精子荧光原位杂交分析男性染色体相互易位携带者的减数分裂分离

目的探讨用精子荧光原位杂交（fluorescence in situ hybridization，HSH）分析男性染色体相互易位携带者减数分裂的分离。方法对4例男性染色体相互易位携带者的精子通过化学方法解聚，在2条易位染色体的断裂点两侧的4个染色体区域中分别选用位于其中3个区域的3个位点探针，进行多色FISH，分析精子染色体组成并推断其分离类型。结果4例染色体相互易位携带者的核型分别为46，XY，t（2；18）（p16；q23）、46，XY，t（4；6）（q34；q21）、46，XY，t（8；13）（q23；q21）和46，XY，t（4；5）（4q31；5q13），其分离结果是：对位分离精子占27．1％～49．4％，邻位-1分离精子占26．9％～37．6％，邻位-2精子占2．7％～15．7％，3：1分离精子占8．6％～33．7％，减数分裂Ⅱ不分离精子占0．2％～1．9％，4：0或二倍体精子占0．1％～0．4％。结论不同的男性染色体相互易位携带者减数分裂的分离结果可能不同，对其行精子FISH分析有助于提供更准确的遗传咨询和行胚胎植入前遗传学诊断的预后估计。     

Increase in aggressiveness of male mice carrying a reciprocal translocation,T(10,13), in the heterozygous state

The agonistic behavior of inexperienced pairs of agouti male mice was determined by counting the bites received from and delivered to the opponent within 24 h. The first 10 min of agonistic encounters was recorded by videotape to analyze the frequency and duration of 10 behavioral traits. Each pair consisted of two F₁ males from the cross of (101/C3H) x NMRI, one of which (test male) was derived from a father carrying a reciprocal translocation, T(10,13), in the heterozygous state, while the other (standard opponent) stemmed from a chromosomally normal father of the same origin. Since the offspring of a heterozygous translocation are segregating into chromosomally normal and translocation mice, the test male was either chromosomally normal or a carrier of the translocation in the heterozygous state. Apart from the translocation, all males were of the same genotypic constitution. Comparing the differences between translocation carriers and chromosomally normal mice relative to their standard opponent, significantly more winners than losers were found among the translocation carriers. The differences of 10 behavioral traits between the test male and his opponent revealed significantly more contacts, pursuits, and attacks and significantly fewer defense and flight responses of the test males when this was a carrier of the heterozygous translocation, T(10,13).Dedicated to Nobel Laureate Professor Dr. Dr. Dr. h.c. mult. Konrad Lorenz on the occasion of his 80th birthday.     

Three cases of mosaicism for balanced reciprocal translocations

Mosaicism for a balanced reciprocal translocation (BRTM) is rare. As far as we know only 26 cases of BRTM, demonstrated in lymphocyte cultures, have been described, five of which had an abnormal phenotype. Prenatally three confirmed cases with a normal phenotypic outcome have been described. Here we present three further cases of BRTM in lymphocyte cultures. The first was detected during a family study, the second after an abnormal karyotype in chorionic villus sampling, and the third because of a history of stillborn children. All three carriers have normal phenotypes. An inventory of the BRTM cases reported so far is made. Am. J. Med. Genet. 79:362–365, 1998. © 1998 Wiley-Liss, Inc.     

Experiences with risk estimates for carriers of chromosomal reciprocal translocations

The risk estimates for individual carriers of ten different familial reciprocal translocations detected among 500 couples with reproductive failures are presented. These were established by application of the empirical data analysed by Stengel-Rutkowski et al. (1988) and the guidelines given in Stene & Stengel-Rutkowski (1988). Different risks were estimated for unbalanced offspring at birth or at second trimester prenatal diagnosis for abortions, or stillbirths/early deaths. These risk estimates varied considerably from translocation to translocation. Carriers of five translocations had risks for offspring with single-segment imbalances. The birth risk figures ranged from 0.1% to 13.8%. Carriers of five other translocations had risks for double-segment imbalances with birth risks ranging from 0% to 3.2%. The estimated risk figures were independent of the method of ascertainment. Among the parents of the index cases we found nine maternal carriers and only one paternal carrier. This presentation illustrates the need for individual risk counselling of each carrier with reciprocal translocation regarding further family planning.     

Effects of prenatal stress on reproduction in male and female mice

Pregnant mice were exposed to heat-restraint stress from Days 14 through 21 of gestation. Feminine receptivity quotients were significantly higher in prenatally-stressed male offspring than in unhandled males; however there were no differences in testes weights or masculine copulatory behavior. Prenatally stressed females exhibited vaginal opening at a later date, had longer estrus cycles and higher median quality receptivity scores than unhandled controls. Prenatal stress had no profound effects on pregnancy, parturition or survival of young. However there was a significantly smaller proportion of parturient postnatally stressed females compared to unhandled controls.     

