卵泡刺激素及戊酸雌二醇对小鼠卵母细胞体外成熟的影响

目的:探讨卵泡刺激素(FSH)、戊酸雌二醇(E2)对小鼠未成熟卵母细胞体外成熟、受精及卵裂能力的影响.方法:对雌性小鼠进行超促排卵后处死小鼠,取出卵巢,刺破卵泡,收集形态较好的未成熟卵母细胞,随机分为6组,在不添加激素的基础培养液中以及分别添加10,50 IU/mL的FSH及1,10,100 mg/L的戊酸E2的培养液中培养24h后,观察各组卵母细胞的成熟情况.对已成熟的卵母细胞进行体外受精,观察卵母细胞受精情况.受精后继续培养48～72 h,观察其卵裂情况.比较各组的成熟率、受精率和卵裂率.结果:①各浓度FSH组及各浓度戊酸E2组与对照组相比,成熟率[生发卵泡破裂率(GVBD率)、MⅡ率]及卵裂率的差别均无统计学意义(P＞0.05).②10,50 IU/mL FSH组以及10 mg/L戊酸E2组的受精率分别为41.18％,40％和42.86％,均高于对照组(19.44％),差异有统计学意义(P＜0.05);1,100 mg/L戊酸E2组与对照组相比受精率差异均无统计学意义(P＞0.05).③各浓度FSH组之间以及各浓度戊酸E2组之间成熟率(GVBD率、MⅡ率)、受精率及卵裂率的差别均无统计学意义(P＞0.05).结论:在小鼠体外成熟培养液中添加10,50 IU/mLFSH及10 mg/L戊酸E2能促进小鼠卵母细胞胞质的成熟,从而提高受精率.     

体外培养成熟技术对未成熟卵母细胞冻融的影响

目的:探索影响未成熟卵母细胞冻融技术的关键因素。方法:A组:来源于体外受精-胚胎移植(IVF-ET)周期中自愿捐献的多余的成熟卵母细胞(MⅡ期),共56个;B组:来源于手术切除的卵巢组织中的未成熟卵母细胞(GV或MⅠ),共67个。不同成熟时期卵母细胞经慢冻快融后培养和体外受精,观察其体外成熟、受精及胚胎发育情况。结果:A组与B组相比,两组冻融后的存活率有显著性差异(60.71%vs 77.61%,P<0.05);A组受精率高于B组,但两组比较无显著性差异(61.76%vs 50.00%,P>0.05);A组与B组相比,两组冻融后的卵裂率和优质胚胎率有显著性差异(76.19%vs 37.50%,47.62%vs 12.50%,P<0.05)。A组获2枚囊胚,而B组没有囊胚培养成功。结论:体外成熟培养技术可能是影响冻融后未成熟卵母细胞发育的关键因素。     

体外窦前卵泡添加微量FSH促卵泡发育及卵母细胞成熟效果显著

体内窦前卵泡是非促卵泡成熟激素(FSH)依赖性的,不同的FSH受体(FSH-R)却在此期的颗粒细胞表达;窦状卵泡是FSH依赖性的,窦形成早期FSH促进颗粒细胞增生,激活芳香化酶,诱导颗粒细胞LH-R表达,从而形成卵泡生长必需的微环境。文献显示,添加FSH和LH是体外培养卵母细胞核成熟的重要保证,且维持卵泡发育的重组FSH(rFSH)最小有效剂量是10IU/L。本研究于体外培养窦前卵泡7个不同时间添加rFSH,探索最佳rFSH调控时间域,获取高质量卵泡和成熟卵母细胞。方法:选取14d雌鼠28只、6周雌鼠2只、有生育能力的雄鼠2只。获取4只14d雌鼠卵巢内早…     

胱硫醚β合成酶基因在小鼠卵巢的定位表达分析

目的了解胱硫醚β合成醚(cystathionine-β-synthase,CBS)在卵巢中的定位表达模式,并推测其在卵母细胞发育中生物学功能。方法利用mRNA原位杂交和 RT-PCR的方法检测CBS在成年小鼠卵巢中的表达。结果原位杂交结果显示,CBS在卵泡细胞中广泛表达;在初级卵泡细胞中,CBS阳性杂交信号强于次级卵泡以及窦卵泡的细胞; 在窦卵泡内部,CBS阳性信号既存在于近窦腔的颗粒细胞,也存在于卵丘细胞中。与经过孕母马血清(PMSG)作用的窦卵泡相比,未受PMSG作用的窦卵泡中,CBS阳性杂交信号尤其集中在靠近窦腔的颗粒细胞以及卵丘细胞。而在卵泡中的整个发育过程中的卵母细胞,都未发现有CBS的表达。利用RT-PCR的方法,以培养的各级卵泡进行的表达分析也证实了CBS表达的广泛性和卵母细胞中不表达的现象。结论 CBS参与了卵母细胞发育成熟的整个过程; 其在卵泡细胞中表达而在卵母细胞中不表达的特点说明了CBS是通过卵泡细胞与卵母细胞的相互作用而影响卵母细胞的生长发育的。     

