CN105660607A - Fertility preserving method of completely immovable sperms - Google Patents
Fertility preserving method of completely immovable sperms Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention discloses a fertility preserving method of completely immovable sperms. The fertility preserving method includes following steps: 1, freezing the completely immovable sperms: collecting the completely sperms, taking a small part out, using laser to identify whether live sperms exist or not, if yes, freeze preserving, and if not, abandoning; 2, evaluating sperm survival state before and after freezing the completely immovable sperms: if the tails of sperms immediately wind up obviously after laser shooting, judging as live sperms, and judging as dead sperms if the tails of the sperms do not wind up and have no response to laser shooting. A circumstance that the completely immovable sperms can be freeze preserved is testified for the first time, and the sperms after being frozen have normal in-vitro fertilization capability. In addition, a method of efficiently freeze preserving fertility of these special groups is developed, and fertilization rate similar to that of fresh immovable sperms can be acquired through laser screening of live but completely immovable frozen sperms for intracytoplasmic sperm injection. Related reports do not appear at home and abroad.
Description
Technical field
The present invention relates to a kind of method that sperm cell fertility preserves, the fertility store method of especially a kind of complete spermatium.
Background technology
At present, the semen cryopreservation technology history of existing more than 200 year. The semen cryopreservation of people can trace back to late 1940s. Semen cryopreservation is mainly used in the preservation of human sperm bank and fertility in China. Domestic and international researcher passes through the decades of the screening to freezing of semen program, freeze-extender, thawing solution etc., has been set up comparatively ripe semen cryopreservation system. The each reproductive center in the whole nation is relatively larger to the demand of Seminal plasma cryoprotective agent, and on market, Seminal plasma Cryoprotectant is almost all monopolized by external biotech firm, and domestic only part scientific research institutions configure poultry semen cryoprotectant voluntarily. Domestic it is applied to clinic but without independently developed human seminal fluid's cryoprotective agent, and existing commercial human seminal fluid's freezen protective system is suitable for the normal semen of people, the preservation effect of few azoospermia is unsatisfactory.
Sperm freezing is to preserve male fertility most efficient method, it has also become requisite technology in Issues of Human Assisted Reproductive Technologies (ART). Many studies have shown that, derive from injection, epididymis punctures the chilled preservation of sperm of (PESA) and testicular biopsy (TESA) and the Clinical Outcome similar to fresh spermatozoa can be obtained after ICSI (ICSI). Traditional viewpoint is thought, the premise of sperm cryopreservation needs the sperm of motion. But, whether have freezen protective to be worth for complete no motion of sperm unclear.
ICSI technology is to treat the promising approach of serious male sterility at present. Have been reported that, apply and can not obtain clinical pregnancy and give birth to healthy offspring after motile row ICSI, but, its rate of fertilization, fetal development potential are significantly lower than motile sperm. Studying confirmation further, motile is not intended that it is dead, and the sperm of its survival still has normal fertility. This prompting, the basis of ICSI is the need for the sperm of survival.So, differentiate the existing state in complete spermatium is crucial how to select survival sperm to carry out fertilization after determining whether applicable freezen protective and thawing how quickly, accurately, efficiently and safely. WHO the 5th edition " human seminal fluid checks and processes workbook " recommend hypo-osmotic swelling test and eosin stains for the vigor of diagnostic assessment sperm. But, both approaches Shortcomings part, the former operates sperm that is cumbersome and that be not suitable for freeze-thaw, even if the latter dyed after be accredited as survival sperm also unrenewable in fertilization. Further study show that, when application laser irradiates sperm uropatagium, the sperm of survival produces unexpected emergency reaction, and sperm tail is curling, and Necrospermia is without this phenomenon, and it is applied in fresh and after freeze thawing complete spermatium and all has similar phenomenon. Studying proof further, laser irradiates sperm tail to sperm head DNA not damaged, and sperm tail does not have tangible meaning in fertilization process. This prompting, laser is probably a kind of effective ways safely and effectively differentiating spermatium existing state.
