JP2005074586A - Improved method for intracytoplasmic sperm injection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an injection pipette for inducing mechanical damage to a head membrane of a sperm and an ICSI method employing the same. <P>SOLUTION: An inner diameter of the injection pipette is structured to be smaller than the maximum diameter of the head of a sperm. Numerous short spikes are formed at the tip of the pipette. The pipette is structured to capture the sperms by the tip and thereafter to inject them into ooplasma, and to be recovered after mixing the sperms with the ooplasma. Thus, fertility is improved by preventing the mechanical damage of the sperms and facilitating the passage of an ooplasmic membrane. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an intracytoplasmic sperm injection (hereinafter referred to as “ICSI”), more specifically, an improved ICSI method capable of solving the problems of the conventional ICSI technique, and this This is related to the production of an injection pipette for ICSI.

  ICSI is a method of artificially injecting sperm into the egg cytoplasm and is widely used in many animals, including humans, for the purpose of overcoming male infertility or to study the fundamental mechanism of fertilization. Since the first ICSI was developed [Palermo et al, Lancet. 340, 1992, p17], this method has been used to achieve very high fertilization success rates, especially in humans [Hsu et al, Fertil. Steril. 72, 1999, p679].

ICSI is not only a very effective tool for overcoming male infertility for a specific sentence, but also a useful tool for discriminating and studying what is necessary and not necessary in the fertilization process [Yanagimachi , In: Knobil E., Neill D , The Physiology of Reproduction, 2 nd ed, New York (eds.):. Raven Press, 1994, p189]. Moreover, ICSI can be used as a useful tool for sperm-mediated gene transfer (hereinafter abbreviated as 'SMGT') (Smith KR, Anim. Biotechnol. 10, 1998, p 1; US Patent No. 6,376,743).

  One of the cellular exocytosis required for normal fertilization in the body to pass through the ZP after the ha sperm binds to the zona pellucida (hereinafter abbreviated as 'ZP') and fuses with the egg membrane One form of acrosome reaction occurs. The substantial fusion site is the postacrosomal region of the sperm head that merges with the egg membrane under in vivo conditions. After the second polar body protrudes, the maternal chromosome condenses to form a female pronucleus (hereinafter abbreviated as 'FPN'). Gamete membrane fusion occurs in front of the posterior region of the acrosome, whereas the sperm head is invaded by the cortical tongue-like process and introduced by phagocytic action. In single sperm fertilization, the head of the sperm binds first and the tail is generally introduced in a zipper-like fashion [Sathananthan AH In: Handbook of in Vitro Fertilization. Trounso A. Gardner DK ( eds). Boca raton, FL: CRC, p237]. In fact, the sperm cell membrane in the upper part of the equatorial region of the acrosome is preferentially fused with the egg cell membrane in eutherian mammals [Bredford et al. In: Poste G., Nicolson GL, eds. Membrane Surface Reviews (Membrane fusion ), Amsterdam: North-Holland. 5, 1978, p65], after fusion, the sperm head is invaded and the entire tail is introduced into the egg cytoplasm.

  As mentioned earlier, research on assisted reproduction technology that elucidates the normal fertilization mechanism and mimics the process is actively underway, one of which is ICSI. In other words, ICSI avoids the recognition, binding, passage and fusion processes of sperm and ovum and arbitrarily changes the series of fertilization processes.

  Traditional ICSI methods that mimic the normal fertilization process in vivo generally involve the process of damaging the tail of the sperm, the process of completely sucking and capturing the sperm into the injection pipette, and mixing the egg cytoplasm with the sperm, and then reincubating the egg Comprised of the process of injection into the cytoplasm.

  In addition, research to increase the fertilization rate by the conventional ICSI method has been continued, and it has been selected mainly focusing on appropriately treating the tail and tail membrane that give motility to sperm. In other words, sperm immobilization and cell membrane disruption are necessary for successful fertilization because of the ease of manipulating sperm with motility and the release of soluble substances in sperm that induce egg cell activity. [Svalander et al. Fertil. Steril. 63, 1995. p828; Vanderzwalmen et al. Hum. Reprod. 11, 1996, p540; Dozortsev et al. Hum. Reprod. 12, 1997, p2792]. That is, when the injection pipette mechanically damages the sperm tail, the cell membrane of the tail is destroyed, and thus cytoplasmic factors in the ovum that induce decondensation of the sperm head can be introduced into the sperm [ Flaherty et al. Reprod. Fertil. Dev. 7, 1995, p197].

