Role of mitotic control in spermatogenesis

OBJECTIVE: To study the correlation between the incidence of sex chromosome aneuploidies in the somatic cells and spermatozoa in karyotypically normal infertile men and fertile donors. DESIGN: A prospective, phase two, controlled study. SETTING: A teaching Hospital Reproductive Medicine and Medical Genetics Units. PATIENT(S): Ten patients with idiopathic oligozoospermia and 10 sperm donors with proven fertility, all with a normal karyotype 46, XY. INTERVENTION(S): Multicolor fluorescence in situ hybridization (FISH) of peripheral blood lymphocytes and spermatozoa using a probe cocktail containing the alpha satellite DXZ1 for the X centromere, DYZ1 for the heterochromatic region of the long arm of the Y, and cosmids D21S259, D21S341, and D21S342 for Down syndrome critical region of chromosome 21. MAIN OUTCOME MEASURE(S): The incidence of chromosome X, Y, and 21 aneuploidies in peripheral lymphocytes and spermatozoa in both groups. RESULT(S): The incidence of aneuploidies related to chromosomes X, Y, and 21 were significantly higher in peripheral lymphocytes and spermatozoa of infertile men compared with donors. There was a positive correlation between the incidence of chromosome aneuploidies in the somatic cells and sperm in all men. CONCLUSION(S): These findings provide suggestive evidence for the importance of mitosis in spermatogenesis and the role of mitotic instability in unexplained oligozoospermia.     

Absence of sperm sex chromosome aneuploidies in an X0/XYY man

OBJECTIVE: To determine appropriate genetic counseling of patients with a mosaic karyotype who wished to undergo assisted reproduction technology.DESIGN: Case report.SETTING: A tertiary center for assisted reproduction technology.PATIENT(S): A male with a mosaic karyotype X0/XYY.INTERVENTION(S): Analysis of ejaculated spermatozoa by using fluorescence in situ hybridisation with probes directed against chromosomes X, Y, and 18.MAIN OUTCOME MEASURE(S): Degree of sex chromosome aneuploidies in spermatozoa.RESULT(S): Levels of sex chromosome aneuploidies in spermatozoa were normal. On the basis of these findings, the couple proceeded to assisted reproduction technology without preimplantation genetic diagnosis and conceived a healthy male baby.CONCLUSION(S): Sex chromosome mosaicism in men does not necessarily lead to high levels of abnormal spermatozoa. Sex chromosome aneuploidies may be eliminated in the testes through the selective degeneration of abnormal spermatogenic cells.     

Comprehensive meiotic segregation analysis of a 4-breakpoint t(1;3;6) complex chromosome rearrangement using single sperm array comparative genomic hybridization and FISH

Complex chromosomal rearrangements (CCR) represent rare structural chromosome abnormalities frequently associated with infertility. In this study, meiotic segregation in spermatozoa of an infertile normospermic carrier of a 4-breakpoint t(1;3;6) CCR was analysed. A newly developed array comparative genomic hybridization protocol was used, and all chromosomes in 50 single sperm cells were simultaneously examined. Three-colour FISH was used to analyse chromosome segregation in 1557 other single sperm cells. It was also used to measure an interchromosomal effect; sperm chromatin structure assay was used to measure chromatin integrity. A high-frequency of unbalanced spermatozoa (84%) was observed, mostly arising from the 3:3 symmetrical segregation mode. Array comparative genomic hybridization was used to detect additional aneuploidies in two out of 50 spermatozoa (4%) in chromosomes not involved in the complex chromosome rearrangement. Significantly increased rates of diploidy and XY disomy were found in the CCR carrier compared with the control group (P < 0.001). Defective condensation of sperm chromatin was also found in 22.7% of spermatozoa by sperm chromatin structure assay. The results indicate that the infertility in the man with CCR and normal spermatozoa was caused by a production of chromosomally unbalanced, XY disomic and diploid spermatozoa and spermatozoa with defective chromatin condensation.     