Traditional viewpoint is thought, the sperm only moved is suitable for freezen protective. But, the activity ratio of some infertile patients sperm is extremely low, and even all of sperm does not all move completely, is more common in the sperm motionless disease of fibril hair and operation on testis takes smart patient. For the former, process conventional clinically is the complete spermatium abandoning injection, testicular needle aspiration trial searching motile sperm of changing one's profession of selecting a time. For the latter, if puncturing is out that complete spermatium also very likely goes out of use, row testicular biopsy art again of selecting a time. Repeated puncture certainly will add chance and the financial burden that patient's long term complication occurs, and therefore, how effectively preserving these patient's fertility is problem worth thinking deeply about.
Conventional sperm freezing method uses the cryovial of 2ml, and seminal fluid and freezing liquid are by putting into liquid nitrogen freezing preservation after 1: 1 dilution proportion after liquid nitrogen is stifling again. Owing in refrigerating process, the factor such as intracellular ice crystal formation can cause a degree of Sperm lesion, adopt the frozen rare sperm anabiosis rate of freezing method traditional at present and the response rate relatively low, after recovery, spermatid membrane damage is serious, causes being not used to follow-up assisted reproductive therapy. Also some reports are had to utilize the empty zona pellucida freezing rareness sperm of the mankind and mice, but the more difficult acquisition of human oocyte zona pellucida, thereby increases and it is possible to the problem that there is pollution, it is difficult to popularization and application. Freezing ring also has and is applied to rare sperm freezing, but sperm to load difficulty bigger. Clinically, the motility of sperm that often some patient obtains is very micro-, even completely motionless. Therefore, a kind of method that need to urgently seek freezing spermatium disease patient's fertility completely stable, efficient.
Theoretical basis:
Sperm membrane integrity is closely related with sperm metabolism, acrosome reaction, capacitation and sperm-egg fusion. Additionally, sperm membrane function integrity is also closely related with germ cell implantation, Embryonic limb bud cell. Sperm tail cell membrane belongs to semipermeable membrane, has the ability of osmotic balance inside and outside film that is automatically adjusted. Laser has heat effect, when low-energy laser shoots motile sperm afterbody, is likely occurred protein denaturation owing to local temperature raises and causes the mobility of sperm tail cell membrane, permeability changes. When laser irradiates sperm uropatagium, heat effect makes the osmotic pressure inside and outside sperm uropatagium uneven, and the cell membrane of sperm of living is in complete state, possesses the ability of osmotic balance inside and outside cell membrane that regulates, and hydrone can by entering in film outside film; And the permeability of Necrospermia uropatagium changes, losing the ability of osmotic pressure inside and outside film that regulates, hydrone cannot be introduced in film. This characteristic of laser differentiates have motile sperm to provide new approaches for ICSI for embryologist from complete spermatium.
The processes such as human sperm needs through mixing with cryoprotective agent in refrigerating process, freezing cooling, ultra-low temperature storage and defrosting rewarming in advance. During this, the physical and chemical effect of experience directly acts on sperm affects the anabiosis rate of sperm. Affecting the main cause of sperm freezing and recovery: one is the formation of intracellular ice crystal, two is solute damage.-5 DEG C～-80 DEG C is the dangerous warm area that intracellular ice crystal is formed, and the formation of ice crystal damages spermatid film and organelle, even results in cell death. Extracellular icing may result in intraor extracellular and is hyperosmotic state and causes cell injury, i.e. solute damage. In refrigerating process, liquid can form crystal and vitreous body two states. Liquid is actually state cured with which kind of in refrigerating process, is mainly determined by the rate of temperature fall in the character of itself and refrigerating process. In glass freezing, due to freezing liquid small volume, so cooling is very rapid. In cooling procedure rapidly, the viscosity of liquid increases, and when viscosity reaches marginal value, cure occurs, forms irregular stiff glassy solids, transparent state, be prevented effectively from the formation of intraor extracellular ice crystal. Realizing vitrification cooling have to be rapid, and this just requires that the volume of freezing liquid is little as much as possible, and the heat conductivility of freezing carrier to be got well. Therefore, the vitrification method only needing trace freezing liquid can reduce the cryolesion in sperm freezing-course of defrosting to greatest extent.
