Improvement of in vitro Cytoplasmic Maturation of Denuded Porcine Oocytes by Coculture with Follicular Mural Granulosa Cells

[Objective] This study aimed to improve the in vitro maturation quality of denuded porcine oocytes and provide scientific basis for establishing a stable and efficient denuded oocyte culture system. [Method] The first polar body extrusion rate, oocyte glutathione （GSH） content, positive rate of brilliant cresyl blue （BCB） staining and development potential of activated oocytes or fertilized oocytes were employed as main indicators to investigate the effects of follicular mural granulosa cell （MGC） coculture on cytoplasmic maturation of cumulus cell-removal oocytes （Denuded Oocyte, DO）. [Result] According to in vitro maturation results, compared with DO group, the first polar body extrusion rate of porcine oocytes in DO＋MGC group was not significantly different, but the nuclear maturation process was improved and was more similar to that in COC （cumulus-oocyte complex） group. Detection of GSH content in mature oocytes showed that there was no significant difference between DO＋ MGC group （optical density of 1 053.67） and COC group （optical density of 1 426.00） or between DO＋MGC group and COC＋GC group （optical density of 1 541.00）, however, GSH content in mature oocytes of DO group （optical density of 724.67） was significantly lower than that of COC group and COC＋GC group （P0.05）. Detection of glucose-6-phosphate dehydrogenase （G6PDH） activity showed that there was no significant difference in BCB positive oocyte rate between DO ＋MGC group （88.26% ） and COC group （92.75%） or between DO＋MGC group and DO group （82.86% ）, however, BCB positive oocyte rate of DO group was significantly lower than that of COC group （P0.05）. Furthermore, the cleavage rate and blastocyst rate of activated mature oocytes derived from DO ＋MGC group （94.98% and 43.67% , respectively） were significantly higher than those from DO group （52.54% and 8.97%, respectively） （P0.05）, and were not significantly different compared with those from COC group （97.11% and 38.30%, respectively）. In addition, the cleavage rate of fertilized oocytes derived from DO＋MGC group （72.65%） showed no significant difference compared with that from DO group （63.59%）, but the blastocyst rate of DO＋MGC group was significantly higher than that of DO group （9.88%） （P0.05）. [Conclusion] MGC coculture can significantly improve the in vitro cytoplasmic maturation quality of denuded porcine oocytes, thereby enhancing the subsequent developmental potential.

Effects of Electroacupuncture on Expression of PI3K/Akt/Foxo3a in Granulosa Cells from Women with Shen(Kidney) Deficiency Syndrome Undergoing in vitro Fertilization-Embryo Transfer

Objective: To observe the effects of electroacupuncture(EA) on reproductive outcomes in women with Shen(Kidndy) deficiency syndrome after in vitro fertilization-embryo transfer(IVF-ET),and explore the underlying molecular mechanism.Methods: Sixty-six infertile patients with Shen de?ciency syndrome undergoing IVF-ET were divided into EA or control groups according to a random table,33 cases in each group.Before undergoing IVF,patients in the EA and control groups received EA therapy and placebo needle puncture,respectively,for 3 menstrual cycles.Shen de?ciency syndrome scores were assessed.Other outcome measures included the number of retrieved oocytes and fertilization,high-quality embryo and clinical pregnancy rates.Follicular ?uid was collected on the day of oocyte retrieval,and granulosa cell expression of phosphatidylinositide3-kinases(PI3 K),serine-threonine kinase(Akt) and forkhead box O3(Foxo3 a) m RNA were measured by reverse transcribed and quantitative real-time polymerase chain reaction.Results: Syndrome scores for pre-versus post-treatments decreased significantly(16.53±1.75 to 8.67±1.61) in the EA group(P<0.05),but showed no signi?cant change in the control group(17.18±1.58 to 14.74±1.58).A signi?cant difference in score change was found between the EA and control groups(P<0.05).High-quality embryo and clinical pregnancy rates were both increased in the EA group compared with the control group [69.15%(195/282) vs.60.27%(176/292) and 66.67%(22/33) vs.42.42%(14/33),respectively,P<0.05].The fertilization rate was equivalent in EA and control groups.No difference was found in the number of retrieved oocytes between the two groups.Granulosa cell expression levels of PI3 K and Akt m RNA were signi?cantly increased in the EA group compared with the control group,while the expression of Foxo3 a was reduced(all P<0.05).Conclusions: For infertile patients with Shen de?ciency syndrome undergoing IVF,EA for tonifying Shen as an adjunct treatment may alleviate clinical symptoms and improve the high-quality embryo rate.The EA-induced mechanism may involve regulation of PI3 K/Akt/Foxo3 a expression in granulosa cells to improve the developmental microenvironment of oocytes and inhibit granulosa cell apoptosis,possibly contributing to the improved clinical pregnancy rate(Registration No.Chi CTR 1800016217).

