Chromatin and transcriptome changes in human myoblasts show spatio-temporal correlations and demonstrate DPP4 inhibition in differentiated myotubes.

Although less attention was paid to understanding physical localization changes in cell nuclei recently, depicting chromatin interaction maps is a topic of high interest. Here, we focused on defining extensive physical changes in chromatin organization in the process of skeletal myoblast differentiation. Based on RNA profiling data and 3D imaging of myogenic (NCAM1, DES, MYOG, ACTN3, MYF5, MYF6, ACTN2, and MYH2) and other selected genes (HPRT1, CDH15, DPP4 and VCAM1), we observed correlations between the following: (1) expression change and localization, (2) a gene and its genomic neighbourhood expression and (3) intra-chromosome and microscopical locus-centromere distances. In particular, we demonstrated the negative regulation of DPP4 mRNA (p < 0.001) and protein (p < 0.05) in differentiated myotubes, which coincided with a localization change of the DPP4 locus towards the nuclear lamina (p < 0.001) and chromosome 2 centromere (p < 0.001). Furthermore, we discuss the possible role of DPP4 in myoblasts (supported by an inhibition assay). We also provide positive regulation examples (VCAM1 and MYH2). Overall, we describe for the first time existing mechanisms of spatial gene expression regulation in myoblasts that might explain the issue of heterogenic responses observed during muscle regenerative therapies.

How much, if anything, do we know about sperm chromosomes of Robertsonian translocation carriers?

In men with oligozoospermia, Robertsonian translocations (RobTs) are the most common type of autosomal aberrations. The most commonly occurring types are rob(13;14) and rob(14;21), and other types of RobTs are described as 'rare' cases. Based on molecular research, all RobTs can be broadly classified into Class 1 and Class 2. Class 1 translocations produce the same breakpoints within their RobT type, but Class 2 translocations are predicted to form during meiosis or mitosis through a variety of mechanisms, resulting in variation in the breakpoint locations. This review seeks to analyse the available data addressing the question of whether the molecular classification of RobTs into Classes 1 and 2 and/or the type of DD/GG/DG symmetry of the involved chromosomes is reflected in the efficiency of spermatogenesis. The lowest frequency value calculated for the rate of alternate segregants was found for rob(13;15) carriers (Class 2, symmetry DD) and the highest for rob(13;21) carriers (Class 2, DG symmetry). The aneuploidy values for the rare RobT (Class 2) and common rob(14;21) (Class 1) groups together exhibited similarities while differing from those for the common rob(13;14) (Class 1) group. Considering the division of RobT carriers into those with normozoospermia and those with oligoasthenozoospermia, it was found that the number of carriers with elevated levels of aneuploidy was unexpectedly quite similar and high (approx. 70%) in the two subgroups. The reason(s) that the same RobT does not always show a similar destructive effect on fertility was also pointed out.

Chromosome (re)positioning in spermatozoa of fathers and sons - carriers of reciprocal chromosome translocation (RCT).

BACKGROUND: Non-random chromosome positioning has been observed in the nuclei of several different tissue types, including human spermatozoa. The nuclear arrangement of chromosomes can be altered in men with decreased semen parameters or increased DNA fragmentation and in males with chromosomal numerical or structural aberrations. An aim of this study was to determine whether and how the positioning of nine chromosome centromeres was (re)arranged in the spermatozoa of fathers and sons - carriers of the same reciprocal chromosome translocation (RCT). METHODS: Fluorescence in situ hybridization (FISH) was applied to analyse the positioning of sperm chromosomes in a group of 13 carriers of 11 RCTs, including two familial RCT cases: t(4;5) and t(7;10), followed by analysis of eight control individuals. Additionally, sperm chromatin integrity was evaluated using TUNEL and Aniline Blue techniques. RESULTS: In the analysed familial RCT cases, repositioning of the chromosomes occurred in a similar way when compared to the data generated in healthy controls, even if some differences between father and son were further observed. These differences might have arisen from various statuses of sperm chromatin disintegration. CONCLUSIONS: Nuclear topology appears as another aspect of epigenetic genomic regulation that may influence DNA functioning. We have re-documented that chromosomal positioning is defined in control males and that a particular RCT is reflected in the individual pattern of chromosomal topology. The present study examining the collected RCT group, including two familial cases, additionally showed that chromosomal factors (karyotype and hyperhaploidy) have superior effects, strongly influencing the chromosomal topology, when confronted with sperm chromatin integrity components (DNA fragmentation or chromatin deprotamination).