  In this way, the conventional ICSI technique immobilizes motility and physically cuts, removes and impacts the sperm tail to cause damage to the cell membrane of the sperm tail, piezo pulse impact, suction and release I have been able to use methods such as repeating. In addition, many researchers have to reduce sperm motility because of the operations described above, and reduce the sperm motility despite its unfavorable effects (polyvinylpyrrolidone, hereinafter referred to as PVP). (Abbreviation) has been used. The viscosity of the PVP solution drastically reduces sperm motility and has been repurposed to regulate the movement of fluid into and out of injection pipettes, and many researchers have been using it until recently. We consider the use of PVP as an important factor for ICSI's success.

  Also, the injection pipette used in the conventional ICSI technique has a sufficient diameter to allow sperm to move freely into and out of the pipette, so it must be made larger than the diameter of the sperm head to be operated. Don't be. However, when the diameter of the injection pipette is larger than 8 μm, the cytoplasm of the egg is easily destroyed during sperm injection [Payne D., Reprod. Fertil. Dev. 7, 1995, p185].

  ICSI, one of the assisted reproductive technologies for injecting sperm into the egg cytoplasm, has shown a high fertilization rate since it was first developed in 1992, and has been widely used in human infertility. Because sexuality exceeds the speed of fine manipulation, sperm motility needs to be reduced for sperm injection, and the sperm tail is powerless to prevent separation from the injection pipette before sperm injection Must be converted. In addition, inducing decondensation of the injected sperm in the egg cytoplasm and damaging the plasma membrane of the sperm tail or cutting the tail to minimize the amount of media flowing in. is necessary.

  As mentioned above, the use of PVP in conventional ICSI techniques, tail scoring, tail cutting, etc. are very different from normal fertilization environment, and the injection pipette diameter is larger than the sperm head thickness, so PVP is included. There are drawbacks in that the amount of medium injected increases and the destruction of egg cells occurs. According to the research results that have already been clarified, the injection pipette is too large for the low survival rate of fertilized eggs fertilized by the conventional ICSI method [Dozortsev et al., Zygote, 1998, 6, p143]. It has also been shown that excessive media inflow surrounds the sperm head membrane and interferes with contact with substances in the sperm head [Hsu et al., Fertil. Steril. 1999, 72, p679].

  In particular, in the case of normal fertilization, despite the site where the sperm head is fused and decondensed, the existing ICSI technique has a limit that has been intensively studied only on the tail membrane wound.

  The present inventor tried a new approach method that can solve the problems of the conventional ICSI method and increase the fertilization rate, and obtained a good result, so that the present invention was completed by clarifying the result. It was.

The purpose of the present invention is to provide a new ICSI injection pipette for ICSI and a new ICSI method using the same, in order to eliminate the problems of the conventional ICSI technique as mentioned above and increase the fertilization rate. In other words, it provides a new ICSI method that is closer to the normal fertilization process.
Specifically, an object of the present invention is to provide an injection pipette that induces mechanical damage to the sperm head membrane and an ICSI method using the same.

  The modified ICSI method of the present invention can be practiced using the characteristic injection pipette of the present invention.

Specifically, the injection pipette of the present invention forms an injection pipette having an inner diameter not larger than the maximum diameter of the sperm head to be injected into the ovum and having many small spikes at the tip, and the tip of the injection pipette After the sperm is captured, it is injected into the egg cytoplasm and mixed with the cytoplasm in the egg cytoplasm, and then the injection pipette is recovered.
Injection pipette
The injection pipette of the present invention is produced by processing a capillary tube with both ends open, similar to that used in conventional ICSI. Specifically, the injection pipette used in the present invention is configured to have an inner diameter that allows sperm to be injected into an egg to be captured by the tip of the injection pipette. In addition, many short and small spikes can be formed at the tip of the injection pipette of the present invention for capturing sperm.

  More specifically, as shown in FIG. 3, the inner diameter of the injection pipette is not larger than the maximum diameter of the sperm head to be manipulated, so that the sperm does not fully enter the injection pipette and is captured at the tip of the pipette. The In particular, the inner diameter of the pipette of the present invention is preferably such that the equator region of the sperm head is fixed to the tip of the injection pipette.

  On the other hand, the other end is formed to be open end, which creates a negative pressure in the current state of the capillary inside the injection pipette, and this makes it possible to capture motility sperm. .

  The injection pipette of the present invention utilizes a capillary tube. Specifically, it can be produced in a drop containing an ovum just before sperm injection after the capillary is pulled out long so that the desired inner diameter is obtained and bent at a desired angle about 1 mm from the tip of the injection pipette (FIG. 1). That is, a narrowly pulled capillary tube is inserted into the entrance of the holding pipette (FIG. 1, A), and the pipette is bent at a point having a desired diameter to form the opening of the injection pipette (FIGS. 1, B, C). After that, break the broken capillary tube in the holding pipette out of the holding pipette. Many small spikes can be formed at the tip of the injection pipette of the present invention.

