Clinical Assisted Reproduction: Cryopreservation of Human Cumulus Cells for Co-cultures and Assessment of DNA Damage After Thawing Using the Comet Assay

Purpose: Cumulus cells have been shown to be beneficial for blastocysts formation in co-cultures but information on cumulus cryopreservation is lacking. The objective was to use the fixed cell comet assay to analyze for DNA damage in cumulus cells after cryopreservation. Methods: Discarded cumulus cells from follicular aspirates obtained during assisted reproduction procedures (N = 4 cases) were pooled and cryopreserved in either 40% ethylene glycol and 0.5 M sucrose, 12:20% glycerol-egg yolk medium, 28% glycerol hypoosmolar medium or control medium. The cells were processed and stored in liquid nitrogen for 48 h. The thawed cells were smeared on glass slides, fixed, stained with acridine orange, embedded in a mini-agarose layer, and electrophoresis carried out. Fluorescent images were analyzed. Results: The cumulus tail moment, a calculated index of DNA damage, was significantly lower for each of the three cryoprotectant when compared with the control. The two cryoprotectants containing glycerol were associated with higher cumulus cell viability. However, the glycerol-egg yolk combination yielded the highest cell viability. Conclusions: The cumulus comet assay demonstrated similar DNA integrity in cells frozen in each of the three cryoprotectants. The glycerol-egg yolk medium had the highest cell viability with little or no DNA damage after freeze-thaw. More studies are needed to examine the long-term effect of the cryoprotectants on thawed cumulus cell viability.     

Protection of cumulus cells following dehydroepiandrosterone supplementation

Background: Growing studies have demonstrated that dehydroepiandrosterone (DHEA) may improve fertility outcomes in poor ovarian responders (PORs). The aim of this study was to compare clinical outcomes and cumulus cell (CC) expression before and after DHEA treatment in PORs undergoing in vitro fertilization (IVF) cycles.

Methods: Six patients with poor ovarian response were enrolled in the study according to Bologna criteria. DHEA was supplied at least 2 months before patients entered into the next IVF cycle. Expression of apoptosis-related genes in CCs was determined by quantitative real-time PCR. Mitochondrial dehydrogenase activity of CCs was assessed by cell counting kit-8 assay.

Results: Metaphase II oocytes, maturation rate, embryos at Day 3, and fertilization rate significantly increased following DHEA treatment. Expression of cytochrome c, caspase 9, and caspase 3 genes in CCs were significantly reduced after DHEA therapy. Additionally, increased mitochondrial activity of CCs was observed following DHEA supplementation.

Conclusions: DHEA supplementation may protect CCs via improved mitochondrial activity and decreased apoptosis, leading to better clinical outcomes in PORs.     

前列腺素E2及其受体在卵泡发育中的作用

颗粒细胞受黄体生成激素（LH）刺激后分泌的前列腺素E2(PGE2),通过旁分泌和自分泌,结合卵丘细胞上的前列腺素受体2,进而激活腺苷酸环化酶,增加胞内cAMP含量并促进透明质酸合酶2和肿瘤坏死因子α介导蛋白6的合成,引起卵丘细胞扩展。PGE2还通过调控颗粒细胞类固醇代谢通路影响孕酮分泌,从而调节黄体的功能。本文就PGE2在卵泡发育的作用进行综述。     

Steroid-depleted polycystic ovarian syndrome serum promotes in vitro oocyte maturation and embryo development

In vitro maturation (IVM) of immature oocytes obtained from patients with polycystic ovarian syndrome (PCOS) is considered as a novel strategy in order to reduce clinical side effects and cost of in vitro fertilization (IVF) technique. The aim of this study was to evaluate the effects of PCOS whole and steroid-depleted serums on in vitro oocyte maturation indices. Patients with PCOS were selected according to the Rotterdam criteria. Cumulus–oocyte complexes and blood serums were collected and pooled. Cumulus cells and immature oocytes were treated with 10% whole or steroid-depleted serums. Stearoyl-CoA desaturase-1 (SCD1) and cyclooxygenase-2 (COX2) expression levels in cumulus cells were evaluated by quantitative PCR. Fatty acid composition of cumulus cells was analyzed using gas–liquid chromatography. Polar body observation was considered as the oocyte maturation index. Oleate (1.28-fold, p?=?.006), SCD1 expression (450-fold, p?=?.001), and COX2 expression (35-fold, p?=?.02) in cumulus cell, as well as oocyte maturation (p?in vitro embryo development (p?相似文献   

