体外受精技术

,单击此处编辑母版标题样式,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,体外,受精技术,体,外,受,精,的,概,念,狭义：,将动物受精过程中的精卵结合在体外环境下完成的现象。包括自然条件下的体外受精(两栖类和鱼类)，和人为培养条件下的体外受精(哺乳动物)。,广义：,经过特殊处理的精子在体外使卵母细胞受精的技术，所以体外受精的胚胎移植后所生婴儿又称为“试管婴儿”。包括卵母细胞的体外成熟和受精卵的体外培养两项配套技术。,优点：,与体内受精相比，所需精子数减少，提高精液利用率。,Example:,流式细胞含仪分离的性控精子只能用于体外受精。,国,内,外,研,究,现,状,1878年，,Schenk(Germany),将体内成熟的家兔和豚鼠卵子与附睾精子放入子宫液中培养，观察到第二极体的排出和卵裂现象； 1945年，张明觉(华裔)偶然获得兔体外受精卵，但无重复性，且无试管动物出生； 1951，张明觉与,Austin(,澳)同时发现精子获能现象；1959年，他首次获得体外受精兔；,80年代以来，对胚胎需求量的加大，促进了家畜体外受精的研究，牛、绵羊、山羊、猪等体处胚相继获得了成功，国内90年代在家畜上也获得了成功； 现已将卵母细胞体外成熟-体外受精-早期胚体外发育-冷冻保存结合在一起，建立工厂化胚胎生产。,国,内,外,研,究,现,状,世界体外受精的首创纪录,动物种类 首创纪录文献,兔,Thibault,(1954),叙利亚仓鼠,Yanaginmachi,chang,(1963),小鼠,Whittingham,(1968),中国仓鼠,Pickworth,Chang(1969),猫,Hammer(1970),土拨鼠,Yanagimachi,(1972),大鼠,Miyamoto,Chang(1973),狗,Mahi,Yanagimachi,(1976),牛,Brackett(1978),猪,Iritani,Niwa,(1977),绵羊,Bondioli,Wright(1980),山羊 花田章(1985),国,内,外,研,究,现,状,我国体外受精的首创纪录,动物种类 首创纪录文献,小鼠 陈秀兰,(1986),兔 范必勤(1987),绵羊 旭日干(1989),牛 旭日干(1989),山羊 钱菊汾(1990),猪 范必勤(1990),水牛 蒋和生(1993),体,外,受,精,技,术,流,程,卵母,细胞的体外成熟,精子的体外获能,卵母细胞的体外受精,受精卵的体外发育,配子的冷冻保存,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的采集与体外培养,1. 离体采集,：30,min,内取卵巢，置生理盐水或,PBS（25-35），3h,内回收。, 卵泡抽吸法：,针头穿刺，一次进针可抽吸卵巢皮质的多个卵泡,优点：回收速度快，不易造成卵母细胞的污染；,缺点：容易损伤卵母细胞周围的卵丘细胞，影响其随后的成熟。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的采集, 卵巢解剖法,：卵巢切为两半，去掉中间的髓质部分，刀片划破卵泡，在培养液中反复冲洗,优点：能保持卵母细胞周围卵泡细胞的完整性，回收率也相对较高；,缺点：回收的速度相对较慢，容易造成污染。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的采集,2. 活体采集(,Ovum Pick-Up,OPU),腔镜法,：腹壁切开一小口，插入腹腔镜，操作杆和穿刺针，配合将卵母细胞抽吸出来,优点：比较直观，容易掌握；,缺点：工作量大，对母牛有损伤，不能频繁手术。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的采集,B,型超声波法：,超声波探头和采卵器插入到阴道子宫颈的一侧穹隆处。术者通过直肠将卵巢贴在探头上，根据,B,超屏幕上所显示的卵泡位置进行穿刺而将卵母细胞抽出,优点：不用手术，操作速度快，对母牛损伤小，可频繁采卵，且亦可对妊娠母牛采卵；,缺点：操作技术较难掌握，并需要昂贵的超声设备。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的筛选,只有周围包被着完整卵丘（,A,级）或部分卵丘脱落（,B,级）(1层以上卵丘细胞）,、,胞浆均质并充满于透明带内的卵母细胞才能进行成熟与胚胎发育的培养。,A,级：卵丘细胞致密，至少有5层以上；,B,级：卵丘细胞为2-4层；,C,级：卵母细胞大部分被包裹；,D,级：卵母细胞少量被包裹；,E,级：裸卵。,A、B,级可用于培养，,C、D,级成熟率低。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟,卵母,细胞成熟目的：,提供可利用的卵母细胞用于体外受精、克隆等；,研究其体内成熟的模型。,卵母细胞体外成熟的特征,生发泡破裂；,染色体凝集；,纺缍体形成；,极体排出；,透明带软化；,卵丘细胞扩展。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟,卵母,细胞成熟培养液,以,TCM-199,较好，可使牛、羊、猪的卵母细胞体外成熟率高达80%以上。,添加成分：,促性腺激素：,FSH、LH,FCS、BSA：,有认为,FCS,比,BSA,好，而发情牛血清或发情羊血清比,FCS,好。,抗生素,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟培养,Oocyte,Collection Medium (OCM),最常用的是碳酸氢钠或,Hepe,s,缓冲的,TCM-199;,2.,Oocyte,Maturation Medium,（1) Bovine steer serum (2),Folltropin,(3),Estradiol,(4) Na,Pyruvate,(5) Glutamine,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟培养,2. 