体外窦前卵泡添加微量FSH促卵泡发育及卵母细胞成熟效果显著

体内窦前卵泡是非促卵泡成熟激素(FSH)依赖性的，不同的FSH受体(FSH—R)却在此期的颗粒细胞表达；窦状卵泡是FSH依赖性的，窦形成早期FSH促进颗粒细胞增生，激活芳香化酶，诱导颗粒细胞LH—R表达，从而形成卵泡生长必需的微环境。文献显示，添加FSH和LH是体外培养卵母细胞核成熟的重要保证，且维持卵泡发育的重组FSH(rFSH)最小有效剂量是10IU／L。本研究于体外培养窦前卵泡7个不同时间添加rFSH，探索最佳rFSH调控时间域，获取高质量卵泡和成熟卵母细胞。     

卵巢组织玻璃化冷冻移植效果及体外受精结局的动物实验研究

目的探讨玻璃化冷冻和组织移植对小鼠卵巢组织的影响。方法将雌性ICR小鼠分为3组:正常对照组(卵母细胞体外培养及受精, n=9)、新鲜移植组(卵巢组织移植后卵母细胞体外培养及受精, n=9)、冷冻移植组(卵巢组织玻璃化冷冻复苏-移植、卵母细胞体外培养及受精, n=9)。同时, 为了更为直接地说明玻璃化冷冻对于卵泡数量的影响和卵巢移植后内分泌功能的改变情况, 本研究还设置了冷冻复苏组(卵巢组织玻璃化冷冻复苏, n=6)和卵巢去势组(卵巢组织切除, n=6)。卵巢组织移植3周后, 通过苏木精-伊红(hematoxylin-eosin, HE)染色计算各组小鼠的卵泡数量、CD31免疫组织化学染色观察卵巢组织新生血管形成、Masson染色观察卵巢组织纤维化、酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测血清性激素水平, 并统计获卵数、体外受精数量和形成囊胚数量。结果新鲜移植组和冷冻移植组小鼠的卵巢组织中总卵泡数量均较正常对照组明显减少(均P<0.001), 且都明显少于冷冻复苏组(均P<0.001);新鲜移植组小鼠卵巢组织CD3...     

MIS,IGF-Ⅱ对人卵巢黄素化颗粒细胞分泌功能的影响

目的:探讨MIS,IGF-Ⅱ对人卵巢黄素化颗粒细胞分泌雌激素的影响。方法:体外受精-胚胎移植(IVF-ET)患者经阴道超声引导下取卵,剥离卵母细胞周围的颗粒细胞作体外培养,培养48h后更换无血清培养基,在有或无卵泡刺激激素(FSR)的作用下,分别加入基因重组人苗勒氏管抑制因子(rhMIS)及人胰岛素样生长因子Ⅱ(rhIGF-Ⅱ)作用于颗粒细胞,于培养后第4、6、8、10天收集培养液测定雌二醇(E2),观察rhMIS及rhIGF-Ⅱ对体外培养的颗粒细胞分泌E2的影响。结果:在无FSH作用下,rhMIS对颗粒细胞分泌E2无明显影响(P>0·05),rhIGF-Ⅱ刺激颗粒细胞分泌E2量增加(P<0·05);加入FSH后,rhMIS的明显抑制颗粒细胞E2分泌量(P<0·05),rhIGF-Ⅱ明显增加颗粒细胞E2的分泌(P<0·05)。结论:MIS和IGF-Ⅱ可单独或协同FSH刺激颗粒细胞甾体激素的分泌,对卵泡的生长发育及卵母细胞的分化成熟起着微观调控作用。     

