The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Sperm RNA preparation for transcriptomic analysis: Review of the techniques and personal experience.

In the last 10 years, several approaches, including microarrays, have been applied to investigate sperm transcript levels. However, success using microarray profiling is highly dependent of the quality of the RNA obtained. Therefore, the development of methods that deliver highly purified and intact RNA is of utmost importance. The three steps used to achieve this goal, purification of spermatozoa, RNA extraction and evaluation of RNA quality, are reviewed. Following that review and preliminary experiments, we processed sperm samples from seven normozoospermic men with a combination of gradient centrifugation and somatic cell lysis. RNA was extracted using the NucleoSpin RNA XS kit (Macherey-Nagel) and its purity checked using the BioAnalyzer. Hybridisation was done on Agilent SurePrint G3 Human GE 8 × 60K V2 microarrays. We identified 900 transcripts among the 1000 high abundance sperm transcripts reported in the literature. These genes are known to be involved in several biological processes, notably spermatogenesis, transcription regulation, cell growth and differentiation, sperm motility and capacitation, fertilisation, and embryogenesis. Therefore, our methodology is highly suitable for sperm transcriptomic analyses and can be used, notably, to compare mRNA profiles between fertile and infertile males.

[Congenital bilateral absence of vas deferens: From diagnosis to assisted reproductive techniques - the experience of three centers]. / Absence bilatérale des canaux déférents : du diagnostic à l'assistance médicale à la procréation. Expérience de 3 centres.

OBJECTIVES: To review the management with assisted reproductive technologies (ART) of men with congenital bilateral absence of vas deferens (CBAVD), associated with cystic fibrosis or not, after surgical retrieval [epididymal aspiration (MESA) or testicular biopsy (TESE)]. METHODS: Multicenter retrospective study made of 2 groups: CBAVD and cystic fibrosis (CF) or CBAVD only (CF-RD). Two centers performed MESA (Brest and Nantes) and one TESE (Rennes). Sperm numeration, motility, vitality, morphology and nuclear maturity were measured in both centers performing MESA. Fertilization rate (TF) and cumulated progressive pregnancy rate by retrieved oocyte (TGC) were compared between centers following ART. RESULTS: Ninety patients underwent surgical retrieval between January 1996 and March 2013, 30 in the CF group and 60 in the CF-RD group. Semen parameters were comparable between groups and centers. Fifty-eight (22 in the CF group and 36 in the CF-RD group) patients received ART between April 1996 and October 2014. TF was 50% and 52% and TGC 26% and 32% in the CF group and CF-RD groups, respectively. The results did not differ between groups but TGC was higher in Rennes than in the other two centers. CONCLUSION: Both semen parameters and ART results are comparable and similar to those reported in the literature. As shown by the results obtained in Rennes, TESE seems to be more effective.

Balanced complex chromosome rearrangement in male infertility: case report and literature review.

Complex chromosome rearrangements (CCRs) are structural rearrangements involving at least three chromosomes and three or more chromosome breakpoints. Generally, balanced CCR carriers have a normal phenotype but they are at a higher reproductive risk. Azoospermia was discovered in the male partner of a couple with primary infertility. Conventional cytogenetics identified a CCR refined by fluorescent in situ hybridisation. The CCR involved three chromosomes, four breakpoints and an insertion. A literature search identified 43 phenotypically normal males referred for reproductive problems presenting a CCR. More males were ascertained because of spermatogenesis failure or disturbances than because of repeated abortions and/or birth of a malformed child. Male carriers of CCR produce a high frequency of chromosomally abnormal spermatozoa due to the aberrant segregation of the rearranged chromosomes. The number of chromosomes and breakpoints involved in the rearrangement, the position of breakpoints, the relative size of the resultant chromosomes and the presence or absence of recombination inside the paired-rearranged segments are presumed to affect the fertility of the carrier. Testicular biopsy should not be performed in males with azoospermia. Intracytoplasmic sperm injection should not be proposed as a procedure for treating the infertility of CCR male carriers as a successful result is unlikely.

DNA fragmentation is higher in spermatozoa with chromosomally unbalanced content in men with a structural chromosomal rearrangement.

