CN111154619A - High-quality sperm in-vitro screening device and using method - Google Patents
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Abstract
The invention discloses a high-quality sperm in-vitro screening device and a use method thereof, relating to the technical field of assisted reproduction, wherein the device comprises a base part and an upper cover part which are matched with each other, wherein the base part comprises a semen sampling area, a vagina primary screening area, a cervix fluid sorting area, a uterus deposition area, an oviduct guide area and a sperm collecting area which are sequentially communicated to form a sperm screening channel; cervical fluid sorting district include cervical canal body and cervical tubule, wherein, cervical tubule be thinner than cervical canal body with the vagina prescreening district, the entry end of cervical tubule with the vagina prescreening district intercommunication, the exit end of cervical tubule with the entry end intercommunication of cervical canal body, the exit end of cervical canal body with uterus deposit district intercommunication, solved the not high enough technical problem of sperm quality of external sperm sieving mechanism screening among the prior art, realized effectively improving the beneficial effect of the external screening quality of sperm.
Description
Technical Field
The application relates to the technical field of assisted reproduction, in particular to a high-quality sperm in-vitro screening device and a using method thereof.
Background
To achieve fertility goals for patients with infertility, the most effective way is by Assisted Reproductive Technology (ART) as typified by Artificial Insemination (AI) and in vitro fertilization-embryo transfer (IVF-ET). And sperm screening during ART is critical. Lower quality sperm can affect the outcome of fertilization and the subsequent formation of good quality embryos, while abnormal sperm can increase the incidence of genetic defects and affect the health of offspring.
Common methods for screening high quality sperm in the prior art include simple washing, upstream, density gradient centrifugation, electrophoresis, annexin magnetic bead sorting, hyaluronic acid binding, and the like. However, the above methods have certain limitations and disadvantages, for example, although sperm treated by a simple washing method can effectively remove seminal plasma wrapping the sperm, the treated sperm still contains many impurities such as round cells, dead sperm and weak sperm, and the separation effect is poor; the upstream method is limited to be carried out in an inclined test tube, the distance of sperm swimming upwards is limited, the sperm is contacted with impurities such as seminal plasma, leucocytes, bacteria and the like for a long time, and the quality of the finally collected sperm is limited; the density gradient centrifugation can well remove impurities such as seminal plasma, leucocytes, bacteria and the like in a sample, but a plurality of centrifugation operation processes can generate more Reactive Oxygen Species (ROS) to increase sperm DNA fragments, and the damage and the incompleteness of the sperm DNA can cause ART failure or transfer abnormal genetic substances to offspring. The electrophoresis method, the annexin magnetic bead sorting method, the hyaluronic acid binding method and the like respectively need to treat sperms by means of an electric field, a magnetic field or enzyme substances, and although a part of abnormal sperms can be eliminated by respective characteristics, the additional physicochemical action can cause damage to normal sperms, so that the methods are not commonly applied to the conventional sperm screening process of ART. A common disadvantage of the above methods is that they bypass the natural selection process that must be performed during in vivo fertilization.
In order to increase the natural selection process which must be undergone during in vivo fertilization, a screening channel is arranged, a batch of sperms with better vitality are screened out by prolonging the sperm swimming distance, and the longer swimming path accords with the real swimming distance of the sperms in the female reproductive tract. However, in the natural selection process in vivo, the internal microenvironment of the female reproductive system is quite complex, and besides the physical distance, the internal microenvironment also comprises the fluid state, the temperature, the biochemical secretion, unique structural factors such as vaginal crinkles, cervical tubes, fallopian tubes and the like.
However, in the process of implementing the technical solution in the embodiment of the present application, the inventor of the present application finds that the above prior art has at least the following technical problems:
in the prior art, the screening factor set by the in-vitro sperm screening device is single, and the difference between the screening factor and the natural screening process in the sperm body is large, so that the quality of screened sperms is not high enough.
Content of application
The embodiment of the application provides an in-vitro screening device for high-quality sperms and a using method, and aims to solve the technical problem that in the prior art, the quality of screened sperms is not high enough due to the fact that screening factors set by the in-vitro screening device are single and have a large difference with a natural screening process in sperms, and natural selection structures of female reproductive systems for the sperms are restored to the maximum extent by setting screening channels similar to channels for naturally screening the sperms in vivo, and the screening channels are similar to the channels for naturally screening the sperms in vivo; the in vitro sperm screening process by adopting the device of the embodiment is close to the in vivo natural screening process of the sperm, thereby effectively improving the quality of the in vitro screened sperm.
In order to solve the above problems, in a first aspect, the embodiments of the present application provide an in vitro high-quality sperm screening device, which includes a base portion and an upper cover portion that are capable of being matched with each other, where the base portion includes a semen sampling region, a vaginal primary screening region, a cervical fluid sorting region, a uterine sedimentation region, an oviduct guiding region and a sperm collecting region that are sequentially communicated to form a sperm screening channel;
cervical fluid sorting district include cervical canal body and cervical tubule, wherein, cervical tubule be thinner than cervical canal body with the vagina prescreening district, the entry end of cervical tubule with the vagina prescreening district intercommunication, the exit end of cervical tubule with the entry end intercommunication of cervical canal body, the exit end of cervical canal body with uterus deposit district intercommunication.
Further, cervical fluid sorting zone still including being located the fluid passage pipe of cervical tubule both sides, two fluid passage pipe symmetry be located the both sides of cervical tubule, the exit end of fluid passage pipe with the entry end of cervical canal body be linked together, the entry end of fluid passage pipe to the both sides slope extension of cervical tubule, the entry end of fluid passage pipe be connected with the liquid feeding district, the fluid passage pipe on still be equipped with first switch.
Furthermore, the included angle between the fluid passage tube and the cervical tubule is 15-60 degrees.
Furthermore, the primary vaginal screening area is a groove with an upward opening, and an inlet of the primary vaginal screening area is communicated with the side surface of the semen sampling area; the vagina primary screening district in from semen adding appearance district to the direction interval of cervical fluid sorting district is equipped with a plurality of baffler board, the bottom of baffler board fix on the bottom surface in vagina primary screening district, the top of baffler board vertically upwards extend, just the height of baffler board is less than the degree of depth in vagina primary screening district.