BDNF对小鼠卵母细胞体外成熟及发育能力的影响

目的:观察脑源性神经营养因子(BDNF)对小鼠未成熟卵的体外成熟及发育能力的影响。方法:以α-MEM为基础培养液,添加不同浓度(0、1、5、10 ng/ml)的BDNF以及FSH、FBS培养小鼠未成熟卵,并进行体外受精,观察卵母细胞成熟率、受精率和胚胎发育至囊胚的能力,了解不同培养条件下BDNF对卵母细胞发育能力的影响。结果:当体外成熟培养液中含有FSH和10%的FBS时,与体外成熟对照组比较,BDNF虽然不影响卵母细胞的成熟率和受精率,但BDNF 5 ng/ml组的囊胚形成率(75.00%)显著高于体外成熟对照组(56.63%),而接近体内成熟组(76.92%);当培养液中仅含FBS时,各组间卵母细胞成熟率和受精率没有差异,但与对照组比较,BDNF显著提高囊胚形成率;当培养液中不含FBS、FSH时,虽然无囊胚形成,但BDNF显著提高了卵母细胞的受精率。结论:BDNF能促进小鼠未成熟卵胞质的发育,提高卵母细胞的发育能力。     

MicroRNA调控卵巢颗粒细胞功能的研究进展

正卵巢颗粒细胞在卵泡形成窦腔时,分化为卵丘细胞(cumulus cells, CC)和壁层颗粒细胞(mural granulosa cells,MGC)。CC突起的尖端穿过透明带,终止于卵母细胞膜,从而形成卵丘-卵母细胞复合体。CC通过缝隙连接与卵母细胞进行双向信息传递,进而有助于卵母细胞的成熟、受精与早期胚胎发育[1]。MGC表面存在多种受体,并且能够分泌多种激素和细胞因子,     

去颗粒方法对卵母细胞冻融的影响

目的:探讨去除卵母细胞周围部分颗粒细胞对冻融后卵母细胞发育潜能的影响。方法:将来源于行体外受精-胚胎移植(IVF-ET)患者捐赠的43枚多余成熟卵母细胞(MⅡ期)随机分为A、B两组,在不同条件下进行慢速冷冻法冻融。根据不同的去除卵母细胞周围部分颗粒细胞的方法将卵母细胞分为两组,A组:21枚,透明质酸酶消化法去颗粒;B组:22枚,机械法去颗粒。观察不同的去颗粒方法对冻融后的卵母细胞存活率、受精能力及胚胎早期发育力的影响。结果:A组存活率(52.38%vs 77.27%)、受精率(63.64%vs 70.59%)及卵裂率(57.14%vs 75.00%)均略低于B组,但无显著性差异;两组优质胚胎率(50.00%vs 55.56%)、桑葚胚率(25.00%vs 22.22%)及囊胚率(25.00%vs 11.11%)也均无显著性差异(P>0.05)。结论:不同的去颗粒方法不影响卵母细胞冻融后的发育潜能。     

Effect of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture

ABSTRACT

Information on the role of resistin on steroidogenesis is limited to animal studies. The aim of this study was to investigate the effect of various doses of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture. Granulosa cells were obtained from follicular fluid aspirated from 50 women undergoing in vitro fertilization (IVF) treatment. The cells were cultured for 48 h after a 24 h pre-incubation period. The effect of resistin at dosages 1, 10 and 100 ng/ml alone or in combinations with FSH (10 and 100 ng/ml) on steroidogenesis was investigated. Estradiol and progesterone were measured by radioimmunoassays in culture supernatants at 24 h and 48 h. FSH treatment increased both estradiol and progesterone secretion. Resistin suppressed basal estradiol (at 1 ng/ml) and progesterone secretion (at all concentrations tested). When resistin (all concentrations) was combined with FSH (100 ng/ml), it eliminated the stimulatory effect of FSH on the secretion of estradiol and progesterone. This study indicates an inhibitory effect of resistin on the secretion of estradiol and progesterone by human luteinized granulosa cells in vitro. It is likely that this adipokine locally affects ovarian function in women.

Abbreviations: 3β-HSD: 3β-hydroxysteroid dehydrogenase; CAP1: cyclase-associated protein 1; DCN: decorin; FIZZ: Found in Inflammatory Zones; hCG: human chorionic gonadotropin; IGF1: insulin-like growth factor type 1; IVF: in vitro fertilization; PCOS: polycystic ovary syndrome; RIA: radioimmunoassay; ROR1: receptor tyrosine kinase-like orphan receptor-1; TLR4: Toll–like receptor 4     

hCG注射日孕酮升高对体外受精-胚胎移植的影响

体外受精-胚胎移植（in vitro fertilization-embryo transfer,IVF-ET）过程中,在卵泡晚期人绒毛膜促性腺激素（hCG）注射日孕酮水平升高是否与妊娠结局有相关性,尚无统一的意见。针对这一问题,基于国内外的研究成果围绕hCG注射日孕酮水平升高对IVF结局的影响,首先阐明孕酮主要由卵泡颗粒细胞产生,而促排卵方案中多种因素可导致孕酮水平升高。其次从激素环境、卵母细胞的质量及子宫内膜的容受性方面综述了孕酮升高影响IVF结局的机制。然后,系统阐述了孕酮升高对IVF结局影响的实证研究,并进一步分析孕酮通过改变子宫内膜容受性而最终降低妊娠率。最后,在这些研究基础上,指导临床医生优化促排卵方案或移植方案,尽可能地改善实验室及临床结局,也为下一步研究微刺激/自然周期IVF提供理论依据。     