It has been previously shown that men with chromosomal structural abnormality had a higher rate of sperm DNA fragmentation. We studied 11 male carriers of a chromosomal structural abnormality (seven with a balanced reciprocal translocation, three with a Robertsonian translocation, one with a pericentric inversion) to determine whether spermatozoa with unbalanced chromosomes were more likely to have fragmented DNA. A sequential method combining analysis of DNA fragmentation using the TUNEL assay followed by analysis of meiotic segregation by fluorescent in situ hybridization was performed on the same spermatozoa. A statistically significant higher number of spermatozoa with unbalanced chromosomal content were found to have fragmented DNA for each man. The rate of spermatozoa with DNA fragmentation was higher than the rate of those without fragmented DNA in particular modes of segregation. Our findings provide a better understanding of the mechanisms involved in male infertility ascribable to chromosomal structural abnormality.

The MLL recombinome of acute leukemias in 2013.

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.

Molecular cytogenetic and genetic aspects of globozoospermia: a review.

Infertility is estimated to affect up to 15% of couples of reproductive age. Among the male factors, globozoospermia (also called round-headed sperm syndrome) is a rare type of teratozoospermia accounting for <0.1% of male infertility. Lack of acrosome, whose production is a postmeiotic event in spermatogenesis, and round sperm head are its main characteristics. The acrosomeless spermatozoon is unable to go through the zona pellucida and fuse with the oolemma of the oocyte, and fertilisation failures have been attributed to a deficiency in oocyte activation capacity, even when intracytoplasmic sperm injection (ICSI) is attempted. The pathogenesis of this anomaly is still unclear but genetic factors are likely to be involved. DNA fragmentation rate has been reported for 16 globozoospermic males, usually using the terminal uridine nick-end labelling (TUNEL) assay. Most of the patients had a DNA fragmentation index (DFI) higher than that in fertile men. The rate of aneuploidy for some specific chromosomes was increased in 12 among the 26 globozoospermic males reported in the literature. The same results (high DFI and aneuploidy rates) were observed in infertile males compared to fertile men, notably in those with oligoasthenozoospermia or teratozoospermia, independently of the origins. Mutations or deletions in three genes, SPATA16, PICK1 and DPY19L2, have been shown to be responsible for globozoospermia. Proteins coded by the first two genes localise to the Golgi apparatus and the proacrosomal granules that are transported in the acrosome. It is likely that other proteins involved in the acrosome formation remain to be identified.

Does 45,X/46,XX mosaicism with 6-28% of aneuploidy affect the outcomes of IVF or ICSI?

OBJECTIVE: Several studies have shown an increased frequency of chromosomal aberrations in female partners of couples examined prior to intracytoplasmic sperm injection (ICSI). A retrospective cohort study was performed to determine whether 45,X/46,XX mosaicism affects the outcomes of in vitro fertilization (IVF) or ICSI. STUDY DESIGN: Forty-six women with a 45,X/46,XX karyotype with 6-28% of aneuploidy were compared with 59 control women (46,XX), matched for age, from the female population who underwent IVF or ICSI between 1 January 1996 and 31 December 2006 at the Reproductive Medicine Unit at Brest University Hospital. The outcomes of 254 treatment cycles were compared according to patient karyotype. RESULTS: No difference was found in the number of retrieved oocytes (8.9 ± 5.5 vs 8.5 ± 4.7; p=0.56) or the number of mature oocytes (7.4 ± 4.7 vs 6.9 ± 4.2; p=0.49) between the 45,X/46,XX group and the 46,XX group, respectively. Fertilization rates did not differ between the groups for either IVF or ICSI. In addition, no difference was found in the pregnancy rate by cycle (17.4% vs 18.7%, respectively; p=0.87). The percentage of first-trimester miscarriages was similar in both groups (13.6% vs 12.5%, respectively; p=0.51). CONCLUSION: 45,X/46,XX mosaicism with 6-28% of aneuploidy has no adverse effect on the outcomes of IVF or ICSI among women referred to assisted reproductive technologies.

[Azoospermia: management and results: a series of 90 cases]. / Azoospermie : prise en charge et résultats. À propos de 90 cas.

