Recurrence of an early postzygotic rescue of an inherited unbalanced translocation resulting in mosaic segmental uniparental isodisomy of chromosome 11q in siblings.

Balanced translocations are associated with a risk of transmission of unbalanced chromosomal rearrangements in the offspring. Such inherited chromosomal abnormalities are typically non-mosaic as they are present in the germline. We report the recurrence in two siblings of a mosaicism for a chromosomal rearrangement inherited from their asymptomatic father who carried a balanced t(2;11)(q35;q25) translocation. Both siblings exhibited a similar phenotype including intellectual disability, dysmorphic features, kyphoscoliosis, and cervical spinal stenosis. Karyotyping, fluorescence in situ hybridization and SNP array analysis of blood lymphocytes of both siblings identified two cell lines: one carrying a 2q35q37.3 duplication and a 11q25qter deletion (~90% cells), and one carrying an 11q uniparental isodisomy of maternal origin (~10% cells). We hypothesize that these mosaics were related to a postzygotic rescue mechanism which unexpectedly recurred in both siblings.

Retinol improves in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm during the first wave of spermatogenesis.

Testicular tissue freezing has been proposed for fertility preservation in pre-pubertal boys. Thawed frozen testicular tissue must undergo a maturation process to restore sperm production. The purpose of the current study was to evaluate the ability of retinol to improve the in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm. Testes from pre-pubertal mice, aged 2.5 and 6.5 days post-partum, were cultured on agarose gel at a gas-liquid interphase for 34, 38 and 60 days (D) and for 16, 30 and 36 D respectively. Assessment of basal medium (BM) supplemented with retinol (RE) alone, FSH/LH alone or a combination of both, was performed. Stereological analyses and tissue lesion scoring were performed at the culture time points indicated above. Sperm production was quantified at D30 and D34 after mechanical dissection of the testicular tissues. FSH/LH significantly increased the percentage of round spermatids at D30 and D38, when compared to BM alone. However, RE significantly increased the percentages of round but also elongated spermatids at D30 and D34. Moreover, RE significantly increased the number of spermatozoa per milligram of tissue at D30 and D34 when compared to BM. Therefore, RE improved the in vitro production of spermatids and spermatozoa from pre-pubertal SSCs during the first wave of spermatogenesis. The use of RE could be a useful tool for in vitro spermatogenesis from pre-pubertal human testicular tissue.

Precocious initiation of spermatogenesis in a 19-month-old boy with Hurler syndrome.

Mucopolysaccharidosis type IH (MPS IH) is a rare autosomal recessive lysosomal storage disorder. Haematopoietic stem cell transplantation (HSCT) has been proposed for the treatment of MPS IH patients and offers the possibility to grow into their adulthood. Precocious puberty has been described in few MPS patients. We report, to the best of our knowledge and for the first time, the initiation of the first waves of spermatogenesis fortuitously observed in seminiferous tubules of a pre-pubertal 19-month-old boy, affected by MPS IH and who did not present any clinical signs of precocious puberty. This patient benefited from testicular tissue cryopreservation before HSCT. Seminiferous tubule size, germ cell differentiation and Sertoli cell expression of androgen receptor and anti-müllerian hormone corresponded to the pattern observed in a pubertal boy. The Hurler syndrome may be responsible for the precocious initiation of spermatogenesis. A specific follow-up during childhood may be useful to confirm if such abnormal testis development is common in young boys with MPS IH and if it may lead to precocious onset of puberty in survivors despite HSCT. Furthermore, we have observed that Sertoli cell maturation (up-regulation of AR expression, down-regulation of AMH expression) occurred before the clinical signs of puberty and before the increase of testosterone plasmatic level.

La Mucopolysaccharidose de type IH (MPS IH) est une maladie rare lysosomale de transmission récessive autosomique. L'allogreffe de cellules souches hématopoïétiques (CSH) a été proposée pour le traitement des patients atteints de MPS IH et offre la possibilité d'une survie de ces patients jusqu'à l'âge adulte. La puberté précoce a été décrite chez quelques patients MPS. Nous rapportons, à notre connaissance et pour la première fois, l'initiation de la première vague de spermatogenèse observée fortuitement dans les tubules séminifères d'un garçon de 19 mois, atteint de MPS IH et sans signes cliniques de puberté précoce. Ce patient a bénéficié d'une cryoconservation de tissu testiculaire avant la greffe de CHS. Le diamètre des tubes séminifères, la différenciation des cellules germinales et l'expression du récepteur aux androgènes et de l'hormone antimüllérienne dans les cellules de Sertoli présentent les caractéristiques retrouvées dans le testicule d'un garçon pubère. Le syndrome de Hurler peut être responsable de l'initiation précoce de la spermatogenèse. Un suivi spécifique pendant l'enfance peut être utile pour confirmer si cette maturation prématurée du testicule est fréquente chez les jeunes garçons atteints de MPS IH et si elle peut conduire à une puberté précoce chez les patients malgré la greffe de CSH. En outre, notre observation démontre que la maturation des cellules de Sertoli se produit avant les signes cliniques de puberté et avant l'augmentation de la concentration plasmatique de testostérone.

