CN1803100A - Testicle sperm freeze-storage and revival method - Google Patents
Testicle sperm freeze-storage and revival method Download PDFInfo
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- CN1803100A CN1803100A CNA2005100618715A CN200510061871A CN1803100A CN 1803100 A CN1803100 A CN 1803100A CN A2005100618715 A CNA2005100618715 A CN A2005100618715A CN 200510061871 A CN200510061871 A CN 200510061871A CN 1803100 A CN1803100 A CN 1803100A
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- sperm
- zona pellucida
- ovum
- testicular
- frozen
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- 238000000034 method Methods 0.000 title claims abstract description 19
- 210000001550 testis Anatomy 0.000 title abstract description 14
- 238000001574 biopsy Methods 0.000 claims abstract description 13
- 238000011084 recovery Methods 0.000 claims abstract description 12
- 210000004340 zona pellucida Anatomy 0.000 claims description 48
- 230000002381 testicular Effects 0.000 claims description 26
- 210000004681 ovum Anatomy 0.000 claims description 23
- 102000002322 Egg Proteins Human genes 0.000 claims description 21
- 108010000912 Egg Proteins Proteins 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 238000002347 injection Methods 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 12
- 239000010902 straw Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 230000006872 improvement Effects 0.000 claims description 8
- 239000002002 slurry Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 230000008859 change Effects 0.000 claims description 4
- 210000002863 seminiferous tubule Anatomy 0.000 claims description 4
- 239000012531 culture fluid Substances 0.000 claims description 3
- 230000008627 meiotic prophase Effects 0.000 claims description 3
- 238000005192 partition Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 230000007774 longterm Effects 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 210000003101 oviduct Anatomy 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 238000001816 cooling Methods 0.000 abstract 1
- 230000001086 cytosolic effect Effects 0.000 abstract 1
- 230000035558 fertility Effects 0.000 abstract 1
- 206010003883 azoospermia Diseases 0.000 description 5
- 238000005138 cryopreservation Methods 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000009165 androgen replacement therapy Methods 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 1
- 206010043298 Testicular atrophy Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 201000010788 atrophy of testis Diseases 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 230000007368 endocrine function Effects 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 230000021595 spermatogenesis Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a cooling storage and resuscitation method of testicle sperm, which is characterized by the following: taking the exhausting cytoplasmic transparent tape as carrier; loading the biopsy testicle sperm in the sealed transparent tape; reducing the sperm loss; improving the sperm recovery ratio by 70 percent; aspirating the sperm under the microscopic operation in the ICSI directly; improving the flexibility of non-sperm patient to receive the auxiliary fertility treatment.
Description
Technical field
The invention belongs to the assisted reproduction field, relate to the frozen and method for resuscitation of testicular sperm.
Background technology
Modern Assisted Reproductive Technology ART makes the azoospermia patient also can obtain a small amount of sperm by testis biopsy, and (Intracytoplasmic sperm injection ICSI) treats and the acquisition filial generation to carry out intracytoplasmic sperm injection in the ovum slurry.Because the pregnancy rate of each ICSI treatment cycle is at 35%-40%, in order to obtain gestation, the part needs of patients repeats ICSI treatment for several times.If it is frozen not carry out testicular sperm, each cycle all will be carried out testis biopsy.And testis biopsy is an invasive surgery, and repeatedly repeatable operation is not only brought extra mental pressure and financial burden to the patient, but also may cause the testis tissue fibrosis, make healthy parenchyma of testis more and more difficulty find.Even more serious is that testis biopsy repeatedly can cause nonvolatil injury of testis, comprise the testis blood confession obstacle of part, irreversible atrophy of testis, spermatogenesis degeneration, even endocrine function is lost and the exogenous testosterone replacement therapy of needs.But the sperm freezing that if testis biopsy can be obtained, testis biopsy operation needn't be carried out simultaneously with its spouse's follicle paracentesis, can carry out the preparation of sperm and frozen when the diagnostic testis biopsy, improves the motility of this class patient assisted reproduction treatment greatly.
But it is cryoprotective agent with glycerol egg yolk that routine is frozen smart method, frozen behind 1: 1 dilution proportion seminal fluid of cryoprotective agent, and the preparation after the sperm recovery needs again through eluting, centrifugal process, and many sperms are lost in this process.Testicular sperm quantity is few, and energy is low, behind the conventional cryopreservation resuscitation, often is difficult to find sperm alive to carry out ICSI.It is the clinical FAQs of assisted reproduction that frozen testicular sperm reclaims failure.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, the frozen and method for resuscitation of testicular sperm be provided, realize by following steps:
(1) testicular sperm is frozen: the zona pellucida of finding time with endochylema is a carrier, with the testicular sperm of biopsy pack into the sealing zona pellucida in, zona pellucida stops 5～10 minutes in phosphate buffer (the adding 3% serum) solution of 8～10% glycerol after, be splined on straw, and after exposure is spent the night in the liquid nitrogen steam, directly drop into liquid nitrogen, carry out long-term frozen and preserve;
(2) recovery of testicular sperm: straw is taken out from liquid nitrogen, ice is separated in 30 ℃ of water-baths, then the complete soln in the straw is put into four orifice plates, mirror is sought zona pellucida down, with zona pellucida flushing in the HOF (mHTF) of improvement, change in the mHTF microdroplet of intracytoplasmic sperm injection (ICSI) ware in the ovum slurry, after holding the ovum pin and fixing the sky zona pellucida, with the entry needle sperm in the sucking-off zona pellucida one by one, (polyvmylpyrrolidone PVP) can directly carry out intracytoplasmic sperm injection (ICSI) in the ovum slurry behind the microdroplet to change 10% polyvidon over to.