Summary of the invention
It is an object of the invention to provide the fertility store method of a kind of complete spermatium.
For solving above-mentioned technical problem, the technical scheme is that
The fertility store method of this complete spermatium, is made up of following steps:
1. complete spermatium freezing method:
After collecting complete spermatium, take fraction out, with laser identify if any survival sperm be namely judged to can freezen protective, if without survival sperm, then discard; Residue seminal fluid is drawn to 100～200 μ L (microlitre), puts into special trace sperm freezing pipe, directly use slow-revving centrifuge; Remove supernatant after centrifugal, then add and the sperm freezing liquid of Sperm pellets 1: 1 volume, mixing, the cumulative volume making final suspension is 10 μ L; Will be equipped with the cryovial of sperm suspension and be placed in from liquid nitrogen surface 2-3cm place, stifling 3～5min, then freezen protective in direct plunge into Liquid Nitrogen; During defrosting, directly cryovial is put in the water-bath of preheated 37 DEG C, place 10min; Finally by sperm suspension sucking-off, it is positioned in culture dish, adds a little culture fluid dilution standby ICSI and ICSI;
2. the assessment of the freezing forward and backward sperm existing state of complete spermatium:
The spermatium of acquisition is joined Quinn ' s-1020 namely by seminal fluid operation ware, the good sperm of form is selected under the visual field, at sperm tail near tail tip place, hypomere in sperm tail is irradiated with the diode laser bundle of 1.48 μm (microns), irradiation time is 2ms (millisecond), if after laser shooting, sperm tail occurs obvious end of reel phenomenon immediately, then be judged to survival sperm, to laser shooting reactionless do not occur the end of reel for Necrospermia; Before freezing and calculate the survival rate of spermatium after recovery respectively, with the survival index after freezing, evaluate the refrigerating effect of complete immotile sperm;
The trace of complete spermatium is freezing, and the cumulative volume of its final sperm freezing suspension is only 10 μ L;
Above-mentioned identifies the survival condition application laser before complete spermatium freezing, after freezing.
Survival index is the living spermatozoa percentage before the survival rate/freezing after freezing.
Beneficial effect:
First demonstration that completely not motile can freezen protective, after freezing, these sperms have normal external fertilization ability; Meanwhile, establishing the method that high-efficiency freezing preserves this part specific group fertility, this preserves for complete spermatium disease patient's fertility and provides New view.
Applying method of the present invention, still obtain higher survival rate after collecting the chilled recovery of complete spermatium of injection and testicular biopsy acquisition, overall sperm survival index is 0.83. With laser screening survival but completely motionless frozen sperm can obtain the rate of fertilization similar to fresh spermatium after carrying out ICSI. It is showed no relevant report both at home and abroad.
Detailed description of the invention
Embodiment 1:
The fertility store method of this complete spermatium, is made up of following steps:
1. complete spermatium freezing method:
After collecting complete spermatium, take fraction out, with laser identify if any survival sperm be namely judged to can freezen protective, if without survival sperm, then discard; Residue seminal fluid is drawn to 100 μ L (microlitre), puts into special trace sperm freezing pipe, directly use slow-revving centrifuge; Remove supernatant after centrifugal, then add and the sperm freezing liquid of Sperm pellets 1: 1 volume, mixing, the cumulative volume making final suspension is 10 μ L; Will be equipped with the cryovial of sperm suspension and be placed in from liquid nitrogen surface 2cm place, stifling 3min, then freezen protective in direct plunge into Liquid Nitrogen; During defrosting, directly cryovial is put in the water-bath of preheated 37 DEG C, place 10min; Finally by sperm suspension sucking-off, it is positioned in culture dish, adds a little culture fluid dilution standby ICSI and ICSI;
2. the assessment of the freezing forward and backward sperm existing state of complete spermatium:
The spermatium of acquisition is joined Quinn ' s-1020 namely by seminal fluid operation ware, the good sperm of form is selected under the visual field, at sperm tail near tail tip place, hypomere in sperm tail is irradiated with the diode laser bundle of 1.48 μm (microns), irradiation time is 2ms (millisecond), if after laser shooting, sperm tail occurs obvious end of reel phenomenon immediately, then be judged to survival sperm, to laser shooting reactionless do not occur the end of reel for Necrospermia; Before freezing and calculate the survival rate of spermatium after recovery respectively, with the survival index after freezing, evaluate the refrigerating effect of complete immotile sperm;
The trace of complete spermatium is freezing, and the cumulative volume of its final sperm freezing suspension is only 10 μ L;
Above-mentioned identifies the survival condition application laser before complete spermatium freezing, after freezing.
