In vitro culture and in vitro maturation of mouse preantral follicles with recombinant gonadotropins

OBJECTIVE: To develop an effective method for in vitro maturation of preantral follicles isolated from mice ovarian tissue. DESIGN: Isolated preantral follicles were randomly allocated to designed experimental groups for study. SETTING: University-based research lab. PATIENT(S): Healthy, normal mice. INTERVENTION(S): Superovulation with pregnant mare serum gonadotropin and hCG. MAIN OUTCOME MEASURE(S): Morphological changes and E(2) production were assessed. RESULT(S): To obtain competent oocytes, preantral follicles must be cultured with medium containing insulin and recombinant gonadotropins (i.e., recombinant FSH and recombinant LH), with a change of medium daily. A high initial recombinant LH or recombinant FSH facilitates E(2) secretion, enhances granulosa cell outgrowth, and has earlier antral formation. However, prolonged culture in high-recombinant LH or recombinant FSH triggers early differentiation and luteinization of granulosa cells, which results in low metaphase II oocyte and blastocyst formation. CONCLUSION(S): We have developed a culture system that allows the successful maturation of preantral follicles in vitro. The matured follicles are a physiologically functional unit that not only secrete E(2) but also generate competent oocytes. In a special condition, 90% of the cultured follicles survived, 53.5% of them produced MII oocytes, and 50% of the derived MII oocytes were fertilized and reached the blastocyst stage after culture in vitro.     

Triiodothyronine receptors in porcine granulosa cells

Triiodothyronine has been found to enhance gonadotropin- and insulin-stimulated morphologic luteinization and progesterone production by porcine granulosa cells in culture. The role of triiodothyronine in the above events is not well defined. One possibility is that triiodothyronine effects are direct and are receptor mediated. To test this concept, we undertook this study to investigate the presence of triiodothyronine receptors in granulosa cells aspirated from large (6 to 12 mm), medium (3 to 5 mm), and small (1 to 2 mm) porcine ovarian follicles. Crude nuclei were isolated and tested for iodine 125-triiodothyronine specific binding. Binding was time and temperature dependent and maximal in cells from small follicles at pH 8. Scatchard analysis yielded a mean apparent dissociation constant of 5.5 X 10(-9) mol/L and a mean apparent total number of binding sites of 1.0 pmol/mg of deoxyribonucleic acid. Competition experiments revealed the following relative binding affinities: triiodothyronine greater than L-thyroxine greater than reverse triiodothyronine. Gonadotropins, prostaglandins, epidermal growth factor, and insulin did not compete with 125I-triiodothyronine for binding. We conclude that there are nuclear binding sites in porcine granulosa cells with characteristics expected of a triiodothyronine receptor which might mediate a direct action of triiodothyronine on granulosa cells.     

Effects of androgen on 17 beta-estradiol and progesterone secretion by cultured human granulosa cells

Human granulosa-luteal cells obtained from dominant preovulatory follicles at the time of oocyte retrieval for in vitro fertilization (about 10 h after the peak of the endogenous LH surge) produced large quantities of progesterone (P) (8.7 pg/cell/first 2 days) in a monolayer culture. Although basal production of estradiol (E2) in these granulosa-luteal cells was restricted to small quantities, a marked increase in E2 production occurred in the presence of testosterone (T) (10(-6) M) as aromatizable substrate. The non-aromatizable androgen, 5 alpha-dihydrotestosterone (DHT) (10(-7) M, 10(-6) M) slightly enhanced E2 production in these granulosa-luteal cells and it did not inhibit T aromatization. In contrast, DHT did not increase E2 production in cultured granulosa cells obtained from follicles 2 approximately 5 days before the LH surge. Thus, the response of granulosa cells to exogenous androgen seems to vary at different stages of cell differentiation. Neither T nor DHT elicited significant changes in the production of P by granulosa-luteal cells obtained from preovulatory follicles during the LH surge. However, T (10(-6) M) suppressed hCG-stimulated P production in these cells. The results suggest that androgen enhances the estrogen biosynthesis of granulosa-luteal cells obtained from preovulatory follicles during the LH surge not only by acting as substrate for aromatization but also by participating in some process of estrogen synthesis. Further, it is suggested that androgen has a negative effect on P biosynthesis in these cells.     