Ultrastructural localisation of calcium deposits in the mouse ovary

Follicle-enclosed mouse oocytes contain numerous calcium deposits. The ultrastructural distribution of calcium deposits in the nuclei, mitochondria and cytoplasm of mouse oocytes and granulosa cells of primary, secondary and antral follicles was examined using the combined oxalate-pyroantimonate method. The mitochondria of oocytes from all types of follicles had the highest levels of calcium deposits of all oocyte compartments, with the exception of primary follicles, in which oocyte nuclei contained the same level of calcium deposits as the mitochondria. Calcium deposits in the cytoplasm of oocytes from primary follicles were significantly lower than those in the cytoplasm of oocytes from secondary and antral follicles. Calcium deposits in the cytoplasm of granulosa cells were significantly lower than calcium deposits in the mitochondria of granulosa cells and this difference persisted throughout all categories of follicles. Calcium deposits in the nuclei of granulosa cells did not differ from levels in the mitochondria in primary and secondary follicles. In contrast, the nuclei of granulosa cells from antral follicles had lower levels of calcium deposits than the mitochondria. The differences observed in calcium deposits in various cellular compartments in oocytes and granulosa cells in the follicles of ovaries of adult mice can be attributed to their acquisition of meiotic competence and follicular development.     

Oocyte developmental competence is reduced in sows during the seasonal infertility period

The modern domestic sow exhibits a period of impaired reproductive performance during the late summer and early autumn months, known as 'seasonal infertility'. A reduction in farrowing rate due to pregnancy loss is the most economically important manifestation of seasonal infertility. The aim of the present study was to determine whether there are changes in oocyte developmental competence associated with season. Ovaries were collected in pairs from sows sourced from commercial piggeries and slaughtered 4 days after weaning during winter and summer-autumn. Following oocyte IVM and parthenogenetic activation, the ability of oocytes from large follicles to form blastocysts was greater in winter (54.94 ± 6.11%) than in summer (21.09 ± 5.59%). During winter, the proportion of oocytes developing to the blastocyst stage from large follicles was significantly higher (54.94 ± 6.11%) than those oocytes from small follicles (23.17 ± 6.02%). There was no effect of season on the proportion of oocytes developing to the blastocyst stage from small follicles. There was no effect of follicle size on blastocyst formation from those oocytes recovered during summer. Blastocysts derived from small follicles during summer had the lowest number of cells (24.25 ± 1.48) compared with blastocysts derived from large follicles during winter (37.5 ± 1.3; P < 0.05). The mean progesterone concentration in follicular fluid collected from small follicles was greater in winter than summer (1235.55 ± 164.47 v. 701.3 ± 115.5 nmol L(-1), respectively; P < 0.001). The mean progesterone concentration in the follicular fluid of large follicles was also greater in winter than in summer (2470.9 ± 169.1 v. 1469.2 ± 156.5 nmol L(-1), respectively; P < 0.001). Regression analysis revealed a positive correlation between progesterone concentration and oocyte developmental competence. The results indicate that porcine oocytes fail to reach their full developmental potential during the period of seasonal infertility, suggesting that the pregnancy losses observed at this time of year may be due to reduced oocyte developmental competence.     

获卵困难患者补充肌注hCG的临床效果研究

目的 探讨获卵困难发生的危险因素及有效的方案措施.方法 回顾性分析郑州大学第二附属医院生殖中心2016年12月至2021年8月采用卵泡期长效长方案、黄体期长效长方案和短效长方案行体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)助孕的620例患者的临床资料....     

