微流控芯片优选人类精子研究

目的:探讨微流控芯片技术对精子的优选能力。方法:自行设计和制造微流控芯片,利用芯片对40例人精液标本进行精子分选实验,优化其分选条件,观察芯片处理前后精液各参数变化。同时对其中30例精液标本(A组:a+b级精子<20%组,n=15;B组:a+b级精子≥20%组,n=15)同时用芯片法和密度梯度离心法分选,比较2种方法分离前后精子活力、形态等参数的变化。结果:①优选后精子活力和精子正常形态率都可见显著提高(P<0.001;P<0.01)。②在精子活动力优选上A、B组芯片法均明显优于密度梯度离心法(P<0.01),尤其在A组这种优势更为明显(P<0.001)。而在精子形态优选上,2种方法无显著差异(P>0.05)。结论:微流控芯片技术在优选精子中具有较高的分选效率,且具有操作简单、分选时间短,对精子损伤小的特点,在辅助生殖技术中特别是体外受精中将有良好的应用前景。     

膜联蛋白Ⅳ在卵巢透明细胞癌和浆液性囊腺癌中的差异表达

目的:通过建立基因表达谱,研究透明细胞癌和浆液性囊腺癌之间差异表达基因。方法:利用cDNA芯片建立和分析两者基因表达谱,并对透明细胞癌中上调表达的膜联蛋白Ⅳ,在组织和细胞中进行了定量PCR以及免疫组织化学和Western blot验证。结果:卵巢透明细胞癌和卵巢浆液性囊腺癌基因表达谱比较,差异2倍以上基因共有82个,25个表达上调,57个表达下调。其中有功能的上调基因7个,下调基因12个。组织和细胞中膜联蛋白Ⅳ在mRNA及蛋白水平上的表达与基因芯片结果一致。结论:通过基因芯片可以建立卵巢透明细胞癌和浆液性囊腺癌的差异表达基因谱,膜联蛋白Ⅳ在卵巢透明细胞癌中上调表达可能具有重要意义。     

人精子膜上凝集素受体的研究

从糖专一性相同或不同的24种凝集素中筛选出SML、MDL、CKL和PHA-S四种能与人精子发生强烈反应的凝集素,浓度为0.98μg/ml时就能使精子凝集成块,6 h后,凝集团块也不分散,用机械方法分散团块所得精子已完全失去活动能力;其凝集作用可被凝集素各自的抑制糖所抑制;用FITC标记的SML、CKL、MDL与正常人精子作用后,在荧光显微镜下可观察到各凝集素受体的分布部位各不相同;用去垢剂增溶精子,Brij 58的增溶效果最好,增溶的膜蛋白可强烈地抑制凝集素对兔红细胞的凝集作用。     

应用基因芯片技术筛查葡萄胎相关基因的研究

目的 探讨应用基因表达谱芯片技术,筛选葡萄胎发病及恶性转化相关基因的有效性。方法 应用含4096条基因的cDNA表达谱基因芯片,对2例完全性葡萄胎组织和与葡萄胎孕周接近的2例正常绒毛组织进行基因表达谱分析,比较两种组织中差异表达的基因。结果 2例葡萄胎及2例正常绒毛组织中均有差异表达的基因89条,占所检基因总数的2．2％。与正常绒毛组织比较,葡萄胎组织中基因表达显著增强(上调)的有24条,表达显著降低(下调)的有65条。基因生物学信息分析显示,RASA1、转化生长因子βⅡ型受体、BTG2等与细胞生长抑制有关的基因,在葡萄胎组织中显著下调;而与组织细胞增生和恶性转化及肿瘤转移有关的基因,如胸腺嘧啶核苷激酶1、核苷酸还原酶小亚单位、葡萄糖转运蛋白3、细胞周期蛋白B等基因在葡萄胎组织中显著上调。结论 基因表达谱芯片技术是一种高效的筛查与葡萄胎发病相关基因的有效方法。     