The effect of Robertsonian translocations on the intranuclear positioning of NORs (nucleolar organizing regions) in human sperm cells.

Only a few studies have described sperm chromosome intranuclear positioning changes in men with reproductive failure and an incorrect somatic karyotype. We studied the influence of Robertsonian translocations on the acrocentric chromosome positioning in human sperm cells. The basis of the analysis was the localization of NORs (nucleolar organizing regions) in sperm nuclei from three Robertsonian translocation carriers, namely, rob(13;22), rob(13;15) and rob(13;14), with a known meiotic segregation pattern. All three carriers presented with a similar percentage of genetically normal sperm cells (i.e., approximately 40%). To visualize NORs, we performed 2D-FISH with directly labelled probes. We used the linear and radial topologies of the nucleus to analyse the NORs distribution. We found an affected positioning of NORs in each case of the Robertsonian translocations. Moreover, the NORs tended to group, most often in two clusters. Both in Robertsonian carriers and control sperm cells, NORs mostly colocalized in the medial areas of the nuclei. In the case of the Roberstonian carriers, NORs were mostly concentrated in the peripheral part of the medial area, in contrast to control sperm cells in which the distribution was more dispersed towards the internal area.

Topology of chromosome centromeres in human sperm nuclei with high levels of DNA damage.

Several studies have shown that the 'poor' sperm DNA quality appears to be an important factor affecting male reproductive ability. In the case of sperm cells from males with the correct somatic karyotype but with deficient spermatogenesis, resulting in a high degree of sperm DNA fragmentation, we observed changes in the preferential topology of the chromosome 7, 9, 15, 18, X and Y centromeres. The changes occurred in radial localization and may have been directly linked to the sperm chromatin damage. This conclusion is mainly based on a comparison of FISH signals that were observed simultaneously in the TUNEL-positive and TUNEL-negative sperm cells. The analyzed cells originated from the same ejaculated sample and FISH was performed on the same slides, after in situ TUNEL reaction. Based on the observed changes and previous data, it appears that the sperm nucleus architecture can be disrupted by a variety of factors and has a negative influence on spermatogenesis at the same time. Often, these factors coexist (e.g. chromosomal translocations, aneuploidies, a higher DNA fragmentation, abnormal seminology), but no direct correlations between the factors were observed.

Meiotic and pedigree segregation analyses in carriers of t(4;8)(p16;p23.1) differing in localization of breakpoint positions at 4p subband 4p16.3 and 4p16.1.

PURPOSE: The purpose of this study was to compare meiotic segregation in sperm cells from two carriers with t(4;8)(p16;p23.1) reciprocal chromosome translocations (RCTs), differing in localization of the breakpoint positions at the 4p subband-namely, 4p16.3 (carrier 1) and 4p16.1 (carrier 2)-and to compare data of the pedigree analyses performed by direct method. METHODS: Three-color fluorescent in situ hybridization (FISH) on sperm cells and FISH mapping for the evaluation of the breakpoint positions, data from pedigrees, and direct segregation analysis of the pedigrees were performed. RESULTS: Similar proportions of normal/balanced and unbalanced sperm cells were found in both carriers. The most common was an alternate type of segregation (about 52 % and about 48 %, respectively). Unbalanced adjacent I and adjacent II karyotypes were found in similar proportions about 15 %. The direct segregation analysis (following Stengel-Rutkowski) of the pedigree of carriers of t(4;8)(p16.1;p23.1) was performed and results were compared with the data of the pedigree segregation analysis obtained earlier through the indirect method. The probability of live-born progeny with unbalanced karyotype for carriers of t(4;8)(p16.1;p23.1) was moderately high at 18.8 %-comparable to the value obtained using the indirect method for the same carriership, which was 12 %. This was, however, markedly lower than the value of 41.2 % obtained through the pedigree segregation indirect analysis estimated for carriers of t(4;8)(p16.3;p23.1), perhaps due to the unique composition of genes present within the 4p16.1-4p 16.3 region. CONCLUSIONS: Revealed differences in pedigree segregation analysis did not correspond to the very similar profile of meiotic segregation patterns presented by carrier 1 and carrier 2. Most probably, such discordances may be due to differences in embryo survival rates arising from different genetic backgrounds.

Genetic dosage and position effect of small supernumerary marker chromosome (sSMC) in human sperm nuclei in infertile male patient.