胰岛素对子宫内膜间质细胞分泌IGF-1和IGFBP-1的影响

目的 :了解胰岛素对子宫内膜间质细胞分泌胰岛素样生长因子 1(IGF 1)和胰岛素样生长因子结合蛋白 1(IGFBP 1)的影响。方法 :用含不同浓度雌二醇和胰岛素的培养液培养增生晚期人子宫内膜间质细胞 2 4h后 ,留取培养液测定IGF 1。用含不同浓度孕酮和胰岛素的培养液培养分泌晚期人子宫内膜间质细胞 2 4h后 ,留取培养液测定IGFBP 1。结果 :1.增生晚期间质细胞培养液中IGF 1的浓度 :2 0 μU ml胰岛素组为 1.39±0 .33μg L ,低于 10 0 μU ml胰岛素组 (2 .0 3± 0 .5 3μg L) (P <0 .0 1) ;10 0ng L雌二醇 + 2 0 μU ml胰岛素组为 2 .18± 0 .36 μg L ,低于 10 0g L雌二醇 + 10 0 μU ml胰岛素组 (3.4 2± 0 .75 μg L) (P<0 .0 1)。 2 .分泌晚期间质细胞液中IGFBP 1浓度 :2 0 μU ml胰岛素组为 0 .16± 0 .5 8μg L ,低于对照组 (P <0 .0 1) ,高于 10 0 μU ml胰岛素组 (0 .10± 0 .4 8μg L) (P <0 .0 1) ;10 μg L孕酮 + 2 0 μU ml胰岛素组为 2 .10± 1.17μg L ,低于 10 μg L孕酮组 (P <0 .0 1) ,高于 10 μg L孕酮 + 10 0 μU ml胰岛素组 (0 .5 0± 0 .2 8μg L) (P <0 .0 1)。 结论 :1.胰岛素促进增生期子宫内膜间质细胞分泌IGF 1;2 .胰岛素抑制分泌晚期子宫内膜间质细胞分泌IGFBP 1。     

DNA ploidy of human granulosa cells from natural and stimulated in vitro fertilization cycles

OBJECTIVE: To analyze and compare the DNA ploidy of granulosa cells from natural and gonadotropin-stimulated follicles obtained during IVF. DESIGN: Retrospective analysis of laboratory data. SETTING: University medical center. PATIENT(S): Seventy-three aspirates of dominant follicles from natural IVF cycles and 113 aspirates from gonadotropin-stimulated cycles were analyzed. INTERVENTION(S): Cytospins were prepared and stained by the Feulgen-thionine method. MAIN OUTCOME MEASURE(S): Image DNA analysis was performed on an automated high-resolution image cytometer. DNA content and the number of nuclei with DNA content >5c were measured. RESULT(S): All samples from natural and gonadotropin-stimulated follicles were found to be diploid. Single cells with DNA content >5c were found in follicular fluid samples of four women with natural IVF cycles and in samples of nine women with gonadotropin-stimulated cycles. CONCLUSION(S): DNA ploidy of granulosa cells from natural follicles has not been studied before. In natural samples, granulosa cells were only diploid, without euploid polyploidization. We were unable to confirm DNA aneuploidy of granulosa cells in gonadotropin-stimulated follicles of women undergoing IVF.     

