CN101671651A - Sperm freezing and unfreezing method and sperm freezing and unfreezing device - Google Patents
Sperm freezing and unfreezing method and sperm freezing and unfreezing device Download PDFInfo
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- CN101671651A CN101671651A CN 200810042695 CN200810042695A CN101671651A CN 101671651 A CN101671651 A CN 101671651A CN 200810042695 CN200810042695 CN 200810042695 CN 200810042695 A CN200810042695 A CN 200810042695A CN 101671651 A CN101671651 A CN 101671651A
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Abstract
The invention provides a sperm freezing and unfreezing method with simple and convenient operation, easy searching of sperms, high sperm survival rate and high sperm recovery rate, and also provides adevice using the method. The method and the device have the main advantages that: (1) sperms are put into microdroplets and unfrozen in oil, the sperms cannot be lost, and the sperm recovery rate isapproximate to 100 percent; (2) refrigerating fluid contains glycerin, and the sperm survival rate can reach 90 percent; and (3) the method and the device have more simple, convenient and feasible operation than the prior art. The method and the device well solve the problem of easy failure of sperm recovery after severe oligo-asthenosperms, testicular sperms and epididymal sperms are frozen in the prior sperm freezing technology, and greatly improve the flexibility for azoospermia patients to receive assisted reproductive treatment.
Description
Technical field
The present invention relates to the assisted reproduction field in the medical science, relate to the freezing and defreezing method of a small amount of testicular sperm, epididymal sperm and the device that is used for aforesaid method.
Background technology
Modern Assisted Reproductive Technology ART can be carried out intracytoplasmic sperm injection in the ovum slurry (Intracytoplasmic sperm injection, ICSI) treatment and obtain filial generation to aspermia or oligospermia or azoospermia patient (obtaining a small amount of sperm by testis, epididymis biopsy).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 not to its spermatozoa cryopreservation, each cycle all will be arranged essence or be carried out testis, epididymis biopsy.Aspermia or oligospermia person sperm instability, may can not get the failure of sperm so that this cycle, and testis, epididymis biopsy are invasive surgery, repeatedly repeatable operation is not only brought extra psychological pressure and economical load to the patient, but also may cause testis, epididymis tissue fibrosis, cause irreversible testicular atrophy, spermatogenesis to be degenerated, even endocrine function is lost and the exogenous testosterone replacement therapy of needs.If but the sperm freezing that a small amount of sperm of discharging or testis, epididymis biopsy can be obtained can carry out the preparation of sperm and frozen when usual or diagnostic testis, epididymis biopsy, can improve the handiness of this class patient assisted reproduction treatment greatly.
Chinese patent application provides a kind of frozen and method for resuscitation of testicular sperm for No. 200510061871.5; this invention provides the frozen and method for resuscitation of testicular sperm; the zona pellucida of finding time with endochylema is a carrier; with the testicular sperm of biopsy pack into the sealing zona pellucida in; carry out the interpolation and the wash-out of sperm freezing protecting agent; the sperm sealing is stored in the zona pellucida in the cryopreservation resuscitation process; the recovery of zona pellucida sperm needn't separate and the washing centrifugal process through cumbersome discontinuous density gradient in this method; directly sucking-off sperm under the micrurgy instrument is directly used in ICSI.
But in aforesaid method, with freezing straw, empty zona pellucida, freezing ring etc. load sperm, and many sperms are lost in operating process, cause the sperm quantity that obtains few, and motility is low, behind the conventional cryopreservation resuscitation, often is difficult to find sperm alive to carry out ICSI.Therefore, in clinical manipulation cryopreservation sperm to reclaim failure even abandon freezing be the clinical FAQs of assisted reproduction.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of easy and simple to handle, seek the freezing and defreezing method of sperm, sperm survival rate height, sperm that the sperm rate of recovery is high easily, simultaneously, also provide a cover to be used for the device of aforesaid method.The described step of present method is as described below.
A kind of freezing and defreezing method of sperm, it comprises following steps:
(1) a small amount of sperm is freezing: preparation contains the refrigerating fulid of glycerine, in freezing sheet portion, do a droplet under the constant humidity condition, micrurgy is put into droplet with pin with a small amount of sperm, put freezing cover, slowly enter in the liquid nitrogen steam, expose after 5 minutes, directly drop into liquid nitrogen, carry out long-term frozen and preserve;
(2) thawing of a small amount of sperm: freezing is taken out from liquid nitrogen, insert rewarming in 37 degrees centigrade of mineral oil, mirror is sought sperm down, is transferred in mHTF (HOF) droplet sperm standby with entry needle.