Chromosomes occupy specific distinct areas in the nucleus of the sperm cell that may be altered in males with disrupted spermatogenesis. Here, we present alterations in the positioning of the human chromosomes 15, 18, X and Y between spermatozoa with the small supernumerary marker chromosome (sSMC; sSMC(+)) and spermatozoa with normal chromosome complement (sSMC(-)), for the first time described in the same ejaculate of an infertile, phenotypically normal male patient. Using classical and confocal fluorescent microscopy, the nuclear colocalization of chromosomes 15 and sSMC was analyzed. The molecular cytogenetic characteristics of sSMC delineated the karyotype as 47,XY,+der(15)(pter->p11.2::q11.1->q11.2::p11.2->pter)mat. Analysis of meiotic segregation showed a 1:1 ratio of sSMC(+) to sSMC(-) spermatozoa, while evaluation of sperm aneuploidy status indicated an increased level of chromosome 13, 18, 21 and 22 disomy, up to 7 × (2.7 - 15.1). Sperm chromatin integrity assessment did not reveal any increase in deprotamination in the patient's sperm chromatin. Importantly, we found significant repositioning of chromosomes X and Y towards the nuclear periphery, where both chromosomes were localized in close proximity to the sSMC. This suggests the possible influence of sSMC/XY colocalization on meiotic chromosome division, resulting in abnormal chromosome segregation, and leading to male infertility in the patient.

FISH and array CGH characterization of de novo derivative Y chromosome (Yq duplication and partial Yp deletion) in an azoospermic male.

This study presents a 28-year-old infertile male who was referred to the cytogenetic laboratory for chromosomal analysis after 4 years of regular unprotected intercourse in whom non-obstructive azoospermia was revealed. Standard cytogenetic G-banding was performed on metaphase spreads and a de-novo karyotype 46,X,der(Y)(q11.22;p11.3) was identified. This analysis was followed by flourescence in-situ hybridization(FISH) and array comparative genomic hybridization (aCGH). Finally, the patient's karyotype was identified as 46,X,der(Y)(qter→q11.221::p11.31→qter).ish der(Y) (qter+,pter-,SHOX+,SRY+,Ycen+,DYZ3+;DYZ1+,qter+).arrYq11.221q12(14,448,863-59,288,511) x2, Yp11.32p11.31(104,062-266,388) x0. It is proposed that de-novo derivative monocentric Y chromosome with duplicated region Y qter→q11.221::p11.31→qter with partial deletion of Yp PAR1 region most probably can perturb the conjugation of sex chromosomes during first meiotic division of spermatogenic arrested differentiation (development).

Recurrence risks for different pregnancy outcomes and meiotic segregation analysis of spermatozoa in carriers of t(1;11)(p36.22;q12.2).

Cumulative data obtained from two relatively large pedigrees of a unique reciprocal chromosomal translocation (RCT) t(1;11)(p36.22;q12.2) ascertained by three miscarriages (pedigree 1) and the birth of newborn with hydrocephalus and myelomeningocele (pedigree 2) were used to estimate recurrence risks for different pregnancy outcomes. Submicroscopic molecular characterization by fluorescent in situ hybridization (FISH) of RCT break points in representative carriers showed similar rearrangements in both families. Meiotic segregation patterns after sperm analysis by three-color FISH of one male carrier showed all possible outcomes resulting from 2:2 and 3:1 segregations. On the basis of empirical survival data, we suggest that only one form of chromosome imbalance resulting in monosomy 1p36.22→pter with trisomy 11q12.2→qter may be observed in progeny at birth. Segregation analysis of these pedigrees was performed by the indirect method of Stengel-Rutkowski and showed that probability rate for malformed child at birth due to an unbalanced karyotype was 3/48 (6.2±3.5%) after ascertainment correction. The risk for stillbirths/early neonatal deaths was -/48 (<1.1%) and for miscarriages was 17/48 (35.4±6.9%). However, the probability rate for children with a normal phenotype at birth was 28/48 (58.3±7.1%). The results obtained from this study may be used to determine the risks for the various pregnancy outcomes for carriers of t(1;11)(p36.22;q12.2) and can be used for genetic counseling of carriers of this rearrangement.

Fertilizing potential of ejaculated human spermatozoa during in vitro semen bacterial infection.