Apoptosis and active caspase-3 expression in human granulosa cells

OBJECTIVE: To document the expression of activated forms of caspase-3 in human granulosa cells. DESIGN: Laboratory study. SETTING: In vitro fertilization (IVF) laboratory of the Split University Hospital and laboratory of the Department of Anatomy, Histology, and Embryology. PATIENT(S): Ovarian tissues were obtained from women undergoing hysterectomy/ovariectomy for benign conditions and human granulosa cells were obtained from women undergoing oocyte retrieval for IVF. INTERVENTION(S): Immunostaining of tissue sections and cell smears using antibody to active caspase-3 and terminal deoxynucleotidyl transferase (TdT) assay (TUNEL) for detection of internucleosomal DNA fragmentation. MAIN OUTCOME MEASURE(S): Microscopic evaluation to assess the presence and cellular co-localization of active caspase-3 and TUNEL-positive cells. RESULT(S): In human ovarian tissue, no apoptosis was observed in primordial and primary follicles. Apoptosis in granulosa cells was detected only in atretic antral follicles. Granulosa cells classified as apoptotic on the basis of their morphologic features contained a single condensed nucleus, multiple nuclear fragments, or apoptotic bodies. All apoptotic granulosa cells expressed active caspase-3, but only few contained fragmented DNA detected with the TUNEL method. The expression of active caspase-3 was also demonstrated in human granulosa cells of preovulatory follicles obtained from patients undergoing IVF. CONCLUSION(S): Caspase-3 dependent apoptosis occurs in human granulosa cells and activates when follicles begin to leave the resting pool. After initial formation of the antrum, activation of caspase-3 is a normal physiologic process of the follicle during atresia and luteinization. Higher numbers of granulosa cells positive with caspase-3 than cells positive with TUNEL suggest an earlier activation of caspase-3 compared with the DNA fragmentation detected by TUNEL assay and also a longer detection period of caspase-3 than DNA fragmentation in apoptotic granulosa cells.     

Increased endogenous level of melatonin in preovulatory human follicles does not directly influence progesterone production

OBJECTIVE: To study the role of follicular melatonin on steroid production by human preovulatory follicles. DESIGN: In vivo comparative and in vitro culture studies. SETTING: University hospital. PATIENT(S): Thirty-six patients with tubal and/or male, but not endocrinological, infertility factors. INTERVENTION(S): Follicular fluid collection during IVF. In vitro granulosa cell culture from luteinizing or growing Graafian follicles. MAIN OUTCOME MEASURE(S): Follicular melatonin, P, T, and E(2) concentrations. In vitro P production by granulosa cells. RESULT(S): There was a positive correlation between follicular melatonin and P concentrations. Melatonin did not stimulate in vitro P production by granulosa cells from luteinizing or growing follicles. Melatonin, P, and E(2) concentrations were significantly higher, but T concentrations were lower, in large follicles than in small follicles. CONCLUSION(S): Preovulatory follicles contain a high amount of melatonin compared with that in small immature follicles; melatonin may play an important, but indirect, role in P production by human granulosa cells.     

雌孕激素对体外卵泡黄素化颗粒细胞分泌巨噬细胞集落刺激因子的影响

目的　探讨雌、孕激素对卵泡黄素化颗粒细胞 (颗粒细胞 )分泌巨噬细胞集落刺激因子(M CSF)的调节作用。方法　抽取接受卵母细胞单精子显微注射 (ICSI)的 12例妇女的颗粒细胞 ,在不同浓度 ( 0、1× 10 - 7、1× 10 - 6 、1× 10 - 5 、1× 10 - 4、1× 10 - 3 mol L)的雌二醇或孕酮作用下培养 72h ,采用酶联免疫吸附法测定颗粒细胞培养液中的M CSF含量。结果　颗粒细胞在雌二醇或孕酮为 0mol L(即无雌二醇或孕酮作用———基础状态 )的情况下培养 72h后 ,分泌一定量的雌二醇 [( 1 0 2± 0 2 3 )×10 - 7mol L]、孕酮 [( 1 0 0± 0 12 )× 10 - 5 mol L]和少量的M CSF[( 4 7± 15)ng L] ;在 1× 10 - 6 ～ 1× 10 - 3mol L浓度的雌二醇作用下 ,M CSF分泌增加 ,其分泌随雌二醇浓度的增加而增加 ,较基础状态分别增加 2 3、4 5、6 9和 7 9倍 ;在≥ 1× 10 - 6 mol L各浓度的雌二醇作用下 ,M CSF分泌与基础状态比较 ,差异均有显著性 (P均 <0 0 5) ;在 1× 10 - 7mol L浓度的雌二醇作用下 ,M CSF分泌与基础状态比较 ,差异无显著性 (P >0 0 5) ;在 1× 10 - 7～ 1× 10 - 3 mol L浓度的孕酮作用下 ,M CSF分泌与基础状态比较 ,差异均无显著性 (P >0 0 5)。结论　雌二醇能促进颗粒细胞M CSF的分泌 ,孕酮对颗     