Furthermore, the uterus sedimentation zone comprises a big head end and a tip end, the outer edge of the uterus sedimentation zone is arc-shaped, a straight groove is arranged at the central axis of the uterus sedimentation zone, the straight groove, the cervix fluid sorting zone and the vagina screening zone are collinear, and a first groove is arranged on the bottom of the straight groove, close to the inlet end of the straight groove; the bilateral symmetry of straight flute is equipped with the arc wall, the arc wall along the edge extension of uterus deposit district, the entry end of arc wall with the entry end of straight flute all with the exit end intercommunication of cervical canal body, two the exit end of arc wall all with the exit end of straight flute communicates each other.
Furthermore, two oviduct guide areas are symmetrically arranged on two sides of the uterus deposition area, the inlet end of the oviduct guide area is communicated with one side of the arc-shaped groove, the outlet end of the oviduct guide area is communicated with the sperm collecting area, and a second switch is arranged on the outlet end of the oviduct guide area.
Further, the width of the oviduct guiding region gradually becomes wider from the uterine deposition region to the sperm collecting region.
Furthermore, the sperm collecting area is in a circular groove shape, a second groove is arranged in the middle of the bottom of the sperm collecting area, and a progesterone layer is coated in the second groove.
Furthermore, a heating module is arranged outside the sperm collecting area, the heating module comprises an electric conduction patch enclosed outside the sperm collecting area, the electric conduction patch is connected with a power supply through a lead, and the lead is connected with a thermal resistor, a temperature regulation controller and a switch.
In a second aspect, the present application provides a method for using a high-quality sperm in-vitro screening device, including the following steps:
step A: opening the second switch, closing the first switch, and adding improved human oviduct culture solution containing human serum albumin into the primary vaginal screening area, the uterine deposition area, the oviduct guide area and the sperm collecting area respectively;
b, mixing and re-suspending the completely liquefied semen sample and the improved human oviduct culture solution with the same volume, and then adding the mixture into the semen sample adding area;
and C: when the resuspended semen sample flows into the cervical tubule, adding the improved human oviduct culture solution into the solution adding area on the fluid passage tube, and turning on the first switch;
step D: turning on a power switch of the heating module, setting the temperature to be 37.5 ℃ by adjusting the temperature adjusting controller, covering the upper cover, and moving the whole device into a constant-temperature constant-humidity incubator for incubation;
step E: taking the device out of the incubator, opening the upper cover, closing the first switch, covering the upper cover, and moving the device into the incubator again for incubation;
step F: taking out the device from the incubator, opening the upper cover, closing the second switch, and sucking screened sperms from the sperm collecting area by using a Pasteur pipette or a pipettor;
step G: inspecting the collected sperms, comparing the inspected sperms with a sample before screening, and judging the screening effect of the device;
step H: the collected sperm are placed into the incubator and await subsequent conventional in vitro fertilization or intracytoplasmic sperm microinjection procedures.
One or more technical solutions in the embodiments of the present application have at least one or more of the following technical effects:
1. the screening channel of the device comprises a semen sampling area, a primary vaginal screening area, a cervical fluid sorting area, a uterine deposition area, an oviduct guide area and a sperm collecting area, and the natural selection structure of the female reproductive system on the sperms is restored to the maximum extent and is similar to a channel for naturally screening the sperms in vivo; and the cervical fluid sorting area well restores a female reproductive system in structural form, so that the in-vitro sperm screening process by adopting the device of the embodiment is similar to the in-vivo natural sperm screening process, and the quality of in-vitro screened sperm is effectively improved.
2. The fluid passage pipe of this application embodiment can simulate the inside liquid flow environment of women reproductive system (the internal environment of reproductive tract is in mobile state rather than static), fine reduction women reproductive system in the liquid flow state for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm and further approach, thereby further improve the sperm quality of external screening.
3. The primary screening zone of vagina has certain length, wherein violently the baffler of establishing has simulated the horizontal line wrinkle wall in the vagina, can carry out preliminary separation and screening to seminal plasma, dead sperm, the sperm of motionless or original place motion and circular immature sperm cell in the semen sample, the fine reduction female reproductive system in physiology structure, the at utmost reduces female reproductive system and selects the nature of sperm, is similar with the passageway of internal natural screening sperm, make and adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm and further be close to further improve the sperm quality of external screening.
4. Uterus deposit district be down the pear shape, long 7cm, the width of widest department is 4cm, the axis department in uterus deposit district be equipped with first recess, the axis both sides are equipped with the arc and protrudingly. The length and width of the uterus deposition area simulate the size of a real uterus, and natural selection of female reproductive systems for sperms is restored to the maximum extent. The arc-shaped groove and the straight groove are separated by the arc-shaped protrusion, and the arc-shaped protrusion can play a role in saving the using amount of a culture solution and preventing the sperm from being excessively diluted.
5. This application embodiment two the arc wall and one the width of straight flute is all narrower, can form extremely simple miniflow channel system, when 3 strands of liquid flows are intersected promptly, because viscous force is greater than inertial force, the form that the liquid flows will keep the laminar flow and not torrent (original direction parallel flow and not mix), at this moment, the better sperm of motility will enter into the parallel liquid stream of both sides through the diffusion in the cervical canal body of central authorities, and impurity such as round cell that still exists after the less sperm of vigor and the prescreening will keep original liquid flow direction, deposit in the first recess on uterus sedimentation district axis. Uterus deposition district fine reduction women reproductive system in physiology structure, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
6. The embodiment of the application oviduct guide zone length be 3 ~ 4cm, also be according to the actual length in the anatomy, combine the long uterus deposit district of 7cm, can follow the distance and effectively filter fast, the sperm of vigor strong, this embodiment the device in physical distance, the physical structure fine reduction female reproductive system, the natural selection of at utmost reduction female reproductive system to the sperm is similar with the passageway of internal natural screening sperm, makes to adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm and further be close to further improve the sperm quality of external screening.