OBJECTIVE: To assess the in vitro effect of three bacterial isolates (Escherichia coli, serotype O75:HNT, Staphylococcus haemolyticus, and Bacteroides ureolyticus) and/or leukocytes on sperm motility, subcellular changes in sperm plasma membranes, and sperm fertilizing potential. DESIGN: An in vitro model of semen bacterial infection. SETTING: Basic research laboratory. PATIENT(S): Healthy normozoospermic volunteers and healthy blood donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm plasma membrane stability was evaluated with a LIVE/DEAD Sperm Viability Kit and with the merocyanine 540 (M540) test both performed using flow cytometry. An oxiSelect TBARS Assay Kit was used for quantitative measurement of malondialdehyde content. Functional ability of spermatozoa was assessed by hypo-osmotic swelling (HOS) test and sperm penetration assay (SPA). RESULT(S): The incubation of sperm with bacteria and/or leukocytes was associated with the reduction of their fertilizing potential demonstrated in both the HOS test and SPA, and this effect can be considered as a natural consequence of diminished motility and sperm membrane injury of lipid bilayers. Bacteroides ureolyticus demonstrated the most significant detrimental effect on sperm structure and function. CONCLUSION(S): Sperm motility and lipid sperm membrane status might be the earliest and the most sensitive indicators of sperm damage with negative consequences for male factor fertility, which can be attributed to both bacteria and leukocytes action.

Sperm FISH and chromatin integrity in spermatozoa from a t(6;10;11) carrier.

Complex chromosome rearrangements (CCRs) are structurally balanced or unbalanced aberrations involving more than two breakpoints on two or more chromosomes. CCRs can be a potential reason for genomic imbalance in gametes, which leads to a drastic reduction in fertility. In this study, the meiotic segregation pattern, aneuploidy of seven chromosomes uninvolved in the CCR and chromatin integrity were analysed in the ejaculated spermatozoa of a 46,XY,t(6;10;11)(q25.1;q24.3;q23.1)mat carrier with asthenozoospermia and a lack of conception. The frequency of genetically unbalanced spermatozoa was 78.8% with a prevalence of 4:2 segregants of 38.2%, while the prevalence of the adjacent 3:3 mode was 35.3%. Analysis of the aneuploidy of chromosomes 13, 15, 18, 21, 22, X and Y revealed an approximately fivefold increased level in comparison with that of the control group, indicating the presence of an interchromosomal effect. Sperm chromatin integrity status was evaluated using chromomycin A3 and aniline blue staining (deprotamination), acridine orange test and TUNEL assay (sperm DNA fragmentation). No differences were found when comparisons were made with a control group. We suggest that the accumulation of genetically unbalanced spermatozoa, significantly increased sperm aneuploidy level and decreased sperm motility (20%, progressive) were not responsible for the observed lack of reproductive success in the analysed infertile t(6;10;11) carrier. Interestingly, in the case described herein, a high level of sperm chromosomal imbalance appears not to be linked to sperm chromatin integrity status.

Cytogenetic and molecular analyses of de novo translocation dic(9;13)(p11.2;p12) in an infertile male.

BACKGROUND: Whole arm t(9;13)(p11;p12) translocations are rare and have been described only a few times; all of the previously reported cases were familial. RESULTS: We present here an infertile male carrier with a whole-arm reciprocal translocation dic(9;13)(p11.2;p12) revealed by GTG-, C-, and NOR-banding karyotypes with no mature sperm cells in his ejaculate. FISH and genome-wide 400 K CGH microarray (Agilent) analyses demonstrated a balanced chromosome complement and further characterised the abnormality as a dicentric chromosome (9;13): dic(9;13)(pter→p11.2::p12→qter),neo(9)(pter→p12→neo→p11.2). An analysis of the patient's ejaculated cells identified immature germ cells at different phases of spermatogenesis but no mature spermatozoa. Most (82.5%) of the germ cells were recognised as spermatocytes at stage I, and the cell nuclei were most frequently found in pachytene I (41.8%). We have also undertaken FISH analysis and documented an increased rate of aneuploidy of chromosomes 15, 18, X and Y in the peripheral blood leukocytes of our patient. To study the aneuploidy risk in leukocytes, we have additionally included 9 patients with non-obstructive azoospermia with normal karyotypes. CONCLUSIONS: We propose that the azoospermia observed in the patient with the dic(9;13)(p11.2;p12) translocation was most likely a consequence of a very high proportion (90%) of association between XY bivalents and quadrivalent formations in prophase I.

Chromatin structure analysis of spermatozoa from reciprocal chromosome translocation (RCT) carriers with known meiotic segregation patterns.

