Frequency of aneuploidy in sperm from patients with extremely severe male factor infertility

BACKGROUND: A protocol for the chromosomal analysis of sperm samples with a severely reduced number of sperm cells was designed. METHODS: A severe male factor condition was the main cause of infertility for 38 couples: 27 were oligoasthenoteratospermic (OAT) and 11 with non-obstructive azoospermia underwent testicular sperm extraction (TESE). A two-round fluorescence in situ hybridization (FISH) protocol was performed with probes specific for the chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The recording of the position of each sperm cell at the microscope allowed diagnosis of each spermatozoon for the nine tested chromosomes. RESULTS: A mean number of 122+/-78.5 sperm were diagnosed per patient with an incidence of total abnormalities corresponding to 13.4%. chi2-tests for the observed frequencies and goodness-of-fit test were highly significant in all cases. A significantly higher proportion of total aneuploidy was detected in 79% of the tested samples compared to the normal population. Testicular sperm were significantly more prone to aneuploidy than ejaculated sperm. CONCLUSIONS: The designed FISH protocol for the analysis of severe OAT and TESE sperm samples is reliable, implying that the studied sample is representative of the original population. In view of the high incidence of aneuploidy in most severe OAT and TESE sperm, the FISH analysis of pathological sperm samples can be routinely performed in order to estimate the chances of the paternal contribution to aneuploidy in the resulting embryos.     

Segregation of a supernumerary del(15) marker chromosome in sperm

Supernumerary marker chromosomes (SMC) can be associated with both normal and abnormal phenotypes. In addition, SMC are found at higher frequency in males with infertility. We identified a SMC, characterized as a del(15)(q11.2) chromosome, in a phenotypically normal male. Using fluorescence in situ hybridization (FISH), we examined the segregation of the del(15) chromosome in sperm from this patient. Only 6.23% of sperm nuclei showed disomy using a chromosome 15 alpha-satellite FISH probe, instead of the expected 50%. In addition, FISH analysis showed no increase for non-disjunction of chromosome 18, excluding an interchromosomal effect for this chromosome. The significant decrease in sperm bearing the del(15) may be due to tissue-specific mosaicism or a result of some form of selection against the del(15) during spermatogenesis. This finding provides a basis for the observation that SMC(15) are less likely to be inherited from a paternal carrier.     

双色FISH检测人精子染色体非整倍体方法的建立

目的　建立用双色FISH检测人精子染色体非整倍体的方法。方法　采用双色荧光原位杂交 (FISH)方法取适量精子标本用EDTA/PBS处理 ,然后用二硫苏糖醇 (DTT) ,使精子去凝集。固定后滴片 ,然后与双色荧光直接标记探针杂交。结果　在OLYMPUS荧光显微镜下可以清楚看到精子头部的蓝色杂交信号 ,头部有 1个绿色荧光杂交信号的精子为X染色体精子 (X精子 ) ,有 1个红色荧光杂交信号的精子为Y染色体精子 (Y精子 )。精子头部有 2个荧光杂交信号的精子为染色体数目异常精子。结论　双色荧光原位杂交 (FISH)方法可以用于测定人精子染色体非整倍体率的变化。     

Chromosome segregation in an infertile man carrying a unique pericentric inversion, inv(21)(p12q22.3), analysed using fluorescence in situ hybridization on sperm nuclei: significance for clinical genetics. A case report

We report the case of a 40-year-old patient referred to our centre after 3 years of infertility. Karyotyping with the aid of fluorescence in situ hybridization (FISH) analysis showed a unique pericentric inversion of chromosome 21:46,XY,inv(21)(p12q22.3). This type of intrachromosomal structural rearrangement can lead to chromosome imbalance in offspring by producing unbalanced gametes if an odd number of crossover events occur within the inverted segment. Therefore, partial trisomy/monosomy with clinical consequences can be observed in the progeny of carriers. Semen samples from the inversion carrier were analysed by FISH using a combination of probes [a subtelomeric 21q probe and a locus-specific Down's syndrome critical region (DSCR) probe] to evaluate the proportion of recombinant chromosomes. Sperm-FISH analysis of 3400 spermatozoa revealed a 67.4% rate of balanced chromosomes (normal or inverted). The frequencies of recombinant chromosomes with duplication of the long arm and deletion of the short arm, and vice versa, were 11.2 and 21.4%, respectively. The risk for the couple of conceiving a child with an unbalanced chromosome 21 is estimated to be around 32%. This case study shows the utility of sperm-FISH analysis in the genetic counselling of a pericentric inversion in a male carrier to assess the frequency of recombinant chromosomes and therefore evaluate the probability of having a normal conception.     