7. The sperm collecting area described in the embodiment of the present application is a circular groove with a diameter of 1cm, progesterone (main chemical secreted by oocytes or granulosa cells) is coated in the second groove, or a proper amount of follicular fluid/cumulus granulosa cells of female patients collected during the ovum-taking operation is added, so that the chemical inducer for attracting sperm is released from the oocytes and surrounding cumulus cells at the sperm-egg combining site, thereby eliminating sperm lacking chemotactic response capability, and finally screening to obtain healthy high-quality sperm. The device of this embodiment fine reduction women reproductive system in inside microenvironment factors such as biochemical secretion, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
8. The electric conduction heat patch of the embodiment of the application can generate heat after being electrified, so that the temperature of the sperm collecting area is kept at 37.5 ℃. The temperature adjusting controller can adjust the current passing through the lead, so that the temperature of the sperm collecting area is kept at 37.5 ℃. The heating module simulates gradient difference presented by the temperature inside the female reproductive system (under the normal physiological state, the temperature of the vagina, cervix, uterus and oviduct is gradually increased from outside to inside), namely the temperature gradient formed in the oviduct guide area further guides sperms to flow to the collection area, and eliminates the sperms lacking temperature response capability, thereby further improving the quality of the sperms screened in vitro. The device of this embodiment fine reduction women reproductive system in the temperature, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
In conclusion, the device provided by the embodiment can better restore (rather than bypass) the process of natural screening in the sperm body, effectively reduce the damage to the sperm caused by the in vitro operation process, and can screen the sperm in a limiting manner by fully considering internal microenvironment factors such as physical distance, liquid flow state, temperature, biochemical secretion and the like in the female reproductive system and unique physiological structural factors such as vaginal crinkle wall, cervical canal, oviduct and the like, so that the whole screening process is more comprehensive and natural, and high-quality sperm with strong mobility, normal morphological function and good DNA integrity can be screened more effectively. Meanwhile, the device has the advantages of reasonable design, simple structure, easy production, low cost and reliable effect.
The foregoing description is only an overview of the technical solutions of the present application, and the present application can be implemented according to the content of the description in order to make the technical means of the present application more clearly understood, and the following detailed description of the present application is given in order to make the above and other objects, features, and advantages of the present application more clearly understandable.
Drawings
FIG. 1 is a schematic diagram of the structure of the base part of an in vitro high-quality sperm screening apparatus according to an embodiment of the present invention;
fig. 2 is a schematic structural diagram of an upper cover part of a high-quality sperm in-vitro screening device in the embodiment of the invention.
Description of reference numerals: the sperm injection device comprises a base part 1, a semen sampling region 11, a vaginal primary screening region 12, a blocking plate 121, a cervical fluid sorting region 13, a cervical tubule 131, a fluid passage tube 132, a liquid adding region 133, a first switch 134, a uterine deposition region 14, a first groove 141, an arc-shaped protrusion 142, an oviduct guide region 15, a sperm collecting region 16, a second groove 161, a second switch 162, a dish edge 17, a heating module 18, an electric heat conduction patch 181, a thermal resistor 182, a temperature regulation controller 183, a switch 184, a lead 185, a power supply 186 and an upper cover part 2.
Detailed Description
The embodiment of the application provides an in vitro sieving mechanism of high quality sperm and application method for solve among the prior art because the screening factor that in vitro sperm sieving mechanism set up is single, and it is great with the internal natural screening process difference of sperm, thereby lead to the technical problem that the sperm quality of screening is high inadequately.
In order to solve the technical problems, the technical scheme provided by the application has the following general idea: the screening channel similar to the channel for naturally screening sperms in the body is arranged, so that the natural selection structure of the female reproductive system for the sperms is restored to the maximum extent and is similar to the channel for naturally screening the sperms in the body; the in vitro sperm screening process by adopting the device of the embodiment is close to the in vivo natural screening process of the sperm, thereby effectively improving the quality of the in vitro screened sperm.
The technical solutions of the present application are described in detail below with reference to the drawings and specific embodiments, and it should be understood that the specific features in the embodiments and examples of the present application are detailed descriptions of the technical solutions of the present application, and are not limitations of the technical solutions of the present application, and the technical features in the embodiments and examples of the present application may be combined with each other without conflict.
Example 1
Fig. 1 is a schematic structural view of a base portion of a high-quality sperm in-vitro screening apparatus according to an embodiment of the present invention, and fig. 2 is a schematic structural view of an upper cover portion of the high-quality sperm in-vitro screening apparatus according to the embodiment of the present invention. As shown in figures 1 and 2, the device comprises a base part 1 and an upper cover part 2 which can be matched with each other, wherein the base part 1 comprises a semen sample adding area 11, a vaginal primary screening area 12, a cervical fluid sorting area 13, a uterine sedimentation area 14, an oviduct guiding area 15 and a sperm collecting area 16 which are communicated in sequence to form a sperm screening channel.
Specifically, the base part 1 is a concave dish simulating the anatomical structure of the female reproductive system, and the upper cover part 2 is a plane dish cover. The primary vaginal screening area 12, the cervical fluid sorting area 13, the passage (i.e. straight groove and arc groove) for sperm flowing in the uterine deposition area 14 and the oviduct guiding area 15 are square grooves with square cross sections. Sperm travel along the sperm sorting channel, from the semen application region 11 to the sperm collection region 16. The base part 1 and the upper cover part 2 are made of transparent and high-temperature-resistant medical-grade high-molecular material (such as polypropylene) through one-step molding, and the base part 1 and the upper cover part 2 are matched in shape (namely, when the base part 1 and the upper cover part 2 are horizontally placed, the projection shapes on the horizontal plane are the same), so that the upper cover part 1 can be completely covered when the upper cover part 2 covers the base part 1.
Cervical fluid sorting zone 13 include cervical tube body and cervical tubule 131, wherein, cervical tubule 131 be thinner than cervical tube body with the vagina primary screening district 12, promptly the groove width of cervical tubule 131 be less than the groove width of cervical tube body with the groove width of vagina primary screening district 12, the entry end of cervical tubule 131 with vagina primary screening district 12 intercommunication, the exit end of cervical tubule 131 with the entry end intercommunication of cervical tube body, the exit end of cervical tube body with uterus sedimentation district 14 intercommunication.
