Added value of buccal cell FISH analysis in the diagnosis and management of Turner syndrome.

STUDY QUESTION: Is there an added diagnosis value of buccal cell FISH analysis compared with blood lymphocyte chromosomal investigations in patients with Turner syndrome (TS)? SUMMARY ANSWER: Buccal cell FISH analysis, a non-invasive technique, modified the chromosomal results obtained with the blood karyotype in 17 patients (12%) of our cohort. WHAT IS KNOWN ALREADY: Few studies have evaluated buccal cell FISH analysis and compared them with blood karyotype in patients with TS. STUDY DESIGN, SIZE, DURATION: A prospective, monocentric cohort study was conducted in a rare diseases centre (CMERC) between July 2017 and August 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 142 adult patients with TS, and at least 5% 45,X cells in a previous blood karyotype, were recruited. All the patients' files were included in the CEMARA database. This national database has been declared to the French data protection agency (CNIL approval number 1187326). In compliance with French law, consent regarding non-opposition to collect and use the data was obtained from each patient. A FISH analysis on a buccal smear was performed. MAIN RESULTS AND THE ROLE OF CHANCE: The percentage of 45,X cells was identical between the two tissues in only 32.4% of cases. The discrepancy was higher than 41% for 12% of the cohort. The percentage of 45,X cells was higher in blood in 53 (37.3%) patients, and higher in buccal cells in 43 (30.3%) of cases. In 17 (12%) cases, the blood karyotype had to be reconsidered in regard to the buccal cell analysis. LIMITATIONS, REASONS FOR CAUTION: It would have been interesting to evaluate karyotypes in cells from other tissues such as cells from skin biopsy or from the urinary tract and even from blood vessels or gonads in case of surgery and to compare them with each patient's phenotype. However, most of the time, these tissues are not available. WIDER IMPLICATIONS OF THE FINDINGS: Although blood lymphocyte karyotype remains the gold standard for the diagnosis of TS, buccal cell FISH analysis is an efficient tool to evaluate the global chromosomal constitution in these patients, thus allowing them to have better care and follow-up. For instance, identifying a Y chromosome can prevent the occurrence of a gonadoblastoma, as gonadectomy should be discussed. On the other hand, finding normal XX cells in a patient with a previous diagnosis of homogenous 45,X TS, may be psychologically helpful and relevant for gynaecological care. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was sought for the study. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Impact on testicular function of a single ablative activity of 3.7 GBq radioactive iodine for differentiated thyroid carcinoma.

STUDY QUESTION: What are the consequences of radioactive iodine (RAI) therapy for testicular function? SUMMARY ANSWER: A single activity of 3.7 GBq RAI for differentiated thyroid carcinoma (DTC) treatment in young men transiently altered Sertoli cell function and induced sperm chromosomal abnormalities. WHAT IS KNOWN ALREADY: Few studies, mainly retrospective, have reported the potential impacts of RAI on endocrine and exocrine testicular function. STUDY DESIGN, SIZE, DURATION: A longitudinal prospective multi-center study on testicular function performed in DTC patients before a single 131I ablative activity of 3.7 GBq (V0) and at 3 months (V3) and 13 months (V13) after treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Forty male patients, aged 18-55 years, with DTC participated. Hormonal analysis included FSH, LH, testosterone and inhibin B serum levels at V0, V3 and V13. Furthermore, sperm parameters, DNA fragmentation and sperm chromosomal abnormalities were evaluated at each time points. The differences in all parameters, between V0-V3, V0-V13 and V3-V13, were analyzed, using a Wilcoxon test. MAIN RESULTS AND THE ROLE OF CHANCE: Prior to RAI administration, all patients had normal gonadal function. At V3, a statistically significant increase in FSH levels and a decrease in inhibin B levels were observed and sperm concentration, as well as the percentage of morphologically normal spermatozoa, were significantly decreased (P < 0.0001). These modifications were transient as both sperm concentration and normal morphology rate returned to baseline values at V13. However, at this later time point, FSH and inhibin B levels were still impacted by RAI administration but remained in the normal range. Although no DNA fragmentation was observed at V3 nor V13, our study revealed a statistically significant increase in the number of sperm chromosomal abnormalities both at V3 (P < 0.001) and V13 (P = 0.01). LIMITATIONS, REASONS FOR CAUTION: Among the 40 patients included in the study, only 24 had all the parameters available at all visits. WIDER IMPLICATIONS OF THE FINDINGS: Prospective studies with longer term follow up would be helpful to determine whether the chromosome abnormalities persist. These studies would be required before sperm banking should be suggested for all patients. However, sperm preservation for DTC patients who require cumulative radioiodine activities higher than 3.7 GBq should be proposed. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Programme Hospitalier de Recherche Clinique, AP-HP (No. P040419). The authors report no conflict of interest in this work. TRIAL REGISTRATION NUMBER: NCT01150318.