成熟培养时间,一般22,h-24h，,但猪40,h-44h，,马30,h-36h，,兔仅12,h-15h,3.,成熟培养的温度和气相环境,人和啮齿类动物：37,牛、猪、羊和马等：3839。,pH：7.7,好于7.1，7.4，8.0,渗透压：330,Osm,/kg,好于300，320,气相： 5%,CO,2,5%CO,2,5%O,2,90%N,2,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟培养,4.,IVM,系统, 开放培养系统,1,ml-2ml,成熟培养液直接置于平皿内或培养板内，放入50枚-200枚卵母细胞培养，上面不盖石蜡油。,优点：简单，对培养液中的脂溶性物质没有若影响，能同时培养大量细胞,缺点：渗透压变化较大，容易污染,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟培养, 微滴培养系统,将成熟培养液在做成50-500,l,的微滴，覆石蜡油，将卵母细胞置于其中培养。,优点：渗透压比较稳定，不易污染；,缺点：对脂溶性物质有稀释作用，成本高，培养结果易受石蜡油质量的影响。, 密闭培养系统,置,12,ml,培养液的试管中，胶塞，在培养箱或恒温水浴中培养；若采用培养皿或微滴培养，则可将其装入密闭的塑料袋内。,优点：不要,CO,2,培养箱，方便运输,缺点：,pH,不如前者稳定。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟,卵母,细胞成熟的标志,第一极体的排出、卵丘细胞的扩展可作为成熟的标志，而生发泡破裂(,GVBD),是卵母细胞恢复减数分裂的主要标准，因此常用地衣红染色才可判断。,牛卵母细胞体外成熟标准：,1级成熟卵：,卵丘细胞完全扩展，卵丘细胞至少向外扩展3倍于裸卵直径；,2级成熟卵：,卵丘细胞中等扩展，卵丘细胞至少向外扩展2倍于裸卵直径；,3级成熟卵：,卵丘细胞轻度假扩展，卵丘细胞仍紧紧粘附于透明带上。,1级和2级卵通常均已排出第一极体。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞体外成熟的质量评定, 形态观察：,细胞质均匀、没有空泡, 固定染色法 ：,0.1%透明质酸酶 醋酸酒精或醋酸甲醇（1:3）固定24,h48h 1%,间苯二酚兰（,Lacmoid,）,或1%地衣红（,Orcein,）,的40,%醋酸溶液染色, 受精和胚胎发育潜能的评定,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟,影响卵母细胞成熟的因素,卵母细胞的来源：,不同种类的动物、不同生理阶段的动物(小牛低于成年牛)、卵母细胞的质量等；,（1）动物种类与年龄：,牛、羊效果较好，而马、猪较差。,小牛卵母细胞的平均直径（118.041.15,m）,比成年母牛的（122.840.74,m）,小。,体外成熟的小牛卵母细胞体外受精后发育潜力也低于成年卵母细胞,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟,（2）卵母细胞的形态,A、 76.2%； B、67%；,C、22%； D、11.4%,（3）,卵巢贮存时间与温度,30,C,对成熟和胚胎发育不利(,First & Parrish）,绵羊的卵巢贮存在22,C,数小时对成熟无明显影响，但体温条件下则会使卵母细胞的质量下降 (,Moodie,& Graham ),体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟,2. 激素,目前大多数都离不开,FSH,或,LH,或两者的混合物，但仍有争议,GH,能显著提高精子的受精率，促进卵裂及胚泡的形成,甲状旁腺激素相关蛋白（,PTHrP,）,胰岛素,激活素,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外成熟,3. 生长因子,目前已经证实与卵母细胞成熟有关的生长因子有：上皮细胞生长因子,（,EGF）、,转化生长因子,和,（TGF-，）、,成纤维细胞生长因子（,FGF）、,胰岛素类生长因子和（,IGF-，）,4.,血清,胎牛血清,（,FBS）、,发情牛血清（,OCS）、,发情前期母牛血清,（,POS）、,超排母牛血清,（,SCS）、,公牛血清（,SS）。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外保存,1. 37保存,应考虑保存时间与卵老化,2.低温保存,0-10,兔:10，受精能力显著降低。,体,外,胚,胎,生,产,技,术,要,点,卵母细胞的体外保存,3. 冷冻保存,借鉴胚胎冷冻方法，现已建立了几种卵母细胞冷冻程序 ：,A,慢速冷冻、慢速解冻法,B,慢速冷冻、快速解冻法,C,快速冷冻、快速解冻法,D,一步冷冻法,E,玻璃化法,F,超快速冷冻法,体,外,胚,胎,生,产,技,术,要,点,影响卵母细胞成熟的因素,培养条件：,温度：37,C,较好，但各种动物最适温度不同，如兔子低于体温3,C,较好，牛35-39,C,都可，但38-39,C,最合适；,培养基：水、血清（无血清、有血清)、,无血清时添加,BSA,等；有血清时如胎牛血清、发情牛血清、公牛血清、超排牛血清等；,体,外,胚,胎,生,产,技,术,要,点,精子的分离与洗涤,洗涤的目的：,去掉精浆中抑制精子获能的因子和死精子，或洗去防冻剂及卵黄等成分，以获得活力好的精子。,精子分离及洗涤方法,悬浮法(,Swim-up)：,利用精子上游的特性将高活力的精子与死精子及精浆成分分开。,将精液置于获能液或受精液中，培养箱中孵育龄10-30分钟，吸取上层液体，再离心洗涤1-2次即可。,精子的分离、洗涤与体外获能,体,外,胚,胎,生,产,技,术,要,点,精子的分离、洗涤与体外获能,离心洗涤法：,对于,活力好的精子,直接将其放入获能液或受精液中，离心洗涤若干次便可。,BSA/,Percoll,密度梯度离心法：,利用形态正常精子密度高于异常精子的原理。如将,BSA,制成不连续的密度梯度或将,Percoll,制成30-40%或45-90%梯度，经平衡离心从而得到高受精力的精子。