卵母細胞玻璃化冷凍液配方的改良研究及臨床應用
發(fā)布時間：2018-09-08 06:07
【摘要】：第一部分改良玻璃化冷凍液對體外成熟卵母細胞冷凍復蘇后發(fā)育潛能的影響 目的針對目前卵母細胞玻璃化冷凍效果不穩(wěn)定的問題,參考常用的兩種卵母細胞玻璃化冷凍液配方,設計一種保持滲透性不變而降低各種滲透性冷凍保護劑濃度的玻璃化冷凍液配方,利用ICSI患者捐贈的未成熟卵母細胞進行玻璃化冷凍方法的研究,驗證新型改良卵母細胞玻璃化冷凍液對體外成熟卵母細胞玻璃化冷凍效果的影響。 方法收集常規(guī)ICSI患者捐贈的未成熟卵母細胞進行體外成熟培養(yǎng),將18～24小時體外成熟的卵母細胞作為研究對象。隨機分為4組,其中三組采用不同的方法進行冷凍保存,分別為B組(EG/DMSO法)、C組(EG/PROH法)和D組(EG/DMSO/PROH法),解凍后進行授精和胚胎培養(yǎng),A組為對照組,培養(yǎng)成熟后直接進行授精和胚胎培養(yǎng)。以每解凍卵母細胞為分母,觀察計算存活率、受精率、優(yōu)胚率、囊胚形成率和優(yōu)質(zhì)囊胚率。 結果不同玻璃化冷凍配方對體外成熟卵母細胞的發(fā)育潛能均有較大的影響,其受精率、優(yōu)胚率和囊胚形成率均顯著低于新鮮組(P0.01),盡管D組的存活率、2PN率、優(yōu)胚率、囊胚形成率及優(yōu)質(zhì)囊胚率高于B組和C組,但統(tǒng)計學分析并無顯著性差異(P0.05)。D組的存活率最高(84.8%),與B(77.2%)、C組(80.0%)比較沒有顯著性差異(P0.05)。D、B、C各組的2PN率分別為46.7%、42.4%和40.0%,優(yōu)胚率分別為5.7%,2.2%和2.2%,囊胚形成率為7.6%,2.2%和3.3%(P0.05),僅D組形成了優(yōu)質(zhì)囊胚,與A組比較沒有顯著性差異(1.9%和6.7%,P0.05)。 結論改良卵母細胞玻璃化冷凍液可以改善體外成熟卵母細胞的發(fā)育潛能,有助于獲得更多的優(yōu)質(zhì)囊胚。 第二部分改良玻璃化冷凍對體外成熟卵母細胞形態(tài)及超微結構的影響 目的卵母細胞作為人體最大的細胞,細胞結構特點決定了其對低溫和高滲環(huán)境更加敏感,同慢速冷凍一樣,玻璃化冷凍也可能導致卵母細胞超微結構的改變。針對卵母細胞骨架系統(tǒng)中的微絲分布和電鏡下超微結構形態(tài),進一步驗證改良卵母細胞玻璃化冷凍液對體外成熟卵母細胞解凍后形態(tài)和超微結構的影響。 方法收集常規(guī)ICSI患者捐贈的未成熟卵母細胞進行體外成熟培養(yǎng),將體外成熟的卵母細胞用兩種冷凍方案冷凍解凍后進行形態(tài)學和超微結構評價。實驗組分為傳統(tǒng)組(EG/DMSO冷凍法)和改良組(EG/DMSO/PROH冷凍法),對照組為新鮮體外成熟卵母細胞,直接固定后行超微結構觀察。每組卵母細胞均進行共聚焦顯微鏡下微絲分布觀察和透射電鏡下超微結構觀察。 結果倒置鏡下,冷凍兩組均有部分卵母細胞存在周邊空泡狀結構。改良組的空泡發(fā)生率為18.7%,遠遠低于傳統(tǒng)組(66.7%)(P=0.000)。改良組的微絲分布正常率(43.8%)略高于傳統(tǒng)組(33.3%),兩者比較沒有顯著性差異(P0.05)。改良組和傳統(tǒng)組的微絲分布正常率均低于對照組(71.1%)(分別是P0.05和P0.01),透射電鏡結果顯示,無論是傳統(tǒng)組還是改良組,冷凍復蘇后卵母細胞中的空泡狀結構略多于新鮮卵母細胞。