Specifically, the cervical tubule 131 is a square groove with a groove width of 5mm and a length (along the sperm swimming direction) of 2.5cm, and the inlet of the cervical tubule 131 is a bell mouth; the cervical canal body and the primary vaginal screening area 12 are grooves. Cervical canal fluid sorting zone 13 through cervical canal body and cervical tubule 131 simulation cervix elongated channel to carry out further restriction screening to the sperm, make the device of this embodiment carry out external sperm screening and the internal natural screening process of sperm further approach, thereby further improve the sperm quality of external screening.
In summary, the screening channel of the device of this embodiment includes a semen sampling area 11, a primary vaginal screening area 12, a cervical fluid sorting area 13, a uterine deposition area 14, an oviduct guiding area 15 and a sperm collecting area 16, so as to restore the natural selection structure of the female reproductive system to sperm to the maximum extent, similar to the channel for natural sperm screening in vivo; and the cervical fluid sorting area 13 well restores the female reproductive system in structural morphology, so that the process of in vitro sperm screening by adopting the device of the embodiment is similar to the process of natural screening in sperm bodies, thereby effectively improving the quality of in vitro screened sperm.
Further, cervical fluid sorting zone 13 still include and be located the fluid passage pipe 132 of cervical tubule both sides, two fluid passage pipe 132 symmetry be located the both sides of cervical tubule 131, the exit end of fluid passage pipe 132 with the entry end of cervical canal body be linked together, the entry end of fluid passage pipe 132 to the slope of the both sides of cervical tubule 131 extension, the entry end of fluid passage pipe 132 be connected with liquid feeding area 133, fluid passage pipe 132 on still be equipped with first switch 134.
Specifically, the width of the fluid passage tube 132 is 7-8 mm, and the liquid feeding area 133 is a circular groove with a diameter of 1 cm. The first switch 134 is a first insertion plate, the fluid passage tube 132 is provided with a first insertion groove, the first insertion groove is perpendicular to the fluid passage tube 132, the first insertion plate is slidably arranged in the first insertion groove, and the first insertion plate completely covers the cross section of the fluid passage tube 132 to cut off the liquid in the fluid passage tube 132.
Furthermore, the included angle between the fluid passage tube 132 and the cervical tubule 131 is 15-60 °, the included angle is too small to form intersection, and the included angle is too large to cause the inertia force to be larger than the viscous force, so that the microfluidic channel system cannot be successfully constructed.
In this embodiment, the fluid passage tube 132 can simulate the fluid flowing environment inside the female reproductive system (the environment inside the reproductive tract is in a flowing state rather than a static state), and the female reproductive system is well restored in a fluid flowing state, so that the process of in vitro sperm screening and natural sperm in vivo sperm screening performed by the device of this embodiment is further similar, and the sperm quality of in vitro screening is further improved.
Furthermore, the semen sample adding area 11 is a circular groove, a notch on the top surface of the circular groove forms a sample adding port, and the diameter of the circular groove is 3 cm.
Further, the primary vaginal screening area 12 is a groove with an upward opening, and the inlet of the primary vaginal screening area 12 is communicated with the side surface of the semen sampling area 11; the semen sampling area 11 is equipped with a plurality of separation plates 121 in the primary vaginal screening area 12 at intervals towards the direction of the cervical fluid sorting area 13, the bottom end of the separation plates 121 is fixed on the bottom surface of the primary vaginal screening area 12, the top end of the separation plates 121 vertically extends upwards, and the height of the separation plates 121 is lower than the depth of the primary vaginal screening area 12 (namely, the height of the separation plates 121 is lower than the edge 17 of the base part 1).
Specifically, the length of the primary vaginal screening area 12 is 2-3 cm, the groove width is 2cm, the barrier plates 121 are thin plates, the barrier plates 121 are transversely arranged in the middle of the primary vaginal screening area 12, the number of the barrier plates 121 is 3-5, and the top ends of the barrier plates 121 are linear or curved.
In this embodiment, the primary vaginal screening zone 12 has a certain length, wherein the horizontally arranged barrier plate 121 simulates the horizontally moving wrinkle wall in the vagina, can perform primary blocking and screening on seminal plasma, dead sperm, sperm which does not move or moves in place and round immature sperm cells in a semen sample, and can restore the female reproductive system well in physiological structure, restore the natural selection of the female reproductive system on the sperm to the maximum extent, similar to the channel for naturally screening the sperm in vivo, so that the device of this embodiment is adopted to perform in vitro sperm screening and the natural screening process in the sperm in vivo further approach to further improve the sperm quality of in vitro screening.
Further, the uterus deposition area 14 comprises a big head end and a tip end, the outer edge of the uterus deposition area is arc-shaped, a straight groove is arranged at the central axis of the uterus deposition area 14, the straight groove, the cervix fluid sorting area 13 and the vagina screening area 12 are collinear, and a first groove 141 is arranged on the bottom of the straight groove and close to the inlet end of the straight groove; the bilateral symmetry of straight flute is equipped with the arc wall, the arc wall along the edge extension of uterus sedimentation district 14, the entry end of arc wall with the entry end of straight flute all with the exit end intercommunication of cervical canal body, two the exit end of arc wall all with the exit end of straight flute communicates each other.
Specifically, uterus deposit district 14 be the shape of falling the pear, long 7cm, the width of widest department is 4cm, uterus deposit district 14 the axis department be equipped with first recess 141, the axis both sides are equipped with arc protrusion 142. The length and width of the uterus deposition area simulate the size of a real uterus, and natural selection of female reproductive systems for sperms is restored to the maximum extent. The arc-shaped groove and the straight groove are separated by the arc-shaped protrusion, and the arc-shaped protrusion can play a role in saving the using amount of a culture solution and preventing the sperm from being excessively diluted.
In this embodiment, the two arc-shaped grooves and one straight groove are narrow in width, and can form a very simple microfluidic channel system, that is, when 3 liquid flows intersect, because the viscous force is greater than the inertial force, the liquid flow form will keep laminar flow rather than turbulent flow (original direction parallel flow without mixing), at this time, the sperm with better motility in the central cervical canal body will enter the parallel liquid flows at both sides through diffusion, and the sperm with poor motility and the impurities such as round cells still existing after primary screening will keep the original liquid flow direction, and deposit in the first groove 141 on the central axis of the uterine deposition region 14. Uterus deposition district fine reduction women reproductive system in physiology structure, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
Further, two oviduct guiding areas 15 are symmetrically arranged on two sides of the uterine deposition area 14, the inlet end of the oviduct guiding area 15 is communicated with one side of the arc-shaped groove, the outlet end of the oviduct guiding area 15 is communicated with the sperm collecting area 16, and a second switch 162 is arranged on the outlet end of the oviduct guiding area 15.