CB-FISH法检测人体内嵌合的21号染色体非整倍体

目的　探讨 2 1三体患者体内二倍体细胞增多的机理。方法　用松胞素 - B诱导的双核细胞荧光原位杂交技术 ,对人体内 2 1号染色体非整倍体和培养细胞中 2 1号染色体分离异常的发生率进行检测。结果　 2 1三体患者体内 ,二倍体细胞率 (16 .90‰± 10 .70‰ )远高于 2 1四体细胞率 (0 .42‰± 0 .6 4‰ ) (P=0 .0 0 0 )。在培养的正常人和 2 1三体患者细胞中 ,2 1号染色体不分离率 (3.6 9‰± 2 .5 0‰和 8.2 2‰±5 .5 4‰ )显著高于丢失率 (0 .10‰± 0 .30‰和 0 .43‰± 0 .49‰ ) (P=0 .0 0 0 )。结论　 2 1三体患者体内较高比例的二倍体细胞源于因三体细胞中 2 1号染色体分离异常而形成的二倍体细胞的逐渐积累     

Ovarian senescence in the rhesus monkey (Macaca mulatta)

BACKGROUND: A decline in fertility is evident in human females past their middle thirties. This 'reproductive senescence', marked by a sharp decline in pregnancy rates, may be attributed to reductions in numbers of available oocytes and their quality. Because Old World primates exhibit ovarian morphology and physiological control and timing of menstrual cycles closely resembling those of humans, the current study investigated the rhesus macaque as a potential model for human reproductive senescence. METHODS: Ovaries collected from females aged 1-25 years and divided into five age groups were analysed histologically. RESULTS: General ovarian morphology demonstrated significant changes as the females approached menopause. The proportions of primordial and primary follicles all demonstrated significant differences across age groups (primordial: 77.1, 79.9, 69.7, 62.9, 55.1%; primary: 21.5, 18.8, 28.5, 35.2, 43.1% for age groups 1 to 5 respectively; P<0.0001 for both). Samples from females approaching or undergoing the menopausal transition (aged 20-25 years) demonstrated evidence of ovarian senescence, having scattered and atretic follicles, low numbers of primordial follicles and reduced stromal tissue. CONCLUSION: This study supports the value of the rhesus monkey as a model for reproductive ageing because its ovary undergoes follicular reservoir depletion similar to that seen in humans.     

Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH)

The use of chromosome specific DNA probes labelled with fluorochromesand especially the combination of several probes has been usedto indirectly study the chromosome constitution in condensedsperm nuclei by fluorescence in-situ hybridization (FISH), andhas allowed to include this test in the protocol of study ofinfertile males. Still, if the test is to be valid, severalstrict conditions must be met, and some specific characteristicshave to be taken into account. This becomes evident when comparingearlier results with more recent ones. The basic technical factorsto be taken into account are the methods of chromatin decondensation,the number of spermatozoa and of individuals to study, the useof internal controls, the scoring criteria, the specificityof the probes and the possible existence of polymorphisms thatmay interfere with the detection of flourescent signals. Inthe last 7 or 8 years, a large number of papers has been published,describing the incidence of aneuploidies in controls, in individualsin whom a tendency to non-disjunction was suspected and in infertilemales. Studies in controls have shown a considerable intra-and inter-individual variability in the frequency of aneuploidies,the tendency of some chromosomes to undergo non-disjunction(chromosome 21 and the sex chromosomes) and the importance of-satellite polymorphisms when using centromere probes. In thecontrol population, the frequency of aneuploidy per haploidset has been estimated at 6%. The incidence of aneuploidiesin sperm nuclei for some of the chromosomes more frequentlyinvolved in trisomies is considerably higher than the incidenceof these trisomies established through epidemiological datausing the global incidence of chromosome abnormalities duringthe peri-implantation stage. In infertile males and in maleswith sex-chromosome abnormalities (usually with very low numbersof spermatozoa) the results show an increased incidence of sexchromosome aneuploidies and diploid (multi-aneuploid?) spermnuclei. The results could be related to the higher incidenceof chromosome abnormalities (especially sex-chromosome aneuploidies)observed in children conceived by intracytoplasmic sperm injection(ICSI).     

