), entrance pool (2), first outlet pond
(3), second outlet pond (4), the first adding mouth (8), the second adding mouth (9) and the punching of the 3rd adding mouth (10) place；
(5) after being cleaned with ultrasonic cleaning machine, cleaned with deionized water, dried up with nitrogen；
(6) dimethyl silicone polymer layer is bonded with slide layer with plasma generator, obtains the essence based on microflow control technique
Son screening and bionic device in vitro fertilization.
7. the screening of the sperm based on microflow control technique and bionic device in vitro fertilization according to claim any one of 1-6
Method of work, it is characterised in that：Comprise the following steps：
(1) after using the pipeline of deionized water rinsing first (5), second pipe (6) and the 3rd pipeline (7), dried in 60 DEG C of baking ovens；
(2) use sample injector from the first adding mouth (8) filling cervical mucus into the first pipeline (5), filling total amount is 10nl；
(3) respectively with sample injector from the second adding mouth (9) and the 3rd adding mouth (10) filling Oviductal Fluid to second pipe (6) and
In 3rd pipeline (7), filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool (1)；
(5) follicle culture liquid and ovarian cumulus compound, follicle culture liquid are added in first outlet pond (3) and second outlet pond (4)
Addition is 350 μ l；
(6) the μ l of semen sample 100 are added in entrance pool (2)；
(7) bionic device surface is covered with diaphragm seal, that is, covers central pool (1), entrance pool (2), first outlet pond (3), second
Outlet bath (4), the first adding mouth (8), the second adding mouth (9) and the 3rd adding mouth (10), then by bionic device body (100)
It is horizontally placed in 37 DEG C of wet box；
(8) observed per 10min under inverted microscope once, monitor sperm running situation；Sperm reach first outlet pond (3) and
Continue 2~3d of culture in second outlet pond (4) and with after ovum fertilization, taking out embryonated egg, obtain fertilization embryo.
8. the screening of the sperm based on microflow control technique and bionic device in vitro fertilization according to claim any one of 1-6
Method of work, it is characterised in that：Comprise the following steps：
(1) after using the pipeline of deionized water rinsing first (5), second pipe (6) and the 3rd pipeline (7), dried in 60 DEG C of baking ovens；
(2) use sample injector from the first adding mouth (8) filling cervical mucus into the first pipeline (5), filling total amount is 10nl；
(3) respectively with sample injector from the second adding mouth (9) and the 3rd adding mouth (10) filling Oviductal Fluid to second pipe (6) and
In 3rd pipeline (7), filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool (1)；
(5) sperm nutrient solution is added in first outlet pond (3) and second outlet pond (4), addition is 350 μ l；
(6) 100 μ l liquefied semen samples are added in entrance pool (2), bionic device surface is covered with diaphragm seal, that is, covers
Central pool (1), entrance pool (2), first outlet pond (3), second outlet pond (4), the first adding mouth (8), the second adding mouth (9) and
3rd adding mouth (10), then bionic device body (100) is horizontally placed in 37 DEG C of wet box；
(7) sperm cultures of the 5 μ l containing sperm is taken after 50min-120min from first outlet pond (3) and/or second outlet pond (4)
Liquid, and carry out sperm count.
9. sperm screening and the work side of bionic device in vitro fertilization according to claim 8 based on microflow control technique
Method, it is characterised in that：The semen sample in entrance pool (2) is suctioned out after step (7), rejoins 100 new μ l semen samples,
Repeat the above steps.