The presence of reciprocal chromosome translocations (RCTs), as well as sperm chromatin disturbances, is known to exert negative influence on male fertility. The aim of this study was to identify an association between chromosome structural rearrangements in male RCT carriers and sperm seminological parameters (concentration, motility, morphology), chromatin status (fragmentation and maturity), meiotic segregation pattern and observed chromosomal hyperhaploidy. Sperm samples originated from ten male RCT carriers with reproductive failure/success. TUNEL assay (DNA fragmentation) and chromomycin A3 (CMA3)/aniline blue (AB) staining (chromatin maturity) were used to analyze sperm chromatin status while fluorescent in situ hybridization (FISH) was applied to observe meiotic segregation patterns and hyperhaploidy in spermatozoa. We found that the mean level of sperm DNA fragmentation in the RCT carrier group (18.0 ± 11.9%) was significantly higher (p=0.0006) than the mean of the control group (7.5 ± 4.3%). There was no correlation observed between sperm DNA fragmentation levels (5.6-38.0%) and the frequency of genetically normal/balanced gametes (34.3-62.4%), sperm seminological quality or revealed reproductive failure. In contrast, a correlation between the frequencies of genetically normal/balanced spermatozoa and of gametes with mature chromatin was observed (CMA3: R=0.4524, p=0.2604; AB: R=0.5238, p=0.1827). A statistically significant increase in the hyperhaploidy level of selected chromosomes in all analyzed RCT carriers was documented but was not correlated to sperm seminology or fertility status. Further evaluation and additional assays toward sperm chromatin quality assessment in RCT carriers is suggested to explain the complexity of genomic structural rearrangements and its possible relevance to reproductive success or failure.

Characterisation of nuclear architectural alterations during in vitro differentiation of human stem cells of myogenic origin.

Cell differentiation is based on a synchronised orchestra of complex pathways of intrinsic and extrinsic signals that manifest in the induced expression of specific transcription factors and pivotal genes within the nucleus. One cannot ignore the epigenetic status of differentiating cells, comprising not only histones and DNA modifications but also the spatial and temporal intranuclear chromatin organisation, which is an important regulator of nuclear processes. In the present study, we investigated the nuclear architecture of human primary myoblasts and myocytes in an in vitro culture, with reference to global changes in genomic expression. Repositioning of the chromosomal centromeres, along with alterations in the nuclear shape and volume, was observed as a consequence of myotube formation. Moreover, the microarray data showed that during in vitro myogenesis cells tend to silence rather than induce gene expression. The creation of a chromosome map marked with gene expression changes that were at least 2-fold confirmed the observation. Additionally, almost all of the chromosomal centromeres in the differentiated cells preferentially localised near the nuclear periphery when compared to the undifferentiated cells. The exceptions were chromosomes 7 and 11, in which we were unable to confirm the centromere repositioning. In our opinion, this is the first reported observation of the movement of chromosomal centromeres along differentiating myogenic cells. Based on these data we can conclude that the myogenic differentiation with global gene expression changes is accompanied by the spatial repositioning of chromosomes and chromatin remodelling, which are important processes that regulate cell differentiation.

In situ reconstruction of humoral immune response against sperm: comparison of SCID and NOD/SCID mouse models.

PROBLEM: Comparison of two types of immunocompromised mouse strains (SCID and NOD/SCID) for production of human antisperm antibodies (AsA). METHOD OF STUDY: Human peripheral blood lymphocytes (PBL) were grafted to mouse peritoneal cavity and sensitized with natively glycosylated and N-deglycosylated sperm extracts. RESULTS AND CONCLUSION: NOD/SCID mice inoculated with hu-PBLs exhibited higher AsA titres with a tendency for greater sperm agglutination than human AsA raised in SCID mouse model. A comparison between 'native' and deglycosylated sperm extracts revealed higher agglutination titres by sera induced with the latter ones. Inhibitory effect of human polyclonal AsA in sperm penetration assay, however, produced opposite results to that for agglutination. Western immunoblotting was used to evaluate reactive sperm antigens prior to and after in situ sensitization showing multiple bands different from positive reactions brought by original sera of in vivo primed AsA-positive males. It seems that in situ generated AsA recognized sperm entities different from those revealed by blood donor's sera samples.

Comparison of chromosome centromere topology in differentiating cells with myogenic potential.