用双色荧光原位杂交检测人精子染色体非整倍体率

目的检测人精子染色体非整倍体率。方法采用双色荧光原位杂交（ＦＩＳＨ）方法,取少量精标本经洗后制片,用二硫苏糖醇（ＤＴＴ）和二碘水杨酸锂（ＬＩＳ）处理,使精子头部染色质去凝集。然后,与生物素标记的α卫星Ｘ染色体特异ＤＮＡ探针（ＤＸＺ１）和地高辛标记的α卫星Ｙ染色体特异ＤＮＡ探针（ＤＹＺ３）进行原位杂交。用ＣＹ３－链亲和素、山羊抗链亲和素检测Ｘ染色体探针杂交信号;用鼠抗地高辛抗体、与荧光素结合的兔抗鼠抗体检测Ｙ染色体探针杂交信号。结果在Ｎｉｋｏｎ荧光显微镜下可以清楚看到精子头部的杂交信号,头部有１个红色荧光杂交信号的精子为Ｘ染色体精子（Ｘ精子）,有１个绿色荧光杂交信号的精子为Ｙ染色体精子（Ｙ精子）。精子头部有２个荧光杂交信号的精子为染色体数目异常精子。若用１条常染色体探针和１条性染色体探针进行ＦＩＳＨ,可以区别头部有２个相同颜色荧光杂交信号的精子属非整倍体精子或二倍体精子。结论双色荧光原位杂交（ＦＩＳＨ）方法,可以用于测定接触致突变剂和非整倍体诱导剂后,人精子染色体非整倍体率的变化。     

Opioids and attachment in rhesus macaque (Macaca mulatta) abusive mothers

This study investigated the role of the endogenous opioid system in maternal and affiliative behavior of group-living rhesus macaque (Macaca mulatta) mothers with a history of abusive parenting. Eighteen mothers received an injection of the opioid antagonist naltrexone or saline for 5 days per week for the first 4 weeks of the infant's life. After treatment, mother-infant pairs were focally observed. Naltrexone did not significantly affect infant abuse or other measures of maternal behavior. Naltrexone increased the amount of grooming received by mothers from other group members and reduced the mothers' rate of displacement activities such as scratching, yawning, and self-grooming. These results concur with previous primate studies in suggesting that opioids mediate the rewarding effects of receiving grooming and affect anxiety-related behaviors.     

Morphology assessment and fluorescence in situ hybridization of the same spermatozoon using a computerized cell-scanning system

BACKGROUND: Poor sperm morphology is statistically associated with an increase in the incidence of chromosome abnormalities. Our aim was to examine the possible correlation between chromosomal aberrations and sperm morphology in the same cell. METHODS: 12349 spermatozoa from 7 teratozoospermic and one globozoospermic patients, and from 3 fertile donors were analyzed using a system which scans for cell morphology and chromosomal ploidy in the same cell using digital technology. RESULTS: Chromosomal aberrations were detected in 5.3% of teratozoospermic cases and in 6.7% in the globozoospermic patient compared with 1.6% in donors (P < 0.0001). Chromosomal aberrations were more common in abnormally formed sperm compared with normal spermatozoa: 4.5% vs 1.3% in the teratozoospermic group and 2.0% vs 0.3% in the control group (NS), especially frequent among sperm with two heads or two tails (52.1-77.2%) or extreme head deformations (10.6-11.1%) irrespective of grouping, and in mild amorphous heads in the globozoospermic patients (20.2%). The frequency of chromosomal aberrations in morphologically normal sperm was comparable whether derived from teratozoospermic or normospermic patients. CONCLUSIONS: The computerized cell-scanning system demonstrated the relationship between chromosomal aberrations and sperm morphology in the same spermatozoon. The incidence of chromosomal aberrations was positively linked to abnormal sperm morphology, the more severe the abnormality, the higher the incidence of aneuploidy.     