10. the screening of the sperm based on microflow control technique and bionic device in vitro fertilization according to claim any one of 1-6
Method of work, it is characterised in that：Comprise the following steps：
(1) after using the pipeline of deionized water rinsing first (5), second pipe (6) and the 3rd pipeline (7), dried in 60 DEG C of baking ovens；
(2) use sample injector from the first adding mouth (8) filling cervical mucus into the first pipeline (5), filling total amount is 10nl；
(3) respectively with sample injector from the second adding mouth (9) and the 3rd adding mouth (10) filling Oviductal Fluid to second pipe (6) and
In 3rd pipeline (7), filling total amount is 10nl；
(4) the μ l of sperm nutrient solution 10 are added in central pool (1)；
(5) Oviductal Fluid is added in first outlet pond (3) and second outlet pond (4), contains an ovarian cumulus in the Oviductal Fluid
Compound, Oviductal Fluid addition are 350 μ l；
(6) 100 μ l liquefied semen samples are added in entrance pool (2), bionic device surface is covered with diaphragm seal, that is, covers
Central pool (1), entrance pool (2), first outlet pond (3), second outlet pond (4), the first adding mouth (8), the second adding mouth (9) and
3rd adding mouth (10), bionic device body (100) is then horizontally placed at 37 DEG C, cultivated in 5%CO2 incubators；
(7) sperm is whether there is with micro- sem observation first outlet pond (3) and/or second outlet pond (4) after 1h, is observed after 4~6h
Whether there is second polar body discharge or female-male pronucleus is formed；If second polar body discharge or female-male pronucleus are formed, embryonated egg is formed, this
When embryonated egg can be suctioned out, continue to cultivate；If formed without second polar body discharge or female-male pronucleus, continue to observe 2h, until
Form embryonated egg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710944615.3A CN107475078A (en) | 2017-10-12 | 2017-10-12 | A kind of sperm screening based on microflow control technique and bionic device in vitro fertilization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710944615.3A CN107475078A (en) | 2017-10-12 | 2017-10-12 | A kind of sperm screening based on microflow control technique and bionic device in vitro fertilization |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107475078A true CN107475078A (en) | 2017-12-15 |
Family
ID=60606276
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710944615.3A Pending CN107475078A (en) | 2017-10-12 | 2017-10-12 | A kind of sperm screening based on microflow control technique and bionic device in vitro fertilization |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107475078A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110172390A (en) * | 2019-05-30 | 2019-08-27 | 南京精竞生物科技有限公司 | A kind of high motile sperm screening micro fluidic device |
| CN110551618A (en) * | 2019-09-27 | 2019-12-10 | 中国科学技术大学 | Sperm sorting unit |
| US20200123484A1 (en) * | 2018-10-22 | 2020-04-23 | National Tsing Hua University | Integrated chip and method for sperm sorting, oocyte incubation, and in vitro fertilization |
| CN111154619A (en) * | 2020-01-17 | 2020-05-15 | 浙江大学 | High-quality sperm in-vitro screening device and using method |
| CN111855657A (en) * | 2020-07-22 | 2020-10-30 | 中国科学技术大学 | Sperm motility evaluation method based on energy gradient |
| TWI710630B (en) * | 2018-10-22 | 2020-11-21 | 國立清華大學 | Integrated chip and method for sperm sorting, oocyte incubation and in vitro fertilization |
| WO2021190644A1 (en) * | 2020-03-27 | 2021-09-30 | 于毅欣 | Method for screening sperm during artificial insemination |
| CN113528310A (en) * | 2021-06-16 | 2021-10-22 | 复旦大学 | Bionic micro-fluidic chip for simulating cervical microenvironment and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242055A (en) * | 2011-06-03 | 2011-11-16 | 博奥生物有限公司 | Method for evaluating sperm activity and screening sperms and special microfluidic chip device for same |
| CN102352305A (en) * | 2011-09-27 | 2012-02-15 | 广州市第一人民医院 | Sperm optimizing microflow control device and sperm optimizing method |
| CN103451186A (en) * | 2013-08-28 | 2013-12-18 | 暨南大学 | Interference fragment of colipase-like 2 (Clpsl2) gene and application of interference fragment |
| CN105936890A (en) * | 2016-06-27 | 2016-09-14 | 广西壮族自治区妇幼保健院 | Method using cumulus cell compound to screen sperms |
| CN106222067A (en) * | 2016-06-27 | 2016-12-14 | 浙江星博生物科技股份有限公司 | A kind of micro-fluidic chip for sperm sorting |
-
2017
- 2017-10-12 CN CN201710944615.3A patent/CN107475078A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242055A (en) * | 2011-06-03 | 2011-11-16 | 博奥生物有限公司 | Method for evaluating sperm activity and screening sperms and special microfluidic chip device for same |