Expression of mRNAs encoding oestrogen receptor (ER) alpha and ERbeta, androgen receptor and progesterone receptor during gonadal and follicular development in the marsupial brushtail possum (Trichosurus vulpecula)

The objective of the present study was to determine which ovarian cells express mRNAs for oestrogen (ERalpha and ERbeta), androgen (AR) and progesterone (PR) receptors during ovarian and follicular development in the brushtail possum. Expression of ERalpha and/or ERbeta mRNA was observed from birth, initially in cells of the blastema, then in the medullary cords from Day 20. ERalpha was expressed in the oocytes and granulosa cells of secondary and antral follicles. Preovulatory follicles did not express ERalpha mRNA, although their oocytes were not examined for any gene. ERbeta mRNA was observed in oocytes at all follicular stages examined, but was not consistently observed in granulosa or theca cells. Expression of AR mRNA before Day 40 was very faint; thereafter, expression was observed in the medullary cords, peaking between Days 60 and 120. Oocytes, granulosa cells and theca of secondary and antral, but not preovulatory, follicles expressed AR mRNA. PR mRNA was expressed throughout the gonad by Day 20. Granulosa cells of some secondary and antral follicles and theca of antral follicles expressed PR mRNA. Thus, the expression of mRNAs encoding steroidogenic receptors in a time- and cell-specific manner supports a role for steroids in the process of ovarian follicular formation and growth.     

两种长效促性腺激素释放激素激动剂在早卵泡期超长方案中助孕结局比较

目的:比较两种长效促性腺激素释放激素激动剂（gonadotropin-releasing hormone agonist,GnRH-a）在早卵泡期超长方案中对体外受精/卵胞质内单精子注射（ in vitro fertilization/intracytoplasmic sperm injection,IVF...     

地塞米松对PCOS患者超促排卵中血清E2水平影响的临床研究

目的:探讨地塞米松对多囊卵巢综合征(PCOS)患者体外受精-胚胎移植(IVF-ET)周期控制性超促排卵(COH)中血清雌二醇(E2)水平的影响。方法:对26例首次行IVF-ET(A组)但未妊娠、COH中血清促黄体生成素(LH)水平处于1.2 IU/L以上而E2水平低的PCOS患者,再次行IVF-ET(B组)的COH中,于启动促性腺激素(Gn)时给予地塞米松1.5mg/d,口服至取卵日。比较两组患者人绒毛膜促性腺激素(HCG)日血清E2水平及IVF-ET结局参数的变化。结果:①B组HCG日E2水平和E2/直径14 mm以上卵泡数的比值均显著高于A组(P<0.05);②B组26例患者有11例(42.31%,11/26)临床妊娠,A组无1例妊娠;③两组获卵数、成熟卵子数、受精率、卵裂率差异均无统计学意义(P>0.05);④B组所有患者均能耐受,无不良反应。结论:地塞米松可以改善PC0S患者COH中LH水平处于1.2 IU/L以上而E2水平低的状态,进而提高IVF-ET妊娠率。     

高孕激素状态下促排卵治疗的卵泡晚期应用低剂量人绒毛膜促性腺激素对多囊卵巢综合征患者体外受精结局的影响

目的:探讨在高孕激素状态下促排卵治疗（progestin-primed ovarian stimulation,PPOS）的卵泡晚期应用低剂量的人绒毛膜促性腺激素（human chorionic gonadotropin, hCG）替代人绝经期促性腺激素（human menopausal gonadotropin,hM...     

添加两种不同培养成分对人未成熟卵体外成熟培养的影响

目的:研究添加两种不同培养成分对人未成熟卵母细胞体外成熟培养(IVM)的效果。方法:选择接受ICSI治疗的77名不孕患者共收集未成熟卵177枚,随机分A组(激素组)33例,添加0.075 IU/ml FSH及0.075 IU/ml LH至基础IVM培养液中;B组(成熟卵泡液组)44例,50%基础IVM培养液与50%人成熟卵泡液配成。将两组收集的未成熟卵母细胞放入不同培养液中培养48 h,每24 h倒置显微镜下观察卵母细胞的形态;对MII卵进行ICSI,ICSI操作后继续培养,ICSI后16～20 h进行受精观察,24及48 h分别进行卵裂观察及胚胎评分。结果:①B组未成熟卵母细胞48 h成熟率和MI期卵母细胞48 h成熟率显著高于A组,差异有统计学意义(P<0.05),两组培养液GV期卵母细胞24和48 h成熟率比较差异无统计学意义(P>0.05)。②B组2PN卵裂率显著高于A组,差异有统计学意义(P<0.05),两组2PN受精率及可利用胚胎率差异无统计学意义(P>0.05)。结论:人成熟卵泡液内可能含有一些未知的有利于卵母细胞成熟的因子,深入研究卵泡液的成分,进一步明确其促卵母细胞成熟机制对开发新的IVM培养液、改善IVM的临床结局有重要意义。