畸形精子症患者精子染色体非整倍体的研究

目的:研究畸形精子症患者精子染色体的非整倍体率。方法:应用18号、X和Y染色体着丝粒探针,采用荧光原位杂交(FISH)技术比较畸形精子症患者(畸精组,n=18)和生育力正常且精子正常形态率、浓度、活力等均正常男性(对照组,n=5)精子中18号、X和Y染色体的非整倍体率。结果:畸精组共计数精子58 178条,对照组共计数精子16 369条。畸精组和对照组杂交效率分别为97.5%和98.3%;染色体非整倍体类型主要有二体(XX18、YY18、XY18、Y1818和X1818)和二倍体(1818XX、1818YY、1818XY)。畸精组和对照组的18号染色体二体率分别为0.29±0.16%和0.03±0.02%,性染色体二体率分别为0.65±0.24%和0.05±0.02%,二倍体率分别为0.14±0.12%和0.04±0.03%。18号、X和Y染色体非整倍体率组间均有统计学差异(P<0.05)。结论:与生育力和精液各参数均正常男性相比,畸形精子症患者18号、X和Y染色体非整倍体率明显升高。     

不同源性及不同参数精子对ICSI结局的影响

目的:研究不同源性精子、不同参数精子及冻融精子对ICSI结局的影响。方法:接受ICSI治疗的510对不育夫妇共进行了517个周期,分为6组。射出精液少弱畸精子症组(A组)82例,共进行85个周期;射出精液严重少弱精子症组(B组)170例,共进行174个周期;附睾细针穿刺抽吸精子组(C组)108例,共进行108个周期;睾丸细针穿刺抽吸精子组(D组)71例,共进行71个周期;冻融射出精子组(E组)34例,共进行34个周期;冻融附睾睾丸精子组(F组)45例,共进行45个周期;比较其妊娠结局。结果:6组患者一般情况比较无统计学差异(P>0.05),6组患者的受精率、优胚率、临床妊娠率、早期流产率无统计学差异(P>0.05)。结论:不同源性精子、不同参数精子及冻融精子对ICSI结局无明显影响。     

Y染色体长臂缺失及不分离不育男性1例报道

目的:报道1例Y染色体长臂缺失合并不分离的男性无精子症患者。方法:常规染色体核型分析,荧光原位杂交以确定核型。PCR-STSs检测以确定Y染色体断裂点,并行睾丸活检。结果:细胞遗传学和FISH证实患者为嵌合体,核型为45,X/46,X,del(Y)/47,X,del(Y)del(Y)。分别占27%,68%,5%。C带显示患者Yq12全部丢失。PCR-STSs检测AZFa存在,AZFb和AZFc区域全部丢失,断裂点位于sY88和sY95之间及sY88以下。睾丸病理显示精曲小管中只有支持细胞,没有生精细胞。未见卵巢组织。结论:患者无精子症、睾丸体积小与病理结果一致,其原因是由于Yq11.2的缺失。     

附睾降解性不活动精子症的研究

目的 :探讨精子不活动是否由附睾降解引起。方法 :本文选择 5例精液中无活动精子的不育患者 ,应用睾丸精子活力检测、精子头 -尾膜完整性结合试验和透射电镜观察 ,探讨其精子不活动的原因。结果 :5例患者睾丸活检组织所分离的睾丸精子 ,经孵育后的活动率为 2 %～ 1 1 %。睾丸精子组中头膜 -尾膜均完整的精子率显著高于射出精子组 ( P<0 .0 1 )。睾丸精子未见明显的降解 ,但透射电镜显示射出精子的浆膜、核等结构表现显著的降解变化。结论 :本组患者的精子可能经历了病理性附睾降解 ,引致精子丧失活动性 ,对这类患者采用活动的睾丸精子作辅助生育治疗有可能改善成功率。     