Dynamic medium produces caprine embryo from preantral follicles grown in vitro

The aim of this study was to develop a dynamic culture medium containing FSH, LH and EGF to promote the in vitro development of oocytes obtained from goat preantral follicles to complete maturation and to improve the capacity of these oocytes for in vitro fertilization (IVF) and embryo production. For experiment I, preantral follicles were cultured for 18 days in medium supplemented with increasing concentrations of FSH (T1 - control) or in control medium added LH alone or in association with EGF: T2 (LH 50 ng/ml), T3 (LH 50 ng/ml + EGF 50 ng/ml), T4 (LH 50 ng/ml + EGF 100 ng/ml), T5 (LH 100 ng/ml), T6 (LH 100 ng/ml + EGF 50 ng/ml) and T7 (LH 100 ng/ml + EGF 100 ng/ml). For experiment II, preantral follicles were cultured only in the culture medium used in T7, and after 18 days, their oocytes underwent in vitro maturation (IVM) followed by IVF. At the end of the culture period, T3, T4 and T7 had a positive influence on the daily follicular growth rate. Oocytes grown in T4 and T7 had a meiosis resumption percentage significantly superior to the other treatments. Two embryos were obtained, in which preantral follicles in medium supplemented with 100 ng/ml LH and 100 ng/ml EGF (T7). In conclusion, our sequential culture system was able to promote the in vitro growth of preantral follicles, promoting their oocyte maturation and caprine embryo production from preantral follicles.     

Concentration of vascular endothelial growth factor released by cultured human luteinized granulosa cells is higher in women with polycystic ovaries than in women with normal ovaries

To test the hypothesis that increased serum levels of vascular endothelial growth factor (VEGF) in women with polycystic ovaries or the polycystic ovary syndrome (PCOS) result from excess release by ovarian granulosa cells.Prospective study.Academic research setting.Twenty women undergoing IVF treatment, of whom 10 had normal ovaries and 10 had polycystic ovaries.Human granulosa lutein cells were isolated from follicular fluid obtained on the day of oocyte retrieval. Release of VEGF was assessed after co-incubation of granulosa lutein cells with gonadotropins and insulin. Serum and follicular fluid concentrations of VEGF were measured.Release of VEGF from granulosa lutein cells and serum levels of VEGF.Incubation with human hCG, and luteinizing hormone increased release of VEGF into the culture medium. Insulin alone did not increase release of VEGF, but addition of insulin increased hCG-stimulated release of VEGF. Serum and follicular fluid VEGF concentrations and the amount VEGF released from granulosa lutein cells obtained from women with polycystic ovaries or PCOS and those who developed the ovarian hyperstimulation syndrome were greater than those from granulosa lutein cells obtained from women with normal ovaries and those who did not develop the ovarian hyperstimulation syndrome.The amount of VEGF released by granulosa lutein cells is gonadotropin dependent and is augmented by insulin. The increased circulating concentrations of VEGF in women with PCOS may not only be due to an increased number of actively secreting granulosa lutein cells but also due to increased secretory capacity of each granulosa cell.     

The same luteinized granulosa-luteal cells from normal and superovulated human chorionic gonadotropin (hCG)-stimulated cycles contain hCG and insulin-like growth factor binding protein-1

OBJECTIVE: To determine whether hCG and IGFBP-1 appear in the same or different cells and in what sequence. DESIGN: Retrospective analysis of laboratory data. SETTING: University medical center. PATIENT(S): Twenty-five women undergoing IVF-ET with natural cycles and 25 women having stimulated IVF-ET. INTERVENTION(S): Cells were obtained from dominant follicles in women with natural cycles and from the follicles from hMG- and hCG-stimulated cycles. MAIN OUTCOME MEASURE(S): Detection and localization of hCG and IGFBP-1 in granulosa-luteal cells using double immunocytochemical staining. Measurement of hCG and IGFBP-1 in follicular fluid and serum. RESULT(S): Three types of hCG staining were found: on the cell surface, on the cell surface and in the cytoplasm, and in the cytoplasm alone. IGFBP-1 stained diffusely in the cytoplasm and was found only in those cells that were luteinized and contained hCG. IGFBP-1 and hCG were colocalized in the same cells. There was a positive correlation between follicular fluid hCG and IGFBP-1 levels, but only in natural IVF-ET cycles. CONCLUSION(S): HCG-driven luteinization is required for IGFBP-1 synthesis to take place in granulosa cells.     