  In the present invention, such a configuration of the injection pipette offers many advantages for sperm capture and sperm injection. The negative pressure of injection pipettes makes sperm capture easier and eliminates the need to use another chemical, PVP, to reduce sperm motility.

  In addition, by having a diameter that allows the sperm to be fixed with the pipette tip without being completely inside the injection pipette, the diameter is smaller than the injection pipette used in conventional ICSI techniques, thus reducing the destruction of the egg cytoplasm. At the same time, the amount of medium that flows into the egg cytoplasm with the sperm can be reduced. Also, it is not necessary to cut the sperm tail to reduce the amount of media flowing in, so the ICSI technique of the present invention provides an environment similar to normal sperm penetration that occurs in the body.

  Moreover, the spike at the tip of the injection pipette can easily damage the sperm head membrane and pass through the egg cytoplasmic membrane.

In particular, the injection pipette of the ICSI technique of the present invention can be used in a wide range without being limited to manipulation or capture of a specific species of sperm by appropriately changing its diameter.
ICSI technique of the present invention The modified ICSI method of the present invention is characterized by the use of an injection pipette as described above.

  More specifically, the reselected ICSI method of the present invention uses the characteristic injection pipette of the present invention to capture motility intact sperm at the tip of the injection pipette and inject it into the cytoplasm, and then recover the injection pipette including. The method of the present invention also includes the step of mixing the injected sperm and egg cytoplasm.

Hereinafter, each process will be specifically described in detail.
Sperm Capture The sperm can be captured by bringing the injection pipette of the present invention close to the tail or head of a sperm having mobility. Alternatively, it is possible when a sperm with mobility is close to the injection pipette. More preferably, the sperm can be captured by bringing the motility sperm close to the injection pipette with an inverted microscope and a micromanipulator.

  Thus, it is a feature of the injection pipette of the present invention in which the negative pressure in the current state of the capillary tube is able to easily capture the sperm without reducing the sperm motility.

  In the present invention, even if the sperm moves too fast, the sperm can be easily captured, and when the injection pipette approaches the tail of the sperm that swims quickly around the boundary of the droop, the equatorial part of the sperm head is the injection pipette. It is easily aspirated until it is fixed to the tip of the.

For sperm capture by negative pressure of the injection pipette, the sperm head may be aspirated first (head-to-head) into the injection pipette and the tail may be aspirated first (tail-tip) and captured. There is also.
Sperm injection in the egg cytoplasm The sperm captured by the injection pipette can be injected into the egg cytoplasm by the ICSI technique of the present invention. That is, the captured sperm is injected directly into the egg cytoplasm with the injection pipette. Specifically, as shown in FIG. 5, sperm injection can be performed side by side with a holding pipette that fixes an egg and an injection pipette that captures the sperm. In particular, the large number of spikes formed at the tip of the injection pipette of the present invention allows easy passage through the egg cytoplasmic membrane.

  After injecting sperm into the egg cytoplasm with an injection pipette, the sperm can be removed from the injection pipette by adjusting the pressure with the mouth and applying negative pressure and positive pressure in sequence with the injection pipette.

  As described above, positive and negative pressures are sequentially applied to mix sperm with egg cytoplasm. Is it preferable to suck and discharge by mouth while looking at the microscope? ? By adjusting the pressure in this way, sperm can be mixed with the egg cytoplasm 2 to 3 times repeatedly.

  Sperm thus mixed with cytoplasmic material is separated from the injection pipette when the injection pipette is removed from the egg cytoplasm, and only the sperm remains in the egg cytoplasm. This is due to the viscosity of the egg cytoplasm that causes the sperm to move along with the injection pipette. It is because going out is prevented.

Thus, direct injection of captured sperm into the egg cytoplasm is completed with mechanical damage to the sperm head membrane during passage through ZP and the egg membrane, and negative pressure in the injection pipette within the cytoplasm. The mixture of damaged sperm and cytoplasmic material can be completed without excessive flow of medium into the cytoplasm and excessive flow of cytoplasmic material into the injection pipette.
Sperm Head Membrane Damage The sperm injection method of the present invention characteristically causes sperm head damage. In other words, when motile sperm is trapped at the sharp tip of an injection pipette, it will cause fine mechanical damage to the head membrane, as shown in FIGS. 3 and 5, along with the injection pipette, ZP and egg cell membrane. More damage can be done to the sperm head membrane while passing through.

