应用荧光原位杂交技术对精子染色体非整倍体的研究

目的 探讨人类精子染色体数目异常与自然流产的相关性。方法 采用多色荧光原位杂交（fluorescence in situ hybridization,FISH)方法，利用CEP（chromesome enumeration DNA probes)X、Y、18及LSI（locus specific identifier DNA probes)13、21两种探针混合液与二硫苏糖醇处理过的自然流产者丈夫的精子核杂交，记数杂交信号，与对照组进行比较。结果 经卡方检验，自然流产组丈夫精子染色体X、Y、18、13、21双体型及其它数目异常明显高于正常对照组（P＜0．005），说明流产者丈夫精子染色体异常是流产的重要原因之一。结论 FISH技术是一种快速、准确、简单、实用性强的检测精子染色体的好方法。     

应用荧光原位杂交技术对精子染色体非整倍性的研究

目的探讨人类精子染色体数目异常与自然流产的相关性. 方法采用多色荧光原位杂交(fluorescence in situ hybridization, FISH)方法,利用CEP(chromesome enumeration DNA probes)X、Y、18及LSI(locus specific identifier DNA probes)13、21两种探针混合液与二硫苏糖醇处理过的自然流产者丈夫的精子核杂交,记数杂交信号,与对照组进行比较.结果经卡方检验,自然流产组丈夫精子染色体X、Y、18、13、21双体型及其它数目异常明显高于正常对照组(P<0.005),说明流产者丈夫精子染色体异常是流产的重要原因之一.结论 FISH技术是一种快速、准确、简单、实用性强的检测精子染色体的好方法.     

精子染色质结构与不明原因复发性流产的相关性

目的通过精子染色质结构分析(SCSA),探讨其与不明原因复发性流产(URSA)的相关性。方法分析URSA患者60例配偶的精液情况,检测精子二项SCSA参数(精子染色质扩散实验计算精子DNA碎片指数,DFI;CMA3染色法检测染色质包装质量)。结果 URSA流产患者SCD小光晕和无光晕精子(精子DNA碎片)比值平均为(26.7±10.6)%,URSA患者组明显高于对照组(11.9±5.9)%,(P0.001),而大光晕和中光晕精子比值URSA患者组明显低于对照组(P0.001);URSA组CMA3染色阳性率(31.36%)明显高于对照组(19.13%)。结论精子染色质结构异常与不明原因复发性流产发生具有的相关性。     

精子染色质结构异常对体外受精-胚胎移植结局的影响

目的 探讨精子染色质结构异常对体外受精-胚胎移植(in vitro fertilization and embryo transfer,IVF-ET)的影响.方法 对IVF-ET治疗的不育夫妇136对,分别检测精子DNA碎片化和染色质包装缺陷,并分析精子染色质结构参数与受精及临床妊娠之间的关系.结果 精子DNA碎片化阳性率和染色质包装缺陷阳性率均与受精率呈负相关(r值分别为-0.198,P＜0.05,和-0.389,P＜0.01);在未获得临床妊娠的患者,精子DNA碎片化阳性率和染色质包装缺陷阳性率均高于获得临床妊娠的夫妇(分别为10.74%vs.5.40%,P＜0.01和23.58%vs.11.83%,P＜0.01).结论 IVF-ET治疗失败与精子染色质结构异常有关,精子染色质结构检测有助于评估IVF-ET失败的风险,以制订最佳治疗方案.

Abstract：

Objective To explore the effects of sperm chromatin structure abnormalities on the outcome of in vitro fertilization and embryo transfer (IVF-ET). Methods Sperm DNA fragmentation and chromatin packaging defects were assessed in 136 couples undergoing IVF-ET because of infertility. The relationship between sperm DNA fragmentation, chromatin packaging defects and fertilization rate and clinical pregnancy was evaluated. Results Both sperm DNA fragmentation and chromatin packaging defect had a negative correlation with fertilization rate (r= -0. 198, P＜0. 05, and r= -0. 389, P＜0. 01,respectively). Both parameters were higher in couples who failed to achieve pregnancy than those who achieved clinical pregnancy (10. 74% vs. 5. 40%, P＜0. 01 and 23. 58% vs. 11. 83%, P＜0. 01,respectively). Conclusion Abnormality of sperm chromatin structure is one of the reasons for IVF-ET failure. Examination of sperm chromatin structure is helpful in predicting he risk of IVF-ET failure and optimizing treatment of infertility.     