猪裸卵体外胞质成熟研究

[目的]改善猪裸卵的体外成熟质量,为建立稳定有效的裸卵培养体系提供科学依据。[方法]以第一极体排出比例、卵母细胞谷胱甘肽含量、亮甲酚蓝阳性率以及早期胚胎发育潜能等为主要衡量指标研究了卵泡壁颗粒细胞共培养对猪裸卵胞质成熟的影响。[结果]体外成熟结果表明,相对于DO组,DO+MGC组卵母细胞排除第一极体的比例无显著差异,但核成熟的进程得到了改善,更接近于COC组;成熟卵母细胞GSH含量检测结果表明,DO+MGC组平均每个卵母细胞谷胱甘肽含量(光密度1 053.67)与COC组(1 426.00)和COC+GC组(1 541.00)差异不显著,但DO组(724.67)显著低于COC组和COC+GC组(P<0.05);G6PDH活性检测结果表明,DO+MGC组BCB阳性卵母细胞比例(88.26%)与COC组(92.75%)以及DO组(82.86%)差异均不显著,但DO组显著低于COC组(P<0.05);成熟卵母细胞孤雌激活后的发育结果表明,DO+MGC组卵裂率(94.98%)和囊胚率(43.67%)显著高于DO组卵裂率(52.54%)和囊胚率(8.97%),与COC组卵裂率(97.11%)和囊胚率(38.30%)差异不显著(P<0.05);成熟卵母细胞体外受精后的发育结果表明,DO+MGC组卵裂率(72.65%)与DO组卵裂率(63.59%)无显著差异,但DO+MGC组囊胚率(17.79%)显著高于DO组(9.88%)(P<0.05)。[结论]卵泡壁颗粒细胞共培养可以显著改善裸卵体外成熟的胞质成熟质量并进而提高后续发育潜力。

人卵丘颗粒细胞和壁层颗粒细胞体外培养方法比较

目的:建立有效分离、提纯及培养人卵丘颗粒细胞的方法,并与人壁层颗粒细胞的体外培养相比较。方法:收集卵胞浆内单精子显微注射取卵时的卵泡液和直接机械分离卵母细胞所得的卵丘颗粒细胞复合物,直接将卵丘颗粒细胞复合物接种于培养皿中培养。用密度梯度离心法分离卵泡液中的人壁层颗粒细胞。用免疫荧光法检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)的表达;用CCK-8法检测细胞生长曲线;用ELISA检测这2种颗粒细胞分泌雌激素的能力。结果:直接法所得人卵丘颗粒细胞培养24 h后可贴壁,体外培养生长状态与人壁层颗粒细胞相似;免疫荧光检测显示两者均表达FSHR;CCK-8实验结果表明,两者体外培养生长曲线相似;ELISA结果显示两者分泌雌激素能力相当。结论:利用机械切割获得人卵子周围卵丘颗粒细胞复合物直接培养的方法操作简单,获得的人卵丘颗粒细胞具有与人壁层颗粒细胞相似的生长状态、生长曲线以及雌激素分泌能力,可作为人颗粒细胞亚群体外培养方法的补充。

体外受精-胚胎移植术联合补肾、疏肝对不孕症患者活化素受体样激酶5的影响

目的观察体外受精—胚胎移植术(IVF-ET)分别联合补肾调经方、逍遥散方治疗不孕症的临床疗效及可能机制。方法 40例不孕症患者辨证分为补肾组、疏肝组各20例,另设单纯控制性超排卵20例为对照组。各组均接受IVF-ET治疗,补肾组同时服用补肾调经方,疏肝组同时服用逍遥散方。治疗后检测各组患者卵巢颗粒细胞中活化素受体样激酶5(ALK5)mRNA的表达,观察患者获卵数、受精率、优质胚胎率和临床妊娠率。结果对照组优质胚胎率、临床妊娠率分别为29.3%、45.0%,补肾组分别为50.3%、65.0%,疏肝组分别为50.6%、60.0%,补肾组、疏肝组优质胚胎率均高于对照组(P<0.05),各组临床妊娠率以及获卵数、受精率比较差异均无统计学意义(P>0.05)。补肾组、疏肝组ALK5 mRNA表达均高于对照组(P<0.05)。结论补肾调经方、逍遥散方均可以上调不孕症患者颗粒细胞膜受体ALK5 mRNA的表达,促进颗粒细胞的增殖,从而调节卵巢功能而治疗不孕症。