Effects of vitamin A on in vitro maturation of pre-pubertal mouse spermatogonial stem cells.

Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7), 9 (D9) and 11 (D11) days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re) or retinoic acid (RA) alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type), intra-tubular cell death and proliferation (PCNA antibody) and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7 °C, -8 °C or -9 °C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10(-6)M and retinol at 3.3.10(-7)M, as well as retinol 10(-6)M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8 °C, after 9 days of organotypic culture using 10(-6)M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10(-6)M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8 °C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular tissue.

[Cryopreservation of testicular tissue in children]. / Cryopréservation du tissu testiculaire chez l'enfant.

The toxicity of cancer therapies can affect all organs and tissues. Some treatments damage spermatogonial stem cells (SSCs), with a risk of infertility. Storage and reimplantation of frozen testicular tissue is a recent approach tofertilitypreservationfor young boys. However, thawed frozen prepubertal testicular tissue must undergo a maturation process to restore sperm production. This process, currently being studied in animal models, can be achieved by in vivo transplantation of SSCs into seminiferous tubules or by testicular grafting, possibly following in vitro maturation.

Concomitant unilateral and synchronous bilateral testis cancer in azoospermic dizygotic twins: differential management of fertility preservation.

OBJECTIVE: To optimize fertility preservation management in unilateral or bilateral testicular cancer. DESIGN: Case series. SETTING: Urology department and reproductive biology laboratory. PATIENT(S): Dizygotic azoospermic twins presenting unilateral and bilateral synchronous testicular tumors. INTERVENTION(S): Testicular sperm extraction (TESE) and orchiectomy. MAIN OUTCOME MEASURE(S): Semen analysis, histologic diagnosis. RESULT(S): No spermatozoa were cryopreserved for the first case, because fertility preservation was proposed after orchiectomy. Spermatozoa were retrieved after TESE for his brother with bilateral tumor. CONCLUSION(S): Clinicians should be aware of the need to recommend sperm banking before treatments may alter spermatogenesis. TESE may be the sole option for fertility preservation in bilateral testicular cancer.

Ovarian tissue thawing: a comparison of two conditions.

Two different protocols to thaw cryopreserved human ovarian cortex have been evaluated using a histological analysis. The slower one, based on a progressive dilution of cryoprotectants, seems to maintain an optimal follicle morphology.

Neutrophil but not eosinophil inflammation is related to the severity of a first acute epidemic bronchiolitis in young infants.

Acute bronchiolitis is the main cause of emergency visits and hospitalizations in infants. Recent data suggest that neutrophil- and eosinophil-mediated inflammations were part of bronchiolitis pathophysiology. Apart from the defined risk factors, few was known on the underlying pathophysiology, which might point out the differences observed in the severity of the disease. The aim of this study was to assess whether the clinical severity of acute epidemic bronchiolitis in young infants might be related to a specific underlying inflammatory process. Total and differential cell counts, IL-8, eotaxin, eosinophil cationic protein (ECP) and albumin levels were assessed at the time of admission in bronchial secretions from 37 infants (median age 17 wk) with acute bronchiolitis. Outcome severity variables were: hypoxemia, Silverman score, tachypnea, feeding alteration, and duration of hospitalization. Neutrophils predominated, and eosinophils were present in 54% of the infants. IL-8 levels strongly correlated with ECP and albumin levels. Albumin levels were correlated with ECP and eotaxin levels. IL-8 levels were higher in infants with hypoxemia and inversely related with SaO(2) levels. IL-8 and albumin levels significantly rose with respiratory rate, and Silverman score. IL-8, albumin and ECP levels were significantly higher in infants hospitalized >/=7 days. Furthermore, IL-8 levels were correlated with the duration of hospitalization. Neither cell counts nor eotaxin levels were related to the severity criteria studied. This study suggests that IL-8-associated airway inflammation significantly contributed to the severity of acute epidemic bronchiolitis.

From spermatocytes to spermatozoa in an infertile XYY male.

Sex chromosome distribution and aneuploidy as well as germ cell degeneration were evaluated in meiotic and post-meiotic cells from an infertile XYY male. Sex chromosome distribution was assessed by multicolour fluorescence in situ hybridization on meiotic preparations. Post-meiotic cell aneuploidy was characterized by a method combining multicolour fluorescence in situ hybridization and immunocytochemistry using the proacrosin-specific monoclonal antibody (mAb 4D4). TUNEL assay was carried out on seminiferous tubules to evaluate germ cell degeneration. At the prophase stage of the first meiotic division, 63.64% of cells at the pachytene stage carried three sex chromosomes. The ratio of X-bearing to Y-bearing spermatids and spermatozoa differed significantly from 1 : 1 with an excess of Y-bearing spermatids and spermatozoa. The frequency of hyperhaploid XY spermatids was increased in the XYY male, as well as the incidence of YY, XY and disomic 18 ejaculated spermatozoa. A preferential elimination of germ cells by apoptosis occurred in spermatocytes I. The persistence of the extra Y chromosome during meiosis of an XYY male is associated with a high rate of spermatocyte I degeneration and a low rate of aneuploid spermatozoa.