  This provides conditions similar to the normal fertilization process in the body, and can release fertilizing substances that induce egg cell activity and increase the fertilization rate. From the fact that the fertility rate increases as the mechanical damage to the tail of the sperm increases with the use of conventional ICSI techniques [Palermo et al. 1996, Hum Reprod. 11, p1023], the fertilization rate of the present invention This is the basis for improvement.

  As mentioned earlier, the injection pipette of the present invention facilitates the capture of motility sperm by its negative pressure, so it does not require the use of chemical substances such as microinjectors and PVP, providing an environment closer to normal fertilization To do. In addition, the injection pipette of the present invention is closer in diameter than the injection pipette used in the conventional ICSI technique, so there is also an advantage of reducing the amount of foreign substances such as culture fluid injected into the egg cytoplasm with less damage to the egg cytoplasm. is there. In particular, fertilization rate can be improved by inducing mechanical damage to the sperm head membrane during the sperm injection process.

  The improved ICSI injection pipette of the present invention and the ICSI method using the same have improved the fertilization success rate, such as eliminating the problems of the conventional ICSI method and showing the highest MPN formation rate and blastocyst development rate I will provide a.

  Hereinafter, embodiments will be described in detail through specific embodiments of the present invention. This example should be significant that the technical idea of the present invention is illustrated and the scope of the present invention is not limited to this example.

[Example 1]: Production of a pipette for the selected ICSI of the present invention Holding pipette Glass tube for producing the holding pipette used in the present invention (G-1, Production source: Narishige, Tokyo, Japan) ) Had an outer diameter (OD) of 1 mm and an inner diameter (ID) of 0.9 mm. The holding pipette was stretched with a micropuller (micropuller, PC-10, manufacturer: Narishige, Tokyo, Japan) until the glass pipette section of about 5 mm in length had an external diameter (OD) of about 120 μm. The programs used were single pulling, heat 101 and no weight.

  After making the glass tube narrow, the tip of the glass tube was cut by gently rubbing with the end of another narrow glass tube at the point where the tip was fit. The cut tip must be vertical and flat. Non-homogeneous and obliquely cut pipettes were discarded. The holding pipette was rolled with the heat of a platinum-iridium filament of microforge (MF-900, production source: Narishige, Tokyo, Japan).

The ID of the rounded portion of the holding pipette was about 30-40 μm, and it was possible to securely fix the egg. Thereafter, the holding pipette was bent at an angle of about 30 on the filament.
Injection pipette
The glass pipette section of about 5mm length of a glass tube with a thin wall with an OD of 1.0mm and an ID of 0.9mm (G-1, production source: Narishige, Tokyo, Japan) is connected to a micropuller (PC- 10, production source: Narishige, Tokyo, Japan). The program used was double pulling, heating 1; 67 (heat 1; 67), heating 2; 80 (heat 2; 80) and one light weight.
The sharp tip of the drawn glass tube was bent at an angle of about 30 ° with the heat of a platinum-iridium filament of microforge (MF-900, production source: Narishige, Tokyo, Japan).

  Then insert the tip of the sharp glass tube into the holding pipette entrance as shown in Fig. 1 (Fig. 1, A) and bend the tip of the inserted glass tube little by little (B, C). Bent until it breaks. This process was carried out in a drop containing the egg and the glass tube fragments remaining inside the holding pipette were discarded.

[Example 2]: Preparation of sperm sorting semen having motility In this example, frozen-thawed semen was used. The semen straw was thawed for 1 minute at 39 ° C. Gently place 100 μl of semen under an Eppendorf tube containing 1 ml of DPBS (Gibco 141900-144, Grand Island, NY) containing 0.1% polyvinyl alcohol (hereinafter PVA). The lid was closed for the process and placed in a 39 ° C., 5% CO ² incubator. Fifty minutes later 0.5 ml of the medium was taken from the upper layer of the Eppendorf tube, placed in a 2.5 ml Eppendorf tube, and centrifuged at 350 × g for 3 minutes. The upper layer solution was removed, and the sperm pellet was stored in a constant temperature bath at 39 ° C until just before ICSI.
Selection of motile sperm A small amount of sperm pellet was moved to the center of the sperm drop. Sperm quickly moved from the center of the dish bottom toward the drop boundary. Before filling the dish with mineral oil, tap the dish lightly on the table to make a thin drop. As a result, it was possible to start sperm swimming up and down in a narrow space of the boundary region.

