Triple-color Fluorescence Research Articles

Нyperdiploid мultiple мyeloma-cytogenetic and clinical aspects

In the current research the attention is focused on the possibilities of identifying a hyperdiploidy myeloma clone (HdMC) by using a triple-color fluorescence “in situ” hybridization (FISH) probe. The cytogenetic results from the bone marrow aspirates of 26 patients with newly diagnosed multiple myeloma admitted in the Hematology Department of our hospital during the period from March to September 2023 have been analyzed. The group consists of 12 female and 14 male patients with an average age of 67 years. A FISH probe for establishing hyperdiploidy myeloma clone was used as well as the most common methods for detecting genetic aberrations affecting the long and short arms of the 1 and 14q32 chromosome rearrangements. According to the results, two subgroups of patients have been established. The first subgroup consists of the patients with positive FISH probes for hyperdiploidy myeloma and/or 14q34 rearrangements and 1q25/1p36, while the second one consists of patients, negative for all the three probes listed above. A comparison between the demographic, laboratory data and the ISS (International Staging System) stage of the two subgroups has been made. The collected data suggests that the use of the triple-color FISH probe, as well as some other factors in the analyzed information, increases the probability of detecting a HdMC by 23%.

Triple-color fluorescence co-localization of PD-L1-overexpressing cancer exosomes.

Programed cell death ligand 1 (PD-L1) is a protein biomarker overexpressed on exosomes derived from tumor cells. It plays an important role in tumor diagnosis, screening, evaluation of therapeutic efficacy, and prognosis. In this study, a facile method is presentedto detect PD-L1-overexpressing cancer exosomes with high specificity and sensitivity. First, gold nanospheres (GNSs) were attached to the bottom of an eight-well chambered slide by electrostatic adsorption, forming the detection substrate. Then, Cy5-labeled CD63 aptamers (i.e., the capture probes) were modified on the GNSs by Au-S bond. After adding samples containing target exosomes which were stained by membrane dyes DiI in advance, FAM-labeled PD-L1 aptamers (i.e., the immunoprobes) were added to recognize PD-L1 on the target exosomes. By triple-color fluorescence co-localization (TFC) of the Cy5, DiI, and FAM channels, highly sensitive and reliable detection of the PD-L1-overexpressing exosomes was achieved in the concentration range 7.78 × 101 to 7.78 × 104 particles/mL with a detection limit down to 6 particles/mL. The advantages of the proposeddetection method include the following; first, the detection substrate is easy to prepare and convenient to clean. Second, the TFC strategy can completely exclude nonspecific reaction sites and thus significantly improves the accuracy. Such a facile and reliable detection method holds a great potential in exosome-based cancer theranostics. In this paper, we proposed a triple-color fluorescence co-localization (TFC) strategy to significantly improve the reliability of exosome detection and the detection substrate is easy to prepare and convenient to clean. In addition, the LOD is down to 6 particles/mL, which is quite low compared with other detection methods.

A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.

Karyotypes of Three Exotic Cucurbit Species Based on Triple-Color FISH Analysis

Cytogenetic investigations based on chromosome composition provide insight into basic genetic and genomic characteristics of a species that in turn facilitate species identification and breeding programs. Tandem repeats (TRs) like the 45S rDNA, 5S rDNA and telomeric repeats are ubiquitous in nuclear genomes and are good cytogenetic markers for karyotyping. In this study, we analyzed the karyotypes of three exotic cucurbit species, namely Cucumis melo var. flexuosus (L.) Naudin (2n=24), Melothria pendula L. (2n =24) and Trichosanthes anguina L. (2n=22), based on the cytogenetic distribution of the 45S, 5S and Arabidopsis-type telomeric TRs through triple-color fluorescence in situ hybridization. T. anguina had larger chromosomes (3.2-5.4 µm) compared to C. melo var. flexuosus and M. pendula (1.5-2.2 µm and 1.8-2.5 µm). One and two pairs of 5S and 45S rDNA signals were observed in C. melo var. flexuosus, respectively; while M. pendula and T. anguina had four and three pairs of 45S rDNA, respectively, and two pairs of 5S rDNA. Co-localized signals of 5S and 45S rDNA were observed in M. pendula and T. anguina, but not in C. melo var. flexuosus. Telomeric repeats were observed at chromosome ends of all chromosomes. This information will be useful in future cytogenomic and phylogenetic studies facilitating cucurbit breeding programs.

