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1、Declining ovarian reserve adversely influences granulosa cell viability,卵巢储备能力的下降对颗粒细胞活性的不良影响,引言 实验过程 统计学分析 结论 讨论,Diminished ovarian reserve(DOR)has been shown to be associated with a poor response to ovarian stimulation,lower number of oocytes retrieved during IVF cycles,lower pregnancy rates after
2、 ART cycles,a greater likelihood of cycle cancelation,as well as higher miscarriage and aneuploidy rates. 已经证明：卵巢储备功能下降与促卵巢发育的不良应答有关。表现为：IVF过程中卵母细胞恢复的数量的降低，ART过程中低怀孕率，周期消失的可能性增加，出现流产和胎儿染色体异常的几率增加。,引言,Low ovarian reserve is associated with abnormalities of folliculogenesis,ovulation and oocyte struct
3、ure,and performance. 并且，低的卵巢储备功能与滤泡形成、排卵、卵母细胞的结构与功能的异常都有关系。,A close anatomic and functional relationship exists between somatic and germ cells of the ovarian follicle.A bidirectional communication between these two cell types is imperative for reproductive physiology. 卵巢滤泡的体细胞和生殖细胞在解剖上和功能上有密切联系。从生殖生
4、理学上来说，这两种细胞的相互协调依赖的双向作用是极其必要和重要的。,The granulosa cells(GC) of women with DOR have been shown to have decreased steroidogenic potential,decreased proliferation, and increased apoptotic changes.The genesis of these defects in the granulosa cell is unclear. 已经证明，卵巢储备功能下降的妇女，他们的颗粒细胞具有以下缺陷：类固醇合成可能性的降低；增生率
5、的降低；细胞凋亡改变的几率的增加。但是，造成颗粒细胞这些缺陷的起源却不清楚。,Moreover,GCs are a heterogeneous population of cells,consisting of two distinct phenotypes. 此外，颗粒细胞是一种异质细胞群，包括两种不同的表型：卵丘颗粒细胞(cumulus GCs)和壁颗粒细胞（mural GCs）,The “cumulus“GCs,which lie in proximity to the oocyte and are intimately involved in the modulation of the
6、 milieu surrounding the developing oocyte and the process of ovulation, exhibit a higher rate of proliferation,lower steroidigenic capacity,and minimal expression of LH receptors compared with the peripheral “mural“GCs. 围绕在卵母细胞周围的卵丘颗粒细胞与周围环境的调节以及排卵过程有密切关系，与外围的壁颗粒细胞相比，显示出更高的增生率，低的类固醇合成能量和极低的促黄体生成素受体的
7、表达水平。,Owing to the proximate relationship between the GCs and the oocytes within developing ovarian follicles,impaired GC function is likely to adversely influence oocyte physiology and thus potentially the reproductive capacity of the respective oocyte. 在卵泡发育过程中，由于颗粒细胞与卵母细胞之间存在密切联系，颗粒细胞功能的受损可能对卵母细胞
8、生理和单个卵母细胞繁殖能力造成不良影响。,The present study was undertaken to test the hypothesis that GCs,because of their close contact with the oocyte,would demonstrate lower cell counts and viability in association with declining ovarian reserve,as reflected by historical maximum basal FSH levels.We furthermore aime
9、d to assess the influence of ovarian reserve on each GC subtype,i.e.,mural and cumulus cells. 我们的研究想证实这个假说：由于颗粒细胞与卵母细胞的密切联系，细胞数量降低和活性降低与卵巢储备功能下降有关，这些可从基础促卵泡素值的历史最大值反应出来。此外，我们的目的在于估测卵巢储备功能对颗粒细胞的每一种亚型（如：卵丘颗粒细胞和壁颗粒细胞）的影响。,MATERIALS AND METHODS实验过程,Patients 针对患者的实验步骤 Mural GC Isolation 提取分离壁颗粒细胞 Cumulus
10、 GC Isolation 提取分离卵丘颗粒细胞,Patients,Infertile women undergoing IVF at the Montefiore Institute for Reproductive Medicine and Health between November 1, 2006, and March 31, 2007, (n =24) were prospectively enrolled in this study of GC parameters (cell count and viability) and ovarian reserve status as
11、reflected by the historical maximum early follicular-phase serum FSH level obtained on cycle day 1-3 for each patient. 2006年11月1日-2007年3月31日在Montefiore生殖医学健康研究所接受IVF的24名不孕妇女接受了GC参数（细胞数与细胞活性）实验，卵巢储备状态通过滤泡早期血清FSH水平的历史最大值测出，选择在每位患者周期的1-3天来测值。,This study was reviewed and approved by the Institutional Re
12、view Board at Montefiore Medical Center, and an informed written consent was obtained from each of the participants. 此研究由Montefiore医学中心研究评审小组批准与评审，并且同每一名参与者（实验对象）都签订了同意书。,The GCs were prospectively collected from 24 patients undergoing IVF; both mural and cumulus GCs were isolated from 22 patients,
13、and cumulus GCs only were collected from 2 patients. 如预期所做：我们从接受IVF的24名患者中收集到GC，其中，从22名患者体中分别分离出卵丘颗粒细胞和壁颗粒细胞，另外两名患者只收集了卵丘颗粒细胞。,Suppression of spontaneous ovulation during COH was achieved using a GnRH agonist in 19 of 24 cycles (lupron; TAP Pharmaceutical Products, Lake Forest, IL) and GnRH antagoni
14、st in 5 of 24 cycles (ganirelix acetate; Organon,Roseland, NJ).Controlled ovarian hyperstimulation (COH) was achieved using recombinant FSH and/or hMG. 针对控制性超速排卵（COH）过程中需求自发排卵的抑制，19名患者的周期采用促性腺激素释放激素兴奋剂（GnRHa）,另外5名采用促性腺激素释放激素的拮抗剂。联合运用FSH和/或hMG（尿促性素）进行控制性超速排卵。,Serial assessments of follicular response
15、 were performed by transvaginal sonogram (TVS) (Aloka 1400, 7.5 MHz, Phillips)and measurement of serum Estradiol (E2) levels. The TVS-guided retrieval of oocytes was performed 34 h after hCG administration。 我们用阴道超声（TVS）来完成对滤泡反应的一系列评估和血清雌二醇水平的测量（TVS：飞利浦牌Aloka1400型号，7.5赫兹）。在服用绒促34小时后导入TVS提取卵母细胞。,Mural
16、 GC Isolation,Follicular fluid aspirates from follicles 14 mm average diameter were collected from all patients in an effort to collect GCs from preovulatory follicles. The first follicle aspirate from each ovary was excluded to eliminate the potential for contamination with vaginal epithelial cells
17、. 从平均直径14 mm的滤泡中抽出滤泡液，以收集排卵前卵泡的颗粒细胞。这些滤泡是从24名患者中收集到的。从每个卵巢提取的第一个卵泡都要清除，以防阴道上皮细胞的污染。,Oocytes were identified and transfered into modified human tubal fluid (mHTF; Conception Technology, San Diego, CA) and 5% plasmanate (Bayer Corporation, Elkhart, IN). Follicular fluid aspirates from each patient were pooled. 卵母细胞析出后，将其转移到试剂人输卵管液（mHTF）里(mHTF由美国圣地亚哥执业资格认证中心认证的生殖技术研究所生产）并且加入5%的人血浆蛋白制剂（此剂由美国埃尔德哈特市的拜耳公司生产）从每个患者提取的卵泡液收集到一起。,