[Example 3] Preparation of in vitro matured eggs Unless otherwise specified, reagents used in this source were those from Sigma manufacturer (Sigma-Aldrich Co., St. Louis, MO, USA).
In vitro ovarian ovaries were collected from heifers at the slaughterhouse, stored in 0.9% (w / v) saline at 35 ° C, and transferred to the laboratory. The ovary was washed three times and a 5 ml single-use syringe was attached with an 18-gauge hypodermic needle for inhalation of immature eggs. As shown in FIG. 2A, many large and small follicles are visible in the ovarian cortex.
The follicle contents were collected in a 15 ml conical tube and the upper layer solution was discarded. Cumulus-oocyte complex (COC) in the precipitate was washed three times with Tyrode's Lactate Hepes (TLH) medium (Table 1) supplemented with 0.01% PVA. COC with homogeneous granular egg cytoplasm and surrounded by three or more dense cumulus cells was selected for in vitro maturation (hereinafter IVM). It can be seen that immature eggs are densely surrounded by cumulus cells as shown in FIG.
IVM medium (Table 2) is 26.2 mM NaHCO ³ , 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 75 μg / ml kanamycin, 20 ng / ml EGF, 5 IU / ml PMSG / hCG (producer: Gibco , Life Technologies Inc., Grand Island, NY, USA). Porcine follicular fluid (hereinafter referred to as “pFF”) inhales 3-8 mm diameter follicles of heifers, centrifuges at 1900 × g for 30 minutes, and 1.2 μm and subsequent 0.45 μm syringe filters (produced by Gelman Sciences, Ann) Harbor, MI, USA) and stored at −30 ° C. until use.
For in vitro maturation, place 30 to 50 immature eggs in a maturation medium supplemented with 10% pFF, and a medium supplemented with hormone for the first 22 hours at 39 ° C and 5% CO ² saturation humidity. Then, the cells were cultured in a 4-well dish, and then cultured for 22 hours in a culture medium without hormones. Mature eggs with well-expanded cumulus cells are observed after 44 hours of IVM, as in FIG. 2C.

インジェクションチェンバーの準備
体外成熟された卵子を0.1%（w/v）ヒアルロニダーゼ？に短く露出した後機械的な ピペッチングで卵丘細胞を除去した。第１極体及び正常な外観の持つ卵子を二回ワシングし0.01% PVAの含む７μｌTLHドラップの中に移し精子ペレトを少しそのドラップへ加えた。

Preparation of injection chamber 0.1% (w / v) hyaluronidase for in vitro matured ova? The cumulus cells were removed by mechanical pipetting after short exposure. The first polar body and the egg with normal appearance were washed twice and transferred into a 7 μl TLH droop containing 0.01% PVA and a little sperm pellet was added to the drup.

[Example 4] Injection of sperm into the egg cytoplasm Equatorial region of the sperm head by approaching the tip of the pipette near the tail or head of a sperm with motility under an inverted microscope and a micromanipulator (LABOVERT FS, Leitz, Germany) Until it is automatically inhaled into the injection pipette. The form in which the inhaled sperm was captured at the tip of the injection pipette is shown in FIG. Only motile sperm were easily captured at the boundaries of the droop (Figure 4).

  As shown in FIG. 5, the captured sperm was transferred to the egg lap and the sperm was directly injected into the cytoplasm. Thereafter, the open end tube was repeatedly inhaled and released 2-3 times through the mouth, and the sperm was mixed with the cytoplasmic components (FIG. 6). When mixing was complete, the injection pipette was removed from the ovum.

[Example 5] Culture of fertilized ovum All in vitro fertilized ovum injected with sperm were added to North Carolina State University-23 (NCSU-23) medium (Table 3) containing 0.4% BSA. 18 hours each in 39 ° C, 5% CO ² , 7% O ² and 88% N ² incubators for confirmation of MPN formation and sperm head decondensation, development into expanded blastocysts and observation of cell number And incubated for 168 hours.

〔実施例６〕MPNの平価及び胚盤胞細胞の計数
ICSI及びIVF18時間後全ての卵子をスライドグラスに移し34℃のマイクロワンプレの上で十分間固定液（アセト酸：エタノール＝1：3）で固定した。以後、45%(v/v)アセト酸溶液に1%(w/v)オルセイン(orcein)を溶かした溶液で染色し400倍率の光学顕微鏡下で精子の脱凝縮及びMPN形成を平価した（図７）。 図7の(A)で二つの前核及び二つの極体が見え、(B)では脱凝縮された精子の頭部及び雌性前核が見える。

[Example 6] MPN parity and blastocyst counting
After 18 hours of ICSI and IVF, all eggs were transferred to a slide glass and fixed with a fixing solution (acetic acid: ethanol = 1: 3) for a sufficient period on a 34 ° C. micro one-plate. Thereafter, staining was performed with a solution of 1% (w / v) orcein dissolved in 45% (v / v) acetic acid solution, and sperm decondensation and MPN formation were normalized under a 400 × optical microscope (Fig. 7). In FIG. 7A, two pronuclei and two polar bodies are visible, and in FIG. 7B, the decondensed sperm head and female pronucleus are visible.