Wherein, the constant humidity condition described in the performing step (1) need be prepared a wet box, and the top of this wet box is provided with the shape of tail oversleeve of an end opening, and the injection shank can enter this wet box inside from this opening, and the bottom of described wet box is made by material transparent.Add water in the described wet box to keep humidity.Described shape of tail oversleeve is made by transparent, softish or resilient film.The bottom of described wet box is made by glass or polystyrene.
Wherein, freezing in the step (1) comprises a bar portion and a slice portion, and described portion is lengthy motion picture shape or groove shape, makes with transparent and harder material for described freezing.Make by glass or polystyrene for described freezing.When being the groove shape for freezing, can preserve moisture at described droplet loam cake oil.
Wherein, in step (1), also be provided with the moistening cotton that is used to keep the constant humidity condition in the freezing cover.
Wherein, in step (2), freezing insert mineral oil after, on freezing, place one and can push down freezing little weight.
Freezing and the thawing apparatus of sperm, wherein, this device is:
A kind of freezing of carrying sperm, described freezing comprises bar portion and sheet portion, and described portion is lengthy motion picture shape or groove shape, makes with transparent and harder material for described freezing.
Wherein, in order to reach better effect, freezing and the thawing apparatus of described sperm also comprises a kind of wet box that can make the constant humidity condition, the top of being somebody's turn to do wet box is provided with the shape of tail oversleeve of an end opening, the injection shank can enter this wet box inside from this opening, and the bottom of described wet box is made by material transparent;
Wherein, described shape of tail oversleeve is made by transparent, softish or resilient film.The bottom of described wet box is made by glass or polystyrene.
Wherein, make by glass or polystyrene for described freezing.
Major advantage of the present invention is: (1) sperm places droplet, thaws in oil, and sperm can not lost, and the sperm rate of recovery is near 100%; (2) contain glycerine in the refrigerating fulid, sperm is deposited activity ratio can reach 90%; (3) operation is simple and easy to do compared to existing technology.The present invention has solved the serious few weak sperm, testis, the frozen back of the epididymal sperm sperm that occur in the present spermatozoa cryopreservation technology well and has reclaimed the difficult problem of failure easily, and the handiness that makes azoospermia patients accept the assisted reproduction treatment greatly improves.
Description of drawings
Fig. 1 is the structural representation of wet box;
Fig. 2 stretches into the interior synoptic diagram of wet box for the injection shank;
Fig. 3 is long flaky freezing structural representation for sheet portion;
Fig. 4 is freezing structural representation of groove shape for sheet portion;
Fig. 5 is the structural representation of freezing cover.
Main symbol implication:
1 wet box 2 bottoms
3 shape of tail oversleeves, 4 injection shanks
6 freezing 7 portions
8 portion's 9 freezing covers
10 moistening cottons
Embodiment
Now according to accompanying drawing also in conjunction with the embodiments, the present invention will be further described.
At first see also Figure 1 and Figure 2, be wet box 1 among the figure, add water in the box to keep humidity, the top of described wet box 1 is provided with the shape of tail oversleeve 3 of an end opening, injection shank 4 can enter described wet box 1 inside from this opening, and the bottom of described wet box 12 is made by polystyrene.Described shape of tail oversleeve 3 is made by transparent plastics.
Seeing also Fig. 3 and shown in Figure 4, is freezing 6 among the figure, and described freezing 6 comprises bar portion and sheet portion, and wherein the freezing sheet portion 7 among Fig. 3 is the lengthy motion picture shape, and the freezing sheet portion 8 among Fig. 4 is the groove shape.
Seeing also shown in Figure 5ly, is freezing cover 9 among the figure, and the bottom that described freezing cover 9 insides divide is provided with moistening cotton 10.
The normal sperm of discharging that takes a morsel is inserted among a spot of improvement HOF (mHTF), the ware of placing sperm is put into wet box 1, do a refrigerating fulid droplet in freezing 6 sheet portion 7, micrurgy is drawn 20 sperms with pin and is put into droplet, at this moment, inspect and freeze preceding survival rate, on freezing 6, put moistening in advance freezing cover 9 then, freezing cover 9 is slowly put into the liquid nitrogen steam, expose after 5 minutes, directly drop into liquid nitrogen, carry out long-term frozen and preserve.
During recovery, take off freezing cover 9 in liquid nitrogen, freezing 6 is inserted in 37 degrees centigrade of mineral oil, slowly move, scissors is cut freezing 6 sheet portion 7, and little weight (not shown) is pushed down freezing 6 near the refrigerating fulid droplet.Siphoning away sperm with entry needle under the inverted microscope, to put into the mHTF droplet standby, inspects the survival rate after freezing this moment.