Chromosome painting using FISH (fluorescence in situ hybridization) with chromosome-6-specific library demonstrates the origin of a de novo 6q+ marker chromosome

Brøndum-Nielsen K, Bajalica S, Wulff K, Mikkelsen M. Chromosome painting using FISH (fluorescence in situ hybridization) with chromosome-6-specific library demonstrates the origin of a de novo 6q+ marker chromosome.
Clin Genet 1993: 43: 235–239. © Munksgaard, 1993
We report the application of chromosome painting using FISH (fluorescence in situ hybridization) to demonstrate the origin of a de novo 6q + marker chromosome. A girl with a mental retardation/multiple malformation syndrome was shown to have the karyotype 46,XX, 6q+. Banding analysis could not determine the origin of the extra chromosomal material. Using FISH with a chromosome-6-specific library we showed that the marker chromosome was completely painted, indicating an origin from chromosome 6. The child's phenotype was compared with previously reported cases with partial chromosome 6 trisomy. A clinically recognized syndrome emerged, although she apparently also demonstrated novel features.     

荧光原位杂交技术研究人类体外未受精卵的17号染色体非整倍体

目的 对比两种不同间期核制备方法的效果，并比较25－30岁和31－35岁这两个女性年龄组、不同的体外受精－胚胎移植（in vitro fertilization-embryo transfer,IVF-ET）指征、不同超排方案等与17号染色体非整倍体率之间的关系。方法 采用0.1% Tween 20/0.01 mol/L HCl和3：1的甲醇：冰醋酸两种方法制备人类未受精卵间期核，选用人类17号染色体端粒探针（17qter），按说明书步骤进行荧光原位杂交并在方法上稍作改进。结果 36个未受精卵中，正常17号单体24枚，二体7枚，三体5枚，非整倍体出现率为33.3%（12／36）；25－30岁和31－35岁这两个女性年龄组、不同IVF指征、不同超排方案的患者的17号染色体非整倍体发生率差异无显著性。结论 两种方法信号检出率均为100％，但前者间期核制备效果较好，易于操作，避免甲醇冰醋酸刺激性气味对环境的污染，卵母细胞17号染色体的非整倍性是造成体外受精失败的重要原因之一。     

组合探针荧光原位杂交检测骨髓增生异常综合征常见染色体异常

目的：评价组合探针荧光原位杂交（fluorescence in site hybridization,FISH)在检测骨髓增生异常综合征（myelodysplastic syndrome,MDS）常见染色体异常中的价值。方法：应用YAC248F5（5q31)、YAC938G5（7q32）、CEP8、YAC912C3（20q12）4种DNA探针，对核型未知的20例MDS患者进行FISH检测－5／5q-、－7／7q-、＋8、20q－等常见染色体异常，并与常规细胞遗传学分析结果相比较。结果：20例MDS患者中，组合探针FISH检出13例有常见染色体异常（其中5例＋8，1例－5／5q-，5例20q-,1例5q-合并20q-，复杂异常1例）；而常规细胞遗传学发现5例常见染色体异常，1例＋21，复杂异常1例，标记染色体1例，正常5例。结论：组合探针FISH是筛查MDS患者常见染色体异常的有效手段。     