Chromosome territories (CT's) constitute the critical element of the intranuclear architecture. Position of these compartmentalized structures plays an important role in functioning of entire genome. Present study was to examine whether the centromeres position of chromosomes 4, X and Y can be changed during differentiation from myoblasts to myotubes. Topological analysis of these centromeres was based on two-dimensional fluorescent hybridization in situ (2D-FISH). During differentiation process the majority of X chromosome centromeres analyzed shifted to the peripheral part of a nucleus and similar phenomenon was observed with one of the chromosome 4 centromeres. Completely different tendency was noticed when investigating the location of the chromosome Y centromeres. Centromeres of this chromosome migrated to the centre of a nucleus. The results obtained demonstrated visible changes in chromosome topology along the myogenic stem cells differentiation.

Positioning of chromosome 15, 18, X and Y centromeres in sperm cells of fertile individuals and infertile patients with increased level of aneuploidy.

Evidence has been accumulating that individual chromosomes in human sperm cells occupy defined, non-random positions. Our earlier study suggested that abnormal spermatogenesis in carriers of reciprocal translocations was reflected in the changes in the intranuclear topology of sperm chromosomes. The purpose of this study was to determine whether the increased level of disomy of sperm chromosomes may be the factor that can disturb topology within the sperm nuclei. The results obtained indicated that within the sperm nuclei of fertile individuals the centromeres of chromosomes 15, 18, X and Y were localized in a small area that may be a fragment of the chromocentre. When compared with the intranuclear positions of the same chromosomes in sperm nuclei of infertile patients with an increased level of aneuploidy, some disturbances in the centromere area were found. In disomic sperm cells (n + 1) centromeres 15,15 or 18,18 or YY (but not X,X) had a shifted average longitudinal position in comparison with normal sperm cells (n = 23).

Interindividual differences and alterations in the topology of chromosomes in human sperm nuclei of fertile donors and carriers of reciprocal translocations.

Recently it has been shown that the nucleus of the human spermatozoon appears to possess a specific architecture. The current prevailing view is that spatial organization of the male genome contains information critical for the spermatozoon's function as well as for early embryonic development. The purpose of this study was to determine whether there are alterations in intranuclear localization of centromeres in spermatozoa of chromosomes associated with particular reciprocal chromosome translocations (RCT). We analyzed the longitudinal and spatial localization of centromeres of selected chromosomes in sperm nuclei of four control males with normal karyotypes as well as in six carriers of reciprocal chromosome translocations: t(1;7), t(7;2), t(7;13), t(7;9), t(9;14), and t(4;13). Our study revealed that chromosomes with translocations may have shifted their intranuclear localization and that these translocations may influence the localization of other chromosomes in sperm nuclei. The chromocenter in sperm nuclei of translocation carriers was widened toward the apical side in comparison with chromocenter sites visible in control males. Our study also revealed interindividual differences in the localization of the Y chromosome centromere in the chromocenter area of sperm from fertile individuals.

Successful pregnancy after preimplantation genetic diagnosis for carrier of t(2;7)(p11.2;q22) with high rates of unbalanced sperm and embryos: a case report.

BACKGROUND: Regarding the literature on the results of preimplantation genetic diagnosis (PGD) in reciprocal chromosomal translocation carriers seems to prevail a view that this method reduces the frequency of miscarriages, and the pregnancy rate is directly proportional to the number of normal spermatozoa. Therefore, we compared the results of sperm karyotype analysis of a carrier of familial t(2;7)(p11.2;q22) with PGD results. The carrier was ascertained as his wife had had two miscarriages. METHODS: Empirical data from a pedigree of t(2;7)(p11.2;q22) carrier was collected. A tri-color fluorescence in situ hybridization method (FISH) was used to show the meiotic segregation pattern in sperm of the proband. PGD of blastomeres from a single ICSI cycle and standard prenatal diagnosis procedures to confirm the PGD results was performed. RESULTS: Meiotic segregation pattern showed only 34.2% of normal/balanced spermatozoa. The high rate (42%) of miscarriages was observed in this family, which could be explained by chromosomal unbalanced karyotypes as a product of fertilization by unbalanced spermatozoa found with a frequency of approximately 66%. The lack of unbalanced progeny at birth suggests a natural selection of unbalanced fetuses. The 37.5% of normal/balanced embryos received after a single ICSI cycle and PGD was similar to the percentage of normal/balanced spermatozoa (34.2%). After 38 weeks a healthy girl with normal karyotype was born. CONCLUSION: The presented study is an optimistic message for translocation carriers showing that even in case with more than 60% of genetically unbalanced sperm there is a reasonable chance for reproductive success.

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.