OBJECTIVE: To report our 15-year experience in managing azoospermic males at the Brest University Hospital. PATIENTS AND METHODS: From 1996 to 2010, 90 azoospermic males were followed: 41 with non-obstructive azoospermia (NOA) and 49 with obstructive azoospermia (OA). Surgical methods proposed for retrieving sperm were Microsurgical Epididymal Sperm Aspiration (MESA) for men with OA and microdissection Testicular Sperm Extraction (mTESE) for those with NOA. RESULTS: Spermatozoa were retrieved in 56.1% of the testicular biopsies for NOA. The embryo transfer rate per cycle for injection intracytoplasmique d'un spermatozoïde (ICSI) with epididymal spermatozoa (OA) was higher to that of ICSI with ejaculated spermatozoa (93.2% vs. 86.6%, P<0.05), but the rate was lower for ICSI with testicular sperm (NOA) (70.2% vs. 86.6%, P<0.01). The rate of clinical pregnancy per embryo transfer was 31.4% following ICSI with epididymal spermatozoa but it was of 24.2% with testicular sperm and 23.1% with ejaculated sperm. CONCLUSION: ICSI are usually difficult in NOA because they are done with very few spermatozoa. When spermatozoa are retrieved from surgical techniques, more than 50% of the OA couples and almost 30% of the NOA couples conceived at least one child.

Aneuploidy and DNA fragmentation in sperm of carriers of a constitutional chromosomal abnormality.

Among various causes responsible for infertility, it has been admitted for a long time that male infertility can be due to impaired spermatogenesis and/or balanced structural chromosomal abnormalities. Sperm DNA fragmentation is also considered as another cause of infertility. Most of the studies on male infertility have concerned either aneuploidy in the sperm of carriers of constitutional chromosomal abnormalities or sperm DNA fragmentation. This review is aimed at analyzing these 2 parameters in the same patients. Furthermore, we present work on the study of these 2 parameters in the same gametes of 4 carriers of a balanced chromosomal abnormality. Meiotic segregation was analyzed by fluorescent in situ hybridization and DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. It was shown that aneuploidy and DNA fragmentation were increased in the sperm of carriers of a balanced chromosomal abnormality. For all 4 carriers of a balanced structural abnormality, there was a 2-5 times higher proportion of spermatozoa with unbalanced chromosomal content and fragmented DNA than among those with normal/balanced content. Moreover, we found a non-random distribution with more gametes with DNA fragmentation when these arose from a particular segregation mode. The mechanism which would tend to explain our results is abortive apoptosis. In conclusion, both meiotic segregation and DNA fragmentation studies should be integrated in the genetic exploration of male carriers of a chromosomal structural abnormality.

Study of aneuploidy and DNA fragmentation in gametes of patients with severe teratozoospermia.

This study investigated meiotic segregation in spermatozoa to determine if severe teratozoospermia should prevent the use of intracytoplasmic sperm injection (ICSI) because of the high production of gametes with chromosomal aneuploidies and analysed DNA fragmentation in gametes from the same semen to determine if DNA integrity was worse in patients with severe teratozoospermia. Sperm samples from 12 infertile patients were studied by fluorescence in-situ hybridization for chromosomes X, Y, 13, 18 and 21 and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling. Four patients with a majority of macrocephalic forms with multiple flagella had more than 99% spermatozoa with abnormal chromosomal content. The other patients (globozoospermia or other abnormalities concerning sperm heads) had no increased aneuploidy or a slightly significant increase (P<0.05). The rate of DNA fragmentation was significantly higher in infertile patients than in the controls (P<0.001; 14.3% versus 1.20%, respectively) but presented important variability. Therefore, ICSI should not be attempted if men have macrocephalic gametes with multiple flagella but morphology is not always a good predictor of chromosomal content, depending upon the kind of teratozoospermia. Evaluation of the rate of aneuploidy and DNA fragmentation in gametes of patients with severe teratozoospermia is recommended.

A study of meiotic segregation of chromosomes in spermatozoa of translocation carriers using fluorescent in situ hybridisation.

