CN107384854B - 人类卵母细胞胞浆成熟优化液 - Google Patents
人类卵母细胞胞浆成熟优化液 Download PDFInfo
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Abstract
本发明公开了一种人类卵母细胞胞浆成熟优化液。本发明以低糖的TCM199组织培养液联合患者自体血清为基础，通过添加一些重要的激素、能量物质及高效抗氧化剂褪黑素(melatonin)开发了本发明。本发明开发了一种适合用于IVF‑ET周期中卵母细胞胞浆的成熟优化，也适合用于周期中未成熟卵母细胞的体外成熟后再利用的培养液。本发明不仅能改善成熟卵母细胞的胞浆成熟度，临床结果证明卵母细胞的优质胚胎率大大提高，也能提高未成熟卵母细胞的发育潜能，证明了IVF‑ET周期中部分未成熟卵母细胞具有再利用价值。
Description
技术领域
本发明涉及人类卵母细胞胞浆成熟优化液，属于生殖医学工程领域。
背景技术
体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)患者经过控制性超促排卵后卵巢上能发育生长多个卵泡，取卵后这些卵子被移入培养液中培养4-6h，随后这些卵子与精子相互作用进行体外受精。为什么取卵后卵子要体外培养一段时间？因为，正常情况下，取卵后这些卵子中的大多数卵子的胞核是成熟的，以排出的第一极体为标志，但胞浆存在不够成熟状态，这时必须在特定培养液中培养一段时间以促进卵子胞浆成熟，卵胞浆不成熟大大降低卵子的受精率，胚胎形成率以及优质胚胎率，最终影响IVF-ET的成功率。因此，取卵后卵母细胞在特定的胞浆成熟液中培养是至关重要的。
另外，控制性超促排卵所获卵母细胞约15％为未成熟卵母细胞，其中4％为GV(Germinal vesicle)期卵，11％约为MI(Metaphase I)期卵。这些未成熟卵母细胞，即体内成熟失败卵母细胞，在发育过程中自始至终一直经受来自优势卵泡的压制，因此，这让我们产生了一些疑问：此类受过优势卵泡压制的未成熟卵母细胞经过体外成熟培养能否发育至成熟阶段？若能发育成熟，它们受精后又能否发育为一名健康的婴儿？1998年，Tucker等将一患者的29枚新鲜卵子冷冻(16枚成熟卵和13枚GV卵)，解冻后仅存活2枚GV卵，体外培养30h后行ICSI(卵胞浆内单精子显微注射技术)，获两枚正常胚胎，移植后孕育一健康女婴。1999年，A.De Vos等将896枚控制性促排卵周期所获的MI卵体外培养4h后获1210枚成熟卵，ICSI后行可用胚胎移植15个周期，获一例健康婴儿分娩。以上报道表明：控制性超排卵周期中部分未成熟卵母细胞通过IVM技术可以获得健康后代。但，后续的大量研究结果显示，此类卵子经成熟培养，体外受精和胚胎体外培养很少能发育至囊胚阶段。这一结果表明：控制性超排卵周期中的未成熟卵母细胞的利用率非常低下。如果这些未成熟卵母细胞能利用一种特定的培养液，经体外成熟，受精，胚胎培养及胚胎移植后能获得健康代，意义是非常重大的。一方面人未成熟卵母细胞体外成熟后可以捐赠给卵子库，或用于研究；另一方面也可以归患者自身应用，提高累积妊娠率。
迄今未止，生殖界仍没有一款专业、高效的人类卵母细胞胞浆成熟优化液。正常情况，取卵后，卵子被移入卵子受精液中培养4-6h，随后行体外受精，胚胎培养和移植。而收集到的未成熟卵子，常规被认为无任何利用价值而被直接丢弃。
发明内容
本发明的目的在于提供了一种专业、高效的人类卵母细胞胞浆成熟优化液，用于IVF-ET周期中卵母细胞胞浆的成熟优化，另外也用于周期中未成熟卵母细胞的体外成熟后再利用。
本发明的人类卵母细胞胞浆成熟优化液，包含有(78％-83％)体积的基础培养液TCM199和(17％-22％)体积的人自体血清，TCM199与人自体血清的总体积为100％；其中还含有丙酮酸钠，FSH，HCG，雌激素，抑菌剂，melatonin。
优选的，所述抑菌剂为双抗。
上述所述的人类卵母细胞胞浆成熟优化液，优选的，以TCM199与人自体血清的总体积为基准，其中还含有(0.0020-0.0025)g/ml丙酮酸钠，(0.070-0.080)IU/ml的FSH，(0.4-0.6)IU/ml HCG，(0.6-1.0)μg/ml雌激素，(0.0050-0.0070)g/100ml双抗，(8-12)μMmelatonin。
上述所述的人类卵母细胞胞浆成熟优化液，优选的，其组分组成为：