精子来源对卵胞浆内单精子注射临床结局及子代出生缺陷的影响

目的:探讨采用射出、经皮附睾穿刺取精术(PESA)及经皮睾丸精子取精术(TESA)获取的精子行卵胞浆内单精子注射(ICSI)的临床结局及子代安全性.方法:回顾分析2004年1月至2011年12月因男性因素于我院生殖中心行[CSI治疗的3079个新鲜周期,按精子来源分为射精组(2199个周期)、PESA组(628个周期)、TESA组(252个周期),比较3组的胚胎发育、妊娠结局及新生儿出生缺陷的情况.结果:射精组受精率最高(78.38％),TESA组受精率最低(72.30％).射精组、PESA组的2PN受精率、卵裂率高于TESA组(74.68％、75.32％ vs 68.22％,98.82％、98.74％ vs 96.89％);PESA组临床妊娠率、胚胎植入率(53.21％、34.31％)显著高于射精组(47.11％、29.09％)及TESA组(48.71％、32.70％) (P＜0.05).PESA组的新生儿体重(2856.63±649.56)显著低于射精组(2991.73±683.19)及TESA组(2906.11 ±638.76) (P＜0.05).3组的分娩率、异位妊娠率、流产率、正常体重儿率、低出生体重儿率、极低出生体重儿率、巨大儿率、新生儿死亡率及出生缺陷率均无显著差异(P＞0.05).结论:PESA、TESA结合ICSI技术安全可行,且对于梗阻性无精症患者,附睾取到精子行ICSI的患者较附睾取不到精子而采用睾丸精子ICSI的患者具有更好的妊娠结局.     

毛细管电泳用于Y染色体微缺失的研究

目的:探讨毛细管电泳在研究男性不育中特发性无精子症和射出精液严重少精子症与Y染色体无精子因子(azoospermiafactor,AZF)缺失的作用。方法:应用多重PCR技术对4例无精子症和29例严重少精子症患者的外周血细胞中Y染色体AZF所在的11.23区的15个位点进行扩增,分别用琼脂糖凝胶电泳和毛细管电泳分离扩增产物,并以6例正常生育男性和2例女性为对照。结果:毛细管电泳发现4例无精子症中3例发生缺失,29例严重少精子症患者中6例发生缺失。其中6例同为SY254(C)、SY242(C)、SY255(C)、SY239(C)、SY152(D)缺失。6例正常生育男性毛细管电泳未发现Y染色体微缺失,而凝胶电泳有2个条带模糊,难以判断。结论:毛细管电泳可提高AZF检测准确性。Y染色体AZFc/DAZ的缺失可能是引起无精子和严重少精子并造成男性不育的重要原因之一。     

98％无头精子一例并文献复习

目的：报道98％无头精子l例并探讨其发生机制,旨为该类患者的诊治提供实验室依据。方法：对此例无头精子患者的睾丸曲细精管病理结构和精子超微结构进行分析;将20％、15％无头精子患者设为对照组,分别对3例无头精子患者的精子进行精子染色质扩散实验（spermnucleusDNAintegritykit,SCD）合并精子荧光原位杂交技术（fluorescenceinsituhybridization,FISH）检测。结果：无头精子患者睾丸曲细精管严重病变;在透射电子显微镜下可见到精子顶体、线粒体、轴丝等程度不同的结构异常：无头精子患者的精子染色体非整倍体率与精子核DNA损伤程度成正比,且随无头精子比例增多而升高。结论：无头精子有病理意义,其发生与精子DNA损伤、精子染色体异常以及编码头尾连接段的基因缺陷有关。     

少量人类精子以空卵透明带为冻贮载体的冷冻保存

目的:探讨空卵透明带作贮存载体冷冻保存少量人类精子。方法:将人或金黄地鼠卵内的细胞成份全部除去,制备成空卵透明带,分别显微注入人睾丸精子、附睾精子和射出精子后冷冻保存,并与正常供精者射出精子作对照。结果:解冻后卵透明带在溶液中容易识别寻找,卵透明带内的精子易于观察。附睾精子组(n=11)与供精者射出精子组(n=7)的冷冻精子活动率和存活率无显著性差异(P>0.05),但睾丸精子组(n=7)这两项参数值均显著低于附睾精子和射出精子组(P<0.01)。含6％、7.5％和9％不同甘油浓度的冷冻保护剂,对冷冻精子活动率无显著性影响(P>0.05);冻贮于人和金黄地鼠空卵透明带内的精子,两者的冷冻精子活动率也无显著性差异(P>0.05〕。结论:空卵透明带是冻贮少量人类精子的合适载体。     

男性不育症精子发生相关基因缺陷的筛查研究

目的:探讨男性不育患者精子发生相关基因缺陷与精子生成的关系。方法:应用多重聚合酶链反应(PCR)扩增分析方法对149例男性不育症患者及100例有正常生育能力的男子进行Y染色体上相关基因检测和常规外周血染色体核型分析。结果:不育症组有11例存在着Y染色体上不同基因片段的微缺失,缺失率为7.38％,染色体异常核型发生率为14.09％;而正常对照组均未发现相应部位的缺失,异常核型发生率为2％。11例存在Y染色体上不同基因片段微缺失者只有1例合并有异常核型,说明两者之间无相关性。结论:提示Y染色体微缺失是引起男性不育的一个重要原因,在进行单精子卵泡浆内注射(ICSI)时应进行Y染色体微缺失的分子检测,以免所生的男性后代亦有与其父亲相同原因的不育问题。     