Deletion of CPEB1 Gene: A Rare but Recurrent Cause of Premature Ovarian Insufficiency.

CONTEXT: Premature ovarian insufficiency (POI) may be secondary to chemotherapy, radiotherapy, or environmental factors. Genetic causes are identified in 20-25% of cases, but most POI cases remain idiopathic. OBJECTIVE: This study aimed to identify new genes involved in POI and to characterize the implication of CPEB1 gene in POI. DESIGN AND SETTING: This was a case report and cohort study replicate conducted in academic medical centers. PATIENTS AND METHODS: A deletion including CPEB1 gene was first identified in a patient with primary amenorrhea. Secondly, 191 sporadic POI cases and 68 familial POI cases were included. For each patient, karyotype was normal and FMR1 premutation was excluded. Search for CPEB1 deletions was performed by quantitative multiplex PCR of short fluorescent fragments or DNA microarray analysis. Gene sequencing of CPEB1 was performed for 95 patients. RESULTS: We identified three patients carrying a microdeletion in band 15q25.2. The proximal breakpoint, for the three patients, falls within a low-copy repeat region disrupting the CPEB1 gene, which represents a strong candidate gene for POI as it is known to be implicated in oocyte meiosis. No mutation was identified by sequencing CPEB1 gene. Therefore, heterozygous deletion of CPEB1 gene leading to haploinsufficiency could be responsible for POI in humans. CONCLUSION: Microdeletions of CPEB1 were identified in 1.3% of patients with POI, whereas no mutation was identified. This microdeletion is rare but recurrent as it is mediated by nonallelic homologous recombination due to the existence of low-copy repeats in the region. This result demonstrates the importance of DNA microarray analysis in etiological evaluation and counseling of patients with POI.

Quality assessment of induced spermatogenesis in hypogonadotrophic hypogonadic men treated with gonadotrophins.

Hypogonadotrophic hypogonadism (HH) is characterized by deficient gonadotrophin secretion, resulting from pituitary or hypothalamic defects. In order to induce spermatogenesis, HH patients are treated with commercially available gonadotrophins. As far as is known, quality and genetic integrity of induced sperm cells have never been investigated, although they represent an important issue, since the ultimate goal of this treatment is to have competent spermatozoa in order to achieve paternity. In order to evaluate the nuclear integrity of induced sperm cells, sperm samples from treated HH patients were compared with sperm samples from normospermic control donors. Sperm cells were analysed by fluorescence in-situ hybridization, using probes specific for chromosomes 13, 21, 18, X and Y, and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Results showed that the rate of aneuploid and diploid sperm cells in patients was not statistically different from controls and that the rate of sperm cells with fragmented DNA was within the normal values. Spermatozoa obtained by gonadotrophin treatment in HH patients are likely to have a balanced chromosomal content and a normal DNA integrity but this conclusion needs to be confirmed by further studies dealing with a greater number of patients.

Birth after TESE-ICSI in a man with hypogonadotropic hypogonadism and congenital adrenal hypoplasia linked to a DAX-1 (NR0B1) mutation.