Different segregation patterns in five carriers due to a pericentric inversion of chromosome 1

Pericentric inversion can produce recombinant gametes; however, meiotic segregation studies on the relationship between the frequency of recombinants and the inverted segment size are rare. Triple-color fluorescence in situ hybridization (FISH) was performed to analyze the meiotic behavior in five inv(1) carriers with different breakpoints. Recombination gametes were absent in Patient 1, whereas the percentages of the recombinants in Patients 2, 3, 4, and 5 were of 9.2%, 15.3%, 17.3%, and 40.9%, respectively. A significant difference was present for the frequencies of the recombinant spermatozoa among the five patients (p < 0.001). For each patient, the frequency of the two types of recombinant gametes (dup(1p)/del(1q) or del(1p)/dup(1q)) did not exhibit a significant difference in comparison with the expected 1:1 ratio (p > 0.05). The meiotic segregation of nine inv(1) carriers (including those presented in this paper) is now available. A significant correlation was discovered between the rate of recombination and the proportion of the chromosome implicated in the inversion (R = 0.9435, p < 0.001). The frequency of the recombinant gametes was directly related to the proportion of the chromosome that was inverted. Sperm-FISH allowed an additional comprehension of the patterns of meiotic segregation and provided accurate genetic counseling.

Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization

With the development of a direct visualization of sex chromosome in a single sperm by fluorescence in situ hybridization (FISH) technique, the frequency of aberration (aneuploidy) in spermatozoa in several mammals has been investigated. However, there is no report in the incidence of X-Y aneuploidy in the sperm population of dogs. Therefore, in this study, the aneuploidy in dog spermatozoa was examined by multicolor FISH using specific molecular probes for canine sex chromosomes and autosome. Semen from eight male Labrador retrievers was used as specimen. For decondensation of sperm nuclei, the specimen was treated with 1 M NaOH for 4 minutes at room temperature. Probes for chromosomes X, Y, and 1, labeled with SpectrumGreen, Cy3 and Cy5, respectively, were hybridized with decondensed spermatozoa. Fluorescence in situ hybridization signals in sperm heads were clearly detected in each specimen, regardless of the sperm donor. The FISH signal of at least one of the three probes was detected in all sperm heads examined. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X and Y chromosomes in spermatozoa of all the eight dogs. Mean percentage of sex chromosome aneuploidy was 0.127% (ranged between 0% and 0.316%). This percentage of canine sex chromosome aneuploidy was lower than the one reported in cattle, horses, river buffalo, and goats sperm, but higher than that observed in mice and sheep.

Study of screening nephroprotective bioactive substances based on triple-color fluorescence probes in Carthami Flos

In this study, an approach based on triple-color fluorescence probes was developed for screening potential nephro-protective bioactive substances. Three fluorescent probes (i. e. FDA, MTR and Hoechst 33342) were used to label HK-2 cells injured by doxorubicin hydrochloride, and cellular fluorescence images were subsequently acquired and analyzed by a cellular-fluorescence image microscopy platform. The established method was applied to screening 53 components of Carthami Flos, and three components C17, C18 and C19 were found to exhibit nephroprotective effects against doxorubicin hydrochloride induced injury on HK-2 cells. Eight compounds (i. e. hydroxysafflor yellow A, 6-hydroxykaempferol-3-O-rutinoside-6-O-glucoside, 6-hydroxykaempferol-3,6-di-O-gluco-side or 6-hydroxykaempferol-6, 7-di-O-glucoside, 6-hydroxykaempferol-3-O-rutinoside, 6-hydroxykaempferol-3-O-glucoside or 6-hydroxykaempferol-7-O-glucoside, rutin, isoquercetin, and kaempferol-3-O-rutinoside) in components C17, C18 and C19 were preliminarily identified by liquid chromatography-mass spectrometry (LC-MS). Isoquercetin, rutin, kaempferol-3-O-rutinoside, and hydroxysafflor yellow A were confirmed by comparing with reference substances, Further study indicated that these four compounds had moderate nephroprotective effects, while isoquercetin showed a significant nephroprotective effect in a dose-dependent manner. These results suggest that isoquercetin, rutin, kaempferol-3-O-rutinoside and hydroxysafflor yellow A might be the nephroprotective bioactive substances in Carthami Flos.

Abstract 5296: Evaluation of HER2 and topoisomera II alpha (TOP2a) expression and gene number in penile carcinoma according to HPV status.