卵巢癌相关基因筛选的初步研究

目的 通过研究人卵巢癌组织和正常卵巢组织中差异表达的基因，筛选出卵巢癌相关基因以用于早期诊断和治疗。方法 以cDNA基因表达谱芯片分析研究卵巢组织样本和正常卵巢组织的基因表达谱，计算机分析后比较两种组织中差异表达的基因。结果 共筛选出差异表达的基因1785条，表达上调基因有40条，下调基因138条，有显著表达差异的基因54条，涉及到11大类基因。结论 卵巢癌同其它恶性肿瘤一样，是多种基因结构和功能改变的结果，有关卵巢癌特异性相关基因及其作用尚有待进一步研究。     

基因表达谱芯片在宫颈癌相关基因筛选中的应用研究

目的探讨基因表达谱芯片技术在高通量筛查肿瘤相关基因群及研究宫颈癌分子病理学变化中的应用价值.方法应用含有9 184条人类全长基因的cDNA表达谱芯片,对3例临床切除的宫颈癌及正常宫颈标本的基因表达谱进行分析.结果在3例宫颈癌和正常宫颈组织中均有差异表达的基因24条,其中在宫颈癌组织中上调基因12条,下调基因12条.结论运用本基因表达谱芯片对基因表达谱进行分析,能够有效筛查出新的宫颈癌相关基因.     

封闭式薄片法对人类微量精子超快速玻璃化冷冻的研究

目的:通过应用封闭式薄片法对人类微量精子进行超快速玻璃化冷冻,旨在寻找一种微量精子冷冻的最佳方法。方法:选取于大连妇儿生殖中心就诊的正常男性精液标本20例,精液上游处理后用于实验。首先用含冷冻保护剂的不同微滴量(0.5μl,1.0μl,3.5μl)对冷冻效果进行比较,以期找到微量精子冷冻的最佳微滴量。同时用无冷冻保护剂法对微量精子进行冷冻,并与冷冻保护剂组进行比较。结果:3组冷冻微滴量解冻后精子的回收率、活动率及存活率比较,差异均无统计学意义(P0.05)。无冷冻保护剂组与冷冻保护剂组的解冻后精子回收率、活动率及存活率比较,差异均无统计学意义(P0.05)。结论:封闭式薄片法超快速冷冻人类微量精子,解冻后可获得较高的精子回收率、活动率与存活率。无冷冻保护剂法冷冻微量精子,解冻后与冷冻保护剂法的结果相似。     

甘油冷冻保护剂及冷冻降温速度对人精子功能及染色体的影响

为研究冷冻对人精子生育力及染色体的影响。本实验选用健康、已生育、２５～３５岁男性供精者的精液,以７．５％的甘油作冷冻保护剂,通过冷冻前后精液常规分析、穿卵试验、染色体核型分析,发现在冷冻－复苏后,精子活动率、穿卵率均有一定程度下降,精子染色体数目和结构异常的发生频率冻贮前后无显著差异。但在活力好、具有生育能力的精子中,携Ｘ－染色体的精子与携Ｙ－染色体的精子所占比例发生了显著改变,由冻前的Ｘ：Ｙ＝５０： ５０变成冻后的 ６０．８５： ３９．１５。     

人冷冻精液巴氏染色法的应用评价

目的:探讨人冷冻精液应用巴氏染色法的染色效果。方法:30例人精液标本冷冻保存后,冷冻精液涂片应用巴氏(Papanicolaou)染色法进行染色,观察精子和非精子细胞的染色效果,检测冷冻精子畸形率。应用透射电镜观察冷冻精子头部(n=8)超微结构的改变。结果:冷冻保存后的精子畸形率,与冷冻前的比较无统计学差异(P>0.05)。冷冻精子头、颈、尾部,各种形态类型精子以及非精子细胞的着色效果与新鲜精液的对比无明显不同。冷冻精液涂片后自然干燥所需的时间较长。透射电镜观察到冷冻精子头部浆膜及顶体发生肿胀和破损。结论:巴氏染色法可以应用于冷冻精液的涂片染色,实际应用时需注意冷冻精液含有甘油等的特点。     