DAX1/NR0B1 mutations are responsible for X-linked congenital adrenal hypoplasia (AHC) associated with hypogonadotropic hypogonadism (HH). Few data are available concerning testicular function and fertility in men with DAX1 mutations. Azoospermia as well as failure of gonadotrophin treatment have been reported. We induced spermatogenesis in a patient who has a DAX1 mutation (c.1210C>T), leading to a stop codon in position 404 (p.Gln404X). His endocrine testing revealed a low testosterone level at 1.2 nmol/l (N: 12-40) with low FSH and LH levels at 2.1 IU/l (N: 1-5 IU/l) and 0.1 IU/l (N: 1-4 IU/l), respectively. Baseline semen analysis revealed azoospermia. Menotropin (Menopur(®):150 IU, three times weekly) and human chorionic gonadotrophin (1500 IU, twice weekly) were used. After 20 months of treatment, as azoospermia persisted, bilateral multiple site testicular biopsies were performed. Histology revealed severe hypospermatogenesis. Rare spermatozoa were extracted from the right posterior fragment and ICSI was performed. Four embryos were obtained and, after a frozen-thawed single-embryo transfer, the patient's wife became pregnant and gave birth to a healthy boy. We report the first case of paternity after TESE-ICSI in a patient with DAX1 mutation, giving potential hope to these patients to father non-affected children. Furthermore, this case illustrates the fact that patients with X-linked AHC have a primary testicular defect in addition to HH.

Fourth case of cerebral, ocular, dental, auricular, skeletal syndrome (CODAS), description of new features and molecular analysis.

Cerebral, ocular, dental, auricular, skeletal syndrome (CODAS, OMIM 600373) is a very rare congenital malformation syndrome. This clinical entity is highly distinctive and associates mental retardation, cataract, enamel abnormalities, malformations of the helix, epiphyseal and vertebral malformations, and characteristic dysmorphic features. Since 1991, only three affected children have been reported. The etiology and pattern of inheritance of CODAS syndrome still remain unknown. We describe a new sporadic case presenting with all the characteristic features of CODAS syndrome associated with previously unreported malformations of the heart, larynx, and liver. All investigations such as karyotype, metabolic screening and array CGH were normal.

[Y chromosome and spermatogenesis]. / Chromosome Y et spermatogenèse.

Human Y chromosome evolution has progressively been directed towards a role in sex determination and reproduction. Cytogenetically visible structural abnormalities have determined long arm chromosomal regions which define the AZF factor that contains genes implicated in the spermatogenic process. By using molecular tools, the AZF factor has been subdivided into three loci, AZFa, b and c, the deletion of which leads to specific spermatogenesis impairments due to the loss of particular genes. Most AZF genes are specifically expressed in testis but their functions are far to be known precisely. Partial deletions of AZF regions have been described. Some of them have allowed to define more precise genotype-phenotype relationships whereas others are considered as variants in relation to Y chromosome polymorphism.

[Y chromosome structural abnormalities and Turner's syndrome]. / Anomalies de structure du chromosome Y et syndrome de Turner.

Although specifically male, the human Y chromosome may be observed in female karyotypes, mostly in women with Turner syndrome stigmata. In women with isolated gonadal dysgenesis but otherwise normal stature, the testis determining factor or SRY gene may have been removed from the Y chromosome or may be mutated. In other women with Turner syndrome, the karyotype is usually abnormal and shows a frequent 45,X/46,XY mosaicism. In these cases, the phenotype depends on the ratio between Y positive and 45,X cell lines in the body. When in mosaicism, Y chromosomes are likely to carry structural abnormalities which explain mitotic instability, such as the existence of two centromeres. Dicentric Y isochromosomes for the short arm (idic[Yp]) or ring Y chromosomes (r[Y]) are the most frequent abnormal Y chromosomes found in infertile patients and in Turner syndrome in mosaic with 45,X cells. Although monocentric, deleted Y chromosomes for the long arm and those carrying microdeletions in the AZF region are also instable and are frequently associated with a 45,X cell line. Management of infertile patients carrying such abnormal Y chromosomes must take into account the risk and the consequences of a mosaicism in the offspring.

[Spermiogenesis: histone acetylation triggers male genome reprogramming]. / Spermiogenèse: l'acétylation des histones déclenche la reprogrammation du génome mâle.