“Balanced” karyotypes in six abnormal offspring of balanced reciprocal translocation normal carrier parents

Among 6800 consecutive blood samples studied for clinical cytogenetic diagnosis, we identified 30 families in which one parent of the proband had a balanced reciprocal autosomal translocation (excluding Robertsonian rearrangements). Twentyeight of the 30 families had a malformed and/or mentally retarded proband: 19 with an unbalanced derived chromosome, 3 with abnormalities involving chromosomes other than those in the translocation, 5 with a “balanced” reciprocal translocation, and 1 with a normal karyotype. We hypothesize that the latter 6 affected probands with “balanced” karyotypes could be abnormal due to submicroscopic deletions and duplications as was originally suggested by Jacobs [1984]. Particularly in these 6 families, 83% of translocation breakpoints were associated with fragile sites, more than expected by chance (P < 0.025). This supports the report of an association between fragile sites and constitutional chromosome breakpoints by Hecht and Hecht [1984]. To explain these findings, we propose that autosomal fragile sites are unstable areas which predispose to breaks and unequal crossing over near the fragile site breakpoints creating minute duplications and deletions. Consequently, newborn infants inheriting a seemingly “balanced” karyotype from a normal parent with a balanced reciprocal translocation may still be at an increased risk of being malformed and/or developmentally delayed because of submicroscopic chromosomal imbalances. © 1995 Wiley-Liss, Inc.     

The detection of viable heritable translocations by chromosome banding procedures

Ethyl methanesulfonate was tested for its ability to induce viable heritable translocations in progeny of male rats given a single IP injection prior to breeding. Reproductively competent Wistar rats were used as the test animals. Males were treated with either 75 or 150 mg/kg EMS or vehicle control. Neonates were used for primary tissue culture; the fibroblasts were harvested for cytogenetic analysis of chromosomes banded by Giemsa banding procedures. Since the cells examined were somatic cells, it was necessary to karyotype only two to three per neonate to ascertain inherited translocations. A reduction in fertility was observed in males treated with EMS. A statistically significant (p<0.05) dose-related increase in heritable translocations was observed in the F₁ generation of treated animals.     

Detection of subtle reciprocal translocations by fluorescence in situ hybridization

Three different subtle reciprocal translocations were detected on long, well-banded chromosomes. The same translocations were examined using fluorescence in situ hybridization (FISH) with chromosome-specific libraries and unique DNA sequences. Our findings show that FISH allows rapid and unequivocal detection and characterization of this type of chromosome rearrangement. This approach is especially useful for prenatal diagnosis when one of the parents is a balanced carrier of such small fragment translocations.     

河南地区586例染色体相互易位的热点断裂区域分析

目的分析河南地区人群染色体相互易位的发生率、常见易位的染色体和核型以及断裂区域,并探讨不同断裂区域对河南地区人群妊娠史或发育史的影响。方法对2016年2月至2018年4月因不孕不育、自然流产、反复流产、死胎等不良妊娠史或发育异常到郑州大学第一附属医院遗传与产前诊断中心遗传咨询和检查的62477例患者进行外周血淋巴细胞染色体核型分析,对染色体相互易位的发生率、常见相互易位的染色体和核型以及断裂区域进行统计。结果62477受检者中共检测出586例染色体相互易位携带者,发生率为0.94%。染色体相互易位群体中,572例染色体相互易位携带者为不孕症患者,发生率为0.92%,14例为发育异常患者,发生率为0.02%。染色体累及次数显示,1号、4号、7号和11号相互易位染色体累及次数最多;核型t(11;22)(q25;q13)累及次数最多。断裂区域分析显示,相互易位染色体共累及出现437个断裂区域,其中11q23、22q13和1p36累及次数最多,且引起携带者不孕不育、流产、胚胎停止发育、先天畸形、发育迟缓/智力低下或表型正常等。结论河南地区染色体相互易位不孕症携带者的发生率为0.92%。断裂区域分析结果表明相互易位累及的染色体及其断裂区域对携带者的妊娠或发育存在影响。探讨相互易位染色体的断裂区域能为携带者提供准确的遗传、生殖和发育咨询,也为阐述断裂区域附近基因功能和寻找新基因及其作用机制提供参考依据。     

Preimplantation genetic diagnosis in female and male carriers of reciprocal translocations: clinical outcome until delivery of 312 cycles