而線粒體和空泡的形態(tài)結構與對照組比較沒有明顯差異,冷凍兩組中卵母細胞皮質(zhì)顆粒的數(shù)量略有減少,且更加繞周邊分布,未觀察到明顯的皮質(zhì)顆粒外排現(xiàn)象。結論改良玻璃化冷凍方案較傳統(tǒng)玻璃化冷凍方案可以更加有效的改善卵母細胞解凍后的光鏡下形態(tài),兩種玻璃化冷凍方案都會影響體外成熟卵母細胞的超微結構。 第三部分不同玻璃化冷凍液對卵母細胞發(fā)育潛能的影響及其臨床結局評價 目的評價一種卵母細胞玻璃化冷凍液配方的優(yōu)劣,最直接的證據(jù)是臨床妊娠結果,本研究回顧性分析了使用不同玻璃化冷凍液玻璃化冷凍卵母細胞的實驗室培養(yǎng)數(shù)據(jù)及臨床結局,為改良冷凍液的推廣應用找到臨床證據(jù)。 方法收集2012年～2013年的卵母細胞解凍的所有患者的數(shù)據(jù),包括自卵使用患者和接受供卵的患者。卵母細胞來源于2008年～2013年不同時間冷凍的卵母細胞。卵母細胞冷凍動機以獲卵數(shù)超過22枚者為多,部分因為取卵日取精失敗。解凍周期患者均采用替代周期方案,卵母細胞胞解凍后行黃體支持。記錄冷凍卵母細胞所用的試劑類型(MC組、KT組和改良組)、解凍卵母細胞使用者的年齡、冷凍存活率、受精率、優(yōu)胚率、囊胚形成率、冷凍和移植胚胎數(shù)、臨床妊娠率及著床率。同時計算每解凍卵母細胞獲得的胚胎利用率。 結果自卵使用數(shù)據(jù)中,卵母細胞的冷凍復蘇率以改良組最高(92.0%),顯著高于其他兩組(MC和KT組分別是88.2%和77.3%)(P0.05)。改良組受精卵分裂后獲得的優(yōu)胚率最高(35.8%),與MC和KT組(29.0%和28.3%)相比有顯著性差異(P0.05)。胚胎移植后的臨床妊娠率與著床率各組之間沒有顯著性差異(P0.05),MC、KT和改良配方組臨床妊娠率分別是37.2%,30.2%和39.6%,著床率分別是21.9%、18.8%和27.4%。但改良組每周期胚胎移植個數(shù)是1.89±0.59,顯著低于MC組(2.28±0.83)(P=0.000),也少于KT組(2.02±0.93)(P0.05)。按照每解凍卵母細胞的效率看,改良配方組的卵母細胞授精后獲得的優(yōu)胚率顯著高于MC組和KT組(P0.01)。每解凍卵母細胞獲得的胚胎利用率高于另外兩組,但沒有顯著性差異(P0.05)。在供卵使用數(shù)據(jù)中僅用到了MC試劑和KT試劑,兩組比較,存活率、受精率、優(yōu)胚率、臨床妊娠率及各項卵母細胞利用率,均沒有顯著性差異(P0.05),供卵組的各項數(shù)據(jù)均比自卵組好,臨床妊娠率可以分別達到50%和43.5%。 結論改良冷凍液配方獲得了較商品化試劑更高的存活率和優(yōu)胚率,在移植更少胚胎的同時,沒有降低臨床妊娠率和著床率。相對于價格昂貴保質(zhì)期超長的商品化試劑,改良冷凍液獲得了更多的可利用胚胎,今后將繼續(xù)觀察改良冷凍液的使用效果,同時追蹤冷凍胚胎的使用結果,以獲得更有價值的冷凍卵母細胞累積利用率指標。
[Abstract]:Part 1 the effect of modified vitrified fluid on the developmental potential of mature oocytes after cryopreservation.