Specifically, in this embodiment, oviduct guide area length be 3 ~ 4cm, also according to anatomical actual length, combine 7cm long uterus sedimentation district 14, can follow from the distance to the sperm fast, the vigor is strong effectively to be screened, this embodiment the device in physical distance, the physical structure fine reduction women reproductive system, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further approach, thereby further improve the sperm quality of external screening.
Further, the second switch is a second inserting plate 134, a second inserting groove is formed in the oviduct guide area 15, the second inserting groove is perpendicular to the oviduct guide area 15, the second inserting plate can be slidably arranged in the second inserting groove, and the second inserting plate 134 completely covers the cross section of the oviduct guide area 15 so as to cut off liquid in the oviduct guide area 15.
Further, the width of the oviduct guiding region 15 gradually widens from the uterine deposition region 14 to the sperm collecting region 16. The length of the oviduct guide area is 3-4 cm; the width (groove width) of the junction 151 of the oviduct guiding region 15 and the uterine deposit region 14 is 2mm, and the oviduct guiding region 15 is gradually widened to 5mm toward both ends.
Furthermore, the sperm collecting area 16 is in the shape of a circular groove, a second groove 161 is arranged in the middle of the bottom of the sperm collecting area 16, and a progesterone layer is coated in the second groove 161.
In this embodiment, the sperm collecting region 16 is a circular groove with a diameter of 1cm, the second groove 161 is coated with progesterone (the main chemical secreted by the oocyte or granulosa cell), or added with a proper amount of follicular fluid/cumulus granulosa cell of a female patient collected during an oviposition surgery, so as to simulate that at the sperm-egg binding site, the oocyte and surrounding cumulus cells release a chemical inducer for attracting sperm, thereby eliminating sperm lacking chemotactic response capability, and finally screening to obtain healthy high-quality sperm. The device of this embodiment fine reduction women reproductive system in inside microenvironment factors such as biochemical secretion, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
Furthermore, a heating module 18 is arranged outside the sperm collecting region 16, the heating module 18 comprises an electric heat conduction patch 181 which is enclosed outside the sperm collecting region 16, the electric heat conduction patch 181 is connected with a power supply 186 through a lead 185, and the lead 185 is connected with a thermal resistor 182, a temperature adjusting controller 183 and a switch 184.
In this embodiment, the electrically conductive heat patch 181 generates heat when energized, so that the temperature of the sperm collection region 16 is maintained at 37.5 ℃. The temperature controller 183 can adjust the current passing through the wire 185 so as to maintain the temperature of the sperm collecting region 16 at 37.5 ℃. The heating module 18 simulates the gradient difference of the temperature inside the female reproductive system (in the normal physiological state, the temperature of the vagina, cervix, uterus and oviduct is gradually increased from outside to inside), namely the temperature gradient formed in the oviduct guide area further guides the sperms to move to the collection area, so that the sperms lacking the temperature response capability are eliminated, and the quality of the sperms screened in vitro is further improved. The device of this embodiment fine reduction women reproductive system in the temperature, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
The use method of the high-quality sperm in-vitro screening device comprises the following steps:
step A: opening the second switch 162, closing the first switch 134, and adding modified Human fallopian tube culture fluid (modified Human tubal fluid, abbreviated as mHTF, available from Irvine Scientific) containing 10% (volume fraction) of Human Serum Albumin (Human Serum Albumin, abbreviated as HSA) into the vaginal primary screening region 12, the uterine deposition region 13, the oviduct guide region 15 and the sperm collection region 16, wherein the total addition amount is 6-8 ml;
b, mixing and re-suspending the completely liquefied semen sample and the equal volume of mHTF containing 10% HSA, and then adding the mixture into the semen sample adding area 11;
and C: when the resuspended semen sample flows into the cervical tubule 131, mHTF containing 10% HSA is added to the liquid addition zone 133 on the fluid pathway tube 132 and the first switch 134 is turned on;
step D: a power switch 184 of the heating module 18 is turned on, the temperature is set to be 37.5 ℃ by adjusting the temperature adjusting controller 183, the upper cover 2 is closed, and the whole device is moved into a constant temperature and humidity incubator with the temperature of 32 ℃ and the concentration of CO2 of 6% for incubation for 30-40 min;
step E: taking the device out of the incubator, opening the upper cover 2, closing the first switch 134, covering the upper cover 2, and then moving the device into a constant-temperature and constant-humidity incubator with the temperature of 32 ℃ and the CO2 concentration of 6% for incubation for 120-150 min;
step F: removing the device from the incubator, opening the cover 2, closing the second switch 162, and using a pasteur pipette or pipettor to aspirate the screened sperm from the sperm collection region 16;
step G: and (3) inspecting the collected sperms for quality indexes such as concentration, vitality, morphology, DNA fragments and the like, comparing the quality indexes with the samples before screening, and judging the screening effect of the device.
Step H: the collected sperm were placed in a constant temperature and humidity incubator at 37 ℃ and 6% CO2 concentration for subsequent routine in vitro fertilization or intracytoplasmic sperm injection (ICSI) procedures.
It should be noted that the device is fully cleaned by deionized water before formal application, subjected to surface hydrophilic treatment (ensuring smooth long and thin pipeline) by a plasma cleaning machine, and finally placed in an oven for drying and then sealed and packaged. Ethylene oxide sterilization is not recommended prior to use of the device, given that residual ethylene oxide can be toxic to sperm.
Experimental verification
Sperm cells screened according to the method described in this example were tested using the apparatus described in this example as follows:
(1) and (3) measuring the concentration of the screened sperms:
and sucking 10 mu l of the sperm sample screened in vitro from the sperm collecting region 16 by using a pipette, loading the sperm sample into a counting pool of a Makler sperm counting plate, observing the distribution of the sperm, and counting the number of the sperm in the square under a 200-time mirror. Selecting 3 different visual fields, repeating counting, ensuring the same number of cells counted for 3 times, respectively calculating the total number of the counted sperms for 3 times, and taking the average value. Sperm concentration N10/N (106/ml) where N is the total number of sperm counted and N is the number of cells.