Abstract BACKGROUND: Penile squamous cell carcinoma (PSCC) is a rare tumor. In specific regions of developing countries, however, it may represent one the most frequent male tumors. Presence of lymph node metastasis (LNM) is the main factor significantly associated with reduced survival, and molecular markers to predict it have been extensively pursued. HER2 is an oncogene part of the EGFR family, often overexpressed in PSCC. HER2 overexpression is associated with poor prognosis and presence of LNM in breast tumors, and it is generally coamplified with topoisomerase IIα (TOP2A), a neighboring gene that is a target for anthracyclines. MATERIAL AND METHODS: We evaluated the frequency of HPV infection in 84 PSCC samples.The presence or absence of HPV DNA was established using generic primers (GP5+/GP6+) specific for the L1 gene of several HPVs. Genotyping of positive samples was done by dot blot hybridization using radioactive probes. Expression of HER2 and TOP2a was evaluated through immunohistochemistry and automatedly quantified using ScanScope XT, Aperio. Sixty cases were also submitted to a triple-color fluorescence in situ hybridization (FISH) assay using probes for the genes of HER2 and TOP2a, and also for the chromosome 17 centromere. Cases showing two signals of each probe were considered non-altered, those showing more than two signals of each probe were considered polysomy and those showing more HER2 or TOP2a signals compared to centromere signals were considered amplified. RESULTS: HPV infection, characterized by detection of viral DNA on the samples, was observed in 22.6% (19) of the cases. Among the positive HPV samples, 51.3% were infected by HPV16, 21.6% by HPV18, and 2.7% by both subtypes concomitantly. In agreement with studies that showed better prognosis of tumors with HPV infection, 5-year and 10-year survival of HPV-positive patients was, respectively, 79.1% and 70.8%, whereas of HPV-negative patients was 60.2% and 38.7% (p=0,018). Immunohistochemistry showed nuclear positivity for TOP2a in 74.2% (106) cases and cytoplasmic expression of HER2 was seen in 20,3% (29) of cases, which was associated with higher histologic grade (p &amp;lt;0,001) and lower overall survival in multivariate analysis (RR=2,966; IC 95% [1,6-5,2]; p&amp;lt;0,001). No membrane staining for HER2 was observed. There was not an association between expression of HER2 and TOP2a (p=0.970), nor with these markers and HPV infection (p=0.177 and p=0.336, respectively). FISH analysis revealed 55 out of 60 cases without gene alterations (91.7%), and five cases (8,3%) presenting polysomy of chromosome 17. No amplification of HER2 or TOP2a genes was observed.CONCLUSIONS: Overexression of TOP2a was frequent and cytoplasmic HER2 associates with poor outcome of PSCC patients. HPV infection seem to have a positive impact in patients’ survival, as reported for other tumors. Citation Format: Alice M. Silva, Lara Termini, Jose Ivanildo Neves, Isabela Cunha, Ademar Lopes, Gustavo Guimaraes, Fernando A. Soares. Evaluation of HER2 and topoisomera II alpha (TOP2a) expression and gene number in penile carcinoma according to HPV status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5296. doi:10.1158/1538-7445.AM2013-5296

Deletions and duplications of the 15q11-q13 region in spermatozoa from Prader-Willi syndrome fathers

Prader-Willi syndrome (PWS) is a genomic disorder mostly caused by deletions of 15q11-q13 region (70%). It has been suggested that the particular genomic architecture of 15q11-q13 region, characterized to be flanked by low copy repeats, could predispose it to Non-Allelic Homologous Recombination (NAHR). However, no studies in gametes of fathers of PWS individuals have been published to date. The objective of the study was to assess the incidence of 15q11-q13 deletions and duplications in spermatozoa from PWS fathers and to appraise the value of the data obtained for the estimation of the risk of recurrence for the syndrome. Semen samples from 16 fathers of PWS individuals and 10 control donors, were processed by triple-color fluorescence in situ hybridization. A customized combination of probes was used to discriminate between normal, deleted and duplicated sperm genotypes. A minimum of 10,000 sperm were scored for every single sample. A significant increase in the frequency of 15q11-q13 deletions and duplications were observed in PWS fathers (0.90 +/- 0.14%) compared with control donors (0.47 +/- 0.07%). Ten out of 16 individuals contributed to this population increase (P < 0.01), suggesting a predisposition for 15q11-q13 reorganizations. Statistical differences were observed in the frequency of 15q11-q13 deletions and duplications in fathers of PWS individuals (0.59 versus 0.31%; P = 0.001), indicating that intra-chromatid NAHR exchanges also substantially contribute to the rearrangements. Results demonstrated the increased susceptibility of some fathers of PWS individuals to generate 15q11-q13 deletions, suggesting that the screening of anomalies in sperm should be advisable as a valuable complement for genetic counseling.