| CN102352305A (en) * | 2011-09-27 | 2012-02-15 | 广州市第一人民医院 | Sperm optimizing microflow control device and sperm optimizing method |
| CN103451186A (en) * | 2013-08-28 | 2013-12-18 | 暨南大学 | Interference fragment of colipase-like 2 (Clpsl2) gene and application of interference fragment |
| CN105936890A (en) * | 2016-06-27 | 2016-09-14 | 广西壮族自治区妇幼保健院 | Method using cumulus cell compound to screen sperms |
| CN106222067A (en) * | 2016-06-27 | 2016-12-14 | 浙江星博生物科技股份有限公司 | A kind of micro-fluidic chip for sperm sorting |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200123484A1 (en) * | 2018-10-22 | 2020-04-23 | National Tsing Hua University | Integrated chip and method for sperm sorting, oocyte incubation, and in vitro fertilization |
| TWI710630B (en) * | 2018-10-22 | 2020-11-21 | 國立清華大學 | Integrated chip and method for sperm sorting, oocyte incubation and in vitro fertilization |
| 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 |
| CN109468226B (en) * | 2018-11-16 | 2023-08-18 | 浙江大学 | In-vitro fertilization device simulating sperm swimming path in natural state and application method thereof |
| CN110172390A (en) * | 2019-05-30 | 2019-08-27 | 南京精竞生物科技有限公司 | A kind of high motile sperm screening micro fluidic device |
| CN110551618A (en) * | 2019-09-27 | 2019-12-10 | 中国科学技术大学 | Sperm sorting unit |
| CN111154619A (en) * | 2020-01-17 | 2020-05-15 | 浙江大学 | High-quality sperm in-vitro screening device and using method |
| CN111154619B (en) * | 2020-01-17 | 2024-01-09 | 浙江大学 | High-quality sperm in-vitro screening device and use method |
| WO2021190644A1 (en) * | 2020-03-27 | 2021-09-30 | 于毅欣 | Method for screening sperm during artificial insemination |
| CN111855657A (en) * | 2020-07-22 | 2020-10-30 | 中国科学技术大学 | Sperm motility evaluation method based on energy gradient |
| CN111855657B (en) * | 2020-07-22 | 2022-12-30 | 中国科学技术大学 | Sperm motility evaluation method based on energy gradient |
| CN113528310A (en) * | 2021-06-16 | 2021-10-22 | 复旦大学 | Bionic micro-fluidic chip for simulating cervical microenvironment and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475078A (en) | A kind of sperm screening based on microflow control technique and bionic device in vitro fertilization | |
| Ludwig et al. | The human female reproductive tract: a scanning electron microscopic atlas | |
| KR20160043929A (en) | Cells, methods and apparatuses for amniotic fluid collection and isolation of cells | |
| Grobstein | The early development of human embryos | |
| KR20190112186A (en) | Method and system for regenerating hair using 3D organoid system of hair follicle stem cells | |
| Smits et al. | Influence of the uterine environment on the development of in vitro-produced equine embryos | |
| CN110408540A (en) | Embryo's changing liquid automatically culture dish | |
| Strug et al. | Making more womb: clinical perspectives supporting the development and utilization of mesenchymal stem cell therapy for endometrial regeneration and infertility | |
| CN109893541A (en) | Application of the excretion body of menses source of human stem cell in the drug of preparation treatment Asherman's syndrom | |
| CN102876573B (en) | Mammal embryo in-vitro culture device and culture method thereof | |
| Bodke et al. | Advancements in microfluidic systems for the study of female reproductive biology | |
| KR20190050360A (en) | Micro Chip Mimicking Female Reproductive Organ and Producing Method Thereof | |
| CN104531612A (en) | Oocyte culture fluid and culture method thereof | |
| CN207313583U (en) | A kind of sperm screening based on microflow control technique and bionic device in vitro fertilization | |
| US9074183B2 (en) | Method and apparatus for regulating optimum flow of semen and separating motile sperms | |
| CN1423692A (en) | Human Muellerian Duct-derived epithelial cells and methods of isolation and uses thereof | |
| CN204569897U (en) | A kind of human sperm preferably with bionic device in vitro fertilization | |
| CN102242063A (en) | Early embryo closed culture device and method for fresh embryo transport | |
| CN109727516A (en) | A kind of emulation takes ootypus type and its application method and application | |
| Singh et al. | Effect of granulosa cells monolayer and oviductal epithelial cells co-culture on cleavage rate and embryo development of in-vitro fertilized goat oocytes | |
| CN109637321B (en) | Ovum-taking model and application thereof | |
| CN104450496B (en) | External fertilization cultivates sieve and external fertilization method | |
| CN111820123A (en) | Artificial insemination method for pteridophyte | |
| CN215895718U (en) | Teaching and training model for vagina ultrasound-guided puncture ovum taking | |
| CN103243069A (en) | Umbilical cord mesenchymal stem cell technology for reproductive health |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |