4种冷冻-解冻方法对家兔卵巢组织形态学的影响

目的:探讨适宜的卵巢组织冻存方案。方法:采用PROH(A2组)及DMSO(B2组)慢速程序化冷冻和DMSO+PROH(C2组)及DMSO+EG(D2组)玻璃化冷冻方法,冻存家兔卵巢组织,解冻复苏后,以相应的4组新鲜组织为对照(A1-D1组),做HE染色,行组织形态学分析。结果:A1-D1组始基卵泡的形态正常率分别为90.1%、91.5%、91.8%、92.2%,其相对应的A2-D2组分别下降为67.6%、69.7%、70.5%、80.1%,差异均有统计学意义(P均<0.05)。冷冻组中A2组始基卵泡形态正常率最高,与B2、C2、D2组比,差异有显著性(P<0.05)。C2、D2组间比,差异无显著性(P>0.05)。各冷冻组合并后形态正常率始基卵泡为72.7%,初级卵泡为55.7%,两者比较有统计学差异(P<0.05)。4个冷冻组中均可见卵巢组织结构受损的表现。结论:4种冷冻解冻方法对卵巢皮质中各级卵泡及卵巢组织结构均造成一定程度的损害,使各级卵泡的形态正常率明显下降,卵巢间质细胞连接变得疏松;PROH慢速程序化冷冻法明显优于DMSO法及玻璃化法,较适合卵巢组织中始基卵泡的保存;冻存卵巢组织对初级卵泡的影响大于始基卵泡。     

新鲜小鼠卵巢组织自体异位移植后卵巢功能的研究

目的 :测定小鼠卵巢组织自身异位移植后动情期血清中 17β 雌二醇 (E2 )和孕酮 (P)的水平 ,称量子宫湿重。在相同条件下 ,与正常同龄小鼠和绝经模型比较 ,探索卵巢组织移植后功能的变化。方法 :将约 10周大的ICR雌性小鼠 4 3只分成 3组 :A组即空白对照组 ,11只 ,不作任何处理 ;B组即去势组 ,16只 ,去除双侧卵巢 ;C组为实验组 ,16只 ,自身卵巢摘除后剪成 3～ 4块种植于腹壁皮下。术后观察阴道涂片变化 ,在动情期断头取血 ,称量子宫湿重 ,并分析子宫内膜组织形态。再用酶联免疫法测定各组血清中E2和P的水平。结果 :B组小鼠的阴道涂片在 1周左右失去正常的周期变化 ,将超过 2周未出现典型动情期者判断为绝经。C组在失去正常周期变化后 ,通常于 8± 3d重新出现动情期。A组 10周动情期小鼠的血清E2 、P的平均水平为 5 3.72pg/ml、9.86ng/ml,子宫湿重平均为 111.0 0mg ;B组分别为 4 2 .17pg/ml、1.5 8ng/ml ,2 4 .4 2mg ;C组分别为 4 7.11pg/ml、11.6 1ng/ml,95 .2 0mg。此外 ,C组子宫湿重及子宫内膜组织形态与正常小鼠无显著差异(P >0 .0 5 )而与B小鼠有明显差异 (P <0 .0 5 )。结论 :小鼠卵巢组织的自身异位移植能够恢复卵巢分泌E2 和P的功能。     

冻存卵巢组织移植成功——首次报道

目的通过卵巢组织冻存技术及自体移植手术保护癌症患者生育力与内分泌功能。方法对1例40岁宫颈鳞癌IIb期患者在放化疗前进行卵巢组织取材、冻存,待其癌症痊愈后进行冻存卵巢组织自体移植手术,随访监测患者的激素水平、卵泡发育情况、月经恢复情况以及绝经相关症状。结果卵巢组织移植4个月后,改良Kupperman评分由20分降低至5分,绝经相关症状基本消失,卵泡刺激素(FSH)降低至22.79 IU/L,此后继续下降至6 IU/L,雌二醇(E_2)由11.8 ng/L升高至84.46 ng/L,B超监测到有卵泡发育且月经恢复。结论卵巢组织冻存移植4个月后卵巢功能恢复正常,证明卵巢组织在临床上移植成功。     

冻存复苏人卵巢皮质在裸鼠体内的发育及卵细胞的提取

目的:探求冻存复苏人卵巢皮质在裸鼠体内的发育及卵细胞的提取。方法:取手术切除的5例患者卵巢皮质,放入1.5 mol/L的二甲基亚砜+10％血清中,应用可控程序冷冻仪逐渐降温,移入液氮中冻存。三个月后,将融解的卵巢皮质移植入89只裸鼠皮下,注射hFSH 12周,刺激卵泡生长。注射hCG 36 h后,用18号针头穿刺提取卵细胞。结果:卵泡直径为3～9 mm,穿刺获19个卵细胞,放入TC-199培养基,加20％胎牛血清,25 mmol/L丙酮酸,75 mIU/mL FSH和LH培养,获15个分裂中期的次级卵母细胞。结论:人卵巢组织冻融后可异体移植生长,且其卵细胞可在体外发育成熟。     

妊娠期肝内胆汁淤积症患者血清丙二醛和维生素E水平测定

目的:探讨妊娠期肝内胆汁淤积症(ICP)患者脂质过氧化和抗氧化状态。方法:分别用硫代巴比妥酸和亚铁嗪显色分光光度法测定30例ICP患者治疗前和治疗后及42例健康孕妇血清MDA和维生素E水平。结果:ICP患者治疗前血清MDA(17.67±12.14nmol/L)和维生素E(2.77±1.58mmol/L)水平均显著高于健康孕妇组(7.57±1.30nmol/L,1.18±0.74mmol/L,P<0.01)。常规治疗1周后,ICP患者血清MDA(15.22±9.58nmol/L)水平略有下降,但仍显著高于健康孕妇组(P<0.01),而维生素E(2.91±1.56mmol/L)水平则略有上升。结论:ICP患者体内脂质过氧化作用明显增强,抗氧化作用代偿性增强严重不足;维生素E是否可用于ICP治疗尚待研究。     

卵巢脂质细胞瘤误诊2例

1病例报告例1 25岁,因出现闭经、多毛、痤疮2年,发现卵巢肿物1年,于2004年11月10日入院。患者既往月经正常。2001年8月足月顺产1女活婴,产后无发热、大出血及刮宫史,哺乳1年半。断奶后无月经来潮,并出现身体多毛、面部痤疮、声音变粗、喉结突出、肥胖等,2年间体重共增加10kg。2003年因闭经就诊我院,查性激素,血清睾酮(T)22·51nmol/L,其余在正常范围,B超检查提示:右卵巢实性占位,2cm×1cm。予人工周期治疗,每月均有撤退性出血,不用药无月经。2004年11月检测性激素:E2 400·11pmol/L,T 42·23nmol/L,其余正常。B超检查提示:右卵巢实性占…     

卵巢恶性肿瘤患者测定血清血管内皮生长因子的临床价值

目的　探讨血清血管内皮生长因子 (VEGF)测定对卵巢恶性肿瘤诊断、病程监测和预后的价值。方法　采用酶联免疫吸附法 (ELISA)对 90例健康妇女 (对照组 )、2 5例卵巢良性肿瘤 (良性组 )及 12 0例卵巢恶性肿瘤 (恶性组 )进行血清VEGF含量分析 ,并进一步对其中 2 5例卵巢恶性肿瘤患者进行手术治疗前后血清VEGF含量的动态观察。结果　 ( 1)恶性组术前血清VEGF含量为 ( 766±12 3 7)mg/L ,显著高于良性组的 ( 5 6± 2 3 )mg/L及对照组的 ( 5 5± 19)mg/L(P =0 0 0 6) ,以 10 0mg/L为界值 ,其诊断的特异性为 87% ,敏感性为 77% ;( 2 )恶性组临床分期Ⅰ～Ⅱ期和高～中分化患者术前血清VEGF含量分别为 ( 198± 2 87)mg/L和 ( 2 80± 5 5 2 )mg/L ,明显低于临床分期Ⅲ～Ⅳ期的 ( 95 5±1716)mg/L和低分化患者的 ( 991± 13 49)mg/L (P <0 0 5 ) ;但血清VEGF含量与组织学类型无关 (P>0 0 5 ) ;( 3 )手术治疗后血清VEGF含量为 ( 118± 110 )mg/L ,较手术治疗前血清VEGF含量 [( 10 74±12 11)mg/L]明显下降 ;( 4)初治患者血清VEGF阳性者 (即VEGF≥ 10 0mg/L)平均总生存期为 2 8个月 ,而血清VEGF阴性者平均总生存期为 3 5个月 (P <0 0 5 )。但COX模型分析结果发现 ,VEGF不是与卵巢恶性肿瘤预后相关的独立因素 (P     

两种玻璃化法冻存小鼠卵巢的研究

目的:探讨2种玻璃化法对小鼠卵巢组织、器官形态和功能保存作用的影响。方法:以改良的DMEM-F12为玻璃化液,分别采用常规玻璃化法(A组)和超速玻璃化法(B组)冻存小鼠卵巢组织及器官,解冻后通过组织学观察、卵巢组织异体、卵巢器官自体肾被膜下移植,观察动情周期恢复率、恢复时间、卵泡发育状况,并分别以新鲜卵巢组织异体移植、卵巢器官自体移植(C组)为对照,评价2种玻璃化法的冻存效果。结果:①A组、B组、C组卵巢组织异体移植小鼠动情周期出现率为100%,出现动情周期分别10.5±5.4d、8.0±2.2d、6.3±1.0d。A组与C组比,差异显著(P<0.05);B组与C组无差异,A组与B组间也无差异(P均>0.05)。②A组、B组、C组卵巢器官自体移植小鼠动情周期出现率为100%,出现动情周期分别为9.4±0.9d、6.9±1.1d、6.1±1.1d,A组与B、C组相比有统计学差异(P<0.05),而B组与C组间无差异(P>0.05)。移植存活的卵巢组织、器官内均可见不同发育阶段的卵泡,形态正常。结论:2种玻璃化法可有效地冻存卵巢组织及器官,但超速玻璃化法效果较优。     

测定血清抑制素B水平预测卵巢储备力及调节控制性超排卵用药的研究

目的探讨血清抑制素B(INHB)水平测定,在评价卵巢储备力和调节控制性超排卵用药中的意义。方法选择96例接受体外受精-胚胎移植(IVF-ET)治疗的患者,根据超排卵用药反应分3组:低度反应组(6例)、中度反应组(72例)、高度反应组(18例)。在经期第3天采用酶联免疫吸附法(ELISA)测定血清INHB水平;采用化学发光法测定血清基础卵泡刺激素(FSH)、黄体生成素(LH)和人绒毛膜促性腺激素(hCG)注射日雌二醇水平;计算周期获卵数、优质胚胎数、周期妊娠率,以调节控制性超排卵用药。结果低度反应组、中度反应组、高度反应组INHB水平分别为(28±20)、(85±42)、(92±34)pg/ml;FSH水平分别为(11·9±5·3)、(7·5±2·6)、(7·2±1·7)U/L;hCG注射日雌二醇水平分别为(2558±2108)、(9366±4472)、(18392±9655)pmol/L;周期获卵数分别为(0·6±0·4)、(8·7±3·6)、(14·3±2·9)个;优质胚胎数分别为(0·4±0·3)、(3·8±1·9)、(4·6±1·7)个;周期妊娠率分别为16·7%、36·1%、61·1%。INHB水平与FSH水平、FSH/LH比值呈负相关(r分别为-0·222,-0·371,P<0·05),与hCG注射日雌二醇水平、周期获卵数、优质胚胎数、周期妊娠率呈正相关(r分别为0·336、0·404、0·323、0·246,P<0·05)。低度反应组、中度反应组、高度反应组在控制性超排卵中,基因重组FSH用量分别为(3808±742)、(3046±709)、(2158±653)U。结论INHB测定可作为预测卵巢储备力的直接指标,在辅助生育治疗中,对指导控制性超排卵用药有重要临床意义。     

卵泡膜纤维瘤分泌抑制素B致继发性闭经和不孕

1例 37岁女性患者 ,主诉继发性闭经和不孕 2年 ,曾测血清 L H16 .3m IU/ ml(正常值 0 .9～ 14.0 m IU/ m l) ,FSH3.8m IU/ ml(正常值 3.7～ 12 .9m IU / ml) ,TSH和 PRL正常。 1998年 2月 B超示子宫和左卵巢正常大小 ,右卵巢 3.8cm× 3.6 cm× 6 .1cm ,内有 2 .8cm× 2 .4cm混合性团块回声 ,疑为卵巢皮样囊肿。1998年 5月复查该团块位于右卵巢后方 ,3.5 cm× 3.7cm× 4.2 cm,又 3个月后复查卵巢内占位增至 2 .6 cm× 3.9cm× 5 .6 cm,呈明显强回声光团。因继发性闭经予注射黄体酮 ,出血第三天测血清 L H2 3.4m IU/ ml,FSH1.7m I…     

Ultrarapid freezing and thawing of hamster oocytes. Morphologic parameters, trypan blue staining and sperm penetration assay for evaluating survival

Nine hundred sixteen hamster oocytes were cryopreserved with the ultrarapid freezing method using five different cryoprotective solutions: 3 mol/L dimethylsulphoxide (DMSO) plus 0.25 mol/L sucrose, 3 mol/L DMSO, 3 mol/L propanediol plus 0.25 mol/L sucrose, 3 mol/L propanediol and 10% glycerol. One hundred eighty fresh oocytes served as controls. The viability of the oocytes was evaluated using morphologic parameters, Trypan blue staining and the sperm penetration assay. The viability rates based on morphologic parameters and Trypan blue staining were 82.3%, 65.0%, 51.4%, 33.0% and 0%, respectively, as compared to 100% in the controls. The sperm penetration rates were 27.0%, 0%, 9.8%, 0% and 0%, respectively, as compared to 94-98% in the controls. Our results indicate that among the various cryoprotective solutions used for ultrarapid freezing, 3 mol/L DMSO plus 0.25 mol/L sucrose gave the best results, with a viability rate of 82.3% and a sperm penetration rate of 27%.     

Developmental capacities of two-cell mouse embryos frozen by three methods

The following three methods were evaluated in order to obtain a most efficient freezing protocol for the preservation of two-cell mouse embryos: (a) slow cooling and slow thawing in 1.5M dimethyl sulfoxide, (b) slow cooling and fast thawing in 1.5M propanediol (PROH), and (c) ultrarapid freezing and fast thawing in either 3.5M DMSO or 3.0M PROH. In the slow-cooling procedures (a and b) ice nucleation (seeding) was induced manually or automatically. With method a, only a slight difference, 51.8% for manual and 58.9% for automatic seeding, was observed in survival rates, while the development to blastocysts was significantly affected: 35.4% with manual and less than 10% with automatic induction (P<0.001). Method b gave high survival (86.2%) and developmental rates (69.0%) with manual seeding compared with automatic seeding (20.7 and 9.8%, respectively;P<0.001). Using protocol c, higher survival and developmental rates were obtained with DMSO (84.8 and 55.9%) than with PROH (39.8 and 19.4%,P<0.001). These results demonstrate that inducing nucleation manually is superior to the use of a highly sophisticated autoseeding system and that method b with manual seeding is most effective in preserving the developmental capacity of twocell mouse embryos after freezing and thawing. There is evidence that this is also true of human embryo cryopreservation.     

Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols

OBJECTIVE: To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. DESIGN: Small follicles (<60 microm in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically. SETTING: Centre hospitalo-universitaire de Biologie de la Reproduction, H?pital Edouard Herriot, Lyon, France. ANIMAL(S): Lambs 5 to 6 months of age. Intervention(s): Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures. Main Outcome Measure(s): Follicular mortality and histologic structure. RESULT(S): For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar. CONCLUSION(S): Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.     

Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum

Chemotherapy and/or radiotherapy protocols have improved the long-term survival of cancer patients. Frequent consequences of antiblastic treatments, used to eradicate malignancies, are the partial loss of ovarian function, which in children and young women can result in permanent sterility. Ovarian tissue cryopreservation implemented before the beginning of treatment may potentially restore fertility. However, the physical effects of cryopreservation can damage oocyte survival and decrease follicular cell integrity and stromal preservation. The aim of this study was to examine the effects of different concentrations of 1,2-propanediol (PROH) and sucrose as cryoprotectants and human serum as protein support. Particular concentrations tested were 1.26, 1.5 and 1.08 mol/l PROH, 0.175, 0.2, 0.224 and 0.3 mol/l of sucrose and 20%, 30% and 40% human serum in the freezing solutions and normal or raised sucrose concentrations in the dilution solutions. Ovarian cortical slices from 13 patients, aged 5-38 years, were cryopreserved using slow freezing-rapid thawing. Tests were conducted using light and transmission electron microscopy. Cryo-damage occurred predominantly in the stromal and follicular cells. The best preservation of morphological characteristics was obtained using the freeze-thaw protocol in which concentrations of cryoprotectants were among the lowest (1.26 mol/l PROH+0.175 mol/l sucrose) with 30% human serum.     