  A surviving egg is (1) one female pronucleus (hereinafter FPN) and one polar body (hereinafter PB) with one MPN, (2) regardless of the presence or absence of FPN. It was classified into three groups: one MPN or one decondensed sperm head (hereinafter DSH) regardless of the presence of one MPN and (3) the presence or absence of FPN.

  After 48 hours or 168 hours of ICSI and IVF, the blastocyst viability and cell number were investigated by dividing and Hoechst 33342 staining, respectively (Fig. 8). In FIG. 8A, a large number of expanded blastocysts are visible, and in FIG. 8B, the nuclei of expanded blastocysts stained with Hoechst 33342 are visible.

Example 7 Statistical Analysis MPN formation and development differences (%) between groups were analyzed using ANOVA after arcsine conversion to maintain uniformity of dispersion. Post hoc analysis was performed to confirm differences between groups using LSD test. The Wilcoxon test with Dunn's method was used for multiple comparisons to determine the presence or absence of significant differences in the number of blastocysts between groups. All analyzes were performed using the SAS (SAS Institute version 8.1) analysis program.
Comparative Experiment [Comparative Example 1]: Preparation of sperm and ovum and in vitro maturation Frozen-thawed boar semen was prepared for fertilization of in vitro matured ova. As a medium for sperm preparation and IVF, PBS without calcium and magnesium and modified Tris-buffered medium (mTBM) were used, respectively. PBS for sperm washing is 136.9 mM / L NaCl, 2.7 mM / L KCl, 1.5 mM / L KH ² PO ⁴ , 8.1 mM Na ² HPO ⁴ , 0.5 mM / L sodium puruvate, 5.6 mM / L glucose and 0.1% (w / v) BSA (Table 4).

For mTBM (Table 5), 113.1 mM / L NaCl, 3 mM / L KCl, 7.5 mM / L CaCl ² , 20 mM / L Tris (T-1410, Trizma base), 11 mM / L glucose, 5 mM / L Contains sodium pyruvate, 4 mM / L anhydrous kapane and 0.2% (w / v) BSA (A-3311, Fraction V, fatty acid free).

The frozen semen was thawed for 1 minute at 39 ° C. and diluted with 10 ml of PBS. The sperm dilution was centrifuged twice at 350 × g for 3 minutes, and the final sperm pellet was diluted again with mTBM to a concentration of 1 × 10 ⁶ sperm / ml.

On the other hand, the ovum after the IVM was gently pipetted with a maturation medium containing 0.1% (w / v) hyaluronidase to remove the expanded cumulus cells and washes twice with mTBM. 20-30 eggs with 5 μl of mTBM solution were placed in a 40 μl fertilization drop filled with mineral oil. Thereafter, 5 μl of sperm dilution was added to each fertilization drop so that the final sperm concentration was 1 × 10 ⁶ sperm / ml, and the cells were cultured in a CO ² incubator for 8 hours.

〔比較実施例２〕：ICSI方法の差による受精効果の比較
従来のICSIテクニックによる受精
従来のICSIのためのインジェクションピペットはヒュマゲン(Humagen, Inc.)(Charlottesville, VA; 10-MIC-S, 30°angled)から購入した。50Ｘ9 mmディッシュの蓋に二つの７μｌドロップを作製した。マイクロマニピュレーターが取り付けられた倒立顕微鏡（Olympus IX50, Melville, NY, USA）を使用しながら精子の尻尾をスコリングし運動性を減らしてインジェクションピペットの中に吸入し（図９のA）卵子を含んでいるドロップに移動した（図９のB）。

[Comparative Example 2]: Comparison of fertilization effect due to difference in ICSI method Fertilization by conventional ICSI technique Injection pipette for conventional ICSI is Humagen, Inc. (Charlottesville, VA; 10-MIC-S, 30 Purchased from ° angled). Two 7 μl drops were made on 50 × 9 mm dish lids. Using an inverted microscope (Olympus IX50, Melville, NY, USA) with a micromanipulator, the tail of the sperm is scored to reduce motility and inhaled into the injection pipette (Fig. 9A) containing the ovum (B in FIG. 9).