,玻璃棉过滤法：,采作小玻璃珠过滤除去死精子。因其成本高，较少采用。,（二）精子获能处理方法,1. 血清白蛋白法,将精子与血清白蛋白溶液进行长时间孵育 。,该法由于需要的时间较长，且诱发精子获能的作用不强，故仅在,IVF,技术研究的初期进行过尝试，具有一定作用，目前仅作为精子获能的一种辅助手段。,2. 高渗盐法,精子表面含有许多被膜蛋白，即所谓的“去能因子”。当用高离子强度溶液处理精子时，这些被膜蛋白将从精子的表面脱落，从而导致精子获能。,由于高离子强度溶液对精子具有渗透打击作用，会影响精子的活力，故目前已废除不用。,（二）精子获能处理方法,3.卵泡液孵育法,卵泡液含有来自血清的大分子物质，并含有诱发精子获能和顶体反应的因子，故在精液中添加一定浓度的卵泡液可诱发精子获能。,10%卵泡液，牛受精分裂率（40.3%,Vs 87.5%）,和囊胚发育率（8.3%,Vs 40.4%）,均显著下降，但加入50%的卵泡内膜细胞条件液则能 。,卵泡液可以用于精子的获能处理，但不能用于,IVF,系统。,（二）精子获能处理方法,4. 钙离子载体法,钙离子载体,A23187,能直接诱发,Ca,2+,进入精子细胞内，提高细胞内的,Ca,2+,浓度，从而导致获能。,0.5,mol/L1.0,mol/L A23187,溶液处理精子5,min，,然后加入等量含1%,BSA,的,BO,溶液终止,A23187,的作用，即可将获能精子加到含卵母细胞的受精滴中进行,IVF。,注意控制,A23187,的工作浓度和作用时间，否则会导致精子的活力下降和死亡。,（二）精子获能处理方法,5. 肝素法,肝素是一种高度硫酸化的氨基多糖类化合物，与精子结合后，能引起,Ca,2+,进入精子细胞内部而导致精子获能。,最初，制备好的精子常用含有肝素（100,mg/L）,的获能液预处理15,min，,然后加到受精滴中进行,IVF。,现直接将肝素（50,mg/L）,添加到受精液中进行受精。,（三）精子获能的检测,1. 精子形态及运动方式的观察,头部膨大，活力增强，超活化,2. 顶体反应的检测,（1）双重染色法,优点：无需昂贵设备，简单易行；,缺点：易发生误判,（2）透射电镜观察,成本高，制片繁琐，可操作性不强。,三、,IVF,培养系统,（一）微滴系统,20-40,l,微滴受精液，覆石蜡油，每滴放入,IVM,的卵母细胞10-20枚及获能处理的精子（1.0-1.5）10,6,个/,ml，,孵育6 -24,h,优点：,受精液及精液的用量均较少,，,IVF,的效果亦较好；,缺点：,结果易受石蜡油质量影响，操作繁琐，成本较高。,（二）四孔培养板系统,每孔加入500,ml,受精液和100-150枚,IVM,的卵母细胞，然后加入获能处理的精子（1.0-1.5）10,6,个/,ml，,孵育6,h-24h。,优点：,操作相对比较简单，受精结果不受石蜡油质量的影响；,缺点：,精子的利用率相对较低，,IVF,效果不如微滴法稳定。,三、,IVF,培养系统,通常以精子穿透率、原核形成率、受精分裂率和囊胚发育率来衡量。其中，囊胚发育率最为可靠。,（一）固定染色法,受精810,h,精子穿透率和多精子受精率；受精18,20,h,双原核形成率,（二）体外培养,受精48,h 2-8,细胞比例,，7,d,囊胚发育率，9,d,囊胚的孵化率,三、,IVF,质量评定,体,外,胚,胎,生,产,技,术,要,点,精子的分离、洗涤与体外获能,Sperm Swim-up,It is slower than the,Percoll,procedure, sometimes it does not give better results.,1. Thaw 6 to 8 straws of,frozen semen,in the,cyto,-thaw for 60 seconds. If possible, use semen from different bulls.,2.,Combine contents of straws in 5 ml Sp-TALP,. Place sample into the,incubator (38.5C) for 5 minutes.,3. Centrifuge semen (200 x g; 5 min) and,discard all but the bottom 1 ml of supernatant.,4. Prepare 4 to 5 test tubes containing 1 ml Sp-TALP. Add approximately,250,m,l of sperm suspension very slowly to the bottom of each tube,using a 20 gauge needle and 1 ml syringe. Place,tubes in incubator (38.5C) for 1 h.,5 At the end of sperm swim-up,aspirate the top 800,m,l from each tube and combine samples.,Centrifuge (1000 rpm) the combined sample for 5 minutes.,Discard all but the bottom 500,m,l of,supernate,.,体,外,胚,胎,生,产,技,术,要,点,精子的分离、洗涤与体外获能,Glass- wool Filtration,This filtration procedure usually requires 10-15 minutes and generally yields nearly 100% viable sperm.,1. Prepare in advance 0.2 ml glass wool columns in 1 ml syringes that are rinsed 10X with,Milli,-Q water and autoclaved.,2. Immediately before starting purification, rinse column