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, the wet box 1 that the ware of this placement convoluted seminiferous tubule is put into, in freezing 6 sheet portion 8, do a refrigerating fulid droplet, adding the mineral oil covering preserves moisture, draw 10 sperms with entry needle and put into droplet, at this moment, inspect and freeze preceding survival rate, on freezing 6, put moistening in advance freezing cover 9 then, freezing cover 9 is slowly put into the liquid nitrogen steam, expose after 5 minutes, directly drop into liquid nitrogen, carry out long-term frozen and preserve.
During recovery, take off freezing cover 9 in liquid nitrogen, freezing 6 is inserted in 37 degrees centigrade of mineral oil, slowly move with rewarming, scissors is cut freezing 6 sheet portion 8, and little weight (not shown) is pushed down freezing 6 near the refrigerating fulid droplet.Siphoning away sperm with entry needle under the inverted microscope, to put into the mHTF droplet standby, inspects the survival rate after freezing this moment.
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, the wet box 1 that the ware of this placement convoluted seminiferous tubule is put into, in freezing 6 sheet portion 8, do a refrigerating fulid droplet, adding the mineral oil covering preserves moisture, draw 20 sperms with entry needle and put into droplet, at this moment, inspect and freeze preceding survival rate, on freezing 6, put moistening in advance freezing cover 9 then, freezing cover 9 is slowly put into the liquid nitrogen steam, expose after 5 minutes, directly drop into liquid nitrogen, carry out long-term frozen and preserve.
During recovery, take off freezing cover 9 in liquid nitrogen, freezing 6 is inserted in 37 degrees centigrade of mineral oil, slowly move with rewarming, scissors is cut freezing 6 sheet portion 8, and little weight (not shown) is pushed down freezing 6 near the refrigerating fulid droplet.Siphoning away sperm with entry needle under the inverted microscope, to put into the mHTF droplet standby, inspects the survival rate after freezing this moment.
Shown in experimental result please see the following form:
| Experiment number | The sperm source | Freeze preceding quantity | Freeze preceding motility rate | Freeze back quantity | Freeze the back |
| Embodiment | |||||
| 1 | The normal sperm of discharging | ??20 | ??100％ | ??20 | ??55％ |
| |
Epididymal sperm | ??10 | ??100％ | ??10 | ??90％ |
| |
Testicular sperm | ??20 | ??100％ | ??20 | ??95％ |
In the above-described embodiments, be that so that keep the state of relative closure in the wet box, wet simultaneously box can be done among a small circle with Stage microscope and move because can pin the injection shank than softish shape of tail oversleeve during in concrete operations in that the shape of tail oversleeve is set above the wet box.It is in order not influence observation that the bottom of described wet box adopts material transparent to make.Described freezing also require with material transparent make be since freezing as the carrier of sperm if opaquely be difficult to look in operation sperm.The little weight of using in the concrete operations is in order to push down freezing usefulness that prevents that it from moving.
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 (16)
1, a kind of freezing and defreezing method of sperm is characterized in that it comprises following steps:
(1) a small amount of sperm is freezing: preparation contains the refrigerating fulid of glycerine, in freezing sheet portion, do a droplet under the constant humidity condition, micrurgy is put into droplet with pin with a small amount of sperm, put freezing cover, slowly enter in the liquid nitrogen steam, expose after 5 minutes, directly drop into liquid nitrogen, carry out long-term frozen and preserve;
(2) thawing of a small amount of sperm: freezing is taken out from liquid nitrogen, insert rewarming in 37 degrees centigrade of mineral oil, mirror is sought sperm down, is transferred in HOF's droplet sperm standby with entry needle.
2, the freezing and defreezing method of sperm as claimed in claim 1 is characterized in that, freezing in the step (1) comprises a bar portion and a slice portion, and described portion is lengthy motion picture shape or groove shape.
3, the freezing and defreezing method of sperm as claimed in claim 2 is characterized in that, when being the groove shape for freezing, can preserve moisture at described droplet loam cake oil.
4, the freezing and defreezing method of sperm as claimed in claim 2 is characterized in that, makes with transparent and harder material for described freezing.
5, the freezing and defreezing method of sperm as claimed in claim 4 is characterized in that, is made by glass or polystyrene for described freezing.
6, as the freezing and defreezing method of any described sperm of claim 1-3, it is characterized in that, constant humidity condition described in the performing step (1) need be prepared a wet box, the top of being somebody's turn to do wet box is provided with the shape of tail oversleeve of an end opening, the injection shank can enter this wet box inside from this opening, and the bottom of described wet box is made by material transparent.