During their post-meiotic maturation, male germ cells undergo an extensive reorganization of their genome, during which histones become globally hyperacetylated, are then removed and progressively replaced by transition proteins and finally by protamines. The latter are known to tightly associate with DNA in the mature sperm cell. Although this is a highly conserved and fundamental biological process, which is a necessary prerequisite for the transmission of the male genome to the next generation, its molecular basis remains mostly unknown. We have identified several key factors involved in this process, and their detailed functional study has enabled us to propose the first model describing molecular mechanisms involved in post-meiotic male genome reprogramming. One of them, Bromodomain Testis Specific (BRDT), has been the focus of particular attention since it possesses the unique ability to specifically induce a dramatic compaction of acetylated chromatin. Interestingly, a mutation was found homozygous in infertile men which, according to our structural and functional studies, disrupts the function of the protein. A combination of molecular structural and genetic approaches has led to a comprehensive understanding of new major actors involved in the male genome reprogramming and transmission.

Underlying karyotype abnormalities in IVF/ICSI patients.

Cytogenetic investigations are performed in couples asking for IVF or intracytoplasmic sperm injection (ICSI) treatment. These serve a diagnostic purpose because male or female infertility might have a chromosomal origin. Chromosomal aberrations found in these patients include numerical abnormalities, such as Klinefelter syndrome, XYY karyotype or Turner syndrome and its variants; sex reversions, such as XX males or XY females; and also structural abnormalities, such as Robertsonian or reciprocal translocations and inversions. Finding the chromosomal origin of infertility in a patient also has a prognostic value because it aids the management of pregnancies obtained after IVF or ICSI and may lead to a proposal of prenatal or preimplantation genetic diagnosis.

Mutations in the protamine 1 gene associated with male infertility.

In elongating spermatids, human sperm chromatin undergoes a complex compaction in which the transition proteins are extensively replaced by the protamine proteins. Several human studies demonstrate that expression of the protamine proteins is altered in some men with male infertility. For this study, we screened the PRM1 (protamine 1) gene for mutations in a large cohort of 281 men seeking infertility treatment. We identified the c.102G>T transversion that results in an p.Arg34Ser amino acid change in two men. One of these patients presented with oligozoospermia associated with increased sperm DNA fragmentation. The second individual was normospermic but together with his partner sought treatment for idiopathic couple infertility. We also identified a novel missense mutation (c.119G>A, p.Cys40Tyr) in a man with oligoasthenozoospermia. These mutations were not observed in control populations. Interestingly, we also detected variants both 5' and 3' to the PRM1 open-reading frame specifically in infertile individuals. Four individuals with unexplained severe oligozoospermia were heterozygote for a c.-107G>C change that is located at -15 bp from the transcription initiation site of the gene. This mutation may influence PRM1 expression. In addition, a c.*51G>C variant was detected in the 3'UTR of PRM1 specifically in a man with severe oligoasthenozoospermia.

Sperm-FISH analysis in a pericentric chromosome 1 inversion, 46,XY,inv(1)(p22q42), associated with infertility.

No phenotypic effect is observed in most inversion heterozygotes. However, reproductive risks may occur in the form of infertility, spontaneous abortions or chromosomally unbalanced children as a consequence of meiotic recombination between inverted and non-inverted chromosomes. An odd number of crossovers within the inverted segment results in gametes bearing recombinant chromosomes with a duplication of the region outside of the inversion segment of one arm and a deletion of the terminal segment of the other arm [dup(p)/del(q) and del(p)/dup(q)]. Using fluorescence in-situ hybridization (FISH), the chromosome segregation of a pericentric inversion of chromosome 1 was studied in spermatozoa of a inv(1)(p22q42) heterozygous carrier. Three-colour FISH was performed on sperm samples using a probe mixture consisting of chromosome 1p telomere-specific probe, chromosome 1q telomere-specific probe and chromosome 18 centromere-specific alpha satellite DNA probe. The frequency of the non-recombinant product was 80.1%. The frequencies of the two types of recombinants carrying a duplication of the short arm and a deletion of the long arm, and vice versa, were respectively 7.6 and 7.2%, and these frequencies were not statistically significant from the expected ratio of 1:1. Sperm-FISH allows the further understanding of segregation patterns and their effect on reproductive failure and allows an accurate genetic counselling.