several times with,Hepes,-TALP and finally with Sperm-TALP to equilibrate column.,3. Frozen-thawed semen (3-5 straws) is washed twice with 10-15 ml Sperm-TALP by centrifugation at 200 x g (10 min) and then,resuspended,in 0.6-0.8 ml IVF-TALP.,4. Sperm suspension is then layered over the wet column and allowed to filter by gravity.,5. The number and viability of filtered sperm is determined.,体,外,胚,胎,生,产,技,术,要,点,精子的分离、洗涤与体外获能,Thaw straw,Layering of sperm onto,Percoll,. After cutting the tip of the straw (Left panel), the contents of the straw are expelled onto the top of the,Percoll,gradient (right panel). Here, removal of the semen is facilitated by using a homemade plunger.,Percoll,gradient: up to bottom45-90%,Removal of sperm from the bottom of the,Percoll,gradient.,Washing sperm in Sp-TALP. The left panel shows the washed and centrifuged sperm. The right panel shows the pellet of sperm remaining in the tube after aspiration of the supernatant.,Percoll,method,Hemacytometer,used for counting sperm.,体,外,胚,胎,生,产,技,术,要,点,精子的分离、洗涤与体外获能,2.精子的体外获能,(1)卵泡液孵育法：,卵泡液含有来自血清的大分子物质，并含有诱发精子获能和顶体反应的因子，故在精液中添加一定浓度的卵泡液可使精子获能。但不能用于体外受精(卵裂率和囊胚率下降)。,(2),钙离子载体法,：钙离子载体,A23187,能诱发,Ca,进入精子细胞内，诱发精子的超活化和顶体反应，从而导致获能。但其作用时间和浓度对于获能非常关键。,体,外,胚,胎,生,产,技,术,要,点,精子的分离、洗涤与体外获能,(,3)肝素法：,它与精子孵育后，能引起,Ca,进入精子内，导致获能。这是被广泛采用的获能法。原来将肝素(100,mg/ml),获能液预处理精液，再受精，现在直接将肝素(50,mg/ml),添加到受精液中受精。,实际操作时，常将多种因素共同使用，如咖啡因-肝素-,BSA,系统。,体,外,胚,胎,生,产,技,术,要,点,精子的分离、洗涤与体外获能,3. 精子获能的检测,(1)精子形态及运动方式改变(常用)：,获能后的精子头部膨大，运动增强。,(2)顶体反应的检测：,双重染色法：,取样固定染色(快绿,FCF,和曙红)涂片干燥观察若头部有厚的蓝绿区为无获能，若出现粉经为获能。,电镜法：,2. 体外授精,授精通常在覆盖下的微滴中进行，微滴以50-100,ul,，,一般每滴中加5-10个卵母细胞，精子最终浓度为1,X10,6,个/,ML。,培养条件：5%,CO,2,、39,C,培养，,BO,液中6-8,h，TALP,液中18-24,h。,体,外,胚,胎,生,产,技,术,要,点,精子的体外获能与体外受精(牛),1.,Remove plates containing matured,oocytes,from the incubator and place on the slide warmer.,2. Add 25 ml sperm preparation and 25 ml PHE mix into each well.,When,pipetting,the sperm, place the pipette in the middle of the sperm suspension rather than on the bottom to avoid grabbing debris that can settle to the bottom of the tube,.,3. Return 4-well plate to incubator for 8-10 h.,Many people do fertilization for 18-20 h. When we were establishing IVF in our lab, 8-10 h gave better results than longer incubation times. Recently, however, we have gotten good results with 18-20 h fertilization times. In addition, longer fertilization times make it easier to remove cumulus cells after fertilization.,To determine the incidence of parthenogenesis, one well should be prepared without sperm, but with PHE. After 8 10 h, place these,oocytes,into a separate culture medium drop and culture for 2 days before looking at rate of parthenogenesis.,体,外,胚,胎,生,产,技,术,要,点,体外受精,体,外,胚,胎,生,产,技,术,要,点,早期胚胎的体外发育,阻滞期：,各种动物的早期胚胎体外发育时都存在阻断期，因此可通过改善培养条件和在培养液中添加生长因子(,TGF-,或,bFGF,等)或细胞外基质因子,克服阻断。