一例伴有der(Y)t(Y;1)异常的多发性骨髓瘤的临床和实验研究

目的 对1例伴有不平衡染色体易位der(Y)t(Y;1)的多发性骨髓瘤(multiple myeloma,MM)患者进行细胞遗传学、中期荧光原位杂交、免疫学及临床研究.方法 采用细胞遗传学G显带行中期染色体核型分析;用1号染色体涂抹探针、Y染色体异染色质区探针进行中期荧光原位杂交检测;免疫分型检测CD38、CD138、ZAP70等的表达及免疫电泳检测免疫球蛋白类型等.结果 细胞遗传学分析结果 发现患者具有高度复杂的异常克隆,其核型为:92,XXYY[3]/49,X,der(Y)t(Y;1)(q12;q21),t(11;14)(q13;q32),+18,+20,+21[47]/49,X,idem,del(13q22),ace[1]/98,XX,der(Y)t(Y;1)×2,+18,+18,+20,+20,+21,+21[10]/46,XY[19].中期荧光原位杂交结果 证实der(Y)t(Y;1)的G显带结果 ,为1q部分三体与Y染色体长臂的不平衡易位.其异常的单克隆免疫球蛋白为IgD,定量6.24 g/L;免疫分型结果 为表达CD38、CD138,不表达ZAP70,考虑为异常克隆浆细胞的表达.结论 Y染色体的结构异常在血液系统肿瘤中非常罕见,本文报道1例发生于多发性骨髓瘤中的伴der(Y)t(Y;1)的核型异常、实验室及临床特点.     

Mosaicism for a small supernumerary ring X chromosome in a dysmorphic, growth-retarded male: mos47,XXY/48,XXY, + r(X)

Supernumerary ring X [r(X)] chromosomes are often found in patients with Turner syndrome. The phenotypic effects of the r(X) chromosome are variable, and largely depend on the presence or absence of the X inactivation (XIST) locus. Ring(X) chromosomes in males are rare and have been previously reported in only four cases, with 47,XY, + r(X) or mos47,XY, + r(X)/46,XY karyotypes. These patients all had developmental delay and dysmorphic features. We describe a 2.5-year-old male patient with facial dysmorphia, growth retardation, microcephaly, global developmental delay, and microphallus. Cytogenetic analysis from peripheral blood lymphocytes and fibroblasts identified mosaicism for two cell lines: mos48,XXY, + r(?X)/47,XXY. Fluorescence in situ hybridization (FISH) with an X chromosome paint showed the ring chromosome to be X chromosome derived. This is the first case of an r(X) chromosome described in a 47,XXY patient. FISH analysis of the r(X) chromosome with an XIST probe showed that the XIST locus was absent. Functional disomy of genes in the r(X) chromosome most likely accounts for the abnormal phenotype in the proband.     

Ultrastructural nuclear defects and increased chromosome aneuploidies in spermatozoa with elongated heads

BACKGROUND: Cellular and molecular mechanisms leading to elongated sperm heads are not known. We have analysed the nuclear status of spermatozoa with elongated heads. METHODS: Fourteen men with at least 30% of spermatozoa with an elongated nucleus were studied and compared with five fertile men as controls. Sperm morphology was analysed by a quantitative ultrastructural analysis. Sperm chromosomal content was assessed by three-colour fluorescence in-situ hybridization (chromosomes X, Y, 18). Y chromosome microdeletion and karyotype were analysed. RESULTS: Elongated sperm head rates of the patients were 46.9% (30-75 versus 0-2% in the control group) by light microscopy and 34.4% by electron microscopy. In all patients, the chromatin was poorly condensed in elongated sperm heads (50% of elongated nuclei). No anomalies of sperm biochemical markers were found. All the men showed normal karyotype (46,XY) and absence of Y chromosome microdeletion. Aneuploidy rates of gonosomes and chromosome 18 were significantly increased in patients (1.64- and 3.6-fold, P = 0.006 and 0.026, respectively). CONCLUSIONS: This study demonstrates that impaired chromatin compaction and slightly increased chromosome aneuploidies are found in spermatozoa with an elongated head, suggesting possible mechanisms such as meiotic non-disjunctions or spermiogenesis anomalies.     