体外成熟卵母细胞体外受精方式的探讨

目的:探讨经体外成熟卵母细胞是否可采用常规IVF方式授精。方法:自愿捐献的常规废弃的未成熟卵母细胞经体外成熟后的卵母细胞206个,随机分为3组:A组(n=69),授精时尽可能保存卵丘细胞并行常规IVF授精;B组(n=68),授精前将卵丘细胞完全拆除后行常规IVF授精;C组(n=69),将卵丘细胞完全拆除后行ICSI授精。常规IVF授精的精/卵个数比约为50 000∶1。比较3组卵子的受精率、卵裂率和优质胚胎率。结果:A组与C组在受精率(82.61%vs 85.51%)、卵裂率(94.74%vs 94.92%)、优质胚胎率(42.59%vs 46.43%)3个方面均无统计学差异(P>0.05),A组、C组的受精率、卵裂率均显著高于B组(14.71%,50.00%)(P<0.01),B组没有优质胚胎出现。结论:体外成熟后的卵母细胞在卵丘细胞较完整时可进行常规IVF方式授精。     

Differences between mature ovarian and oviductal oocytes: a study using the golden hamster

To investigate whether mature ovarian oocytes are physiologically identical with recently ovulated oocytes, hamster oocytes collected from ovaries approximately 1 h before ovulation were compared with the oocytes which had been in the oviduct for 3-4 h. Although these two groups of oocytes were at the metaphase of the second meiosis, there were quantitative differences between the two with respect to (i) sensitivity of cumulus matrix to hyaluronidase and spermatozoa (oviductal greater than ovarian), (ii) size of the perivitelline space (oviductal greater than ovarian), (iii) viscosity of the ooplasm (ovarian greater than oviductal), (iv) responsiveness to Ca2+-ionophore (oviductal greater than ovarian), and (v) time needed in completing meiosis (ovarian greater than oviductal). The most prominent difference was found in the zona pellucida. The zona of the oviductal oocyte was 'heterogeneous' in its optical density and had a stronger acrosome-reaction-inducing ability than that of the ovarian oocyte. When cultured in artificial media (e.g. F-10 medium with serum) for 4 h, the ooplasm of ovarian oocytes became like that of oviductal oocytes. However, their zonae remained unchanged. Zonae of ovarian oocytes became like those of oviductal oocytes only when they were exposed to ampullary and/or isthmic fluids. The zona-altering factors in the oviductal fluid (oviductal glycoproteins), which are apparently integrated into the native zona, may act to enhance the various functions of the zona.     

经后增殖方含药血清对超排卵大鼠卵巢COCs及CCs中GDF9与Bim表达的影响

目的 观察经后增殖方含药血清对超排卵大鼠卵巢的卵丘-卵母细胞复合体(COCs)及卵丘细胞(CCs)中生长分化因子9(GDF9)及Bim表达的影响,探讨其治疗效果及作用机制.方法 制备大鼠超排卵卵巢模型,收集COCs和CCs,分别与雌性SD超排卵大鼠空白血清和经后增殖方药物血清在体外共同培养,各分3组:空白血清组(空白组)、含药血清组(对照组)、含药血清+ GDF9受体阻断剂组(实验组),共6组.24 h后收集COCs和CCs,实时荧光定量PCR检测GDF9和Bim mRNA表达水平,蛋白质印迹法检测GDF9蛋白表达水平.结果 (1)对照组COCs中GDF9 mRNA表达水平明显高于空白组和实验组(P＜0.05);对照组和实验组COCs中Bim mRNA表达水平均明显低于空白组(P＜0.05);对照组COCs中GDF9蛋白表达水平明显高于空白组(P＜0.05),亦高于实验组,但差异无统计学意义(P＞0.05).(2)CCs中GDF9 mRNA表达水平在3组间的差异性比较结果与COCs中一致;对照组和实验组CCs中Bim mRNA表达水平均明显低于空白组(P＜0.05);对照组CCs中Bim mRNA表达水平明显低于实验组(P＜0.05);对照组CCs中GDF9蛋白表达水平明显高于空白组和实验组(P＜0.05).(3)对照组COCs中GDF9 mRNA表达水平明显高于CCs中(P＜0.05),其余组内COSs和CCs比较差异无统计学意义(P＞0.05).结论 经后增殖方舍药血清能提高COCs和CCs中GDF9的表达水平,维持COCs和CCs中Bim的低水平表达,抑制细胞凋亡,促进卵泡发育;COCs较剔除卵母细胞的CCs更有利于GDF9 mRNA的表达.     