Sperm were injected into an egg fixed with a holding pipette from 6 o'clock or 12 o'clock and mixed with a small amount of cytoplasm [Martin, Biol. Reprod., 2000, 63, p109] (FIG. 9).
Fertilization with ICSI of the present invention The reselected ICSI of the present invention fertilized an egg as described in Example 4.
Comparison of fertilization effect by each ICSI method (Experiment 1)
Experiment 1 compared the MPN formation of eggs fertilized by different methods. Fertilized eggs fertilized by each method were cultured, and MPN formation rate and sperm head decondensation were evaluated 18 hours after fertilization. The IVF group also examined polysperm fertilization.
The survival, division and blastocyst formation rate of fertilized eggs were observed and compared at 24 hours, 48 hours and 168 hours after fertilization. The number of blastocyst cells was observed as described in [Example 6].
Comparison of the effect of sperm capture by the ICSI method of the present invention (Experiment 2)
Experiment 2 examined the development of MPN and blastocysts due to differences in sperm capture type.
As described in [Example 4], the sperm capture type captured at the tip of the reselected injection pipette of the present invention has two forms. That is, when the sperm whose tail was first captured as seen in A in Fig. 10 is injected into the egg cytoplasm (the sperm head is visible outside the injection pipette, X200) and as seen in B in Fig. 10 There is a case where the head is injected into the egg cytoplasm of a previously captured sperm (the arrow indicates the sperm head and the tail is at the end of the injection pipette, X200). Motile sperm were always captured at the sperm drop boundary (FIG. 4).
Results of Comparative Experiments Experiment 1: Differences by Fertilization Method Table 6 shows the MPN formation rates by the two ICSI methods and IVF. In the IVF group, polysperm fertilization was found in 22 eggs, which showed two or more male pronuclei or sperm heads (not shown in Table 6).

  The normal fertilization (46.7%) and MPN formation rate (50.7%) of the newly selected ICSI method were significantly higher than those of the conventional ICSI method (21.3% and 27.9%) (P <0.001>). In the modified ICSI method of the present invention, the decondensation of the sperm head containing MPN (80.0%) is the conventional ICSI (55.7%) and IVF group (63.5%) if polysperm fertilization with IVF group is excluded The value was significantly higher (P <0.001>).

他の２種類のICSI方法及びIVFでの受精卵の生存率、分割率及び胚盤胞形成率は表７に示してある。本発明の改選されたICSI方法による卵子の生存率（71.7%）は従来のICSI方法による生存率（48.1%）に比べて有意的に高い値を示した(P〈0.001〉。また、本発明の改選されたICSI方法による分割率（60.6%）は従来のICSI方法（48.7%）及びIVFグルプ（53.2%）に比べ有意に高い値であった（P〈0.001〉。
IVFグルプでの拡張胚盤胞への発達率（19.4%）が従来のICSI10.5%）及び 改選されたICSI（17.5%）に比べ有意に高い値を示しているが（P〈0.001〉、 多精子受精卵子の胚盤胞への発達を考えると本発明の改選されたICSI方法はIVFグルプとほぼ同じいかやや高い率と考えられる。また、グルプの間で胚盤胞の細胞数には有意差が観察されなかった。

Table 7 shows the survival rate, division rate, and blastocyst formation rate of fertilized eggs with the other two ICSI methods and IVF. The survival rate (71.7%) of the eggs by the re-selected ICSI method of the present invention was significantly higher than the survival rate (48.1%) by the conventional ICSI method (P <0.001>). The split rate (60.6%) of the newly selected ICSI method was significantly higher than that of the conventional ICSI method (48.7%) and IVF group (53.2%) (P <0.001>).
Although the IVF group has a significantly higher rate of development to expanded blastocysts (19.4%) than the conventional ICSI (10.5%) and the newly selected ICSI (17.5%) (P <0.001>), Considering the development of multisperm fertilized ova into blastocysts, the modified ICSI method of the present invention is considered to have a somewhat higher rate than the IVF group, and the number of blastocyst cells between groups is No significant difference was observed.

実験２．精子捕獲形態
本発明の改選されたICSI方法に置いて、頭部が先に捕獲された場合と尾部が先に捕獲される場合の二つグルプ間のMPN形成率は表８に示した。 尾部が先に捕獲された場合(55.4, 59.8, 78.3%)は頭部が先に捕獲された場合(45.1, 54.9, 74.4%)の間に正常受精、MPN形成及び脱凝縮では有意差が無かった。

Experiment 2. Sperm Capture Form Table 8 shows the MPN formation rate between two groups when the head is captured first and the tail is captured first in the modified ICSI method of the present invention. When the tail was captured first (55.4, 59.8, 78.3%), there was no significant difference in normal fertilization, MPN formation and decondensation compared to when the head was captured first (45.1, 54.9, 74.4%). It was.