80％TCM199+0.0022g/ml丙酮酸钠+0.075IU/mlFSH+0.5IU/ml HCG+0.8μg/ml雌激素+20％人自体血清+0.0060g/100ml双抗+10μM melatonin。
本发明中所述双抗可以为常用的青霉素和链霉素，其用量比可以为1:1，双抗的用量表达是为青霉素和链霉素的各自用量，如(0.0050-0.0070)g/100ml双抗是指含(0.0050-0.0070)g/100ml青霉素、(0.0050-0.0070)g/100ml链霉素；0.0060g/100ml双抗是指含0.0060g/100ml青霉素、0.0060g/100ml链霉素。
本发明以低糖的TCM199组织培养液联合患者自体血清为基础，通过添加一些重要的激素、能量物质及高效抗氧化剂褪黑素(melatonin)开发了本发明。本发明中，丙酮酸钠为能量物质，为卵子成熟提供能量；FSH，HCG和雌激素促进卵子核及胞浆成熟；双抗，抑制培养液中细菌生长；melatonin,抗氧化剂，清除培养过程中培养液中产生的氧活性物质(ROS)，另外也促进卵子胞浆成熟。
本发明开发了一种适合用于IVF-ET周期中卵母细胞胞浆的成熟优化，也适合用于周期中未成熟卵母细胞的体外成熟后再利用的培养液。不仅能改善成熟卵母细胞的胞浆成熟度，临床结果证明卵母细胞的优质胚胎率大大提高，也能提高未成熟卵母细胞的发育潜能，证明了IVF-ET周期中部分未成熟卵母细胞具有再利用价值。经安徽医科大学第一附属医院生殖医学中心对本发明的应用发现，IVF/ICSI临床妊娠率上升达15％，ICSI周期中未成熟卵母细胞再利用方面，已出生3例健康的试管婴儿。
本发明应用了患者自体血清，添加了褪黑素(melatonin)，褪黑素不仅能清除体外培养过程中产生的ROS，而且可以高效地促进卵母细胞核及胞浆成熟。本发明应用于试验中对未成熟卵母细胞进行体外培养，那些未成熟卵母细胞均为去除了颗粒细胞后的裸卵，这也打破了人类未成熟卵子体外成熟培养必须含颗粒细胞的理论。本发明带来了多项理论突破：①人类未成熟卵母细胞体外成熟培养可以去除颗粒细胞，②超排卵周期中一些未成熟卵母细胞可以被再次利用，③可以应用于IVF或ICSI周期优化MII卵子的胞浆成熟度，以达到提高优质胚胎率的目的。
具体实施方式
下述实施例是对于本发明内容的进一步说明以作为对本发明技术内容的阐释，但本发明的实质内容并不仅限于下述实施例所述，本领域的普通技术人员可以且应当知晓任何基于本发明实质精神的简单变化或替换均应属于本发明所要求的保护范围。
实施例1
本发明的人类卵母细胞胞浆成熟优化液的组成如下：
80％TCM199(V/V)+0.0022g/ml丙酮酸钠+0.075IU/ml FSH+0.5IU/ml HCG+0.8μg/ml雌激素+20％(V/V)人自体血清+0.0060g/100ml双抗+10μM melatonin。
其中：
1.基础培养液：TCM199‐‐‐100ml装，批号为M4530，低糖，不含HEPEAS，公司Sigma；
2.双抗：选用PENICILING(青霉素)和STREPTOMYCIN SULFATE(链霉素)，批号分别为：LOT17H1214；LOT58H0442，公司SIGMA；
3.丙酮酸钠：选用PYRUVIC ACID SODIUM(a‐ketopropionic acid)，P‐2256，25mg，批号：LOT19H0645，公司SIGMA；
4.雌激素：β‐ESTRADIOL(17β‐Estradiol)(雌激素),E‐2758,250mg,批号：052K13705，公司SIGMA；
5.FSH/HCG：FSH‐促卵泡成熟激素，HCG‐人绒毛膜促性腺激素；选用瑞士产Gonal‐F/Profasi，Swithland；
6.血清：选用患者自体血清；
7.Melatonin，褪黑素，公司Sigma。
(1)成熟卵母细胞胞浆成熟优化的实施如下：
由安徽医科大学第一附属医院生殖医学中心从受试者为来我们中心寻求IVF治疗的不孕症患者，年龄均小于35岁。患者经常规长或短方案促排卵，当B超监测卵巢上有1或2枚直径大于18mm的优势卵泡，患者被注射10000IU的HCG，36h后行超声监测下过阴道穿刺取卵，取卵后，卵母细胞移入人类卵母细胞胞浆成熟优化液体外培养4-6小时，随后行IVF或ICSI授精，18～22小时后观察卵母细胞受精状况，受精卵被挑出和依次入分裂期胚胎培养液和囊胚培养液行分裂期胚胎培养和囊胚培养。体外环境均为37℃、6％CO2及饱和湿度条件下培养。观察和记录卵母细胞存活状况、受精状况、胚胎发育状况及囊胚发育状况。本实例共收集受试者卵母细胞300枚，受精225枚，受精率约75％，卵裂222枚，卵裂率约98.7％，获得优质囊胚109枚,优质囊胚率约49.1％左右，胚胎移植后临床妊娠率55％左右。