Cryptic association of e antigen with different morphologic forms of hepatitis B surface antigen

When highly purified HBsAg particles, separated by rate zonal centrifugation into populations differing in predominant size, were tested for HBeAg, the e1 specificity was detected preferentially in association with particle fractions containing large filaments and Dane particles. These results were obtained both by agar gel diffusion and by radioimmunoassay for e antigen. The e antigen activity present in these fractions was potentiated by prior treatment of particles with Tween 80, suggesting cryptic localization of e1 specificity within or under the outer membrane. The HBeAg released by detergent treatment from a purified preparation composed predominantly of small-particle forms of HBsAg was separated by electrofocusing into a peak of nonparticulate e antigen in the pH range of 5.7--6.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major polypeptides in this preparation with approximate molecular weights of 25,000, 55,000, and 70,000. Furthermore, two additional peaks of e antigen activity were detected which migrated in association with HBsAg particles at isoelectric points of 4.4 and 5.5--5.6. The major portion of e antigen remained in association with particles after further purification by rate zonal centrifugation.     

Correlation between in vitro maturation and expression of LH receptor in cumulus cells of the oocytes collected from PCOS patients in HCG-primed IVM cycles

BACKGROUND: The aim of this study was to investigate whether in vitro maturation (IVM) and blastocyst development of oocytes collected following HCG-primed IVM cycles of PCOS patients are correlated with their cumulus cell (CC) patterns and further to investigate mRNA expression of the receptors for FSH, LH and epidermal growth factor (EGF) in the CCs with each pattern. METHODS: Patients who underwent IVM were primed with 10,000 IU of HCG 36 h before oocyte aspiration. The isolated cumulus-oocyte complexes were divided into three groups according to the CC patterns: oocytes with dispersed CCs (group A), oocytes with compacted CCs (group B) and oocytes with sparse CCs (group C). Oocyte maturation and blastocyst development were compared among three groups. The expression of the mRNA for FSH, LH and EGF receptors in group A and B was analysed by semi-quantitative RT-PCR. RESULTS: The maturation rate of group A was significantly higher than those of group B and C. The rate of blastocysts in group A was significantly higher than those of group B and C. mRNA expression of the LH receptor in group A was more abundant than that of group B. CONCLUSIONS: These results suggest that the presence of dispersed CCs at oocyte collection may be positively correlated with the rates of oocyte maturation and blastocysts in HCG-primed IVM cycles. In addition, the expression of LH receptor in CCs may be correlated with the CC pattern of oocytes at collection.     

Effect of cyclic AMP on the isolated human oocyte -- cumulus complex

Cyclic AMP (cAMP) is one of the key regulators of the resumptionof oocyte meiosis (oocyte maturation) in several mammalian species.In this study we have examined the role for cAMP in regulationof spontaneous maturation of human oocytes. Oocytes were obtainedin connection with gynaecological operations and cultured for24 or 48 h. A significantly larger proportion (77%) of oocytescultured in the presence of 1 mM dibuturyl-cAMP (dbcAMP) for24 h were arrested in meiosis compared to oocytes cultured incontrol medium (25%). In addition, dbcAMP stimulated progesteroneaccumulation during 48 h of incubation and inhibited monolayerformation of the cumulus cells attached to the oocyte. The datapresented in this study demonstrate that dbcAMP delayed thespontaneous maturation of human oocytes and stimulated progesteroneproduction by the cumulus cells. These results indicate thatcAMP may be important also for regulation of meiosis in humanoocytes and confirm results obtained in other species.