性激素对人卵巢颗粒细胞凋亡的作用

目的通过探讨雌、雄激素对人卵巢颗粒细胞凋亡的作用,阐明其调节人类卵泡闭锁的分子机制。方法应用细胞培养、选择性ＤＮＡ抽提和琼脂糖凝胶电泳技术分析闭锁卵泡和发育中卵泡颗粒细胞的凋亡状况,及雌激素（１μｇ／ｍｌ）、雄激素（１μｇ／ｍｌ）对体外培养发育中的卵泡颗粒细胞凋亡的作用;用Ｎｏｒｔｈｅｒｎ印迹杂交技术检测经雌、雄激素刺激后发育中的卵泡颗粒细胞ｂｃｌ－２基因ｍＲＮＡ表达的变化。结果闭锁卵泡颗粒细胞均出现凋亡,ｂｃｌ－２基因ｍＲＮＡ表达下降,雌激素则可致相反的效应。结论卵泡闭锁的本质可能是其细胞发生凋亡;雄激素促进卵泡闭锁、雌激素对抗雄激素作用及促进卵泡发育的机理,可能与调节内源性核酸内切酶的活化和ｂｃｌ－２基因表达有关     

Progesterone production from granulosa cells of individual human follicles derived from diabetic and nondiabetic subjects

Insulin and insulin-like growth factors have been implicated in the stimulation of ovarian steroidogenesis. To assess the effect of diabetes mellitus on this process, a comparison was made of progesterone production by cultured granulosa cells (50,000 cells/well) from 11 individual follicles of nondiabetic and 6 individual follicles of diabetic women. Diabetic metabolic control was fair [HbA1C 6.8, 8.7 (nl 5.0-7.5)]. Cells were collected by laparoscopic follicular aspiration after ovulation induction and isolated by Percoll gradient centrifugation. Progesterone production was measured after culture with hCG (10 IU/mL) or insulin (100 microU/mL). In both nondiabetic and diabetic groups on day 4, hCG significantly stimulated progesterone production (1,686 +/- 1,268 ng/mL to 4,123 +/- 2,825 ng/mL and 1,059 +/- 249 ng/mL to 1,506 +/- 245 ng/mL, respectively). In nondiabetic follicles, insulin also stimulated progesterone production on days 4 (2366 +/- 1032 ng/mL to 3699 +/- 1582 ng/mL; P less than .05) and 7 (987 +/- 475 ng/mL to 1858 +/- 929 ng/mL; P less than .05); this response was not noted in diabetic granulosa cells. We suggest that insulin-stimulated progesterone production by granulosa cells isolated in the presence of fair diabetic metabolic control is impaired.     

抗苗勒氏管激素(AMH)与多囊卵巢综合征(PCOS)卵泡发育异常的关系

抗苗勒氏管激素(AMH)主要抑制始基卵泡进入生长阶段,在小窦卵泡中表达最高。多囊卵巢综合征(PCOS)主要表现为卵巢持续性无排卵。PCOS患者过高的LH和LH/FSH比值、胰岛素水平促进了雄激素的分泌,高雄激素进一步影响GC产生AMH,导致AMH水平异常增高,高水平的AMH通过降低GC对FSH敏感性,抑制P450芳香化酶表达,雄激素向雌激素转化受阻,E2水平降低,致使体内雄激素堆积,高雄激素又促进AMH生成,形成一个恶性循环,最终导致优势卵泡选择受阻,卵泡发育停滞在小窦状卵泡阶段。     

Testosterone levels in pregnant women correlate with the insulin response during the glucose tolerance test