Objective To study the unstable effect of vitrified oocytes cryopreservation and to design a vitrified cryoprotectant formula which can keep the osmotic property unchanged and reduce the concentration of various osmotic cryoprotectants. The immature oocytes donated by ICSI patients were used for vitrification. The freezing method was studied to verify the effect of the new modified vitrification solution on the vitrification of mature oocytes in vitro.
Methods Immature oocytes from ICSI patients were collected and cultured in vitro. The oocytes matured in vitro for 18-24 hours were divided into four groups randomly. Three groups were cryopreserved by different methods, namely, group B (EG/DMSO), group C (EG/PROH) and group D (EG/DMSO/PROH). Sperm and embryo culture, group A as control group, fertilization and embryo culture were carried out directly after maturation. The survival rate, fertilization rate, good embryo rate, blastocyst formation rate and high quality blastocyst rate were observed and calculated with each thawed oocyte as denominator.
Results Different vitrification refrigeration formulas had significant effects on the development potential of oocytes matured in vitro. The fertilization rate, the rate of superior embryo and blastocyst formation were significantly lower in group D than in group C (P 0.01). Although the survival rate, the rate of 2PN, the rate of superior embryo, the rate of blastocyst formation and the rate of superior blastocyst formation in group D were higher than those in group B and group C, there was no significant difference in statistical analysis. (P 0.05). The survival rate of group D was the highest (84.8%) and there was no significant difference between group B (77.2%) and group C (80.0%) (P 0.05). The 2PN rates of group D, B and C were 46.7%, 42.4% and 40.0%, respectively. The excellent embryo rates were 5.7%, 2.2% and 2.2%, and blastocyst formation rates were 7.6%, 2.2% and 3.3% (P 0.05). 0.05).
Conclusion Modified vitrification solution can improve the development potential of oocytes matured in vitro and help to obtain more high quality blastocysts.
The second part is the effect of modified vitrification on the morphology and ultrastructure of in vitro maturation oocytes.
Objective As the largest cell in human body, the characteristics of cell structure determine that oocytes are more sensitive to hypothermia and hyperosmotic environment. Like slow freezing, vitrification may also lead to ultrastructural changes of oocytes. Effect of oocyte cryopreservation on morphology and ultrastructure of mature oocytes in vitro after thawing.
Methods Immature oocytes from ICSI patients were collected and cultured in vitro. The oocytes were frozen and thawed in two freezing methods. The experimental group was divided into traditional group (EG / DMSO freezing method) and improved group (EG / DMSO / PROH freezing method). The control group was fresh oocytes matured in vitro. Ultrastructure of oocytes was observed after direct immobilization. Microfilament distribution and ultrastructure of oocytes were observed under confocal microscope and transmission electron microscope.
Results Under inverted microscope, some oocytes in both groups had peripheral vacuoles. The incidence of vacuoles in the improved group was 18.7%, which was much lower than that in the traditional group (66.7%) (P = 0.000). The normal distribution of microfilaments in the modified group (43.8%) was slightly higher than that in the traditional group (33.3%). There was no significant difference between the two groups (P 0.05). The normal cloth rate was lower than that of the control group (71.1%) (P 0.05 and P 0.01). Transmission electron microscopy (TEM) showed that there were slightly more vacuoles in oocytes after cryopreservation than fresh oocytes in both traditional and modified groups. Conclusion Modified vitrification is more effective than traditional vitrification in improving the morphology of oocytes after thawing. Both vitrification schemes can affect the in vitro maturation of oocytes. Ultrastructure.
The third part is the effect of different vitrification refrigerants on the developmental potential of oocytes and the evaluation of their clinical outcomes.
Objective To evaluate the advantages and disadvantages of a vitrified oocyte cryopreservation solution, the most direct evidence is the clinical pregnancy results. This study retrospectively analyzed the laboratory culture data and clinical outcomes of vitrified oocytes frozen with different vitrified cryopreservation solutions.