7, the freezing and defreezing method of sperm as claimed in claim 6 is characterized in that, adds water in the described wet box to keep humidity.
8, the freezing and defreezing method of sperm as claimed in claim 6 is characterized in that, described shape of tail oversleeve is made by transparent, softish or resilient film.
9, the freezing and defreezing method of sperm as claimed in claim 6 is characterized in that, the bottom of described wet box is made by glass or polystyrene.
10, the freezing and defreezing method of sperm as claimed in claim 1 is characterized in that, wherein, in step (1), also is provided with the moistening cotton that is used to keep the constant humidity condition in the freezing cover.
11, the freezing and defreezing method of sperm as claimed in claim 1 is characterized in that, in step (2), freezing insert mineral oil after, on freezing, place one and can push down freezing little weight.
12, a kind of freezing and thawing apparatus that is used for the sperm of the described method of claim 1 is characterized in that this device is:
A kind of freezing of carrying sperm, described freezing comprises bar portion and sheet portion, and described portion is lengthy motion picture shape or groove shape, makes with transparent and harder material for described freezing.
13, the freezing and thawing apparatus of sperm as claimed in claim 12, it is characterized in that, freezing and the thawing apparatus of described sperm also comprises a kind of wet box that can make the constant humidity condition, the top of being somebody's turn to do wet box is provided with the shape of tail oversleeve of an end opening, the injection shank can enter this wet box inside from this opening, and the bottom of described wet box is made by material transparent.
14, the freezing and thawing apparatus of sperm as claimed in claim 13 is characterized in that, described shape of tail oversleeve is made by transparent, softish or resilient film.
15, the freezing and thawing apparatus of sperm as claimed in claim 13 is characterized in that, the bottom of described wet box is made by glass or polystyrene.
16, the freezing and thawing apparatus of sperm as claimed in claim 12 is characterized in that, is made by glass or polystyrene for described freezing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810042695 CN101671651B (en) | 2008-09-09 | 2008-09-09 | Sperm freezing and unfreezing method and sperm freezing and unfreezing device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810042695 CN101671651B (en) | 2008-09-09 | 2008-09-09 | Sperm freezing and unfreezing method and sperm freezing and unfreezing device |
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| Publication Number | Publication Date |
|---|---|
| CN101671651A true CN101671651A (en) | 2010-03-17 |
| CN101671651B CN101671651B (en) | 2013-05-29 |
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| CN 200810042695 Expired - Fee Related CN101671651B (en) | 2008-09-09 | 2008-09-09 | Sperm freezing and unfreezing method and sperm freezing and unfreezing device |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105850986A (en) * | 2016-06-06 | 2016-08-17 | 上海市第人民医院 | Efficient and safe DPV freezing system and method for human oligoasthenozoospermia |
| 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 |
| CN109082363A (en) * | 2018-07-23 | 2018-12-25 | 上海市第人民医院 | A kind of preferred sperm freezing culture device |
| CN110432261A (en) * | 2019-09-02 | 2019-11-12 | 李娜 | A kind of micro sperm freezing and defreezing method and its automatic fixture device |
| WO2022161195A1 (en) * | 2021-02-01 | 2022-08-04 | 深圳拜尔洛克生物技术有限公司 | Thaw recovery system and method for biomaterial |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005533501A (en) * | 2002-07-22 | 2005-11-10 | エックスワイ，インコーポレイテッド | Sperm cell manipulation system |
| CN101219074B (en) * | 2008-01-25 | 2010-09-29 | 重庆市畜牧科学院 | Method for producing and thawing grain type frozen semen of pig |
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2008
- 2008-09-09 CN CN 200810042695 patent/CN101671651B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105850986A (en) * | 2016-06-06 | 2016-08-17 | 上海市第人民医院 | Efficient and safe DPV freezing system and method for human oligoasthenozoospermia |
| CN105850986B (en) * | 2016-06-06 | 2019-03-12 | 上海市第一人民医院 | A kind of the people's rareness sperm DPV refrigeration system and freezing method of highly effective and safe |
| CN109082363A (en) * | 2018-07-23 | 2018-12-25 | 上海市第人民医院 | A kind of preferred sperm freezing culture device |
| 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 |
| CN110432261A (en) * | 2019-09-02 | 2019-11-12 | 李娜 | A kind of micro sperm freezing and defreezing method and its automatic fixture device |
| WO2022161195A1 (en) * | 2021-02-01 | 2022-08-04 | 深圳拜尔洛克生物技术有限公司 | Thaw recovery system and method for biomaterial |
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| CN101671651B (en) | 2013-05-29 |
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