基因芯片筛选上调miRNA-205表达后HeLa细胞相关基因

目的：应用基因表达谱芯片技术筛查上调微小RNA-205（miR-205）表达后人宫颈腺癌细胞株HeLa细胞中差异表达基因,并探讨其功能及上调miR-205致癌机制。方法：应用含有23 232条人类全长基因的cDNA表达谱芯片,对上调miR-205表达的HeLa细胞及HeLa细胞原株的基因表达谱进行分析。结果：与HeLa细胞原株相比,上调miR-205表达的HeLa细胞中有差异表达的基因共172条,其中34条上调,138条下调。结论：基因芯片技术能够有效筛查出上调miR-205表达后HeLa细胞中差异表达基因,miR-205引发宫颈癌是多因素多基因共同作用的结果。     

Fresh and frozen-thawed human sperm bind in a similar pattern to the zona pellucida in the hemizona assay.

This study evaluated the impact of sperm cryopreservation on sperm quality. The HZA was used to test the binding capacity of fresh as opposed to frozen-thawed sperm from 12 donors. Fresh and frozen-thawed sperm motility was 47% +/- 1.5% and 24% +/- 3.8% (mean +/- SE), respectively. However, the number of sperm cells attached to the hemizonae was 75 +/- 12.0 and 74 +/- 11.9, respectively. We conclude that cryopreservation results in a reduced number of motile sperm cells but does not adversely affect the ability of rescued sperm cells to bind to the ZP. The study also supports the use of frozen-thawed rather than fresh donor sperm for control in the HZA procedure.     

Seven pregnancies and deliveries from non-mosaic Klinefelter syndrome patients using fresh and frozen testicular sperm

Purpose: The aim of this study was to investigate the feasibility of using frozen-thawed testicular sperm as well as the timing of testicular sperm extraction (TESE) in patients with non-mosaic Klinefelter syndrome. Methods: Intracytoplasmic sperm injection (ICSI) was performed in six of 17 (35%) patients whose sperm was recovered by TESE. Multiple biopsies of both testes were performed on the day of oocyte retrieval in all but one of the six patients. Results: Seven pregnancies and deliveries were achieved in five couples, and one couple was unsuccessful. Five pregnancies were achieved using fresh motile sperm, and two were achieved using frozen-thawed sperm. Sperm cryopreservation was not possible in one of the five couples because of the small number of recovered sperm, and possible in four other couples for subsequent ICSI. One woman whose husband had TESE performed prior to ovarian stimulation did not become pregnant. This may be due to the attainment of only a few immotile sperm following the frozen-thawed procedure. Conclusion: The outcome of ICSI using fresh or frozen-thawed testicular sperm in patients with non-mosaic Klinefelter syndrome was identical; however, TESE should be performed on the day of oocyte retrieval until such time as a procedure with a higher sperm yield from TESE is available. Moreover, an improved recovery procedure after cryopreservation-thawing of a single spermatozoon must be developed.     

Comparison of in-vitro outcomes from cryopreserved oocytes and sibling fresh oocytes

In Italy, the restrictive IVF law generalizes the indication for oocyte freezing for surplus oocytes in 78.5% of in-vitro assisted reproductive cycles. With a view to understanding better what the prospects for intracytoplasmic sperm injection (ICSI) on frozen-thawed oocytes might be, the consequences of freeze-thaw procedures on fertilization, cleavage rates and embryo quality obtained from frozen-thawed oocytes were studied and compared with the results obtained from sibling fresh oocytes. Eleven IVF and 29 ICSI on 76 and 169 fresh oocytes were performed and the corresponding 40 ICSI on 221 sibling frozen-thawed oocytes. There was no difference in terms of fertilization rate between fresh and sibling frozen-thawed oocytes. The cleavage rate (98.0 and 94.4% with fresh oocytes in IVF and ICSI; 77.3% with frozen-thawed oocytes in ICSI; P < 0.001) and embryo quality (grade I embryos over total embryos: 36.7 and 22.2% with fresh oocytes in IVF and ICSI; 12.1% with frozen-thawed oocytes in ICSI; respectively P < 0.001 and P < 0.05) were statistically lower after oocyte cryopreservation. The significant decrease in meiotic spindle retrieval rate before freezing (62.4%) and after thawing procedures (43.4%; P < 0.001) suggests that cryoconservation induces irreversible damage to microtubule repolymerization. The consequences of oocyte cryopreservation procedures on embryo development are reviewed.     