Survival index is the living spermatozoa percentage before the survival rate/freezing after freezing.
Embodiment 2:
The fertility store method of this complete spermatium, is made up of following steps:
1. complete spermatium freezing method:
After collecting complete spermatium, take fraction out, with laser identify if any survival sperm be namely judged to can freezen protective, if without survival sperm, then discard; Residue seminal fluid is drawn to 200 μ L (microlitre), puts into special trace sperm freezing pipe, directly use slow-revving centrifuge; Remove supernatant after centrifugal, then add and the sperm freezing liquid of Sperm pellets 1: 1 volume, mixing, the cumulative volume making final suspension is 10 μ L;Will be equipped with the cryovial of sperm suspension and be placed in from liquid nitrogen surface 3cm place, stifling 5min, then freezen protective in direct plunge into Liquid Nitrogen; During defrosting, directly cryovial is put in the water-bath of preheated 37 DEG C, place 10min; Finally by sperm suspension sucking-off, it is positioned in culture dish, adds a little culture fluid dilution standby ICSI and ICSI;
2. the assessment of the freezing forward and backward sperm existing state of complete spermatium:
The spermatium of acquisition is joined Quinn ' s-1020 namely by seminal fluid operation ware, the good sperm of form is selected under the visual field, at sperm tail near tail tip place, hypomere in sperm tail is irradiated with the diode laser bundle of 1.48 μm (microns), irradiation time is 2ms (millisecond), if after laser shooting, sperm tail occurs obvious end of reel phenomenon immediately, then be judged to survival sperm, to laser shooting reactionless do not occur the end of reel for Necrospermia; Before freezing and calculate the survival rate of spermatium after recovery respectively, with the survival index after freezing, evaluate the refrigerating effect of complete immotile sperm;
The trace of complete spermatium is freezing, and the cumulative volume of its final sperm freezing suspension is only 10 μ L;
Above-mentioned identifies the survival condition application laser before complete spermatium freezing, after freezing.
Survival index is the living spermatozoa percentage before the survival rate/freezing after freezing.
Clinical verification:
Early stage, we are to 9 example testicular needle aspiration and the patient without motile, differentiate that motionless but survival sperm is for ICSI, has 6 example patients to obtain clinical pregnancy (pregnancy rate 66.67%) with laser, wherein have 1 example to be born the child of health. Confirm that laser selects survival but the effectiveness of spermatium. Subsequently, patient's (1 example injection seminal fluid, 3 example testicular needle aspiration) of the 4 complete spermatiums of example after informed consent, is left and taken a small amount of sperm suspension and carries out trace freezen protective by us.
Sperm suspension is divided into two parts: a for counting the survival rate of sperm before freezing, another part is freezing for trace.
Before this 4 example patient's sperm freezing, average viability is 50.98%, and after defrosting, average viability is 42.36%, and total sperm survival index is 0.83 (this value is average living spermatozoa percentage before living spermatozoa percentage/freezing average after thawing). In order to verify the fertility of freeze-thaw sperm, we adopt laser method to select the sperm (sperm namely survived) of the end of reel that failed MII ovum of being fertilized is carried out ICSI checking. Through patient's informed consent voluntary donations/contributions, obtaining 61 pieces of unfertilized MII ovum altogether, wherein, 34 pieces are used for fresh spermatium, and 27 pieces for freezing spermatium. Fresh group of normal fertilization embryo 24 pieces, rate of fertilization is 70.59%; 19 pieces of normal fertilization of freezing group, rate of fertilization is 70.37%; The rate of fertilization of two groups compares no significant difference.