Carriers of reciprocal translocations (rcp) are known to be at risk for reproductive difficulties. Preimplantation genetic diagnosis (PGD) is one of the options these carriers have to try in order to fulfil their desire to have a child. In the present study, we retrospectively looked at the results of 11 years (1997-2007) of PGD for rcp in our center to improve the reproductive counseling of these carriers. During this period 312 cycles were performed for 69 male and 73 female carriers. The mean female age was 32.8 years, the mean male age 35.8 years. Most carriers were diagnosed with a translocation because of fertility problems or recurrent miscarriages, and most of them opted for PGD to avoid these problems. In 150 of the 312 cycles, embryo transfer (ET) was feasible and 40 women had a successful singleton or twin pregnancy. This gives a live birth delivery rate of 12.8% per started cycle and of 26.7% per cycle with ET. Owing to the large number of abnormal embryos, PGD cycles for rcp often lead to cancellation of ET, explaining the low success rate when expressed per cycle with oocyte pick-up. Once ET was feasible, the live birth delivery rate was similar to that of PGD in general at our center. PGD is therefore an established option for specific reciprocal translocation carriers.     

基于二代测序的植入前遗传学检测在71例染色体易位家系中的应用

目的探讨基于二代测序(next generation sequencing,NGS)的植入前遗传学检测(preimplantation genetic test,PGT)对于染色体易位携带者实现正常生育的意义。方法对71对染色体易位携带夫妇(60例为平衡易位,11例为罗氏易位)行PGT周期的超促排卵和卵母细胞浆内单精子注射,培养至囊胚阶段,活检囊胚滋养外胚层细胞行全基因组扩增(whole genome amplification,WGA),用NGS技术对扩增产物进行基因组序列分析,选择正常或平衡胎胚植入,并对检测结果和胚胎着床、妊娠情况进行分析。结果71对夫妇共进行92个周期,活检胚胎303枚,301枚囊胚活检成功,成功诊断胚胎287个,诊断成功率为95.3%(287/301),获得可供移植的整倍体胚胎共85个,其中18个周期无可用胚胎,周期取消率19.5%。平衡易位组获得整倍体胚胎67个,罗氏易位组获得整倍体胚胎18个,PGT后胚胎着床率分别为89.3%(42/47)和88.8%(8/9),早期流产率分别为25.5%(12/47)和22.2%(2/9),继续妊娠率+活产率分别为63.8%(30/47)和66.6%(6/9),产前诊断结果与PGT结果一致。结论基于NGS的PGT可准确的对胚胎进行染色体病诊断,避免反复流产和非意愿性终止妊娠,并获得理想的临床妊娠率。     

Acrylamide: Induction of heritable translocations in male mice

Acrylamide (AA), known to induce dominant lethals in male rodents, was studied in the mouse heritable translocation test by using intraperitoneal injections on 5 consecutive days. Matings on days 7-10 following the last injection yielded a high frequency of translocation carriers in the F₁ male population, which demonstrated that acrylamide is an effective inducer of translocations in postmeiotic germ cells. As an inducer of both dominant lethals and heritable translocations in late spermatids and early spermatozoa, AA is similar to alkylating agents such as ethylmethanesulfonate and ethylene oxide. However, AA′s chemical structure, the nature of adducts formed with DNA, and its lack of mutagenicity in bacteria suggest a different mechanism as the basis for AA′s germ cell mutagenicity.     

Analysis using dual-colour fluorescencein situ hybridization of meiotic chromosome segregation in male mice heterozygous for a reciprocal translocation

Meiotic chromosome behaviour was investigated in male mice heterozygous for the translocation T(7;16)67H. At metaphase I, chain-of-four quadrivalents were present in approximately 80% of the spermatocytes; the bulk of remaining cells contained a ring quadrivalent, with only a few having either a trivalent plus univalent configuration or two bivalents. A low rate of non-disjunction, approximately 5%, was found through analysis of C-banded metaphase II spermatocytes. Using fluorescencein situ hybridization with differentially labelled whole chromosome paints, a wide array of segregation products were observed at metaphase II, depending on whether they arose from alternate, adjacent I, adjacent II orientation at metaphase I or were uninformative for alternative/adjacent I because of the presence of a chiasma in an interstitial pairing segment. Some 62% of the cells fell into this latter category, with only small proportions clearly arising through alternate (1.8%) or adjacent I (0.7%) orientations. Approximately 30% of the cells contained the products of adjacent II orientation. Consideration of the data suggested that most of these cells arose from metaphase I cells that contained a chain quadrivalent. Ring quadrivalents appeared predominantly to orientate in an alternate/adjacent I manner.for publication by J. S. (Pat) Heslop-Harrison     