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。     

Sperm ultrastructure and 18, X,Y aneuploidies in a man with a 46 XY, 47 XY + 18 mosaic karyotype: case report

Purpose To describe sperm morphology and meiotic segregation in the case of a man with a 46 XY/ 47 XY + 18 mosaic karyotype. Materials and methods A 25-year-old man came to our Centre for semen analysis. Morphological sperm evaluation was performed by light and electron microscopy; meiotic segregation was examined by fluorescence in situ hybridisation (FISH) technique using probes for chromosomes 18, X and Y. PCR was carried out on DNA from peripheral blood lymphocytes to analyze Y microdeletions. Results Mathematically elaborated transmission electron microscopy data highlighted a low number of sperm devoid of ultrastructural defects, and the presence of characteristics of apoptosis and immaturity. FISH showed the presence of aneuploidies of chromosome 18 and sex chromosomes. Conclusions In this case of mosaicism morphological and meiotic spermatogenetic impairment is shown, as well as structural chromosomal alterations. We describe sperm morphology and meiotic segregation in a carrier of 46 XY/ 47 XY + 18 mosaic karyotype.     

Detection of mosaicism for 46,X,i(Y) (q10) in the blood lymphocytes in a phenotypically normal male neonate with prenatally detected 45,X/46, XY at amniocentesis and cytogenetic discrepancy in various tissues

ObjectiveWe present detection of mosaicism for 46,X,i(Y) (q10) in the blood lymphocytes in a phenotypically normal male neonate with prenatally detected 45,X/46, XY at amniocentesis and cytogenetic discrepancy in various tissues.Case reportA 35-year-old, gravida 2, para 1, woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 45,X [8]/46,XY [15]. Simultaneous array comparative genomic hybridization (aCGH) on uncultured amniocytes revealed the result of arr (Y) × 0–1 with 25.493-Mb mosaic deletion of chromosome Yp11.31-q11.23. Prenatal ultrasound findings were unremarkable. The fetus had normal male external genitalia on fetal ultrasound. Following genetic counseling, the pregnancy was carried to 38 weeks of gestation, and a phenotypically normal male baby was delivered without any abnormalities of the male external genitalia. The cord blood had a karyotypes of 46,X,i(Y) (q10)[8]/45,X[3]/46,XY [29], and placenta had a karyotypes of 45,X [25]/46,X,i(Y) (q10)[7]/46,XY [8]. When follow-up at age two months, the neonate was normal in development. The peripheral blood had a karyotypes of 46,X,i(Y) (q10)[8]/45,X[5]/46,XY [27]. Interphase fluorescence in situ hybridization (FISH) analysis on 101 buccal mucosal cells showed normal X and Y signals in 101/101 cells.ConclusionFetuses with 45,X/46, XY at amniocentesis can be associated with mosaicism for 46,X,i(Y) (q10) in the blood lymphocytes, cytogenetic discrepancy in various tissues and a favorable outcome.     

Sperm FISH analysis of a 46,XY,t(3;6)(p24;p21.2),inv (8)(p11;2q21.2) double chromosomal rearrangement

A complex chromosome rearrangement (CCR) can be defined as a structural chromosomal aberration that involves at least three breakpoints located on two or more chromosomes. Highly unbalanced gametes may lead to infertility or congenital malformations. Here is reported a double rearrangement considered as the simplest possible CCR and, in a sense, not a true CCR, meiotic segregation for a 46,XY,t(3;6)(p24;p21.2),inv(8)(p11;2q21.2) male patient referred after his partner had undergone three early miscarriages. Sperm fluorescence in-situ hybridization was used to screen for translocation and inversion segregation and an interchromosomal effect (ICE) for 13 chromosomes not involved in CCR. The malsegregation rates for the reciprocal translocation and pericentric inversion were 61.2% and 1.7%, respectively. ICE analysis revealed that the observed chromosome aneuploidy rates of between 0.1% and 0.8% did not differ significantly from control values. A slight increase in cumulative ICE (P=0.049) was observed in the patient, relative to control spermatozoa (with rates of 4.6% and 3.1%). The sperm DNA fragmentation rate differed significantly from control values (5.0%; P=0.001). Reciprocal translocation had no impact on meiotic segregation of the pericentric inversion in this double rearrangement. No conclusion could be drawn regarding the impact of pericentric inversion on translocation.     