Used biopsy testicular sperm is that convoluted seminiferous tubule is inserted among the HOF (mHTF) of improvement in the step (1), with the preparation of discontinuous density gradient partition method, put into 10% polyvidon (PVP) microdroplet of ovum slurry intracytoplasmic sperm injection (ICSI) ware.
Zona pellucida derives from and is in first meiotic prophase, mid-term ovum or unfertilized egg in the step (1), the zona pellucida preparation is to utilize micromanipulation system to carry out on the hot platform of inverted microscope, ovum is fixed with the ovum pin of holding of internal diameter 15um, entry needle with internal diameter 5um is made a call to a hole at 3 o ' clock positions, and, become empty until zona pellucida with this entry needle suction endochylema.
Zona pellucida stopped 2～3 minutes in HOF (mHTF) culture fluid of improvement, treat that nature recovers shape after, draw testicular sperm with entry needle, be injected into zona pellucida then lentamente, 14-17 sperm of an empty zona pellucida injection.
Characteristics of the present invention are: the sperm sealing is stored in the zona pellucida in (1) middle cryopreservation resuscitation process, seldom causes losing of sperm, and its sperm response rate can be up to 70%, far above conventional freezing preservation method.(2) recovery of zona pellucida sperm needn't separate and the washing centrifugal process through cumbersome discontinuous density gradient, and directly sucking-off sperm under the micrurgy instrument is directly used in ICSI, and efficient obviously improves.(3) the invention solves serious azoospermia, the sperm recovery of the frozen back of the testicular sperm difficult problem of failure easily of lacking that occurs in the present spermatozoa cryopreservation technology, the motility that makes azoospermia patients accept the assisted reproduction treatment greatly improves.
The specific embodiment
The present invention is described further in conjunction with the embodiments.
Embodiment 1
The convoluted seminiferous tubule that biopsy is taken out is inserted among a spot of improvement HOF (mHTF), convoluted seminiferous tubule pulverizes through the elbow tweezers, after the suction pipe piping and druming evenly, prepare testicular sperm with the discontinuous density gradient partition method, put into 10% polyvidon (PVP) microdroplet of ovum slurry intracytoplasmic sperm injection (ICSI) ware.
What zona pellucida derived from donations is in first meiotic prophase, mid-term ovum or unfertilized egg.The zona pellucida preparation is to utilize micromanipulation system to carry out on the hot platform of inverted microscope, and ovum is fixed with the ovum pin of holding of internal diameter 15um, and makes a call to a hole with the entry needle of internal diameter 5um at 3 o ' clock positions, and with this entry needle suction endochylema, becomes empty until zona pellucida.Zona pellucida was stopping 2～3 minutes in the HOF (mHTF) of improvement, after the recovery shape in the culture fluid after the recovery shape, drew testicular sperm with entry needle naturally, was injected into zona pellucida lentamente.14-17 sperm of an empty zona pellucida injection.
After sperm was inserted, zona pellucida was splined on straw stop 5-10min in phosphate buffer (the adding 3% serum) solution of 8～10% glycerol after.Straw directly drops into liquid nitrogen after exposing and spending the night in the liquid nitrogen steam, can carry out long preservation.
During recovery, straw is taken out from liquid nitrogen, 30 ℃ of water-bath 30-60 all melt up to ice second, then the complete soln in the straw is put into four orifice plates, mirror is sought zona pellucida down, zona pellucida washes 2 times in mHTF after, change in the mHTF microdroplet of ICSI ware, after holding the ovum pin and fixing the sky zona pellucida,, can directly carry out ICSI after changing the 10%PVP microdroplet over to the entry needle sperm in the sucking-off zona pellucida one by one.
Embodiment 2
With reference to embodiment 1 method, have 34 zona pellucidas and be used for cryopreservation sperm, 8 frozen testicular sperms of zona pellucida wherein, as reference, frozen few azoospermia of 12 zona pellucidas in addition, 14 frozen eupyrene sperms of zona pellucida.Totally 530 sperms alive inject in the zona pellucida, and frozen preceding average each zona pellucida contains 15.6 sperms, 12.3 sperms alive of average each zona pellucida recovery in recovery back, 1.8 individual Necrospermia, the testicular sperm response rate is 71.7%, and the average sperm response rate is 78.7%, and the result is referring to table 1.