See WHO's manual for human semen examination and treatment laboratory of world health organization 5 th edition (2010 edition).
Counting results show that: the semen sample with the initial concentration of 95 × 106/ml is screened to obtain the semen with the concentration of 3.3 × 106/ml; the semen sample with the initial concentration of 67 × 106/ml is screened to obtain the semen concentration of 0.9 × 106/ml; semen samples with an initial concentration of 181 × 106/ml were screened to give a sperm concentration of 6.1 × 106/ml. The suggestion, through the screening of this device, the vast majority of sperm in the former semen sample have been screened out.
(2) And (3) measuring the sperm motility after screening:
a10. mu.l sample of the in vitro-screened sperm is aspirated from the sperm collecting region 16 by a pipette, and loaded into a counting cell of a Makler (Mark) sperm counting plate, the distribution of the sperm is observed, the numbers of forward movement (PR), non-forward movement (NP) and immobile sperm (IM) are recorded under a 200-fold mirror, and the percentage of each level of sperm is calculated. Different 3 visual fields are selected for repeated counting and statistics, the same grid number is guaranteed to be counted for 3 times, and the average value is taken.
See WHO's manual for human semen examination and treatment laboratory of world health organization 5 th edition (2010 edition).
The activity measurement result shows that: initial PR: 35%, NP: 5%, IM: 60% of semen specimen, wherein PR of the sperm obtained after screening is 100%; initial PR: 40%, NP: 12%, IM: the PR of the sperm obtained by screening a 48 percent semen sample is 100 percent; initial PR: 25%, NP: 6%, IM: the PR of the sperm obtained by screening 69 percent of semen samples is 100 percent; initial PR: 19%, NP: 5%, IM: the sperm sample of 76% has PR 99%, NP 1% and IM 0% after screening. The suggestion, through the screening of this device, can obtain high activity sperm.
(3) Evaluation and analysis of sperm morphology after screening:
sucking 10 μ l of the in vitro screened sperm sample from the sperm collection area 16 with a pipette, dropping the sample onto one end of a slide, moving a second slide along the surface of the first slide at an angle of 45 ° and removing the sperm sample droplet, then slowly pulling back the pull tab along the long axis of the slide to form a sperm smear, placing the coated slide horizontally, drying naturally in air, and then performing repeated sperm morphological analysis with the Diff-quik method: fixing the slide glass in the fixing solution for 10s, erecting the slide glass on absorbent paper to remove the excess liquid, staining in staining solution A (xanthene capable of binding with basic protein in sperm cytoplasm to give pink color), removing the excess liquid, staining in staining solution B (azathiobenzene capable of binding with acidic protein in sperm cytoplasm to give purple color) for 15s, soaking in running water for 10-15 times, air drying, and classifying and counting sperm morphology under 100-fold objective lens of optical microscope. The above method was repeated 1-2 times for counting analysis.
Normal sperm: the head is oval (the size is normal, the length-width ratio is 1.50-1.75, the limit of the top body is clear, the top body accounts for 40% -70% of the head), and the head, the neck, the middle section and the tail are normal.
Defective sperm: the head is conical, pear-shaped, round, amorphous, vacuole-containing, acrosome-free or small acrosome; the neck or middle section is curved, asymmetric, too thick, too thin; the tail part is short, the angle is folded, and the tail part is bent greatly; the middle section has a cytoplasmic droplet.
See WHO's manual for human semen examination and treatment laboratory of world health organization 5 th edition (2010 edition).
The results of sperm morphological evaluation showed that: the semen specimen with the initial morphological normal rate of 9 percent obtains the normal morphological rate of 56 percent of the sperms after screening; the semen specimen with the initial morphological normal rate of 5 percent obtains the normal morphological rate of 45 percent of the sperms after screening; the semen specimen with the initial morphological normal rate of 3 percent obtains the normal morphological rate of 27 percent of the sperms after screening; the semen specimen with the initial morphological normal rate of 1 percent obtains the sperm with the normal morphological rate of 9 percent after screening; the semen specimen with the initial morphological normal rate of 7 percent obtains the sperm with the normal morphological rate of 34 percent after screening. It is suggested that by the screening of the device, higher proportion of morphologically normal sperm can be obtained.
(4) Sperm DNA fragmentation rate determination after screening:
according to the principle of sperm chromatin dispersion experiment, a DNA fragment detection kit of Shenzhen Boruide company is selected, and DNA integrity detection is carried out according to a related instruction manual: putting an EP tube filled with agarose in a water bath kettle at 80 ℃ for 20min to melt the agarose, then putting the tube in a warm box at 37 ℃ for balancing, sucking 60 mu l of sperm sample screened in vitro from a sperm collecting area 16, mixing the sperm sample with the agar, taking 30 mu l of the mixture, loading the mixture on a slide which is provided with a coating and is pre-cooled at 4 ℃ so as to enable the agar to become microgel, embedding the sperm into the gel, immersing the slide without a cover glass into an acidic reaction solution A for 7min, transferring the slide into a reaction solution B for 25min, washing the slide with distilled water for 5min, dehydrating the slide in ethanol with a certain concentration gradient (70% -90% -100%), drying the slide in the air, dyeing the slide with a Wright (Ray) dye solution for 15min, and observing and counting the DNA integrity of the sperm under a 400-fold mirror after drying.
Whether or not a sperm contains DNA debris depends on the extent of diffusion of sperm chromatin.
Sperm containing DNA debris: 1/3 that the width of red halo on the head is less than or equal to the minimum diameter of the head of the sperm after the sperm is dyed
DNA-normal sperm: width of red halo on head after sperm staining > 1/3 of smallest diameter of sperm head
See WHO's manual for human semen examination and treatment laboratory of world health organization 5 th edition (2010 edition).