OBJECTIVE: To evaluate the association between insulin/insulin-like growth factor I (IGF-I) systems and androgen levels in pregnancy. DESIGN: Prospective cohort study. SETTING: Yale University School of Medicine. PATIENT(S): Pregnant women undergoing a 100-gram 3-hour glucose tolerance test (GTT). INTERVENTION(S): Serum samples collected during GTT were analyzed for insulin, androgens, free IGF-I, insulin-like growth factor-binding protein (IGFBP) 1, and estriol. MAIN OUTCOME MEASURE(S): Observing the relationship between insulin/IGFs and androgen levels. RESULT(S): The insulin area under the curve (I(AUC)) during GTT correlated positively with total T and free T, but not with dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), or estriol. The peak insulin values (I(max)) during GTT also correlated positively with total T and free T, but not with DHEA, DHEAS, or estriol. There was no statistically significant correlation of T levels with free IGF-I, IGFBP-1, glucose, DHEAS, or estriol. Multiple linear regression analysis modeling showed that I(AUC) and I(max) did have a statistically significant correlation with free T levels. CONCLUSION(S): This study demonstrates for the first time that I(AUC) and I(max) measured in hyperinsulinemic states such as pregnancy correlate with T levels. In view of the lack of correlation between insulin and DHEAS or estriol, insulin-related T production during pregnancy is likely of ovarian origin.     

Effect of rosiglitazone on insulin resistance, growth factors, and reproductive disturbances in women with polycystic ovary syndrome

OBJECTIVE: To evaluate the effects of rosiglitazone on insulin resistance, growth factors, and reproductive disturbances in women with polycystic ovary syndrome (PCOS). DESIGN: Prospective study. SETTING: Women with PCOS attending as outpatients of the Endocrine Division, Hospital Durand, Buenos Aires. PATIENT(S): Twenty-four insulin-resistant women with PCOS. INTERVENTION(S): Hormonal evaluations and a standardized oral glucose tolerance test before and after a 3-month trial of 4 mg of rosiglitazone daily. MAIN OUTCOME MEASURE(S): Serum LH, FSH, T, IGF-1, IGFBP-1, IGFBP-3, leptin, 17alpha-hydroxyprogesterone, insulin, and glucose concentrations. The area under insulin curve (AUC-insulin), the HOMA index (insulin resistance), the QUICKI index (insulin sensitivity), and the beta-cell function were calculated. Body mass index (BMI) and the waist/hip ratio were evaluated. RESULT(S): A significant decrease was observed in serum fasting insulin, AUC insulin, HOMA index, beta-cell function, IGF-1, LH, and waist/hip ratio. The QUICKI index and IGFBP-1 increased significantly. Serum sex hormone-binding globulin, androgens, leptin, IGFBP-3, and BMI remained unchanged. Twenty-two of 23 females had their menses restored, and three patients became pregnant. One patient was excluded because she became pregnant at the second month. CONCLUSION(S): Associated with the decrease in LH, rosiglitazone improved insulin-resistance parameters and normalized the menstrual cycle, which suggests that this drug could improve the endocrine-reproductive condition in insulin-resistant women with PCOS.     

Anti-annexin V antibodies in patients with early pregnancy loss or implantation failures

OBJECTIVE: To examine the impact of flare (short) vs. down-regulation (long) GnRH agonist (GnRH-a) on serum and follicular fluid (FF) LH and androgen concentrations in women undergoing IVF treatment cycles. DESIGN: Prospective observational study. SETTING: IVF clinic. PATIENT(S): One hundred sixteen ovulatory subjects undergoing IVF. INTERVENTION(S): Fifty-eight ovulatory patients undergoing a down-regulation regimen matched with 58 undergoing the flare regimen as part of an IVF cycle. MAIN OUTCOME MEASURE(S): Serum concentrations of LH, FSH, Progesterone (P4), Androstenedione (A), T, and E(2) on the day of hCG administration were compared between the two groups. In addition, the FF P4, 17OHP4, A, T, and E(2) levels were compared in the two groups. RESULT(S): Serum LH was significantly higher with the flare regimen (15.2 +/- 1.14 IU/L, P<.05) when compared with results with the down-regulation protocol (9.5 +/- 0.77 IU/L). In addition, FF A was significantly higher in the flare protocol (57.3 +/- 13.3 ng/mL, P<.05) compared with in the down-regulation protocol (27 +/- 2.44 ng/mL). Serum and FF P4, 17OH P4, T, and E(2) were not statistically significantly different between the two groups. CONCLUSION(S): Serum LH and FF A are significantly higher in the flare regimen in comparison with the down-regulation regimen. Circulating LH appears to play a role in determining FF A concentration.