In sum, applying method of the present invention, still obtain higher survival rate after collecting the chilled recovery of complete spermatium of injection and testicular biopsy acquisition, overall sperm survival index is 0.83. With laser screening survival but completely motionless frozen sperm can obtain the rate of fertilization similar to fresh spermatium after carrying out ICSI. We prove first completely not motile be can freezen protective, and there is normal external fertilization ability, this is that complete spermatium disease patient's fertility preserves and provides New view. It is showed no relevant report both at home and abroad.
Claims (2)
1. a fertility store method for complete spermatium, is characterized in that: be made up of following steps:
1. complete spermatium freezing method:
After collecting complete spermatium, take fraction out, with laser identify if any survival sperm be namely judged to can freezen protective, if without survival sperm, then discard; Residue seminal fluid is drawn to 100～200 μ L (microlitre), puts into special trace sperm freezing pipe, directly use slow-revving centrifuge; Remove supernatant after centrifugal, then add and the sperm freezing liquid of Sperm pellets 1: 1 volume, mixing, the cumulative volume making final suspension is 10 μ L; Will be equipped with the cryovial of sperm suspension and be placed in from liquid nitrogen surface 2-3cm place, stifling 3～5min, then freezen protective in direct plunge into Liquid Nitrogen; During defrosting, directly cryovial is put in the water-bath of preheated 37 DEG C, place 10min; Finally by sperm suspension sucking-off, it is positioned in culture dish, adds a little culture fluid dilution standby ICSI and ICSI;
2. the assessment of the freezing forward and backward sperm existing state of complete spermatium:
The spermatium of acquisition is joined Quinn ' s-1020 namely by seminal fluid operation ware, the good sperm of form is selected under the visual field, at sperm tail near tail tip place, hypomere in sperm tail is irradiated with the diode laser bundle of 1.48 μm (microns), irradiation time is 2ms (millisecond), if after laser shooting, sperm tail occurs obvious end of reel phenomenon immediately, then be judged to survival sperm, to laser shooting reactionless do not occur the end of reel for Necrospermia; Before freezing and calculate the survival rate of spermatium after recovery respectively, with the survival index after freezing, evaluate the refrigerating effect of complete immotile sperm;
The trace of complete spermatium is freezing, and the cumulative volume of its final sperm freezing suspension is only 10 μ L;
Above-mentioned identifies the survival condition application laser before complete spermatium freezing, after freezing.
2. the fertility store method of a kind of complete spermatium according to claim 1, it is characterised in that: described survival index is the living spermatozoa percentage before the survival rate/freezing after freezing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108935445A (en) * | 2018-08-08 | 2018-12-07 | 贵州医科大学附属医院 | A kind of jelly precision method based on ICSI micromanipulation |
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| CN203290151U (en) * | 2013-01-21 | 2013-11-20 | 郜鸿生物科技（上海）有限公司 | Novel closed-type refrigerating superfine tube carrier for rare sperms |
| CN104823966A (en) * | 2015-05-19 | 2015-08-12 | 上海交通大学医学院附属第九人民医院 | Trace sperm cryopreservation carrier and corresponding freezing and thawing methods thereof |
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2016
- 2016-03-18 CN CN201610157472.7A patent/CN105660607A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN203290151U (en) * | 2013-01-21 | 2013-11-20 | 郜鸿生物科技（上海）有限公司 | Novel closed-type refrigerating superfine tube carrier for rare sperms |
| CN104823966A (en) * | 2015-05-19 | 2015-08-12 | 上海交通大学医学院附属第九人民医院 | Trace sperm cryopreservation carrier and corresponding freezing and thawing methods thereof |
Non-Patent Citations (1)
| Title |
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| 陈焕华等: "应用激光从完全不动精子中筛选存活精子行ICSI后获临床妊娠:附2例报告", 《中华男科学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108935445A (en) * | 2018-08-08 | 2018-12-07 | 贵州医科大学附属医院 | A kind of jelly precision method based on ICSI micromanipulation |
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