Live birth following transfer of a cryopreserved embryo generated from a cryopreserved oocyte and a cryopreserved sperm: Case report

As is the case with non-frozen oocytes, the efficient and successful use of cryopreserved oocytes in human assisted reproduction is, in part, dependent on the ability to apply selection criteria when choosing the ‘best’ embryos for transfer from a cohort. In many cases this, in turn, will necessitate the cryopreservation of non-transferred embryos to minimise the risk of multiple pregnancy. It is therefore important to establish that an embryo, generated by fertilization of a frozen-thawed oocyte, can be capable of surviving subsequent cryopreservation while retaining the potential for normal development. In this case report, we document the delivery of a normal male infant following transfer of a frozen-thawed embryo, generated by the fertilization of a frozen-thawed oocyte by a frozen-thawed sperm.     

Lectin histochemistry of ovarian mucinous cystadenomas.

The lectin histochemistry of ovarian mucinous cystadenoma was studied using a panel of lectins comprising Triticum vulgaris, Lotus tetragonolobus, Arachis hypogaea, Griffonia simplicifolia I, concanavalin A, and Dolichus biflorus. All 23 cases examined in this study stained extensively with T. vulgaris. Of all the lectins, D. biflorus was the least reactive. Concanavalin A stained mainly the perinuclear zone, whereas the other lectins frequently stained the cytoplasm, glycocalyx, and extracellular luminal mucin. This pattern of lectin reactivity resembles endocervical more than intestinal epithelium and suggests that ovarian mucinous cystadenomas are of müllerian nature. The lack of difference in lectin reactivity between cystadenomas with and without goblet/Paneth cells supports a common histogenetic origin of both groups.     

Exogenous gonadotropin administration affects the glycocalyx of rat endometrial epithelial cells during the period of implantation

Purpose Exogenous gonadotropins which cause superovulation are known to effect endometrial morphology, including the glycocalyx of surface epithelial cells. Certain of the carbohydrates in the glycocalyx of surface epithelial cells may be involved in the attachment and implantation of the blastocyst.Methods The effect of exogenous gonadotropins on specific carbohydrates in the glycocalyx of the rat endometrium around the time of implantation was investigated. Lectin-avidin-biotin-ferritin cytochemistry was used to ascertain which carbohydrates were affected. The lectins soybean agglutinin, fucose binding protein and wheat germ agglutinin were used.Results Statistically significantly less lectin was associated with the apical membrane of surface epithelial cells of animals following hyper stimulation than in noninjected pregnant animals.Conclusions The reduction in the carbohydrates contributes to a reduced receptivity of the endometrium for the blastocyst.     

Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection

OBJECTIVE: To determine the feasibility of using frozen-thawed testicular spermatozoa for intracytoplasmic sperm injection. DESIGN: Prospective clinical study. SETTING: A university hospital. PATIENT(s): One hundred seventy-five azoospermic men participating in a routine intracytoplasmic sperm injection program. INTERVENTION(s): The men underwent testicular biopsy for cryopreservation of tissue to be used in consecutive intracytoplasmic sperm injection treatment cycles. Their female partners underwent controlled ovarian hyperstimulation for conventional IVF treatment. MAIN OUTCOME MEASURE(s): Fertilization and pregnancy rates. RESULT(s): In 77% of the patients, spermatozoa could be harvested from the testis by an open testicular biopsy technique and used for intracytoplasmic sperm injection after freezing and thawing of testicular tissue. Histopathologic evaluation revealed a Sertoli cell-only pattern in 21%, maturation arrest in 60%, and hypospermatogenesis in 19% of the patients. In 2. 9% of the patients, carcinoma in situ or a germ cell tumor was detected. In all patients, viable spermatozoa could be visualized after the tissue samples were thawed. One hundred thirty-five intracytoplasmic sperm injection treatment cycles were performed, with a fertilization rate of 45% and a clinical pregnancy rate of 30% per oocyte retrieved. CONCLUSION(s): The use of frozen-thawed testicular tissue allows ovarian stimulation of the female partner to be timed and avoids cancellation of ovum pick-up when spermatozoa cannot be retrieved.     