(2)ICSI周期中未成熟卵母细胞体外成熟的实施如下：
由安徽医科大学第一附属医院生殖医学中心从受试者为来我们中心寻求ICSI治疗的不孕症患者，年龄均小于35岁。患者经常规长或短方案促排卵，当B超监测卵巢上有1或2枚直径大于18mm的优势卵泡，患者被注射10000IU的HCG，36h后行超声监测下过阴道穿刺取卵，取卵后，MII卵用于随后的ICSI受精或胚胎培养，而未成熟卵母细胞(MI/GV)移入人类卵母细胞胞浆成熟优化液体外培养24小时，随后体外成熟的卵母细胞依次行ICSI授精和胚胎体外培养。本实例共收集卵母细胞105枚，成熟86枚，受精68枚，受精率约79.1％，卵裂67枚，卵裂率约98.5％，获得囊胚33枚,优质囊胚19枚，优质囊胚率28.4％左右。
对比例1
本例的卵母细胞成熟培养为常规受精液-I(K-SIFM-50，COOK,AUS)。
(1)成熟卵母细胞胞浆成熟优化的实施如下：
将人类卵母细胞胞浆成熟优化液调整为受精液-I(K-SIFM-50，COOK,AUS),过程同实施例1，本例共收集卵母细胞145枚，受精108枚，受精率74.5％，卵裂102枚，卵裂率94.4％，获得优质囊胚40枚,优质囊胚率约39.2％左右，胚胎移植后临床妊娠率40％左右，临床妊娠率低于实例1，且有显著性统计学意义(P<0.05)。(2)ICSI周期中未成熟卵母细胞体外成熟的实施如下：
将人类卵母细胞胞浆成熟优化液调整为受精液-I(K-SIFM-50，COOK,AUS),过程同实施例1，本实例共收集卵母细胞25枚，成熟17枚，受精13枚，受精率约76.5％，卵裂12枚，卵裂率约92.3％，未获囊胚。
对比例2
本例的卵母细胞成熟培养为常规受精液-II(G-IVFTM PLUS,Vitrolife,Sweden)。
(1)成熟卵母细胞胞浆成熟优化的实施如下：
将人类卵母细胞胞浆成熟优化液调整为受精液-II(G-IVFTM PLUS,Vitrolife,Sweden),过程同实施例1，本例共收集卵母细胞120枚，受精89枚，受精率74.2％，卵裂86枚，卵裂率96.6％，获得优质囊35枚,优质囊胚率约40.7％左右，胚胎移植后临床妊娠率39％左右，临床妊娠率低于实例1，且有显著性统计学意义(P<0.05)。
(2)ICSI周期中未成熟卵母细胞体外成熟的实施如下：
将人类卵母细胞胞浆成熟优化液调整为受精液-II(G-IVFTM PLUS,Vitrolife,Sweden),过程同实施例1，本实例共收集卵母细胞28枚，成熟19枚，受精14枚，受精率约73.7％，卵裂12枚，卵裂率85.7％，未获囊胚。
对比例3
过程同实施例1，但本例的卵母细胞成熟培养为含20％SSS(血清替代物，IrovineScientific，USA)不含褪黑素(melatonin)的成熟液，组分如下：
80％TCM199(V/V)+0.0022g/ml丙酮酸钠+0.075IU/mlFSH+0.5IU/mlHCG+0.8μg/ml雌激素+20％(V/V)SSS+0.0060g/100ml双抗。
(1)成熟卵母细胞胞浆成熟优化的实施如下：
本例共收集卵母细胞135枚，受精101枚，受精率74.8％，卵裂101枚，卵裂率100％，获得优质囊胚41枚,优质囊胚率约40.6％左右，胚胎移植后临床妊娠率39％左右，临床妊娠率低于实例1，且有显著性统计学意义(P<0.05)。
(2)ICSI周期中未成熟卵母细胞体外成熟的实施如下：
本例共收集卵母细胞51枚，成熟19枚，成熟率37.3％，受精16枚，受精率约84.2％，卵裂14枚，卵裂率约87.5％，囊胚2枚，囊胚率14.3％，未获优质囊胚，囊胚率明显低于实例1，呈极显著性统计学意义(P<0.01)。
对比例4
过程同实施例1，但本例的卵母细胞成熟培养为含20％SSS(血清替代物，IrovineScientific，USA)+10μM褪黑素(melatonin)的成熟液，组分如下：