Evaluation of the role of mitotic instability in karyotypically normal men with oligozoospermia

OBJECTIVE: To estimate the incidence of sex chromosome aneuploidies in the somatic cells of karyotypically normal infertile men and fertile donors. DESIGN: A prospective, two-phase, controlled study. SETTING: Reproductive medicine and medical genetics units of a teaching hospital. PATIENT(S): Ten patients with oligozoospermia and 10 sperm donors with proved fertility, all with a normal karyotype 46 XY. INTERVENTION(S): Multicolor fluorescence in situ hybridization (FISH) of peripheral blood lymphocytes using a probe cocktail containing the alpha satellites DXZ1 for the X centromere and DYZ1 for the heterochromatic region of the long arm of the Y and the radiolabeled alpha satellite D18Z1 for chromosome 18. MAIN OUTCOME MEASURE(S): The incidence of sex chromosome aneuploidies in both groups. RESULT(S): A 13-fold increase in sex chromosome aneuploidies was observed in the somatic cells of infertile men with "unexplained" oligozoospermia compared to donors (P=.008). CONCLUSION(S): These findings provide suggestive evidence for the existence of an inherent mitotic instability in men with unexplained oligozoospermia.     

Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR

Quantitative fluorescent polymerase chain reaction (QF-PCR) assays and small tandem repeat (STR) markers have been successfully employed for the rapid detection of major numerical aneuploidies affecting human autosomes. So far, the analysis of chromosomes X and Y disorders has been hampered by the rarity of highly polymorphic markers which could distinguish normal female homozygous PCR patterns from those seen in patients with Turner's syndrome. A new marker (X22) of the X/Y chromosomes has been identified which maps in the Xq/Yq pseudoautosomal region PAR2; used together with the HPRT it allows the rapid diagnosis of numerical aneuploidies of the sex chromosomes. Blood samples from normal male and female subjects and from patients with X and Y chromosome disorders (45,X and 47, XXY) have been tested by QF-PCR with the X22 polymorphic pentanucleotide (12 alleles) together with the HPRT and P39 markers. The samples were also tested by multiplex QF-PCR with STRs specific for chromosomes 21,18,13 and amelogenin (AMXY). Tested by QF-PCR, all samples from normal females were heterozygous for either the X22 or the HPRT marker with fluorescent peak ratios near 1:1, thus allowing a correct, rapid diagnosis of their chromosome complement. Turner's patients (45,X) showed only one X22 and one HPRT fluorescent peak, thus documenting the presence of a single X chromosome. Turner's patients with mosaicism showed a major fluorescent peak for the X22 and HPRT markers and a minor peak revealing the presence of a second minor population of cells. Two 47, XXY cases could also be diagnosed. Multiplex analyses can be performed using simultaneously STR markers for chromosomes 21,18,13 X and Y. The diagnostic value of a third X-linked marker (P39) was also investigated. These results suggest that rapid diagnosis of major numerical anomalies of the X and Y chromosomes can be performed using QF-PCR with a new highly polymorphic X-linked marker, X22, which maps in the Xq/Yq pseudoautosomal region PAR 2. Multiplex QF-PCR tests-using the X22 STR in association with HPRT and, in rare cases, a third P39 marker-allow the rapid diagnosis of major aneuploidies affecting chromosomes 21, 18, 13, X and Y. The X22 marker can also be employed for the detection of fetal cells present in maternal peripheral blood or the endocervical canal.     

Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization-intracytoplasmic sperm injection program.

OBJECTIVE: To evaluate the frequency of disomy (for chromosomes X, Y, and 18) and of diploidy in the spermatozoa of infertile men undergoing intracytoplasmic sperm injection (ICSI). DESIGN: Prospective analysis of sperm nuclei by fluorescence in situ hybridization (FISH). SETTING: University-affiliated IVF-ICSI program. PATIENT(S): Semen samples from 19 patients participating in an IVF-ICSI program. INTERVENTION(S): Semen samples were analyzed and prepared for FISH. MAIN OUTCOME MEASURE(S): Semen parameters were evaluated. The frequency of disomy for chromosomes X, Y, and 18 and the frequency of diploidy were analyzed by FISH. RESULT(S): A total of 9,373 spermatozoa from 19 infertile patients were analyzed and compared with spermatozoa from a control group of 5 healthy men. No differences in the frequency of disomy 18 were found, but statistically significant differences in the incidence of sex chromosome disomy and of diploidy were observed. CONCLUSION(S): The study of sperm nuclei by FISH is useful to improve genetic counseling in infertile patients selected for ICSI.     