但目前的发育率仍然比较低，约为40%，是限制体外受精应用的一个突出问题。各种动物阻断期： 牛： 猪： 羊：,体,外,胚,胎,生,产,技,术,要,点,早期胚胎的体外发育,培养液,添加的成分：,(1)丙酮酸钠和乳酸钠：,作为能量底物。,(2)生物体液和蛋白质：,FCS、NCS、,发情母畜血清、,BSA,等。,(3)激素：,PMSG、FSH、,胰岛素、雌激素等，提高囊胚率。,(4)生长因子及多肽：,IGF，,促进卵母细胞的成熟、受精和早期胚胎发育；,EGF,促进胚胎的分化和囊胚的形成；,TGF,促使牛的胚胎通过早期阻断；,LIF(,白血病抑制因子)提高孵化囊胚发育率，并抑制胚胎干细胞的体外分化。,体,外,胚,胎,生,产,技,术,要,点,早期胚胎的体外发育,2. 胚胎的体外培养系统,常规培养系统：,微滴法和培养板法。,输卵管离体培养法：,克服阻断，但因繁琐不常用。,与体细胞共培养：,如输卵管上皮细胞、滋养层细胞、颗粒细胞、子宫内膜细胞等。,体,外,胚,胎,生,产,技,术,要,点,胚胎的冷冻保存,常规的慢速冷冻法,冷冻液:,1.5,M,甘油+0.25,M,蔗糖+,PBSS,液+0.1,mg/,mlPVP,1.5M,乙二醇+0.25蔗糖+,P BSS,液+0.1,mg/,mlPVP,冷冻方法,：,在室温下，先将牛胚胎放入防冻液中平衡一段时间； 装管； 以每分钟1的速度降温至7； 植冰； 以每分钟0.5的速度降温至35；平衡10分钟后，投入液氮中保存。,体,外,胚,胎,生,产,技,术,要,点,胚胎冷冻保存,2. 玻璃化冷冻,冷冻液：,EFS40：40%,乙二醇+0.3,M,蔗糖+18%聚蔗糖+,PBSS,冷冻步骤：,首先将胚胎放入20%乙二醇溶液（,EFS20）,中平衡5,min；,用移卵管移入玻璃化溶液中平衡10,min；,装管，封口； 将细管投入液氮冷冻保存。,影,响,体,外,受,精,的,因,素,卵母细胞的成熟,精子的体外获能,（一）卵母细胞的成熟度,（二）精子体外获能,（三）公畜的影响,Some instruments used to pick up embryos and,oocytes,.,From left to right are,a 1 cc syringe with an extension of rubber tubing connected to a,Unopette,a,wiretrol,(from Drummond Scientific),the same device as #1 without the rubber tubing extension and,a 5 ml Drummond,Microdispenser,.,Use of the,wiretrol,using two hands (left panel) or one hand (right panel).,Two methods for using the,Unopette,tip. The device on the left panel has been modified to include a piece of rubber tubing between the,Unopette,and syringe to give the technician greater control over the volume aspirated.,(,A,),On a,Petri,-dish (i.e.,Intergrid,plate) place 3,microdrops,of 70,l holding medium side by side.,(B) Place one,blastocyst,into the middle drop,(,C(C-D),Insert the cotton plug side of a 0.25 ml French straw into the wide end of the,pipet,tip.,D( (E) Aspirate one empty drop.,A (E) Aspirate air to create a 0.5 cm column,A (F) Aspirate the,microdrop,containing embryo (make sure that the embryo is aspirated by observing under the microscope;,A (F) Aspirate air to create a 0.5 cm column,h,(G),Aspirate the remaining empty drop until the cotton plug is wet.,R (H) Remove transfer straw from syringe and place on slide warmer until use.,Who should be treated with in vitro fertilization?,In vitro fertilization can be used as an effective treatment for infertility of all causes except for women with infertility caused by an anatomic,problem with the uterus, such as severe intrauterine adhesions. It is generally used in couples who have failed to conceive after at least one year of trying who also have one or more of