Genetic counseling in carriers of reciprocal chromosomal translocations involving long arm of chromosome 16

Families with balanced chromosomal changes ascertained by unbalanced progeny, miscarriages, or by chance are interested in their probability for unbalanced offspring and other unfavorable pregnancy outcomes. This is usually done based on the original data published by Stengel-Rutkowski et al. several decades ago. That data set has never been updated. It is particularly true for the subgroup with low number of observations, to which belong reciprocal chromosomal translocations (RCTs) with breakpoint in an interstitial segment of 16q. The 11 pedigrees from original data together with the new 18 pedigrees of RCT carriers at risk of single-segment imbalance detected among 100 pedigrees of RCT carriers with breakpoint position at 16q were used for re-evaluation of the probability estimation for unbalanced offspring at birth and at second trimester of prenatal diagnosis, published in 1988. The new probability rate for unbalanced offspring after 2 : 2 disjunction and adjacent-1 segregation for the total group of pedigrees was 4 +/- 3.9% (1/25). In addition, the probability estimate for unbalanced fetuses at second trimester of prenatal diagnosis was calculated as 2/11, i.e. 18.2 +/- 11.6%. The probability rates for miscarriages and stillbirths/early deaths were about 16 +/- 7.3% (4/25) and <2% (0/25), respectively. Considering different segment lengths of 16q, higher probability rate (0/8, i.e. <6.1%) for maternal RCT carriers at risk of distal 16q segment imbalance (shorter segment) was obtained in comparison with the rate (0/10, i.e. <4.8%) for RCT at risk of proximal segment imbalance (longer segment). It supports findings obtained from the original data for RCT with other chromosomes, where the probability for unbalanced offspring generally increased with decreasing length of the segments involved in RCT. Our results were applied for five new families with RCT involving 16q, namely three at risk of single-segment imbalance [t(8;16)(q24.3;q22)GTG, ish(wcp8+,wcp16+;wcp8-,wcp16+), t(11;16)(q25;q22)GTG, and t(11;16)(q25;q13)GTG] and two with RCT at risk of double-segment imbalance [t(16;19)(q13;q13.3)GTG, isht(16;19)(q13;q13.3) (D16Z3+,16QTEL013-D19S238E+,TEL19pR-; D16Z3-, D19S238E-,TEL19pR+), and t(16;20)(q11.1;q12)GTG, m ish,t(16;20)(wcp16+,wcp20+;wcp16+,wcp20+)]. They have been presented in details to illustrate how the available empiric data could be used in practice for genetic counseling.     

Benefits and drawbacks of preimplantation genetic diagnosis (PGD) for reciprocal translocations: lessons from a prospective cohort study

Preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridisation probes was carried out for 59 couples carrying reciprocal translocations. Before treatment, 85% of pregnancies had resulted in spontaneous miscarriage and five couples had achieved a healthy live-birth delivery. Following treatment, 33% of pregnancies failed and 21of 59 couples had a healthy live-born child. The accuracy of diagnosis was 92% (8% false abnormal and 0% false normal results). The overall incidence of 2:2 alternate segregation products was 44% however, products consistent with 2:2 adjacent segregation were ∼twice as likely from male heterozygotes, and those with 3:1 disjunction were three times more likely from female heterozygotes. Our results indicate that up to three stimulation cycles per couple would give an ∼50% chance of a successful live birth, with the risk of miscarriage reduced to the level found in the general population. In our study, 87% of all normal/balanced embryos available were identified as being suitable for transfer. We conclude that PGD provides benefit for couples with high-risk translocations by reducing the risk of miscarriage and avoiding a pregnancy with an unbalanced form of the translocation; however, for fertile carriers of translocations with a low risk of conceiving a chromosomally unbalanced offspring, natural conception may be a more viable option.     