睾丸固定钳固定法在经皮附睾穿刺取精术中的应用价值

目的评价睾丸固定钳固定法在经皮附睾穿刺取精术(PESA)中的应用价值。方法选取初步诊断为梗阻性无精子症患者532例,将其随机分为三指固定法组(249例)和睾丸固定钳组(283例),比较两组PESA穿刺精子获取率差异。另根据经阴囊超声附睾头有无扩张以及扩张特征将病例分为附睾头细网状扩张亚组、附睾头管状/多囊管状扩张亚组和附睾头无扩张亚组,比较两种PESA方法对不同附睾头病变穿刺精子获取率的差异。结果三指固定法组穿刺精子获取率为60.64%(151/249),睾丸固定钳组为74.56%(211/283),显著高于三指固定法组(P0.05)。睾丸固定钳组穿刺精子获取率的优势主要由细网状扩张组贡献,该组三指固定法穿刺精子获取率为72.67%(125/172),而睾丸固定钳法为89.90%(178/198),显著高于三指固定法组(P0.05)。管状/多囊管状扩张亚组以及附睾头无扩张亚组2种PESA法穿刺精子获取率都偏低,差异无统计学意义(P0.05)。结论使用睾丸固定钳固定法对附睾头细网状扩张的患者进行PESA穿刺能提高精子获取率。     

Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection.

OBJECTIVE: To determine the incidence of nondisjunction for chromosomes X, Y, and 18 using fluorescence in situ hybridization (FISH) on morphologically normal sperm from infertile men who are candidates for ICSI. DESIGN: After standard hematoxylin staining, sperm with normal morphology were identified using Kruger's strict morphology criteria. The location of each normal-appearing sperm was recorded using an electronic microstage locator. Slides were subsequently subjected to FISH for detection of chromosomes X, Y, and 18 (control probe). Nuclei were relocated and analyzed under the fluorescent microscope. SETTING: University-affiliated IVF and intracytoplasmic sperm injection program. PATIENT(s): Men classified as infertile on the basis of abnormal strict morphology (<4% by Kruger's criteria). For controls, normal fertile men (n=6) were also analyzed. INTERVENTION(S): Semen smears were obtained retrospectively from infertile (n=8) and fertile (n=6) men. MAIN OUTCOME MEASURE(s): Ploidy of each cell was determined according to the number of signals detected for each probe. RESULT(S): Approximately 100-150 morphologically normal sperm were identified and located in each case. Subsequent FISH analysis of these normal sperm showed aneuploidy to range from 1.8% to 5.5% in the infertile group as compared with 0 to 2.6% among the control fertile group. Statistically significant differences in the incidence of aneuploidy for the sex chromosomes as well as for all three (X, Y, and 18) chromosomes was observed. CONCLUSION(S): Although 95% to 98% of the sperm were found to be normal for X, Y, and 18, our findings show that infertile couples undergoing ICSI are likely to be at an increased risk for having a genetically abnormal conceptus as compared with the fertile controls. These results demonstrate that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Hence, observed pregnancy failures among ICSI patients may in part be due to the selection of aneuploid sperm.     

High sex chromosome aneuploidy and diploidy rate of epididymal spermatozoa in obstructive azoospermic men

Purpose: To evaluate the frequencies of sex chromosome aneuploidy and diploidy rate of epididymal spermatozoa from obstructive azoospermic men and its impact on intracytoplasmic sperm injection (ICSI) outcomes. Methods: Epididymal spermatozoa retrieved from 24 obstructive azoospermic men and ejaculated spermatozoa from 24 fertile donors were analyzed using triple color fluorescence in situ hybridization (FISH) techniques, in order to investigate the rates of diploidy and aneuploidy for chromosomes 18, X and Y. Results: Epididymal spermatozoa from obstructive azoospermic men had total sex aneuploidy, disomy 18, and diploidy rates significantly higher than ejaculated spermatozoa from normozoospermic fertile controls (1.44% vs. 0.14%, 0.11% vs. 0.02%, and 0.18% vs. 0.02%, respectively; p < 0.005). There were no statistically significant differences in ICSI outcomes between the patients who had high and low epididymal sperm aneuploidy rate. Conclusions: Epididymal spermatozoa from obstructive azoospermic patients had an elevated sex chromosome aneuploidy and diploidy rate. The increased frequency of chromosomal abnormalities did not have a direct effect on the ICSI outcome.     