the following:,1.,Blocked fallopian tubes or pelvic adhesions,with distorted pelvic anatomy. Women that have had,tubal ligation,and are considering,tubal,reversal surgery,as well as men that are considering,vasectomy reversal surgery,might also consider IVF.2. Severe,male factor infertility,(low sperm count or low motility)3. Failed 2-6 cycles of,ovarian stimulation,with,intrauterine insemination,Who should be treated with in vitro fertilization?,4.,Advanced female age,- over 385.,Reduced ovarian reserve, which means lower quantity (and sometimes quantity) of eggs. A,day 3 FSH and,estradiol,test,and,antral,follicle counts,are often done as screening tests for egg quantity (and quality). Reduced egg quantity and quality is usually treated with either IVF, or with IVF using,egg donation,from another woman.6. Severe,endometriosis,The graph shows live birth rates for all 23 reporting Illinois IVF centers for 2002 These clinics reported live birth rates ranging from 13.5% to 47.4% per egg retrieval for women under 35 Five Illinois IVF programs failed to report their outcome data to the government (US law requires reporting annually),How is in vitro fertilization performed?,1. Basic screening tests are performed on both partners at all IVF clinics.,In general, some testing of ovarian reserve should be done on the female prior to starting the injections. We use,day 3 FSH testing,as well as,antral,follicle counts,for this purpose. The results of these tests give us,some,ability to predict whether her ovaries will respond well to the drugs (make sufficient follicles and eggs). The,number of eggs retrieved correlates strongly with IVF success rates,.,2. Consents are signed by all parties.,3. The woman is stimulated with injected medications to develop multiple egg development. These injections continue for about 8 - 10 days.,How is in vitro fertilization performed?,4. Blood and ultrasound testing is done every 1-3 days to monitor the development of the follicles (egg-containing structures) in the ovaries.,We need to get a minimum number of 3 follicles to develop to maturity in order to be able to proceed with the egg retrieval. About 90% of women under 40 with a normal FSH and normal,antral,follicle counts will develop at least this minimum number of follicles. If this many mature follicles can not be obtained from the stimulation process - we will cancel the cycle (not proceed to egg retrieval).,How is in vitro fertilization performed?,5. When the womans follicles are mature, a,transvaginal,ultrasound-guided,egg retrieval,(egg aspiration) procedure is performed to remove the eggs from the