吸烟对精子DNA碎片的影响

目的探讨男性不育症患者吸烟对精子DNA碎片的影响。方法采用精子染色质扩散（SCD）实验检测383例男性不育患者精子DNA碎片的比率,根据吸烟多少分组并进行比较研究。结果吸烟〉20支/天组患者与吸烟≤20支/天组患者及不吸烟组患者相比DNA碎片率显著升高（P〈0.05）。吸烟≤20支/天组患者与不吸烟组患者相比DNA碎片率没有明显差别。结论吸烟对DNA碎片率有影响,且吸烟量越大、时间越长、精子DNA碎片率越高。     

不育厨师精液质量参数及精子DNA完整性分析

目的探讨不育厨师精液常规质量参数和精子畸形率、精子DNA碎片率等指标。方法对生殖门诊52名不育厨师（观察组）和49名育前体检者（对照）行精液常规和DNA碎片率等检测。结果观察组精液液化异常率、精子畸形率、DNA碎片率都显著高于对照组;精子浓度、前向运动精子比率显著低于对照组。结论厨师职业暴露会影响精液质量,导致男性不育,应加强保护和防范。     

无精子症患者减数分裂遗传重组的研究

目的 探讨无精子症患者精母细胞减数分裂过程中染色体联会与遗传重组的情况.方法 对7名汉族正常人(对照组)和7例汉族无精子症患者,包括2例梗阻性无精子症(obstructive azoospermia,OA)和5例非梗阻性无精子症(non-obstructive azoospermia,NOA)进行睾丸组织精母细胞免疫荧光染色.联会复合体蛋白3(synaptonemal complex protein 3,SCP3)抗体标记联会复合体,DNA修复蛋白(human mutL homologl,MLHl)抗体定位遗传重组位点.结果 OA组和NOA组偶线期细胞比例较对照组均显著增加,差异具有统计学意义(P＜0.05).详细分析粗线期精母细胞MLH1位点数目及分布情况,发现NOA组患者中每个细胞MLH1位点数较对照组显著减少(P＜0.05),常染色体上无MLH1位点的染色体数目较对照组显著增加(P＜0.01),含有至少1条染色体臂上无MLH1位点的细胞比例较对照组增高.分析粗线期精母细胞中SCP3形态时发现,NOA患者中SCP3不完全联会的粗线期精母细胞比例显著高于对照组(P＜0.01).结论 无精子症患者减数分裂过程有延迟现象;NOA患者精母细胞遗传重组MLH1位点的数目和分布出现异常,染色体联会不完全现象增加,这些异常事件可能与NOA患者精子发生障碍有关.     

精子染色质结构分析法(SCSA)的临床应用研究

目的通过两种检测DNA碎片的方法结果的比较,评价精子SCSA法的临床应用价值。方法收集生殖中心门诊94例男性患者精液标本,分别采用精子DNA碎片染色质结构分析法(SCSA)和改良精子染色质扩散法(SCD)两种方法检测DNA碎片指数。按精子DFI指数分为3组(≤15%DFI、15~≤30%DFI和30%DFI),分别比较两种方法的检测结果差异。结果SCSA法与SCD法的检测结果具有明显的正相关性(r=0.758,P0.001)。在≤15%DFI和30%DFI两组中,其结果无显著差异,而在15~≤30%DFI组中,SCSA法检测的结果(20.23±3.90)明显高于SCD法检测的结果(16.96±7.82),差异具有统计学意义(P0.01)结论 SCSA法和SCD法具有较好的一致性,与SCD法相比,SCSA法稳定、快速、灵敏度高,重复性好,更适用于临床门诊检测。     