Detection of TEM-induced reciprocal translocations in F1 sons of CD-1 male mice: comparison of sequential fertility evaluation and cytogenetic analysis

To determine the positive and negative classification error rates associated with the HTA in our laboratory, F1 sons of TEM-exposed CD-1 male mice were evaluated by the sequential fertility method with subsequent cytogenetic analysis. Males who sired three litters of size 10 or less when mated to primiparous females from either the B6C3F1 or the BCF1 strain were classified as partial steriles. When meiotic chromosome analyses revealed the presence of at least two cells containing multivalent figures, males were classified as translocation heterozygotes. When the fertility evaluation and the cytogenetic analysis were compared, normal fertility was observed on 5 of 83 (6.02%) translocation-bearing F1 males mated to B6C3F1 tester females and on 3 of 83 (3.61%) F1 males mated to BCF1 tester females. Thus, the false-negative error rates were 6.02% and 3.61% with these two tester strains. Multivalent figures were not observed in the meiotic chromosomes of 410 F1 males. Of these, 12 (2.93%) had reduced fertility when mated to the B6C3F1 tester strain as did 7 (1.71%) mated to the BCF1 strain. Thus, the false-positive error rates with these two tester strains were 2.93% for the B6C3F1 strain and 1.71% for the BCF1 strain. Our results indicate that non-zero error rates, both false-positive and false-negative, are associated with the sequential mating method HTA. In addition, the magnitude of these error rates was influenced not only by the tester female strain but also by the genotype of the F1 male.     

Effect of reciprocal cross-fostering on aggression of female mice toward lactating strangers

Reciprocal cross-fostering of mice was carried out between an inbred strain (C57 BL/6) in which the female showed a particular form of aggression toward lactating female intruders, and a strain (C3H/He) which proved to lack totally this type of attack. In adulthood, cross-fostered females of the 2 strains and their controls were submitted to a series of aggression tests. They encountered in the test situation either a strange lactating female of their own strain or one of the foster strain. No differences in aggression measures were found between cross-fostered animals and controls, indicating that experiential factors (viz., cross-fostering) were without effect.     

PGD in female carriers of balanced Robertsonian and reciprocal translocations by first polar body analysis.

Preimplantation genetic diagnosis (PGD) using the first polar body (1PB) is a modality of PGD that can be used when the woman is the carrier of a genetic disease or of a balanced chromosomal reorganization. PGD using 1PB biopsy in carriers of balanced chromosome reorganizations has not become generalized. Here, we describe our experience based on the analysis of unfertilized or fresh, non-inseminated control oocytes, by fixing separately the 1PB and the corresponding oocyte, and on the study of six clinical cases of PGD using 1PB biopsy (four Robertsonian translocations and two reciprocal translocations). In fresh oocytes, the chromosome morphology of the 1PB was well preserved, and the results were always concordant for each oocyte-1PB pair. This indicates that the 1PB can be reliably used for the diagnosis of chromosome reorganizations. In these studies the technical problems encountered when performing PGD using 1PB biopsies for chromosome studies are also addressed. Three different strategies of 1PB biopsy (laser beam, partial zona dissection and acid Tyrode's) and two different protocols (intracytoplasmic sperm injection before or after 1PB biopsy) and their effect on the percentage of oocytes diagnosed and the fertilization rate, are discussed. In reciprocal translocation cases, published in the literature or studied by us, in which at least nine oocytes had been diagnosed, a correlation has been found between the frequency of nondisjunction observed and the theoretical recombination rate. To date, PGD by 1PB analysis alone or combined with blastomere biopsies in female carriers of chromosomal rearrangements has been used in 18 cases, with a further six cases reported here. A total of 325 cumulus-oocyte complexes have been obtained, of which 294 were biopsied and 224 were diagnosed. A total of 52 embryos was transferred, 19 of which implanted and 17 produced full-term pregnancies.     

Sex steroids modulate the signals from volatile female odors in the accessory olfactory bulb of male mice

We previously reported that male mice detect volatile female odors via the accessory olfactory system, and that these odors activate granule cells in the accessory olfactory bulb (AOB) with a characteristic pattern. We also reported that sex steroids modulate the attraction of male mice to volatile female odors. The present study investigated hormonal modulation of signals from volatile female odors in the AOB with c-Fos immunostaining. After intact male mice were exposed to volatile female odors, there were more c-Fos positive cells in the caudal granule cell layer (GCL) than in the rostral GCL of the AOB. This effect was observed 3 days but not 7 days after castration, suggesting that hormonal deficiency causes the reorganization of the AOB after 3 days. There was no difference in the number of c-Fos positive cells between the rostral and caudal GCL of castrated male mice treated with 17 beta-estradiol (E). In contrast, there were more c-Fos positive cells in the caudal GCL than in the rostral GCL of castrated male mice treated with dihydrotestosterone (DHT). In both DHT- and E-treated castrated male mice, there was no difference in the number of c-Fos positive cells between the rostral GCL and caudal GCL. This finding suggests that E disrupts the effect of DHT, and that androgen is required for maintaining the intact neuronal network of the AOB. The present study suggests that sex steroids modulate the signals from volatile female odors in the AOB of male mice.