男性不育患者精子染色体畸变及精子DNA完整性分析

目的 探讨男性不育患者精子染色体和精子DNA完整性的改变.方法 精子染色质扩散(sperm chromatin dispersion,SCD)实验分析精子DNA碎片,正常生育男性(对照组)32名,特发性严重少弱精子症患者(idiopathic severe oligoasthenozoospermia,ISOA)19例,妻子不明原因反复自然流产(unexpbined recurrent miscarriage,URM)38例;多色荧光原位杂交(fluorescent in situ hybridization,FISH)技术检测URM妇女丈夫(n=12)、ISOA不育者(n=10)及对照组(n=5)精子13、21、18、X和Y染色体畸变.结果 对照组、ISOA不育者及URM妇女丈夫精子13、18和21染色体数目总体异常的比率分别为1.29%、4.02%和3.91%,而X和Y染色体数目总体异常的比率分别为0.61%、2.03%和1.98%,与对照组比较差异均有统计学意义(P＜0.01).SCD实验分析精子DNA碎片,ISOA不育患者(n=19)及URM妇女丈夫(n=38)精子DNA碎片比例平均为40.7%±17.8%和22.1%±10.3%,均显著性高于对照组(12.1%±5.2%,P＜0.01).FISH精子染色体(13、21、18、X和Y探针)数目畸变率与精子DNA碎片比率呈正相关(r=0.874,P＜0.01,n=27),精子DNA碎片比率与精子密度及前向运动精子率呈负相关(r=-0.571,P＜0.01,和r=-0.616,P＜0.01,n=89),与畸形精子比率呈正相关(r=0.637,P＜0.01,n=89).结论 精子染色体畸变率和精子DNA碎片比率增高,可能是ISOA和妻子URM不育男性的原因之一,精子DNA损伤筛查町能为特发性男性不育患者提供有用的信息.     

Risk factors for endometriosis in the rhesus monkey (Macaca mulatta): a case-control study

The autopsy records between 1980 and 1995 of 399 female rhesusmonkeys (Macaca mulatta) at the Wisconsin Regional Primate ResearchCenter were examined. Spontaneous endometriosis was found in81 (20%) of the animals. The mean (±SD) ages at deathfor animals with and without endometriosis were 20.7±5.5(range 10-35) and 13.4±7.7 (range 4-37) years respectively.Many of the animals had been exposed to experimental procedures,including laparoscopies, hysterotomies and oestradiol implants,and these were examined as possible risk factors for endometriosis.Of the 81 affected animals, 62 were matched to unaffected controlsfor age at death (to within 1 year) and year of death (to within2 years) and the effect of various factors on the developmentof endometriosis was determined using conditional logistic regression.Exposure to three or more oestradiol implants or one or morehysterotomies were both significant risk factors, with estimatedrelative risks of 9.7 (95% confidence interval 2.5-37.2) and5.8 (95% confidence interval 1.6-20.2) respectively. Animalsthat had been exposed to one or more laparoscopies showed noincreased risk for developing endometriosis. These findingsprovide insight into the aetiology of the disease in women.     

Population genetics of the ABO locus within the rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaque hybrid zone

Knowledge of the macaque ABO blood group system has been critical in the development of nonhuman primates (NHPs) as a translational model. Serving not only as a useful homologue of the disease‐linked ABO system in humans, macaque ABO blood groups must be typed in colonies prior to performing experimental procedures requiring blood transfusion or transplantation. While the rates of blood type incompatibility and the distributions of A, B and AB blood groups are known in large samples of rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaques, there is a dearth of blood type data from macaque populations occupying the rhesus‐cynomolgus hybrid zone in Southeast Asia. Using molecular phenotyping, we profiled ABO blood group distributions of 232 macaques from 10 populations in the hybrid zone and compared them to pure blood populations of the two species. We found that while these distributions are significantly different in most populations, there was a lack of differentiation between the hybrid and cynomolgus macaques as well as between the Thai and neighbouring populations. This supports a more expansive model of hybridization between rhesus and cynomolgus macaques than often proposed and highlights the increased need for consideration of population genetic structure in biomedical studies that employ macaques as animal models. Additionally, we report an enrichment of indeterminate blood types in the hybrid populations.