80％TCM199(V/V)+0.0022g/ml丙酮酸钠+0.075IU/mlFSH+0.5IU/mlHCG+0.8μg/ml雌激素+20％(V/V)SSS+0.0060g/100ml双抗+10μM melatonin
(1)成熟卵母细胞胞浆成熟优化的实施如下：
本例共收集卵母细胞125枚，受精94枚，受精率75.2％，卵裂92枚，卵裂率97.9％，获得优质囊胚37枚,优质囊胚率约40.2％左右，胚胎移植后临床妊娠率41％左右，临床妊娠率低于实例1，且有显著性统计学意义(P<0.05)。
(2)ICSI周期中未成熟卵母细胞体外成熟的实施如下：
本例共收集卵母细胞52枚，成熟28枚，成熟率53.8％，受精24枚，受精率约85.7％，卵裂23枚，卵裂率约95.8％，囊胚6枚，囊胚率26.09％，优质囊胚3枚，优质囊胚率13.0％，囊胚率及优质囊胚率明显低于实例1，均呈显著性统计学意义(P<0.05)。
对比例5
过程同实施例1，但本例的卵母细胞成熟培养为含20％自体血清不含剂褪黑素(melatonin)的成熟液，组分如下：
80％TCM199(V/V)+0.0022g/ml丙酮酸钠+0.075IU/mlFSH+0.5IU/mlHCG+0.8μg/ml雌激素+20％(V/V)自体血清+0.0060g/100ml双抗。
(1)成熟卵母细胞胞浆成熟优化的实施如下：
本例共收集卵母细胞150枚，受精115枚，受精率76.7％，卵裂113枚，卵裂率98.3％，获得优质囊胚52枚,优质囊胚率约46％左右，胚胎移植后临床妊娠率45％左右，临床妊娠率低于实例1，且有显著性统计学意义(P<0.05)。
(2)ICSI周期中未成熟卵母细胞体外成熟的实施如下：
本例共收集卵母细胞88枚，成熟67枚，成熟率76.1％，受精51枚，受精率约76.1％，卵裂49枚，卵裂率约96.1％，囊胚12枚，囊胚率24.5％，优质囊胚1，优质囊胚率2％，囊胚率及优质囊胚率明显低于实例1，前者(囊胚率)呈显著性统计学意义(P<0.05)和后者(优质囊胚率)呈极显著性统计学意义(P<0.01).
发明公开了人类卵母细胞胞浆成熟优化液。本发明以低糖的TCM199组织培养液联合患者自体血清为基础，通过添加一些重要的激素、能量物质及高效抗氧化剂褪黑素(melatonin)开发了本发明。本发明开发了一种适合用于IVF-ET周期中卵母细胞胞浆的成熟优化，也适合用于周期中未成熟卵母细胞的体外成熟后再利用的培养液。不仅能改善成熟卵母细胞的胞浆成熟度，临床结果证明卵母细胞的优质胚胎率大大提高，也能提高未成熟卵母细胞的发育潜能，证明了IVF-ET周期中部分未成熟卵母细胞具有再利用价值。
Claims (1)
1.人类卵母细胞胞浆成熟优化液，其特征在于，以TCM199与人自体血清的总体积为基准，其组分组成为：
80%V/V TCM199+0.0022g/ml丙酮酸钠+0.075IU/mlFSH+0.5IU/mlHCG+0.8μg/ml雌激素+20%V/V人自体血清+0.0060g/100ml青霉素+0.0060g/100ml链霉素+10μM褪黑素。
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| CN110547291A (zh) * | 2019-09-27 | 2019-12-10 | 安徽医科大学 | 一种高效抗氧化的人卵母细胞冷冻保护剂 |
| CN111172099B (zh) * | 2020-03-06 | 2023-12-29 | 广州辅康生物科技有限公司 | 配子缓冲液及其制备方法 |
| CN111676188A (zh) * | 2020-07-13 | 2020-09-18 | 扬州大学 | 一种高龄小鼠卵母细胞体外成熟的优化液 |
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| CN115029303B (zh) * | 2022-06-22 | 2023-08-15 | 安徽第肆纪医学科技有限公司 | 一种用于coh周期中人未成熟卵母细胞ivm能量培养液 |
| CN115161267B (zh) * | 2022-08-18 | 2023-06-30 | 中国医学科学院医学生物学研究所 | 一种食蟹猴的未成熟卵母细胞和胚胎的体外培养液及其应用 |
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