[Molecular anomalies of the Y chromosome: Consequences on male fertility]. / Polymorphismes du chromosome Y et fertilité masculine.

Molecular anomalies of the Y chromosome leading to male infertility are mainly microdeletions of the long arm of the Y chromosome. Three recurrently deleted portions of the long arm are the AZFa, AZFb and AZFc (AZF: Azoospermia Factor) regions. Complete deletions of the AZFc region are found in 10% of cases of severe male infertility. In addition to the AZF deletions, certain classes of Y chromosome (haplogroups) may also predispose to male infertility and could be transmitted to future male descents by various Assisted Reproductive Techniques (ART). Since the first discovery of microdeletions, the sequence of the Y chromosome has become available, revealing the mechanisms underlying deletion formation and also resulting in a coherent screening strategy. Recently, partial deletions of the AZF regions have been described. The significance of these deletions in the clinical context remains to be defined.

Haplotypes, mutations and male fertility: the story of the testis-specific ubiquitin protease USP26.

Recently, mutations in the X-linked ubiquitin protease 26 (USP26) gene have been proposed to be associated with male infertility. In particular a 371insACA, 494T>C and 1423C>T haplotype, which results in a T123-124ins, L165S and H475Y amino acid change respectively, has been reported to be associated with Sertoli cell-only syndrome (SCOS) and an absence of sperm in the ejaculate. Here, we demonstrate that two of these changes actually correspond to the ancestral sequence of the gene and that the USP26 haplotype is present in significant frequencies in sub-Saharan African and South and East Asian populations, including in individuals with known fertility. This indicates that the allele is not associated with infertility. The pattern of frequency distribution of the derived haplotype (371delACA, 494T), which is present at high frequencies in most non-African populations could be interpreted as either a result of migration followed by simple genetic drift or alternatively as positive selection acting on the derived alleles. The latter hypothesis seems likely, because there is evidence of strong positive selection acting on the USP26 gene.

[Genetic risks in embryo donation]. / Indications et contre-indications génétiques à l'accueil d'embryon.

Embryo donation is now an acceptable practice which offers new therapeutic possibilities to many infertile couples. It can be proposed for genetic reasons like the existence of hereditary diseases in the families of both members of a couple, leading to a high risk of transmission to a child, or when a male or female sterility is associated with a transmissible genetic abnormality from the other partner. Embryos eligible for donation may have been conceived by in vitro fertilization performed because of a male or female infertility of genetic origin. As a consequence, they are considered at increased risk of carrying a known or unknown genetic abnormality related to parental infertility. A consultation should be provided before donation acceptation for defining exactly the causes of the initial in vitro fertilization and checking the genetic screening in the couple. According to recent guidelines established by the Genetics Commission of the French Federation of CECOS, some embryos could be excluded from donation process.

Is the CAG repeat of mitochondrial DNA polymerase gamma (POLG) associated with male infertility? A multi-centre French study.

BACKGROUND: Recent data emphasized the implication of polymerase gamma (POLG) CAG repeats in infertility, making it a very attractive gene for study. A comparison of POLG CAG repeats in infertile and fertile men showed a clear association between the absence of the usual 10-CAG allele and male infertility, excluding azoospermia. It has also been suggested that the POLG gene polymorphism should be considered as a possible contributing factor in unexplained couple infertility where semen parameters are normal. In this study, we investigated the POLG CAG repeats, in a well-defined population of patients with severe male factor infertility. METHODS: We conducted a large study of POLG CAG repeats in 433 infertile and 91 fertile, normozoospermic and healthy males. In all subjects, phenotypic data, including semen parameters, hormonal status and clinical profiles, were available. RESULTS: Thirteen 'homozygous mutants' (3%) were found among the 433 idiopathic infertile patients. The follow-up of the 13 'homozygous mutant' resulted in pregnancy for more than half of the couples, through assisted reproductive techniques or even spontaneously. In addition, one 'homozygous mutant' was identified in 91 fertile men (1.1%) CONCLUSION: Under our conditions, our study does not confirm any relationship between the polymorphic CAG repeat in the POLG gene and male infertility.