Sperm chromosome analysis and outcome of IVF in patients with non-mosaic Klinefelter's syndrome

OBJECTIVE: The aim of the study was to determine the potential risk for fetal chromosomal anomalies in non-mosaic Klinefelter's syndrome patients undergoing IVF and intracytoplasmic sperm injection. DESIGN: Individually collected spermatozoa were isolated from wet testicular tissue preparations and fixed on glass slides using micromanipulation. Their nuclei were analyzed for chromosomes X, Y, and 18 by fluorescent in situ hybridization. SETTING: Assisted reproductive technology program. PATIENT(S): Consenting patients with non-mosaic Klinefelter's syndrome undergoing testicular biopsy and IVF (fresh specimens) or following such treatment (cryopreserved specimens). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The rates of numerical chromosome abnormalities for chromosomes X, Y, and 18 among spare testicular sperm and the pregnancy outcome following treatment. RESULT(S): Testicular sperm were found in 8 of 20 patients. Four couples became pregnant following embryo replacement. Sperm chromosomes were analyzed in five patients. One hundred and five sperm of 112 analyzed (93.7%) were normal with X to Y ratio of 50:55 (NS) respectively. Among the 112 sperm tested, seven (6.3%) demonstrated chromosomal abnormalities, of which five were related to the sex chromosomes and two to chromosome 18. One set of triplets, one set of twins, and two singletons (four males and three females) with normal karyotypes were born. CONCLUSION(S): Most of the testicular sperm retrieved from Klinefelter's syndrome patients demonstrates a normal pattern of sex chromosome segregation. Therefore, the risk of transmitting numerical sex chromosome abnormalities is relatively low and probably comparable with the rates found in other severe male factor infertility patient groups.     

Sperm chromosome abnormalities in men with severe male factor infertility who are undergoing in vitro fertilization with intracytoplasmic sperm injection

OBJECTIVE: To investigate the potential paternal contribution to the risk of fetal chromosomal anomalies after intracytoplasmic sperm injection (ICSI). DESIGN: Spermatozoa isolated from testicular tissue and ejaculated specimens of consenting patients undergoing testicular biopsy and ICSI were analyzed for chromosomes X, Y, and 18 by FISH. SETTING: Assisted reproductive technology program. PATIENT(S): Consenting patients undergoing testicular biopsy and ICSI, severe oligozoospermic patients, and normal fertile donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The rate of chromosome abnormalities in testicular sperm with regard to the type of azoospermia and ejaculated sperm compared to healthy men. RESULT(S): The mean serum levels of FSH in the groups with nonobstructive azoospermia (n = 9), obstructive azoospermia (n = 10), severe oligozoospermia (n = 9), and the normal donors (n = 6) were 17.5 +/- 8.2 (P<.05), 3.5 +/- 2.6, 14.6 +/- 3.5 (P<.05), and 3.1 +/- 0.4 IU/mL, respectively. The corresponding rates of sperm chromosome abnormalities among these groups were 19.6% (P<.001), 8.2% (P<.001), 13.0% (P<.001), and 1.6%, respectively. The corresponding rates of disomy among these groups were 7.8% (12 of 153 spermatozoa), 4.9% (18 of 367), 6.2% (109 of 1,751), and 1% (5 of 500 spermatozoa), respectively. Errors in chromosomes X and Y were significantly more common than in chromosome 18. CONCLUSION(S): The present findings demonstrate a linkage between gonadal failure (high serum FSH levels) and the occurrence of sperm chromosome aneuploidies. Our findings may explain the increased incidence of sex chromosome abnormalities found after IVF in the severe male factor patient population. Genetic screening during pregnancy or before embryo replacement should be considered carefully.     

Analysis of aneuploidy in mini-percoll gradient centrifuged human sperm for intracytoplasmic sperm injection (ICSI) using fluorescence in situ hybridization

AIM: To study the aneuploidy rates of chromosomes 13, 18, 21, X and Y in Percoll gradient centrifuged sperm from infertile patients with male infertility factor treated by intracytoplasmic sperm injection (CSI) compared with healthy fertile donors and infertile patients with normal semen parameters. METHODS: This case-controlled study was conducted in a university hospital. Semen samples were obtained from three healthy fertile donors, eight infertile patients with normal semen parameters, and 18 infertile patients with male infertility factor. All samples were subjected to mini-Percoll gradient centrifugation before being processed through fluorescent in situ hybridization. The incidences of aneuploidy were compared using Chi-squared test. RESULTS AND CONCLUSIONS: A total of 64949 spermatozoa were analyzed. The disomy rates for chromosomes 13, 18, 21, and X or Y of sperm from patients with male infertility factor were 0.21%, 0.37%, 0.36% and 0.63%, respectively, whereas the diploidy rate was 0.17-0.23%. These incidences were higher than those from men with normal semen parameters. The result suggested that the embryos of patients with male infertility factor treated by ICSI are at increased risk of chromosome abnormalities.     