Evaluation of semen variables, sperm chromosomal abnormalities and reproductive endocrine profile in patients with chronic hepatitis C

To evaluate reproductive endocrine profile, sperm chromosomal abnormalities, and semen quality in patients with chronic hepatitis C. In all, 82 patients with chronic hepatitis C, aged 18-60 years, were recruited for the study; 76 age-matched healthy male volunteers served as controls. All participants provided a medical history and had a complete physical examination and routine semen analysis. Two blood samples were drawn from each participant at 15-min intervals to determine the resting levels of luteinizing-hormone (LH), follicle-stimulating hormone (FSH), prolactin, testosterone, oestradiol, and sex hormone-binding globulin. The hypothalamus-pituitary-testis axis was assessed using the gonadotrophin-releasing hormone (GnRH) test. All participants also received an injection of human chorionic gonadotrophin (hCG) and serum testosterone was determined before the hCG injection and on the third day afterwards. Conventional karyotype analysis and triple-colour fluorescence in situ hybridization for chromosomes X, Y and 18 were conducted in all patients and controls. The mean basal serum levels for LH, FSH, and testosterone in patients with hepatitis C was significantly lower than the mean for normal controls (P = 0.01). The injection of GnRH analogue did not yield significantly higher FSH and LH levels in the patients than in normal controls (P = 0.001). In patients with chronic hepatitis C, the mean total sperm count, motility and normal morphology was significantly lower than in controls (P = 0.001). There was a significantly greater frequency of disomy in men with chronic hepatitis C than controls for chromosomes 18, X, and Y (P = 0.01). Patients with chronic hepatitis C are at risk of showing sperm chromosomal abnormalities, the incidence of which is higher in patients with more advanced disease. Hypogonadotrophic hypogonadism is caused by the selective loss of pituitary gonadotrophin function. Further studies are needed to replicate our results.

Histone H1 is released from myonuclei and present in rimmed vacuoles with DNA in inclusion body myositis

To investigate myonuclear alterations in sporadic inclusion body myositis (s-IBM), we immuno-localized histones in muscles in 11 patients. The examination showed that vacuolar rims were frequently positive for histone H1. In triple-color fluorescence study, the H1-positive products were found on the inner side of an emerin-positive circle with DNA. Moreover, H1-positive materials appeared to be released into the cytoplasm in some vacuoles and myonuclei. The localization of H1 was different from phosphorylated Elk-1, which is a nuclear protein, but abnormally accumulated in the cytoplasm in s-IBM. The results strongly support the hypothesis that rimmed vacuoles are derived from the nucleus. The cytoplasmic H1-release suggests dysfunction of nuclear membranes in an early phase of the nuclear disintegration. We hypothesize that, in s-IBM muscles, compromised nuclear envelope may permit release of some nuclear components such as histone H1 and cannot facilitate the incorporation of others to the nucleus as in pElk-1.

The Analysis of Meiotic Segregation Patterns and Aneuploidy in the Spermatozoa of Father and Son With Translocation t(4;5)(p15.1;p12) and the Prediction of the Individual Probability Rate for Unbalanced Progeny at Birth

Reciprocal chromosomal translocations (RCT) have long been recognized as important etiological factors in reproductive failure. In the present study, the meiotic segregation patterns of the spermatozoa of two related t(4;5)(p15.1;p12) carriers (proband and his father) were compared to the empirical data from a three-generation pedigree for risk assessment. Cytogenetic analysis of the metaphase chromosomes was performed, and triple color fluorescence in situ hybridization (FISH) was applied to the sperm heads. Similar patterns of meiotic segregation were observed for both carriers, despite the finding of teratozoospermia in the proband but not in his father. In addition, an increase of aneuploidy in chromosome 15 in the proband and aneuploidy of chromosomes X and Y in the father were observed. The high rate of miscarriages (6/10 pregnancies and 4/7 pregnancies after ascertainment correction) in this family could be explained by the genetically unbalanced karyotype and fertilization mediated by the unbalanced spermatozoa observed for both men at a frequency of more than 60%. The risk assessment for unfavorable pregnancy outcomes was predicted as 1.6% for unbalanced progeny at birth and about 30% for miscarriage. These figures may be used as guidelines for the genetic counseling of families with similar RCT.