In the infertile male population, there is a 2-20-time higher probability of having a structural chromosomal abnormality than in general population. Generally, these men have a normal phenotype but they can have sperm abnormalities. As they can produce a variable proportion of unbalanced gametes, it is important to evaluate the percentage of unbalanced chromosomal spermatozoa to assess the risk of injecting a chromosomally unbalanced gamete during ICSI procedure. We report here the meiotic segregation analysis of chromosomes in spermatozoa of 12 men with a balanced reciprocal translocation and 4 men with a Robertsonian translocation using a fluorescent in situ hybridisation analysis. The frequencies of normal or balanced spermatozoa ranged from 34.4% to 49.1% in balanced reciprocal translocation carriers. For Robertsonian translocation, the frequencies of normal or balanced spermatozoa ranged from 78.4% to 91.2%. These analyses allow us to define the orientation of genetic counselling according to the results of meiotic segregation obtained. As a last resort, it could then be discussed of the possibility of having recourse to donor spermatozoa or adoption.

45,X/46,XX mosaicism below 30% of aneuploidy: clinical implications in adult women from a reproductive medicine unit.

OBJECTIVE: Turner's syndrome (TS) is well known, but prognosis for 45,X/46,XX mosaicism below 30% of aneuploidy has not been established. We evaluated differences in clinical features and biological parameters between patients with numerical sex chromosome mosaicism diagnosed incidentally and control women. DESIGN: Retrospective observational study of clinical features and biological parameters. METHODS: Standard endocrinological and gynecological examination was done and early-follicular-phase blood values were collected from the medical records of women aged 21-43, who were referred to our ward from 1996 to 2006 because of infertility and were karyotyped. Seventy-one women with sex chromosome mosaicism (45,X/46,XX) ranging from 4 to 28% were assigned a chromosomally normal woman (46,XX) matched according to age (n=71). RESULTS: In group 45,X/46,XX, 8% or more of aneuploidy accounted for a smaller height compared to controls (P=0.01). Body mass index was increased from 6% of aneuploidy (P=0.02) and was positively correlated to the percentage of 45,X cells (P=0.0001); menarche occurred earlier from 10% of aneuploidy (P=0.01) and was inversely correlated to the percentage of 45,X cells (P=0.045). No difference was found between the groups for FSH, LH, estradiol, inhibin B, and TSH values. Spontaneous abortions were more frequent in case of mosaicism (P=0.01), and recurrence was positively correlated to the percentage of aneuploidy (P=0.008). CONCLUSION: Sex chromosome mosaicism is responsible for clinical changes from 6% of aneuploidy, corresponding to the main phenotypical features of TS.

New insights to the MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

Increased aneuploidy rates in spermatozoa of a male carrier of a trisomy 18 mosaicism.

Semen analysis of a 31-year-old infertile man showed a severe oligoteratozoospermia. Karyotyping of peripheral blood lymphocytes showed a 47,XY,+18[13]/46,XY[16] mosaicism. Cultured skin fibroblasts, right and left jugal smears showed 3, 50 and 65% trisomic cells respectively. The aim of the study was to evaluate the aneuploidy rates of chromosomes X, Y, 13, 18 and 21 and the diploidy rate in his spermatozoa by fluorescence in situ hybridization. The rate of disomy 18 was significantly increased in the spermatozoa of the patient (0.68%) compared to the control group (0.06%). A statistically significant difference in the rates of disomy for chromosome 13 (0.46% vs. 0.14%) and the gonosomes (0.78% vs. 0.24%) and diploidy (0.93% vs. 0.34%) was also found between the patient and the control group. However, no significant difference was observed for chromosome 21 (0.34% vs. 0.15%). Our results show evidence of a generalized perturbation of the meiotic mechanism that could lead to an increased risk for a mosaic trisomy 18 infertile male of producing offspring with aneuploidy that is not only on account of the father's mosaicism, but also more particularly because of severe oligoteratozoospermia.

Fluorescence in situ hybridization characterization of ider(20q) in myelodysplastic syndrome.

Isochromosome of the long arm of chromosome 20 with loss of interstitial material [ider(20q)] is a variant of deletion of chromosome 20q and a rare abnormality in myelodysplastic syndrome (MDS). We studied seven cases with an ider(20q) in MDS. Fluorescence in situ hybridization (FISH) studies showed all proximal breakpoints to be consistently located in 20q11.21 band whereas distal breakpoints were variable. Amplification of HCK, TNFRSF6B and DIDO1 genes included in retained regions associated with loss of tumour suppressor genes in deleted regions could explain cell tumour progression and possibly the less favourable prognosis of ider(20q) compared with del(20q).