Analysis of meiotic segregation patterns and interchromosomal effects in sperm from six males with Robertsonian translocations

Purpose To provide more genetic information about meiotic segregation behavior and the possibility of interchromosomal effects (ICE) in spermatozoa from carriers of Robertsonian (Rob) translocations. Materials and methods Meiotic segregation behavior in spermatozoa from six carriers of Rob translocations, four t(13;14), one t(14;22) and one t(13;21), was investigated by dual fluorescence in-situ hybridization (FISH). Aneuploidy for chromosomes 18, X and Y was studied by triple FISH. Results The rate of normal/balanced spermatozoa resulting from alternate segregation ranged from 78.14 to 86.88%. The frequency of unbalanced spermatozoa resulting from adjacent segregation varied between 11.70 and 19.53%. The higher frequencies of aneuploidy for sex chromosome were observed in three Rob translocation carriers. In addition, the increased rates of diploid were found in two t(13;14) carriers. Conclusions Alternate segregation is dominant in the different types of Rob translocations. Some carriers may be at an increased risk for ICE. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An increased aneuploidy for sex chromosome observed in three Robertsonian translocation carriers suggests that an interchromosomal effect is likely in some carriers.     

Mucinous cystadenoma in a female patient with 45,X/46,XY karyotype

The mosaic karyotype of 45,X/46,XY has a wide phenotypic spectrum and there are substantial differences between prenatally and postnatally diagnosed cases. The phenotype varies between normal male to classical Turner syndrome. There is a high risk of gonadal tumor development in the dysgenetic gonads of patients with sex chromosome mosaicism. We report a case of a 24-year-old patient with a pelvic mass and amenorrhea referred to our laboratory for karyotyping. Peripheral blood chromosome analysis showed a mosaic karyotype of 45,X[17]/46,XY[83]. The tumor originated from the left ovary and the right ovary was found to be a streak gonad. The uterus was intact. Pathologic examination of the tumor revealed mucinous cystadenoma. Physical examination of the patient showed signs of Turner syndrome, as short stature (145 cm), short neck and asymmetric shoulders. Her mental state was normal. Y chromosome microdeletion screening involving SRY and ZFY genes was performed and no deletion was found. The patient was informed about the condition during the genetic counseling session.     

1例嵌合型45,X/46,X,r(Y)患者的遗传学分析

目的:应用细胞遗传学和分子生物学技术分析1例嵌合型45,X/46,X,r(Y)患者的核型。方法:应用常规染色体标本制备方法进行G-显带和C-显带;并应用CEPX(DXZ1,Xp11.1-q11.1,Spectrum Green,Vysis)探针、LSI SRY(Yp11.3,Spectrum Orange,Vysis)探针和CEP18(D18Z1,18p11.1-q11.1,Spectrum Aqua,Vysis)与患者的中期分裂相进行荧光原位杂交(fluorescence in situ hybridization,FISH);同时应用PCR技术对患者进行Y染色体微缺失检测。结果:结合G-显带、C-显带、FISH检测结果和Y染色体微缺失的检测结果,确定该患者核型为46,X,r(Y)(p11.3q12)[85]/45,X[15]。Yq11区生精基因微缺失检测未显示该患者存在缺失。结论:细胞遗传学检测结合FISH可以诊断复杂的染色体异常,为患者提供正确的遗传咨询和生育指导。     

Fetal blood sampling demonstrating chimerism in monozygotic twins discordant for sex and tissue karyotype (46,XY and 45,X)

Fetal blood sampling has been used in the genetic work-up of twin gestations for rapid karyotyping. We present a case of twins which on ultrasound evaluation revealed hydrops fetalis in one twin and a normal second twin. Fetal blood sampling revealed the presence of mosaicism for 46,XY/45,X in both twins. HLA antigen testing showed the twins to be identical. The patient elected pregnancy termination. Blood chromosomal analysis after delivery revealed both twins to have 46,XY/45,X mosaicism, but the twin with signs of hydrops fetalis had tissue chromosomes of 45,X and the normal twin had tissue chromosomes of 46,XY. Amniotic fluid chromosomal analysis revealed 46,XY in twin A and 45,X in twin B. This represents a case of identical (monozygotic) twins with sex discordance. In this case, there was the probable occurrence of post-zygotic chromosomal non-disjunction leading to the discordancy of the sex in this set of twins. With the presence of vascular communication in monozygotic twins, there is the possibility of exchange of blood in monozygotic twins and the result of blood chimerism in twins.     