Methods The data of all patients with oocyte thawing from 2012 to 2013 were collected, including those who used the oocytes and those who received the donated oocytes. Patients were treated with alternative cycles and oocytes were thawed for luteal support. The types of reagents used in cryopreservation (MC, KT and modified groups), the age of the users of thawed oocytes, cryopreservation survival rate, fertilization rate, excellent embryo rate, blastocyst formation rate, number of frozen and transplanted embryos, clinical pregnancy rate and implantation rate were recorded. The utilization rate of embryos obtained from each thawed oocyte.
Results The frozen recovery rate of oocytes in the improved group was the highest (92.0%) and significantly higher than that in the other two groups (88.2% and 77.3% in the MC and KT groups, respectively) (P 0.05). The improved group had the highest rate of excellent embryos (35.8%) after cleavage, which was significantly different from that in the MC and KT groups (29.0% and 28.3%). The clinical pregnancy rates of MC, KT and modified formula groups were 37.2%, 30.2% and 39.6%, 21.9%, 18.8% and 27.4% respectively. However, the number of embryo transfer per week in the modified group was 1.89 (+ 0.59), significantly lower than that in the MC group (2.28 (+ 0.83) (P = 0.000) and less than that in the KT group (2.02 (+ 0.93) (P 0.05)). The efficiency of frozen oocytes was significantly higher in the improved formula group than in the MC and KT groups (P 0.01). The utilization rate of frozen oocytes was higher than that in the other two groups, but there was no significant difference (P 0.05). There was no significant difference (P 0.05) in the rate of good embryo, clinical pregnancy and utilization rate of oocytes. The data of donor group were better than that of self-ovulated group. The clinical pregnancy rate could reach 50% and 43.5% respectively.
Conclusion The improved cryogenic liquid formula has higher survival rate and better embryo rate than commercial reagent, and it does not reduce clinical pregnancy rate and implantation rate while transplanting fewer embryos. The use effect and the use result of frozen embryos were tracked to obtain more valuable index of cumulative utilization of frozen oocytes.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R714.8
本文編號：2229602
[Abstract]:Part 1 the effect of modified vitrified fluid on the developmental potential of mature oocytes after cryopreservation.
Objective To study the unstable effect of vitrified oocytes cryopreservation and to design a vitrified cryoprotectant formula which can keep the osmotic property unchanged and reduce the concentration of various osmotic cryoprotectants. The immature oocytes donated by ICSI patients were used for vitrification. The freezing method was studied to verify the effect of the new modified vitrification solution on the vitrification of mature oocytes in vitro.
Methods Immature oocytes from ICSI patients were collected and cultured in vitro. The oocytes matured in vitro for 18-24 hours were divided into four groups randomly. Three groups were cryopreserved by different methods, namely, group B (EG/DMSO), group C (EG/PROH) and group D (EG/DMSO/PROH). Sperm and embryo culture, group A as control group, fertilization and embryo culture were carried out directly after maturation. The survival rate, fertilization rate, good embryo rate, blastocyst formation rate and high quality blastocyst rate were observed and calculated with each thawed oocyte as denominator.
Results Different vitrification refrigeration formulas had significant effects on the development potential of oocytes matured in vitro. The fertilization rate, the rate of superior embryo and blastocyst formation were significantly lower in group D than in group C (P 0.01). Although the survival rate, the rate of 2PN, the rate of superior embryo, the rate of blastocyst formation and the rate of superior blastocyst formation in group D were higher than those in group B and group C, there was no significant difference in statistical analysis. (P 0.05). The survival rate of group D was the highest (84.8%) and there was no significant difference between group B (77.2%) and group C (80.0%) (P 0.05). The 2PN rates of group D, B and C were 46.7%, 42.4% and 40.0%, respectively. The excellent embryo rates were 5.7%, 2.2% and 2.2%, and blastocyst formation rates were 7.6%, 2.2% and 3.3% (P 0.05). 0.05).
Conclusion Modified vitrification solution can improve the development potential of oocytes matured in vitro and help to obtain more high quality blastocysts.