Molecular cytogenetics analysis with whole chromosome paint probes of sperm nuclei from a (13;15) Robertsonian translocation carrier.

Meiotic segregation of a Robertsonian translocation (13;15) was assessed in sperm nuclei using dual-color fluorescent in situ hybridization (FISH) with whole-chromosome paint probes. Most spermatozoa in the (13;15) translocation carrier resulted from alternate segregation. Although an increased frequency of unbalanced gametes was observed, spontaneous pregnancy led to the birth of a boy with a normal karyotype.

Molecular characterization of a 14q deletion in a boy with features of Holt-Oram syndrome.

Holt-Oram syndrome, the major "heart-hand" syndrome is defined by the association of radial defects or triphalangeal thumbs and septal heart defects. The transmission is autosomal dominant and the causative gene has been shown to be TBX5, located on 12q24.1, which encodes a transcription factor. Genetic heterogeneity has been suggested by several reports. We identified a 14(q23.3 approximately 24.2q31.1) deletion in a boy presenting severe bilateral asymmetrical radial aplasia, congenital heart defects, and developmental delay. This deletion, whose size could be estimated to be 9.6-13.7 Mb, was shown to be inherited via his mother's interchromosomal insertion. This is the second report of a chromosome 14 interstitial deletion associated with clinical features of Holt-Oram syndrome. These observations suggest the existence of a new "heart-hand" locus on chromosome 14q.

Primary flagellar abnormality is associated with an increased rate of spermatozoa aneuploidy.

The frequencies of aneuploid and diploid spermatozoa were determined in 3 patients presenting a complete asthenozoospermia due to a primary and specific flagellar anomaly: patients 1 and 2 presented a "stump tail syndrome," more than 50% of spermatozoa with a short flagella, patient 3 had a Kartagener syndrome including situs inversus, sinusitis, and bronchiectassis. No pregnancy was obtained after 3 intracytoplasmic sperm injection (ICSI) attempts in patients 1 and 2. A 3-color fluorescence in situ hybridization analysis was performed on their spermatozoa using centromeric probes for chromosomes X, Y, and 18 and compared with those of 8 fertile males. The frequency of disomic 18 and hyperhaploid XY spermatozoa was not significantly increased in the 3 patients when compared with controls. However, the 3 patients showed elevated frequencies of XX, YY, and diploid spermatozoa. These data add to growing evidence that systematic sperm anomalies of flagella increase the rate of spermatozoa aneuploidy and may also reduce the chances of pregnancy after intracytoplasmic sperm injection.

Comparison of six density gradient media for selection of cryopreserved donor spermatozoa.

The aim of our study was to evaluate the efficiency of 4 density gradient media for motile cryopreserved spermatozoa selection to Percoll (Kabi Pharmacia, Uppsala, Sweden) and to Puresperm (J.C.D. International Laboratory, L'Aigle, France). Puresperm was the new medium chosen in our laboratory in 1996 as the substitute for Percoll. The solutions tested were 3 colloidal silane-coated silica particle media (Isolate, SpermGrad-100, Sil-Select Plus) and iodixanol (Optiprep). Semen parameters analyzed after selection were concentration, motility, and morphology. Semen parameters after Puresperm gradient had similar values compared to Percoll. Optiprep was less efficient with a poor concentration. Isolate had a comparatively better concentration, but the capacity of selection was not satisfactory. SpermGrad-100 and Sil-Select Plus were less effective than Puresperm. In conclusion, Puresperm could be considered a better alternative to Percoll for cryopreserved spermatozoa migration.

Fluorescence in situ hybridisation (FISH) analysis of chromosome segregation and interchromosomal effect in spermatozoa of a reciprocal translocation t(9,10)(q11;p11.1) carrier.

A couple was referred for exploration of repetitive abortions. The man was found to be a carrier of a balanced reciprocal translocation t(9;10)(q11;p11.1). The meiotic segregation of chromosomes 9 and 10 was analysed in 5,157 spermatozoa from this translocation carrier and in 15,255 spermatozoa from three control donors using three-colour fluorescence in situ hybridisation (FISH). The theoretical viability of the different segregation patterns was performed using the computer system HC Forum developed by the Department of Cytogenetics at the Grenoble University Medical School, La Tronche, France. A normal or balanced constitution was found in 56.25% of the analysed spermatozoa. The tertiary 3:1 segregation mode was the most frequently observed (14.37%). The frequencies of adjacent-1, adjacent-2 and 3:1 interchange modes were 12.85, 9.38 and 7.14% respectively. The cumulative frequency of non-viable imbalance was estimated at 20.91% according to the theorical viability of the different segregation patterns. Spermatozoa aneuploidy frequency was also evaluated for chromosomes X, Y and 18, and there was no evidence of interchromosomal effect in spermatozoa from the translocation carrier. FISH analysis of spermatozoa in combination with the viability theorical estimation of the different segregation patterns could be considered a useful tool for genetic counselling in carriers of reciprocal translocation.