The number and the response rate in table 1. zona pellucida after the spermatozoa cryopreservation recovery
| The sperm kind | The zona pellucida number | Smart number alive before frozen | Reclaim the smart number of living | The necrospermia number | Lose sperm count | The smart response rate alive |
| The few sub-eupyrene sperm of azoospermia of testicular sperm | 8 12 14 | 14.1(113/8) 16.5(198/12) 15.6(219/14) | 10.1(81/8) 13.1(157/12) 12.8(179/14) | 3.3(26/8) 1.9(23/12) 0.8(11/14) | 0.8(6/8) 1.5(18/12) 2.1(29/14) | 71.7％ 79.3％ 81.7％ |
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the preferred specific embodiments of front is interpreted as only illustrating, but not limits the scope of the invention by any way.
Claims (4)
1. the frozen and method for resuscitation of testicular sperm is characterized in that realizing by following steps:
(1) testicular sperm is frozen: the zona pellucida of finding time with endochylema is a carrier, with the testicular sperm of biopsy pack into the sealing zona pellucida in, zona pellucida stops 5～10 minutes in the phosphate-buffered liquor of 8～10% glycerol that add 3% serum after, be splined on straw, and after exposure is spent the night in the liquid nitrogen steam, directly drop into liquid nitrogen, can carry out long-term frozen and preserve;
(2) recovery of testicular sperm: straw is taken out from liquid nitrogen, ice is separated in 30 ℃ of water-baths, then the complete soln in the straw is put into four orifice plates, mirror is sought zona pellucida down, zona pellucida is washed in the HOF of improvement, change in the mHTF microdroplet of intracytoplasmic sperm injection ware in the ovum slurry, after holding the ovum pin and fixing the sky zona pellucida, with the entry needle sperm in the sucking-off zona pellucida one by one, can directly carry out intracytoplasmic sperm injection in the ovum slurry after changing 10% polyvidon microdroplet over to.
2. the frozen and method for resuscitation of testicular sperm according to claim 1, it is characterized in that: the testicular sperm of used biopsy is that convoluted seminiferous tubule is inserted among the HOF of improvement in the step (1), with the preparation of discontinuous density gradient partition method, put into 10% polyvidon microdroplet of ovum slurry intracytoplasmic sperm injection ware.
3. the frozen and method for resuscitation of testicular sperm according to claim 1, it is characterized in that: what zona pellucida derived from donations in the step (1) is in first meiotic prophase, mid-term ovum or unfertilized egg, the zona pellucida preparation utilizes micromanipulation system to carry out on the hot platform of inverted microscope, ovum is fixed with the ovum pin of holding of internal diameter 15um, entry needle with internal diameter 5um is made a call to a hole at 3 o ' clock positions, and, become empty until zona pellucida with this entry needle suction endochylema.
4. according to the frozen and method for resuscitation of claim 1 or 3 described testicular sperms, it is characterized in that: zona pellucida is in people's fallopian tube culture fluid of improvement, after treating that nature recovers shape, draw testicular sperm with entry needle, be injected into zona pellucida then lentamente, 14-17 sperm of an empty zona pellucida injection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005100618715A CN1803100A (en) | 2005-12-07 | 2005-12-07 | Testicle sperm freeze-storage and revival method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005100618715A CN1803100A (en) | 2005-12-07 | 2005-12-07 | Testicle sperm freeze-storage and revival method |
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| Publication Number | Publication Date |
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| CN1803100A true CN1803100A (en) | 2006-07-19 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2005100618715A Pending CN1803100A (en) | 2005-12-07 | 2005-12-07 | Testicle sperm freeze-storage and revival method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396945A (en) * | 2014-12-03 | 2015-03-11 | 张丽红 | Spermatozoa cryopreservation and thawing method for detecting sperm DNA (Deoxyribonucleic Acid) integrity |
| CN106962323A (en) * | 2017-03-03 | 2017-07-21 | 中国医学科学院医学生物学研究所 | The sperm cryopreservation and method for resuscitation of non-human primate |
| CN110055212A (en) * | 2019-02-14 | 2019-07-26 | 中国科学院西北高原生物研究所 | A method of embryo is produced using Ovisammon Linnaeus tissue sperm in vitro |
-
2005
- 2005-12-07 CN CNA2005100618715A patent/CN1803100A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396945A (en) * | 2014-12-03 | 2015-03-11 | 张丽红 | Spermatozoa cryopreservation and thawing method for detecting sperm DNA (Deoxyribonucleic Acid) integrity |
| CN106962323A (en) * | 2017-03-03 | 2017-07-21 | 中国医学科学院医学生物学研究所 | The sperm cryopreservation and method for resuscitation of non-human primate |
| CN106962323B (en) * | 2017-03-03 | 2020-12-15 | 中国医学科学院医学生物学研究所 | Sperm cryopreservation and recovery method for non-human primates |
| CN110055212A (en) * | 2019-02-14 | 2019-07-26 | 中国科学院西北高原生物研究所 | A method of embryo is produced using Ovisammon Linnaeus tissue sperm in vitro |
| CN110055212B (en) * | 2019-02-14 | 2022-12-23 | 中国科学院西北高原生物研究所 | Method for producing embryo in vitro by using sperm of pannage testis tissue |
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