男性不育患者精子染色体畸变研究

目的本研究通过精子染色体畸变分析,对正常男性、少弱精子症、严重少精子症和无精子症患者进行精子染色体畸变分析,同时建立单精注射动物卵技术。方法随机选取2004年1月-2005年11月在我院不孕不育专科就诊的男性不育症患者,年龄23～42岁,不育时间1～10年。采用动物卵自然受精或ICSI受精,进行精子染色体畸变分析。结果显示无精子症组的精子染色体畸变率19.67%、断裂率13.12%,与正常对照组比较差异非常显著性（P〈0.01）。其它两组畸变率、断裂率较正常组也有增加,但差异无统计学显著性。结论无精子症组精子染色体畸变率明显增高,这说明男性不育与精子染色体异常存在相关性,在行ICSI治疗前,用此方法进行精子染色体畸变分析是有效的,也是必要的。     

15例体细胞染色体正常的反复流产夫妇丈夫精子染色体分析

为了解体细胞染色体正常的反复流产夫妇丈夫精子染色体改变，作者利用去透明带金黄地鼠卵诱导人精子单倍染色体形成的方法对１５例反复流产夫妇丈夫精子染色体进行分析。共分析１５１２组单倍体核型，其中畸变核型发现９５组，结构异常５４组，数目异常４１组，畸变率为６．２８％。卡方检验：与１５例正常生育男性组的自然畸变率比较，Ｘ２＝４．３８，Ｐ＜０．０５，表明两组间有统计学差异。在分类比较中，对照组与流产组的数目异常的Ｘ２＝７．４９，Ｐ＜０．０１，有显著性差异；而结构异常的Ｘ２＝０．０４，Ｐ＞０．０５．无统计学差异。精子染色体检查在流产病因诊断中具有一定的临床使用价值，为诊断流产病因提供了一种新方法。     

男性不育精子细胞染色质结构流式细胞术分析

目的探讨流式细胞术在评估男性不育中的应用。方法依照WHO诊断标准,将30例正常生育男性和45例男性不育患者的精子标记相应的荧光染料,用流式细胞仪检测精子的染色质状态,并与精液常规检测方法进行比较分析。结果男性不育患者精子DNA碎片率（15.96±8.23）%高于正常生育组（8.05±3.83）%,而且不育组精子畸形率（36.67±11.2）%也高于正常组（30.23±4.75）%,活率和密度（61.62±12.65）（43.25±16.06）%均低于正常组（70.69±4.40）（57.89±21.58）%,且DNA碎片率与精子畸形率呈正相关（r=0.371,P〈0.01）,与精子活率呈负相关（r=-0.349,P〈0.01）。结论流式细胞术与常规检测方法相比,不仅具有快速、精确的优点,还可以了解精子DNA的受损程度,评价精子功能,是一种比传统精液分析更稳定更敏感的评估男性生育力的方法。     

精子DNA碎片率与男性年龄、精子活动力和体外受精结局关系的研究

目的 探讨精子DNA碎片率(DNA fragmentation index,DFI)与男性年龄、精液检查指标、体外受精(in vitro fertilization,IVF)的受精率、优质胚胎率、周期妊娠率和胚胎着床率等关系.方法 随机选取本院生殖中心111例IVF患者,使用流式细胞仪行精子DFI测定,DFI值按不同界...     

Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients

The hypothesis that sperm aneuploidy and diploidy increase as a function of spermatogenesis impairment was addressed. Ejaculated semen samples from a series of men (n = 22) with very low total normal motile count (1 x 10(6)) was analysed in terms of sperm aneuploidy and diploidy by in-situ hybridization and compared with controls (n = 10). Germ cell aneuploidy was also analysed in an additional series of infertile patients presenting unexplained infertility (n = 3), congenital absence of the vas deferens (CAVD) (n = 6) and non-obstructive azoospermia (n = 3) undergoing IVF, microsurgical epididymal sperm aspiration (MESA)/ICSI and testicular sperm extraction (TESE)/ICSI cycles respectively. In-situ hybridization for chromosomes 1, 17, X and Y was performed on ejaculate, epididymal and testicular spermatozoa. Significantly higher sperm aneuploidy and diploidy rates where found (for the four chromosomes analysed) in spermatozoa from oligoasthenoteratozoospermia (OAT) over controls (18 versus 2.28% and 2.8 versus 0.13% respectively; P < 0.001). Testicular germ cells had even higher rates of sperm aneuploidy and diploidy. However, in this group it was difficult to determine whether the cells analysed were dysmorphic spermatozoa or spermatids. The data warrant further investigation on the cytogenetic abnormalities found in most germ cells identified in testicular tissue biopsies of azoospermic patients.     

Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men

Spermatozoa from 32 infertile patients and 13 controls with normal semen parameters were analysed using dual and triple colour fluorescence in-situ hybridization (FISH) techniques, in order to investigate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y. The patients were divided into three groups according to their karyotypes or the karyotypes of their offspring: 15 were infertile men with abnormal semen parameters and normal karyotypes (group 1), 13 were infertile men with abnormal karyotypes and normal or abnormal semen (group 2) and four were infertile men with abnormal semen and normal karyotypes but whose wives conceived a child (or a fetus) with a numerical chromosomal abnormality through an intracytoplasmic sperm injection cycle (group 3). Patients with abnormal semen parameters showed a significantly higher aneuploidy rate for the investigated chromosomes in their spermatozoa compared to controls (P < 0.005). Our data suggest the presence of a correlation between poor semen parameters and an increase in aneuploidy rate of chromosomes 13, 18, 21, X and Y in spermatozoa (r = -0.81071, P < 0.002); therefore the risk of a chromosomal aneuploidy in spermatozoa seems to be inversely correlated to sperm concentration and total progressive motility. Patients with abnormal karyotypes showed a higher incidence of diploidy and chromosomal aneuploidies compared to controls (P < 0.002). This strongly suggests the presence of an interchromosomal effect of the cytogenetic rearrangement. Men who fathered a child with an abnormal karyotype through intracytoplasmic sperm injection did not present a higher aneuploidy rate for the investigated chromosomes in spermatozoa compared to patients with infertility due to a similar male factor but showed higher incidence of chromosomal aneuploidy compared to normal controls.     

Disintegration of chromosomes in dead sperm cells as revealed by injection into mouse oocytes

Intracytoplasmic sperm injection of immotile (dead) ejaculated human spermatozoa has been carried out by several centres for the treatment of infertility caused by severe asthenozoospermia (necrozoospermia). No healthy pregnancies have been reported as yet, suggesting irreversible damage to sperm DNA, centrioles and/or other important structures. We investigated this hypothesis by injection of immotile human spermatozoa obtained from several male infertility patients into mouse oocytes and analysis of the oocyte activation rate and sperm chromosome integrity. Motile spermatozoa of the same sample were used as a control. The proportion of living spermatozoa among the immotile was also assessed in each sample and was related to the results of the mouse oocyte injection test. The oocyte activation rate after injection of immotile human spermatozoa into mouse oocytes was the same or only slightly lower than after injection with initially motile spermatozoa (87-100% versus 100% respectively). The rate of normal sperm chromosome spreads correlated significantly (r = 0.90, P < 0.05) with the proportion of living immotile spermatozoa in a given sample. It varied from 4 to 48% for samples containing respectively 8 and 40% of living spermatozoa. Most of the mouse oocytes injected and activated with immotile human spermatozoa were arrested during a prolonged period of time at the interphase of the first cell cycle (from 22 to 60%). Others underwent a delayed nuclear envelope breakdown but showed signs of abnormal structure of both male and female or only the male pronuclear chromosomes. Our data demonstrate an irreversible damage of chromosomes in dead ejaculated human spermatozoa and provide an experimental basis for recommending the use of testicular or epididymal spermatozoa for treatment of male infertility due to necrozoospermia.      

A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia

BACKGROUND: Mitochondria are vital to sperm as their motility powerhouses. They are also the only animal organelles with their own unique genome; encoding subunits for the complexes required for the electron transfer chain. METHODS: A modified long PCR technique was used to study mitochondrial DNA (mtDNA) in ejaculated and testicular sperm samples from fertile men undergoing vasectomy (n = 11) and testicular sperm from men with obstructive azoospermia (n = 25). Nuclear DNA (nDNA) fragmentation was measured by an alkaline gel electrophoresis (comet) assay. RESULTS: Wild-type mtDNA was detected in only 60% of fertile men's testicular sperm, 50% of their ejaculated sperm and 46% of testicular sperm from men with obstructive azoospermia. The incidence of mitochondrial deletions in testicular sperm of fertile and infertile men was not significantly different, but the mean size of the deletions was significantly less in testicular sperm from fertile men compared with men with obstructive azoospermia (P < 0.02). NDNA fragmentation in testicular sperm from fertile men and men with obstructive azoospermia was not significantly different. CONCLUSION: Multiple mtDNA deletions are common in testicular and ejaculated sperm from both fertile and infertile men. However, in males with obstructive azoospermia, the mtDNA deletions in testicular sperm are of a larger scale.     

Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection

In this study the fertility and outcome of intracytoplasmic sperm injection (ICSI) using megalohead spermatozoa from the ejaculates and testicles was evaluated. Seventeen males with megalohead and pinhead sperm forms in their ejaculate were studied in 22 cycles. A high number of sperm heads without tails and abundant round spermatid forms were commonly observed. Round-headed spermatozoa were seldom accompanied by these severely abnormal spermatozoa. The majority of megalohead spermatozoa were observed to have multiple tails, were predominant in the sample, and were used for ICSI. Ejaculated megalohead spermatozoa were used for ICSI in 15 cycles, while testicular spermatozoa were used in seven cycles where there were no vital spermatozoa or spermatozoa of low vitality in the ejaculate. The same abnormal morphology was observed in the testicles as in the ejaculated spermatozoa in the same males. Mean (+/- SD) low motility 4.7 +/- 5.6% and sperm count (3.8 +/- 4.19 x 10(6)) were common findings in these severely teratozoospermic patients. A low fertilization rate (43.2%) was achieved by using megalohead sperm forms (group I, n = 17) in comparison with the control group (60.2%) which had zero normal sperm morphology according to strict criteria (group II, n = 30) (P <0.01). Furthermore, a low pregnancy rate (9.1%) was obtained in the megalohead sperm group in comparison with the control group (40%) (P <0.05). Low fertilization and pregnancy rates may be due to a high incidence of chromosomal abnormalities from severely defective spermatozoa in the ejaculate. Couples should be counselled and warned about possible low fertilization and pregnancy rates with ICSI when only pinhead and megalohead forms with a high number of sperm heads without tails are present in the ejaculate.     

Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa

BACKGROUND: Sperm DNA damage (fragmentation) is a recently discovered cause of male infertility for which no efficient treatment has yet been found. Previous findings have suggested that clinically relevant sperm DNA damage may occur at the post-testicular level. This study was undertaken to assess the clinical usefulness of ICSI with testicular spermatozoa in this indication. METHODS: The percentage of spermatozoa with fragmented DNA, assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay, and ICSI outcomes were compared in two sequential attempts performed, respectively, with ejaculated and testicular spermatozoa in 18 men with increased sperm DNA fragmentation. RESULTS: The incidence of DNA fragmentation was markedly lower in testicular spermatozoa as compared with ejaculated spermatozoa. No differences in fertilization and cleavage rates and in embryo morphological grade were found between the ICSI attempts performed with ejaculated and with testicular spermatozoa. However, eight ongoing clinical pregnancies (four singleton and four twin) were achieved by ICSI with testicular spermatozoa (44.4% pregnancy rate; 20.7% implantation rate), whereas ICSI with ejaculated spermatozoa led to only one pregnancy which was spontaneously aborted. CONCLUSIONS: These data show that ICSI with testicular spermatozoa provides the first efficient assisted reproduction treatment option for men with high levels of sperm DNA damage.     

Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men

Sperm samples from infertile men with oligozoospermia or teratozoospermiawere studied by multicolour fluorescence in-situ hybridization(FISH) using DNA probes for chromosomes 13 and 21. A total of90 809 sperm nuclei from nine infertile men and 182 799 spermnuclei from 18 control donors were analysed. There was a highlysignificant increase in the frequency of spermatozoa disomicfor chromosome 13 in infertile patients (0.28%) compared tocontrol donors (0.13%) (two-tailed Z statistic P <0.0001and for chromosome 21 (0.48% in infertile men versus 0.37% incontrols, P <0.0001). Also there was a significantly increasedfrequency of diploid spermatozoa in infertile men (0.85%) comparedto control donors (0.66%) (P <0.0001). Our previous studieson these same infertile patients demonstrated increased frequenciesof sperm disomy for chromosomes 1 and XY. This suggests thatinfertile men, who are prime candidates for intracytoplasmicsperm injection, may be at a very small increased risk of aneuploidoffspring.