また、本発明のICSI方法に置いて二つのグルプ間の生存率、分割率及び胚盤胞形成率を表９に示した。尾部が先に捕獲された場合と頭部が先に捕獲されたグルプ間で生存率に差は無かった（79.8及び73.7%）。改選されたICSIでは精子と共に注入される培地の最小量は精子の注入される卵子の生存に影響を与えなかった。
分割率にも有意差が観察されなかった。拡張胚盤胞への発達は頭が先に捕獲された場合が尻尾の先に捕獲された場合より高い（66.2及び57.1%，4.3及び12.5%）値を示したが有意差は無かった。拡張胚盤胞の細胞数では有意差が無かった(39.5 及び32.8)。

In addition, Table 9 shows the survival rate, division rate, and blastocyst formation rate between the two groups in the ICSI method of the present invention. There was no difference in survival between the group with the tail captured first and the group with the head captured first (79.8 and 73.7%). In the re-selected ICSI, the minimum amount of medium injected with sperm did not affect the survival of sperm-injected eggs.
No significant difference was observed in the split ratio. Development into expanded blastocysts was higher (66.2 and 57.1%, 4.3 and 12.5%) when the head was captured first than at the tail, but there was no significant difference. There was no significant difference in the number of cells in expanded blastocysts (39.5 and 32.8).

FIG. 1 shows the production process of the injection pipette of the present invention. FIG. 2 shows changes in porcine immature eggs. FIG. 3 shows the sperm captured at the tip of the injection pipette. (A) When the tail is captured first. (B) When the head is captured first. FIG. 4 shows that motility sperm was captured at the tip of the injection pipette of the present invention. FIG. 5 shows that sperm captured using the injection pipette of the present invention is injected into a mature egg. FIG. 6 shows the process of mixing the injected sperm with the cytoplasm by adjusting the pressure of the injection pipette in the middle of the cytoplasm. FIG. 7 shows a fertilized egg that has been fertilized with the modified ICSI technique of the present invention and then stained with aceto-orcein. (A) Two pronuclei and two polar bodies are visible. (B) Decondensed sperm head and female pronucleus are visible. FIG. 8 shows a blastocyst that has been fertilized with the modified ICSI technique of the present invention and has developed 168 hours after fertilization. (A) Many extended blastocysts are visible. (B) Fluorescent nucleus of expanded blastocyst stained with Bisbenzimide. FIG. 9 shows a method of injecting sperm into a mature egg using a conventional ICSI technique. (A) The sperm tail is scoring under the tip of the injection pipette. (B) Sperm in the injection pipette is injected into the egg cytoplasm. FIG. 10 shows two methods of capturing sperm and injecting them into mature eggs with the injection pipette of the present invention. (A) When the sperm tail is captured first. (B) The sperm head is captured first.

Claims (9)

In the injection pipette used for the intracytoplasmic injection method,
An injection pipette characterized by having an inner diameter larger than the maximum diameter of the sperm head to be injected into the egg cytoplasm or larger than the maximum diameter of the sperm tail.   2. The injection pipette as set forth in claim 1, wherein a large number of spikes are formed at the tip of the pipette.   3. The method for producing an injection pipette according to claim 1 or 2, wherein the narrowed capillary tube is inserted into a holding pipette entrance to a point having a desired diameter, and then the capillary tube is bent and cut.   In the method of injecting sperm into the egg cytoplasm after capturing the sperm with an injection pipette and injecting it into the egg cytoplasm, the injection pipette according to claim 1 or 2 is used. A method for injecting sperm into the egg cytoplasm.   5. The method of injecting sperm into the egg cytoplasm, wherein the sperm head is captured by the tip of the injection pipette according to item 4.   6. Intracytoplasmic sperm injection method characterized in that the specific part of the sperm head to be captured is an equatorial region.   5. The method of injecting sperm into the egg cytoplasm, comprising the step of mixing sperm and egg cytoplasm before collecting the injection pipette.   8. The method for injecting sperm into an egg cytoplasm according to item 7, wherein the mixing step is repeatedly applied to the other end of the injection pipette by pressurizing or depressurizing. 8. A method for injecting sperm into an egg cytoplasm according to item 7, wherein the mixing step includes repeating the release and inhalation 2-3 times by adjusting the other end of the injection pipette with the mouth.

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