Deoxyribonucleic acid analysis of the zinc finger Y gene in 45,X/46,XY subjects

The deoxyribonucleic acid from nine subjects with a 45,X/46,XY karyotype with a cytogenetically intact Y chromosome and phenotypically presenting with bilateral streak gonads, streak and testis, or bilateral scrotal testes along with a control male and female were analyzed for the presence of the zinc finger Y sequence through the molecular probe pDP1007. This particular probe is thought to constitute part of the putative testicular-determining factor gene. All the study subjects demonstrated the presence of zinc finger Y. Laser densitometry studies confirmed a correlation between the intensity of the zinc finger Y band and the percentage of Y cell lines. This study supports the fact that individuals with mixed gonadal dysgenesis and cytogenetically intact Y chromosomes will tend to have intact zinc finger Y sequences.     

Preferential alternate segregation in the common t(11;22)(q23;q11) reciprocal translocation: sperm FISH analysis in two brothers

Segregation behaviour studies in t(11;22) carriers have reported controversial results. Whereas some authors have detected a preponderance of 3:1 products, no evidence of such prevalence was found by others. This study reports a fluorescence in-situ hybridization (FISH) segregation analysis on decondensed spermatozoa in two brothers, carriers of the same t(11;22)(q23;q11) rearrangement. Data revealed a similar meiotic segregation pattern in both carriers, 2:2 Alternate segregation being the most frequent (42.94 and 45%), while 3:1 genotypes were the least frequent in both patients, with percentages around 10%. The production of three chiasmata, based on the presence of G-light bands along the translocated segments and the presence of recombination sites at 11q and 22q distal regions, are proposed as the cause of a preponderance of the Alternate segregation. Interchromosomal effects involving chromosomes 13, 18, 21, X and Y were also evaluated. An increased frequency of sex chromosome disomies was detected in one patient. Reviewing the literature, a relationship between this phenomenon and the involvement of acrocentric chromosomes in the reorganization is suggested. FISH segregation and interchromosomal effects studies in spermatozoa are encouraged to gather information to establish the best approach for preimplantational genetic diagnosis in reorganization carriers.     

Sperm meiotic segregation, aneuploidy and high risk of delivering an affected offspring in carriers of non-Robertsonian translocation t(13;15)

Purpose

To determine the percentage of unbalanced spermatozoa and an interchromosomal effect in two carriers of balanced translocations t(13;15)(q32;q26) and t(13;15)(q32;p11.2).

Methods

Sperm nuclei analysis by fluorescent in situ hybridization for detection of percentage of unbalanced spermatozoa and sperm with disomy of chromosomes X, Y, 8, 18, 21 and diploidy.

Results

The incidence of unbalanced spermatozoa was 50.5 % and 44.6 % in patient 1 (P1) and patient 2 (P2), respectively. Partial disomy of chromosome 13 was detected in 13.4 % and 21.3 % of sperm in P1 and P2, respectively. The unbalanced karyotype der(15)t(13;15) was found previously in a son of P1 and in two adult relatives, and prenatally in the family of P2. This demonstrates a high risk of delivering an affected offspring. Significantly increased frequencies of chromosomes 8, 18, X and XY disomy and diploidy were observed in P2, which might either indicate an interchromosomal effect or be related to his asthenoteratozoospermia.

Conclusions

Since the proportions of unbalanced spermatozoa and the risk of delivering an affected offspring are high, prenatal or preimplantation genetic diagnosis is recommended for such patients.     

Comparison of sex chromosome aneuploidy in spermatozoa of fertile men and those requiring ICSI treatment detected by fluorescence in situ hybridization

OBJECTIVE: To compare the frequencies of aneuploidy for chromosomes X, Y and 18 in spermatozoa of infertile and fertile males, using 3-color fluorescence in situ hybridization. METHODS: Twelve infertile patients who underwent intracytoplasmic sperm injection treatment at Queen's Medical Centre, Nottingham were studied. Three fertile men served as controls. Aneuploidy frequencies in both groups were compared using 2-sample t-tests. RESULTS: A total of 26,615 ad 93,649 cells were scored in the control and infertile groups respectively. The frequencies of diploidy, sex chromosome disomy and chromosome 18 disomy in the fertile (0.11, 0.28 and 0.11%) compared to the infertile males (0.05, 0.18 and 0.06%) were not statistically significantly different. CONCLUSION: Our preliminary data do not indicate an increased risk from paternal origin sex chromosome aneuploidies in ICSI. However, we recommend further investigations of the cytogenetic constitution of spermatozoa from severe male factor patients.