The result of sperm DNA fragmentation rate measurement shows that: the semen sample with the initial DNA fragmentation rate of 19 percent is screened to obtain the sperm with the DNA fragmentation rate reduced to 6 percent; the semen sample with the initial DNA fragmentation rate of 16 percent is screened to obtain the sperm with the DNA fragmentation rate reduced to 4 percent; the semen sample with the initial DNA fragmentation rate of 9 percent is screened to obtain the sperm with the DNA fragmentation rate reduced to 2 percent; the semen sample with the initial DNA fragmentation rate of 26.5 percent reduces the DNA fragmentation rate of the sperm to 6 percent after screening. The device is used for screening to obtain sperms with complete and normal DNA in higher proportion.
Through the verification, the device can effectively screen out high-quality sperms with strong mobility, normal morphological function and good DNA integrity.
In conclusion, the device provided by the embodiment can better restore (rather than bypass) the process of natural screening in the sperm body, effectively reduce the damage to the sperm caused by the in vitro operation process, and can screen the sperm in a limiting manner by fully considering internal microenvironment factors such as physical distance, liquid flow state, temperature, biochemical secretion and the like in the female reproductive system and unique physiological structural factors such as vaginal crinkle wall, cervical canal, oviduct and the like, so that the whole screening process is more comprehensive and natural, and high-quality sperm with strong mobility, normal morphological function and good DNA integrity can be screened more effectively. Meanwhile, the device has the advantages of reasonable design, simple structure, easy production, low cost and reliable effect.
The high-quality sperm in-vitro screening device and the using method thereof are as follows:
1. the screening channel of the device comprises a semen sampling area 11, a primary vaginal screening area 12, a cervical fluid sorting area 13, a uterine deposition area 14, an oviduct guide area 15 and a sperm collecting area 16, and the natural selection structure of the female reproductive system on the sperms is restored to the maximum extent and is similar to the channel for naturally screening the sperms in vivo; and the cervical fluid sorting area 13 well restores the female reproductive system in structural morphology, so that the process of in vitro sperm screening by adopting the device of the embodiment is similar to the process of natural screening in sperm bodies, thereby effectively improving the quality of in vitro screened sperm.
2. The fluid passage pipe described in this embodiment can simulate the liquid flow environment inside the female reproductive system (the environment in the reproductive tract is in a flow state rather than static), and the female reproductive system is well restored in a liquid flow state, so that the process of in vitro sperm screening and natural sperm screening performed by the device described in this embodiment is further similar, and the sperm quality of in vitro screening is further improved.
3. The primary screening zone 12 of the vagina described in this embodiment has a certain length, wherein the barrier plate 121 transversely arranged simulates the transverse wrinkle wall in the vagina, can perform preliminary blocking and screening on seminal plasma, dead sperm, sperm which does not move or moves in place and round immature sperm cells in a semen sample, well restore the female reproductive system in physiological structure, restore the natural selection of the female reproductive system to the sperm to the maximum extent, and is similar to the channel for naturally screening the sperm in vivo, so that the device described in this embodiment is adopted to perform in vitro sperm screening and the natural screening process in the sperm in vivo further approach, thereby further improving the sperm quality of in vitro screening.
4. Uterus deposit district 14 described in this embodiment be the shape of falling the pear, long 7cm, the width of widest department is 4cm, uterus deposit district 14 the axis department be equipped with first recess 141, the axis both sides are equipped with arc protrusion 142. The length and width of the uterus deposition area simulate the size of a real uterus, and natural selection of female reproductive systems for sperms is restored to the maximum extent. The arc-shaped groove and the straight groove are separated by the arc-shaped protrusion, and the arc-shaped protrusion can play a role in saving the using amount of a culture solution and preventing the sperm from being excessively diluted.
5. The width of two arc grooves and one straight groove described in this embodiment is all narrower, can form extremely simple miniflow channel system, when 3 liquid streams intersect, because viscous force is greater than inertial force, the form that the liquid flows will keep laminar flow rather than turbulent flow (original direction parallel flow and not mix), at this moment, the better sperm of motility will enter into the parallel liquid stream of both sides through the diffusion effect in the cervical canal body of central authorities, and impurity such as the less sperm of vitality and the round cell that still exists after the prescreening will keep original liquid flow direction, deposit in first recess 141 on uterus sedimentation zone 14 axis. Uterus deposition district fine reduction women reproductive system in physiology structure, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
6. The length of the oviduct guide area is 3-4 cm in the embodiment, the oviduct guide area is also the actual length in the aspect of anatomy, and the 7cm long uterine deposition area 14 is combined, so that the sperm with high speed and strong vitality can be effectively screened in the aspect of distance.
7. In this embodiment, the sperm collecting region 16 is a circular groove with a diameter of 1cm, and progesterone (a main chemical secreted by oocytes or granulosa cells) is coated in the second groove 161, or a proper amount of follicular fluid/cumulus granulosa cells of a female patient collected during an oviposition surgery is added, so that a chemical inducer for attracting sperm is released from the oocytes and surrounding cumulus cells at the sperm-egg binding site, thereby eliminating sperm lacking chemotactic response capability, and finally screening to obtain healthy high-quality sperm. The device of this embodiment fine reduction women reproductive system in inside microenvironment factors such as biochemical secretion, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
8. The electrically conductive heat patch 181 of this embodiment is capable of generating heat when energized, so that the temperature of the sperm collection area 16 is maintained at 37.5 ℃. The temperature controller 183 can adjust the current passing through the wire 185 so as to maintain the temperature of the sperm collecting region 16 at 37.5 ℃. The heating module 18 simulates the gradient difference of the temperature inside the female reproductive system (in the normal physiological state, the temperature of the vagina, cervix, uterus and oviduct is gradually increased from outside to inside), namely the temperature gradient formed in the oviduct guide area further guides the sperms to move to the collection area, so that the sperms lacking the temperature response capability are eliminated, and the quality of the sperms screened in vitro is further improved. The device of this embodiment fine reduction women reproductive system in the temperature, the natural selection of furthest reduction women reproductive system to the sperm is similar with the passageway of internal natural screening sperm for adopt this embodiment the device carry out external sperm screening and the internal natural screening process of sperm further be close, thereby further improve the sperm quality of external screening.