A comparison of the morphology of testicular, epididymal, and ejaculated sperm from fertile men and men with obstructive azoospermia

Objective: To assess the morphology of testicular, epididymal, and ejaculated sperm.

Design: Morphology of the three types of sperm was assessed by using Tygerberg strict criteria.

Setting: The Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom.

Patient(s): Thirty-two men with obstructive azoospermia and 10 fertile men.

Intervention(s): Trucut needle testicular biopsy and percutaneous epididymal sperm aspiration under local anesthetic.

Main Outcome Measure(s): Percentages of normal sperm and sperm with head, midpiece, and tail defects for testicular, epididymal, and ejaculated sperm. Testicular sperm morphology in men with obstructive azoospermia was compared with that of fertile men.

Result(s): The percentage of normal testicular sperm (4.3%) differed significantly from the percentages of normal epididymal (10.8%) and ejaculated sperm (9.6%). Testicular sperm morphology in men with obstructive azoospermia did not differ from that in fertile men.

Conclusion(s): Tygerberg strict criteria are not suitable for the assessment of testicular sperm morphology.     

Penetration of zona-free hamster oocytes using human sperm aspirated from the epididymis of men with congenital absence of the vas deferens: comparison with human in vitro fertilization.

OBJECTIVES: To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF). DESIGN: Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF. PATIENTS, PARTICIPANTS: Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm. RESULTS: Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71%. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative. CONCLUSIONS: These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm.     

体外受精后多原核受精卵的移植价值

目的:对体外受精周期中产生多原核受精卵运用荧光原位杂交技术(FISH)进行非整倍体率检测,分析其移植价值。方法:应用FITC、Texas red标记的X/Y双色染色体着丝粒部位探针对体外受精(IVF)和单精子显微注射(ICSI)周期中的三原核受精卵(3PN)进行荧光原位杂交和分析。结果:分析有杂交信号的3PN卵子68个,IVF后3PN的三倍体率为92.31%,二倍体率为3.85%,单倍体率为3.85%;共存在3种三倍体核型:XXX、XXY、XYY。ICSI后3PN 的三倍体率为70.83%,二倍体率为25%,单倍体率为4.17%;共存在2种三倍体核型:XXX、XXY。结论:IVF后3PN卵子中多余的原核多为精子来源,没有移植价值;ICSI后3PN中多余的原核非精子来源,ICSI后3PN中二倍体率与IVF相比显著增高,在患者ICSI后无2PN卵子时可考虑行植入前遗传学诊断。     

Chromosomal abnormalities in embryos derived from testicular sperm extraction

OBJECTIVE: To compare the rate of chromosome abnormalities in embryos obtained from karyotypically normal patients with nonobstructive azoospermia undergoing testicular sperm extraction (TESE) to those from patients undergoing intracytoplasmic sperm injection (ICSI) with ejaculated sperm. DESIGN: Retrospective analysis. SETTING: IVF centers. PATIENT(S): Male partners had either nonobstructive zoospermia or oligospermia. INTERVENTION(S) : Preimplantation genetic diagnosis. Chromosome enumeration was performed by fluorescence in situ hybridization (FISH). Embryos classified as abnormal were reanalyzed to study mosaicism. MAIN OUTCOME MEASURE(S): Chromosome abnormalities in embryos. RESULT(S): Embryos from ICSI cycles with ejaculated sperm (group 1) were 41.8% normal, 26.2% aneuploid, and 26.5% mosaic. In contrast, the embryos from ICSI cycles with TESE for nonobstructive azoospermia (group 2) were 22% normal, 17% aneuploid, and 53% mosaic. The difference in mosaicism rate between the two groups of embryos was highly significant. CONCLUSION(S): The present study results indicate a high incidence of mosaicism in embryos derived from TESE in men with a severe deficit in spermatogenesis. Sperm derived from TESE for nonobstructive azoospermia may have a higher rate of compromised or immature centrosome structures leading to mosaicism in the embryo.