follicles.,The average duration of this procedure is 5-6 minutes (at our facility).,6.,The embryos are cultured in the,laboratory,for 2-6 days.,7. The,embryo transfer procedure,is done which places the embryos in the womans uterus where they will hopefully implant and develop to result in a live birth. This is like a Pap smear for the woman. There should be no discomfort.,8. If there are leftover embryos (of sufficient quality) beyond the number that is transferred, many couples prefer to have them frozen (,cryopreserved,) for use in a future cycle.,Embryo,cryopreservation,can be used for another attempt at having a baby if the fresh cycle fails - or as an attempt to have another child if the fresh cycle is successful.,How is in vitro fertilization performed?,5. When the womans follicles are mature, a,transvaginal,ultrasound-guided,egg retrieval,(egg aspiration) procedure is performed to remove the eggs from the follicles.,The average duration of this procedure is 5-6 minutes (at our facility).,6.,The embryos are cultured in the,laboratory,for 2-6 days.,7. The,embryo transfer procedure,is done which places the embryos in the womans uterus where they will hopefully implant and develop to result in a live birth. This is like a Pap smear for the woman. There should be no discomfort.,8. If there are leftover embryos (of sufficient quality) beyond the number that is transferred, many couples prefer to have them frozen (,cryopreserved,) for use in a future cycle.,Embryo,cryopreservation,can be used for another attempt at having a baby if the fresh cycle fails - or as an attempt to have another child if the fresh cycle is successful.,Incubators are often stacked like this to save lab spaceThe colored tape on the door of the upper unit is used to identify the location of a patients eggs or embryos in the incubator before the door is openedThis reduces the amount of time that the door has to remain open,A hood like this keeps the air circulating in a certain pattern so dirt, dust, etc. does not fall into the open dishes while they are out of the incubators,On each side are micromanipulator controllers used for ICSI and assisted hatching On the far right is our video and printing equipment,The stage (where the dish containing your embryos will be placed) of our microscope is heated to 37,o,C,The dishes on the left contain freshly aspirated follicular fluidThe dish under the microscope with the light on it is being searched for,eg,The,cryopreservation,area of our lab1: liquid nitrogen supply tank, 2: backup embryo freezer, 3: main embryo freezer, 4 & 5: Storage tanks for frozen embryos and sperm,