The second part is the effect of modified vitrification on the morphology and ultrastructure of in vitro maturation oocytes.
Objective As the largest cell in human body, the characteristics of cell structure determine that oocytes are more sensitive to hypothermia and hyperosmotic environment. Like slow freezing, vitrification may also lead to ultrastructural changes of oocytes. Effect of oocyte cryopreservation on morphology and ultrastructure of mature oocytes in vitro after thawing.
Methods Immature oocytes from ICSI patients were collected and cultured in vitro. The oocytes were frozen and thawed in two freezing methods. The experimental group was divided into traditional group (EG / DMSO freezing method) and improved group (EG / DMSO / PROH freezing method). The control group was fresh oocytes matured in vitro. Ultrastructure of oocytes was observed after direct immobilization. Microfilament distribution and ultrastructure of oocytes were observed under confocal microscope and transmission electron microscope.
Results Under inverted microscope, some oocytes in both groups had peripheral vacuoles. The incidence of vacuoles in the improved group was 18.7%, which was much lower than that in the traditional group (66.7%) (P = 0.000). The normal distribution of microfilaments in the modified group (43.8%) was slightly higher than that in the traditional group (33.3%). There was no significant difference between the two groups (P 0.05). The normal cloth rate was lower than that of the control group (71.1%) (P 0.05 and P 0.01). Transmission electron microscopy (TEM) showed that there were slightly more vacuoles in oocytes after cryopreservation than fresh oocytes in both traditional and modified groups. Conclusion Modified vitrification is more effective than traditional vitrification in improving the morphology of oocytes after thawing. Both vitrification schemes can affect the in vitro maturation of oocytes. Ultrastructure.
The third part is the effect of different vitrification refrigerants on the developmental potential of oocytes and the evaluation of their clinical outcomes.
Objective To evaluate the advantages and disadvantages of a vitrified oocyte cryopreservation solution, the most direct evidence is the clinical pregnancy results. This study retrospectively analyzed the laboratory culture data and clinical outcomes of vitrified oocytes frozen with different vitrified cryopreservation solutions.
Methods The data of all patients with oocyte thawing from 2012 to 2013 were collected, including those who used the oocytes and those who received the donated oocytes. Patients were treated with alternative cycles and oocytes were thawed for luteal support. The types of reagents used in cryopreservation (MC, KT and modified groups), the age of the users of thawed oocytes, cryopreservation survival rate, fertilization rate, excellent embryo rate, blastocyst formation rate, number of frozen and transplanted embryos, clinical pregnancy rate and implantation rate were recorded. The utilization rate of embryos obtained from each thawed oocyte.
Results The frozen recovery rate of oocytes in the improved group was the highest (92.0%) and significantly higher than that in the other two groups (88.2% and 77.3% in the MC and KT groups, respectively) (P 0.05). The improved group had the highest rate of excellent embryos (35.8%) after cleavage, which was significantly different from that in the MC and KT groups (29.0% and 28.3%). The clinical pregnancy rates of MC, KT and modified formula groups were 37.2%, 30.2% and 39.6%, 21.9%, 18.8% and 27.4% respectively. However, the number of embryo transfer per week in the modified group was 1.89 (+ 0.59), significantly lower than that in the MC group (2.28 (+ 0.83) (P = 0.000) and less than that in the KT group (2.02 (+ 0.93) (P 0.05)). The efficiency of frozen oocytes was significantly higher in the improved formula group than in the MC and KT groups (P 0.01). The utilization rate of frozen oocytes was higher than that in the other two groups, but there was no significant difference (P 0.05). There was no significant difference (P 0.05) in the rate of good embryo, clinical pregnancy and utilization rate of oocytes. The data of donor group were better than that of self-ovulated group. The clinical pregnancy rate could reach 50% and 43.5% respectively.
Conclusion The improved cryogenic liquid formula has higher survival rate and better embryo rate than commercial reagent, and it does not reduce clinical pregnancy rate and implantation rate while transplanting fewer embryos. The use effect and the use result of frozen embryos were tracked to obtain more valuable index of cumulative utilization of frozen oocytes.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R714.8
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