The effects of age on the incidence of aneuploidy rates in spermatozoa of oligoasthenozoospermic patients and its relationship with ICSI outcome

The development of intracytoplasmic sperm injection (ICSI) for treatment of infertility as a result of severe male factor has improved the chances of achieving pregnancy in many infertile couples. However, concerns have been raised regarding the safety of this technique, because natural sperm selection is bypassed. In the present study, 25 oligoasthenozoospermic patients who were divided into two groups according to age: group A, 20-34 (n = 10) and group B, 35-50 (n = 15), were included. Pooling the data of the three semen parameters that were tested (volume, concentration and progressive motility) no statistically significant difference between the two age groups was found. A total of 50 883 decondensed spermatozoa was analysed using the dual and triple colour fluorescence in situ hybridization to estimate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y in the two age groups. There was a significantly higher incidence of disomy for chromosome 21 compared to the other autosomes (chromosomes 13 and 18) in both age groups. The disomy rate of XY was significantly higher in the younger subject group (0.1%) compared to the older group (0.05%, p < 0.05). Statistically significant differences in the mean number of clinical pregnancies and abortions were not observed between the two age groups. The aneuploidy rates for all the analysed chromosomes did not differ significantly, both between and within the two age groups, and as a result there seems to be no effect of male age on chromosome numbers in the spermatozoa and on the ICSI outcome.

Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.

Morphologically normal spermatozoa of patients with secretory oligo-astheno-teratozoospermia have an increased aneuploidy rate.

Normal morphology is a major criterion for selecting spermatozoa to be injected. Given that teratozoospermia is one of the most critical parameters associated with sperm aneuploidy, the purpose of this study was to evaluate the aneuploidy rate of morphologically normal spermatozoa of patients with oligo-astheno-teratozoospermia (OAT). Ten patients with secretory OAT and six age-matched normozoospermic men with a normal karyotype were enrolled. After assignment to normal or abnormal category, the location of each spermatozoon was recorded using an electronic microstage locator. Slides were then subjected to triple-colour fluorescence in situ hybridization for chromosomes X, Y and 12. OAT patients had a lower number of morphologically normal and abnormal spermatozoa carrying the X chromosome, compared with normozoospermic men. They also exhibited increased XY and XX disomy rates. Morphologically abnormal spermatozoa from normozoospermic men also had an increased XX disomy rate compared with normally shaped spermatozoa obtained from the same men. The total sperm aneuploidy rate of morphologically abnormal spermatozoa of normozoospermic men was 4.4-fold higher than that of spermatozoa with normal morphology. The total aneuploidy rates of spermatozoa with normal or abnormal head shape from OAT patients were similar to each other and to that of abnormally shaped spermatozoa from normozoospermic men, but they were higher than the rate found in normally shaped spermatozoa of normal men. Normally shaped spermatozoa of OAT patients have an increased aneuploidy rate.

Diagnostic value of CD45RO expression on circulating T lymphocytes of fetuses and newborn infants with pre-, peri- or early post-natal infections.

We examined the expression of the CD45RO antigen, which characterizes the antigen primed/memory phenotype of T lymphocytes, as a marker for congenital infection in blood samples of newborns and fetuses. CD45RO expression on T cells was determined by triple-colour fluorescence flow cytometry. In total 537 blood samples of newborns and infants up to an age of 3 months and 89 fetal blood samples from gestational weeks 19-31 were analysed. Of the newborns and infants, 74 had a clinically, serologically and/or antigenically evident infection, and four of the fetuses had a confirmed intra-uterine infection. In 35 infants with acute predominantly bacterial infections such as sepsis or pneumonia, 17 (48.6%) had elevated CD45RO(bright) expression. In 39 infants with proven pre-, peri- or early post-natal infections with toxoplasmosis, cytomegalovirus (CMV), rubella, herpes simplex virus (HSV) or human herpes virus type 6 (HHV6), 25 (64.1%) exhibited enhanced CD45RO(bright) expression. Three of four fetuses with confirmed intra-uterine infection (three with CMV, one with parvovirus B19) exhibited elevated CD45RO(bright) expression. The specificity of the CD45RO assay for detecting microbial infections was 94.6% for newborns and infants up to 3 months and 90.6% for fetuses. It is concluded that elevated numbers of CD45RO(bright) T cells in infants up to 3 months of age strongly suggest an infection. However, the sensitivity of the CD45RO assay is not sufficient to enable the test to be used as a general marker for prescreening infants to detect pre-, peri- or early post-natally acquired infections.