In conclusion, the device provided by the embodiment can better restore (rather than bypass) the process of natural screening in the sperm body, effectively reduce the damage to the sperm caused by the in vitro operation process, and can screen the sperm in a limiting manner by fully considering internal microenvironment factors such as physical distance, liquid flow state, temperature, biochemical secretion and the like in the female reproductive system and unique physiological structural factors such as vaginal crinkle wall, cervical canal, oviduct and the like, so that the whole screening process is more comprehensive and natural, and high-quality sperm with strong mobility, normal morphological function and good DNA integrity can be screened more effectively. Meanwhile, the device has the advantages of reasonable design, simple structure, easy production, low cost and reliable effect.
Example 2
This example differs from example 1 in that: the primary vaginal screening zone 12, the cervical fluid sorting zone 13, the channels for sperm flowing in the uterine deposition zone 14 (i.e. straight and arc grooves) and the tubal guiding zone 15 are all semicircular grooves (semicircular in cross section), and the groove widths (widths) listed in example 1 are the corresponding diameters in this example, for example: the cervical tubule 131 is thinner than the cervical tube body and the primary vaginal screening area 12, namely, the diameter of the cervical tubule 131 is smaller than the diameter of the cervical tube body and the diameter of the primary vaginal screening area 12, and the cervical tubule 131 is a semicircular groove with the diameter of 5mm and the length of 2.5 cm.
Other technical schemes of the embodiment are the same as those of the embodiment 1.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, 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 or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. The utility model provides an external sieving mechanism of high quality sperm which characterized in that: the device comprises a base part and an upper cover part which can be matched with each other, wherein the base part comprises a semen sampling area, a vaginal primary screening area, a cervical fluid sorting area, a uterine deposition area, an oviduct guide area and a sperm collecting area which are sequentially communicated to form a sperm screening channel;
cervical fluid sorting district include cervical canal body and cervical tubule, wherein, cervical tubule be thinner than cervical canal body with the vagina prescreening district, the entry end of cervical tubule with the vagina prescreening district intercommunication, the exit end of cervical tubule with the entry end intercommunication of cervical canal body, the exit end of cervical canal body with uterus deposit district intercommunication.
2. The in vitro screening device of claim 1, wherein said fluid sorting area further comprises fluid passage tubes located at two sides of said cervical tubule, two of said fluid passage tubes are symmetrically located at two sides of said cervical tubule, the outlet end of said fluid passage tube is connected to the inlet end of said cervical canal body, the inlet end of said fluid passage tube extends obliquely towards two sides of said cervical tubule, the inlet end of said fluid passage tube is connected to a liquid feeding area, and said fluid passage tube is further provided with a first switch.
3. The in vitro high-quality sperm screening device of claim 2, wherein the included angle between said fluid passage tube and said cervical tubule is 15 ° to 60 °.
4. The in vitro high-quality sperm cell screening device of claim 3 wherein said primary vaginal screening zone is an upwardly open groove and the inlet of said primary vaginal screening zone is in communication with the side of said semen application zone; the vagina primary screening district in from semen adding appearance district to the direction interval of cervical fluid sorting district is equipped with a plurality of baffler board, the bottom of baffler board fix on the bottom surface in vagina primary screening district, the top of baffler board vertically upwards extend, just the height of baffler board is less than the degree of depth in vagina primary screening district.
5. The in vitro high-quality sperm screening device according to claim 4, wherein said uterine cavity comprises a large end and a pointed end, said uterine cavity has an outer edge in the shape of a circular arc, said uterine cavity has a straight groove at its central axis, said straight groove, said cervical fluid sorting region and said vaginal screening region are collinear, and a first groove is formed on the bottom of said straight groove near the entrance end of said straight groove; the bilateral symmetry of straight flute is equipped with the arc wall, the arc wall along the edge extension of uterus deposit district, the entry end of arc wall with the entry end of straight flute all with the exit end intercommunication of cervical canal body, two the exit end of arc wall all with the exit end of straight flute communicates each other.
6. The in vitro high-quality sperm screening device of claim 5, wherein two said oviduct guiding areas are symmetrically arranged on both sides of said uterine deposition area, the inlet end of said oviduct guiding area is communicated with one side of said arc-shaped groove, the outlet end of said oviduct guiding area is communicated with said sperm collecting area, and the outlet end of said oviduct guiding area is provided with a second switch.
7. The in vitro high-quality sperm cell screening device of claim 6, wherein said oviduct guide region has a width that gradually widens from said uterine deposit region to said sperm collecting region.
8. The in vitro high-quality sperm screening device according to claim 7, wherein said sperm collection area is in the form of a circular groove, a second groove is provided in the middle of the bottom of said sperm collection area, and a progesterone layer is coated in said second groove.
9. The apparatus according to claim 8, wherein a heating module is provided outside the sperm collection area, said heating module comprises an electrically conductive patch enclosed outside said sperm collection area, said electrically conductive patch is connected to a power source via a wire, and said wire is connected to a thermal resistor, a temperature controller and a switch.
10. The method of using a high quality sperm cell in vitro screening device according to any one of claims 1 to 9, comprising the steps of:
step A: opening the second switch, closing the first switch, and adding improved human oviduct culture solution containing human serum albumin into the primary vaginal screening area, the uterine deposition area, the oviduct guide area and the sperm collecting area respectively;
b, mixing and re-suspending the completely liquefied semen sample and the improved human oviduct culture solution with the same volume, and then adding the mixture into the semen sample adding area;
and C: when the resuspended semen sample flows into the cervical tubule, adding the improved human oviduct culture solution into the solution adding area on the fluid passage tube, and turning on the first switch;
step D: turning on a power switch of the heating module, setting the temperature to be 37.5 ℃ by adjusting the temperature adjusting controller, covering the upper cover, and moving the whole device into a constant-temperature constant-humidity incubator for incubation;
step E: taking the device out of the incubator, opening the upper cover, closing the first switch, covering the upper cover, and moving the device into the incubator again for incubation;
step F: taking out the device from the incubator, opening the upper cover, closing the second switch, and sucking screened sperms from the sperm collecting area by using a Pasteur pipette or a pipettor;
step G: inspecting the collected sperms, comparing the inspected sperms with a sample before screening, and judging the screening effect of the device;
step H: the collected sperm are placed into the incubator and await subsequent conventional in vitro fertilization or intracytoplasmic sperm microinjection procedures.
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