Cumulative live birth rates after transfer of cryopreserved ICSI embryos

A cohort follow-up study was designed to assess the efficacy of an intracytoplasmic sperm injection cryopreservation programme through analysis of cumulative live birth rates in successive frozen-thawed cycles in a tertiary referral centre. There were 2013 patients and they underwent 2680 frozen-thawed embryo transfer cycles. The follow-up period was between 1992 and 2001. Only frozen-thawed embryo transfer cycles up to the fourth trial were included. Crude cumulative live birth rates were calculated in five age subgroups, i.e. <30, 30-34, 35-37, 38-39 and >/=40 years old and in surgically or non-surgically retrieved sperm subgroups. Expected cumulative live birth rates were calculated only for the total number of patients. Outcome measure was a live birth occurring after 25 weeks of gestation. Overall, the expected cumulative live birth rate was as high as 26.7% after four cycles while the crude cumulative delivery rate was 10.5%. Multiple cryopreserved embryo transfer cycles increase the chance of a couple to achieve a live birth.     

Strategies for fertility preservation in female and male cancer survivors

Improved survival rates and quality of life following modern cancer treatment have resulted in a growing number of patients requesting maintenance of reproductive capacity, both before and after completion of treatment. Several established options are currently available. In men, sperm banking should be offered as soon as the diagnosis of any malignant disease is established, irrespective of the expected cryosurvival rate. In such cases, conception can be achieved with frozen-thawed spermatozoa following either intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI). In women, depending on the type of cancer, the presence of an adequate ovarian reserve, and time to delay cancer treatment, in vitro fertilization (IVF) with embryo cryopreservation constitutes a valid alternative. On the other hand, cryopreservation of mature oocytes following IVF/ICSI offers some advantages, but it is still limited by its low success rate. Emerging and exciting techniques of germ cell/gonadal tissue cryopreservation (banking) followed by autotransplantation have been clinically explored, particularly in women. Novel cryotechnologies of ovarian and testicular tissue have proven efficacious and new transplantation strategies promise improved results. However, only one live birth following autotransplanted frozen-thawed ovarian tissue has been established and there are still no pregnancies reported from autotransplanted cryopreserved testicular tissue in the human. Efficiency and safety of these techniques needs to be demonstrated. Methods for gamete in vitro maturation also need further development. Gonadal tissue cryopreservation and transplantation should be considered experimental in humans for the present time until greater evidence regarding efficacy and safety is accrued.     

Intentional cryopreservation of epididymal spermatozoa from percutaneous aspiration for dissociated intracytoplasmic sperm injection cycles

BACKGROUND: To investigate the possibility of cryopreservation of spermatozoa obtained from percutaneous epididymal sperm aspiration (PESA) in patients with obstructive azoospermia and the feasibility of intentional dissociation of PESA and intracytoplasmic sperm injection (ICSI) cycles. METHODS: Fifty-six patients with obstructive azoospermia underwent diagnostic PESA before ovarian stimulation. If spermatozoa were found, they were frozen for subsequent ICSI. The outcome was compared with 17 fresh PESA/ICSI cycles. RESULTS: Among the 56 patients, diagnostic PESA obtained spermatozoa in 51 patients. The mean motility of the spermatozoa decreased from 15.2% to 4.2% after freezing and thawing. These patients underwent 96 frozen PESA/ICSI cycles. The rates of fertilization, implantation and clinical pregnancy for frozen-thawed spermatozoa (71.6, 14.0 and 40.6%, respectively) were similar to those for fresh spermatozoa (69.2, 13.2 and 41.2%, respectively). CONCLUSIONS: Sufficient numbers of spermatozoa can be obtained for cryopreservation through PESA and the spermatozoa work well after thawing. The strategy of performing diagnostic PESA before ovarian stimulation and freezing the recovered spermatozoa for subsequent ICSI is feasible for patients with obstructive azoospermia.