Frequency of recombinant and nonrecombinant products of pericentric inversion of chromosome 1 in sperm nuclei of carrier: by FISH technique.

Meiotic segregation products of carriers with pericentric inversion are very important for assessing the risk of unbalanced forms and appropriate genetic counseling. We investigated the incidence of recombinant and nonrecombinant products of chromosome 1 with pericentric inversion, in the sperm nuclei of the carrier by using triple color fluorescence in situ hybridization (FISH). The centromere specific and telomere specific probes for chromosome 1 were used. In the segregation analysis, 1,636 sperm nuclei were analyzed; 82.5% of the sperms were including normal or inverted chromosome 1, and the dup(p)/del(q) and del(p)/dup(q) recombinant products in sperm nuclei of our carrier were 8.7 and 7.3%, respectively. The number of recombinant products may be dependent on the formation of an inversion loop, which the number of the formation of chiasmata results in the different number of normal/balanced and recombinant products. The use of FISH, using different probe combination, in sperm nuclei has proved to be an accurate approach to determine the meiotic segregation patterns and could help to better establish a reproductive prognosis and genetic counseling.

Absolute polymorphic teratozoospermia in patients with oligo-asthenozoospermia is associated with an elevated sperm aneuploidy rate.

Infertile patients with abnormal sperm parameters have an increased sperm aneuploidy rate, despite a normal blood karyotype. The evaluation of sperm chromosome aberrations in patients with teratozoospermia only has shown a rate similar to that found in patients exhibiting oligo-astheno-teratozoospermia, which suggests that teratozoospermia is the critical parameter associated with aneuploidy. However, it is not known which alteration of the sperm morphology is associated with chromosome aberrations. The few cases reported so far have shown an association with the presence of abnormal head morphology and particularly with enlarged heads. We report the sperm aneuploidy rate of 3 patients with oligo-asthenozoospermia who have absolute teratozoospermia (100% abnormal forms) and a different percentage of sperm head abnormalities. Fourteen healthy men with normozoospermia served as control subjects. Sperm aneuploidy and diploidy rates were calculated by using triple-color fluorescence in situ hybridization (FISH) for chromosomes 12, X, and Y, and double-color FISH was used for chromosomes 8 and 18. Patient K53, who had the highest number of spermatozoa with enlarged heads (54.3%), also had the highest aneuploidy and diploidy rates. The other 2 patients, K56 and K61, had sperm aneuploidy and diploidy rates lower than those of patient K53 but still well above the range found in normal men. Sperm chromosome abnormalities were intermediate in patient K61 and lower in patient K56, who had the lowest rate of spermatozoa with enlarged heads (18.9%). These data add further evidence that patients with teratozoospermia have an increased sperm aneuploidy rate and that this is particularly high in presence of an elevated percentage of spermatozoa with enlarged heads. For this reason, germ cells exhibiting this abnormality should not be used in in vitro fertilization programs.

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

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. 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. 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. 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.

Aneuploid and unbalanced sperm in two translocation carriers: evaluation of the genetic risk.

Translocation carriers have an increased risk of reproductive failure or affected offspring, because of the production of unbalanced gametes by meiotic segregation or the possible presence of interchromosomal effects (ICE). We therefore performed an analysis of meiotic segregation using the human-hamster IVF technique, and an aneuploidy assay for chromosomes 6, 18, 21, X and Y, using dual and triple-colour fluorescence in-situ hybridization, in two translocation carriers, t(1;13)(q41;q22) and t(3;19)(p21;p13.3). Sperm chromosome complements were analysed by whole chromosome painting. The frequencies observed for alternate, adjacent I, adjacent II and 3:1 segregations were, for t(1;13), 41.6, 41.6, 14.5 and 2.3% respectively, and for t(3;19), the frequencies were 39.1, 35.9, 21.8 and 3.2% respectively. More than 20,000 sperm per subject were analysed in the aneuploidy assay. Disomy 21 was found to be higher than other autosome disomies. Evidence for a possible ICE was found only in t(3;19). This study has shown that unbalanced sperm are more frequent than aneuploid sperm in the total sperm population. However, data in the literature suggest that the importance of each aberrant population seems to be more significant for embryo viability than would be expected from the increases in the percentages of abnormal sperm.