The evaluation of various culture media in combination with dimethylsulfoxide for ultrarapid freezing of murine embryos.

Bicarbonate-buffered HTF medium, Medicult, and T6 are as effective as PB1 medium when used in combination with DMSO in ultrarapid freezing of two-cell mouse embryos. However, the use of phosphate-buffered T6 results in reduced in vitro development and inner cell mass size as compared with bicarbonate- and Hepes-buffered T6 when used with 3.5 M of DMSO. Hence, the use of this media for ultrarapid freezing should be avoided when this concentration of DMSO is used.     

小鼠卵母细胞慢速冷冻的初步研究——附人妊娠分娩1例报道

目的:探讨不同浓度蔗糖联合丙二醇(PROH)冷冻保护剂对小鼠卵母细胞冻融的影响。方法:①观察不同蔗糖浓度(0.1mol/L、0.2mol/L、0.3mol/L)联合应用1.5mol/L的PROH对小鼠卵母细胞复苏率的影响。②对1例采卵日未能取得精子的不孕症患者,应用0.3mol/L蔗糖联合1.5mol/LPROH将其卵母细胞用慢速冷冻方法冷冻保存,随后解冻并移植。结果:①随着蔗糖浓度的提高,卵母细胞复苏率也随之升高,0.3mol/L蔗糖组复苏率达74%。②1例患者卵母细胞复苏后行卵胞浆内ICSI-ET,获临床妊娠,妊娠中期羊水染色体核型分析正常,妊娠35周因妊高征剖宫产1健康男婴。结论:对卵母细胞慢速冷冻中,适当提高蔗糖浓度可获得较满意的复苏率、受精率及卵裂率,胚胎移植后能够出生健康新生儿。     

Successful ultrarapid freezing of unfertilized oocytes

Successful application of ultrarapid freezing techniques to unfertilized murine oocytes has not been reported. In an effort to improve results, preovulatory murine oocytes were exposed to three ultrarapid freezing protocols involving varying sucrose concentrations (0.25, 0.5, and 1.0M) and 3.5M dimethyl sulfoxide (DMSO) as cryoprotectants prior to direct immersion in liquid nitrogen. Postthaw morphology and rates of in vitro fertilization and embryo development were compared with those obtained after freezing oocytes employing two established programmed cooling techniques. The rates of fertilization and development to the blastocyst stage in vitro of oocytes undergoing ultrarapid freezing after exposure to 3.5M DMSO and 0.5M sucrose were similar or superior to those obtained with programmed cooling techniques. Of oocytes which appeared morphologically normal postthaw, only those which underwent ultrarapid freezing with 0.25 or 0.5M sucrose and 3.5M DMSO reached the blastocyst stage at rates similar to those of controls. Ultrarapid freezing may represent a viable option for successful murine oocyte cryopreservation.Presented in part at 44th Annual Meeting of the American Fertility Society, October 10–13, 1988, Atlanta, GA.     

Pregnancies after transfer of ultrarapidly frozen human embryos

Purpose: We report our experience of freezing human embryos using an ultrarapid freezing method. Methods: The patients were superovulated. Oocytes were inseminated and cultured in HTF + 10% serum. A maximum of three embryos was transferred and the rest of the embryos were frozen ultrarapidly after a 3-min equilibration period in PB1 + 3.5 MDMSO + 0.25 Msucrose. Embryos were thawed in a 37°C water bath for 6 sec, then cultured in PB1 + 20% serum for 10 min. The surviving embryos were transferred into patients on the same day of thawing. Results: Sixty-three embryos were thawed. of which 52 embryos (83%) survived with at least one intact blastomere. Nineteen frozen-thawed embryo transfers were made. The mean embryos per transfer was 2.7. Three pregnancies (16%/transfer) were established. One miscarriage occurred in the eighth week of pregnancy. Two pregnancies went to term and three healthy infants were born. Conclusions: The present data demonstrate that ultrarapid freezing is a method worth consideration in the area of human embryo freezing.