Susana Fernandes

Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia

Genomic imprinting marks in the male germ line are already established in the adult germinal stem cell population. We studied the methylation patterns of H19 and MEST imprinted genes in sperm of control and oligozoospermic patients, by bisulphite genomic sequencing. We here report that 7 out of 15 (46.7%) patients with a sperm count below 10 x 10(6)/ml display defective methylation of H19 and/or MEST imprinted genes. In these cases, hypomethylation was observed in 5.54% (1.2-8.3%) and complete unmethylation in 2.95% (0-5.9%) of H19 clones. Similarly, for the CTCF-binding site 6, hypomethylation occurred in 4.8% (1.2-8.9%) and complete unmethylation in 3.7% (0-6.9%) of the clones. Conversely, hypermethylation occurred in 8.3% (3.8-12.2%) and complete methylation in 6.1% (3.8-7.6%) of MEST clones. Of the seven patients presenting imprinting errors, two had both H19 hypomethylation and MEST hypermethylation, whereas five displayed only one imprinted gene affected. The frequency of patients with MEST hypermethylation was highest in the severe oligozoospermia group (2/5 patients), whereas H19 hypomethylation was more frequent in the moderate oligozoospermia (2/5 patients). In all cases, global sperm genome methylation analysis (LINE1 transposon) suggested that defects were specific for imprinted genes. These findings could contribute to an explanation of the cause of Silver-Russell syndrome in children born with H19 hypomethylation after assisted reproductive technologies (ART). Additionally, unmethylation of the CTCF-binding site could lead to inactivation of the paternal IGF2 gene, and be linked to decreased embryo quality and birth weight, often associated with ART.

Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex chromosomes, including the gene DMRT1.

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a mans risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10−3), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10−3), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10−5). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.

Novel FOXF1 Mutations in Sporadic and Familial Cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Imply a Role for its DNA Binding Domain

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA‐binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

Nonoptical Massive Parallel DNA Sequencing of BRCA1 and BRCA2 Genes in a Diagnostic Setting

The introduction of the benchtop massive parallel sequencers made it possible for the majority of clinical diagnostic laboratories to gain access to this fast evolving technology. In this study, using the Ion Torrent Personal Genome Machine, we present a strategy for the molecular diagnosis of hereditary breast and ovarian cancer and respective analytical validation. The methodology relies on a multiplex PCR amplification of the BRCA1 and BRCA2 genes combined with a variant prioritization pipeline, designed to minimize the number of false‐positive calls without the introduction of false‐negative results. A training set of samples was used to optimize the entire process, and a second set was used to validate and independently evaluate the performance of the workflow. Performing the study in a blind manner relative to the variants in the samples and using conventional Sanger sequencing as standard, the workflow resulted in a strategy with a maximum analytical sensitivity ≥98.6% with a confidence of 95% and a specificity of 96.9%. Importantly, no true variant was missed. This study presents a comprehensive massive parallel sequencing–Sanger sequencing based strategy, which results in a high analytical sensitivity assay that provides a time‐ and cost‐effective strategy for the identification of mutations in the BRCA1 and BRCA2 genes.

Gene expression pattern of IGF2, PHLDA2, PEG10 and CDKN1C imprinted genes in spontaneous miscarriages or fetal deaths.

Genomic imprinting is defined as an epigenetic modification that leads to parent-of-origin specific monoallelic expression. Some current research on the fetal control growth has been focused on the study of genes that display imprinted expression in utero. Four imprinted genes, two paternally expressed (IGF2 and PEG10) and two maternally expressed (PHLDA2 and CDKN1C), are well known to play a role in fetal growth and placental development. Pregnancy loss in the general reproductive population is a very common occurrence and other genetic causes beyond chromosomal abnormalities could be involved in spontaneous miscarriages or fetal deaths, such as alteration of expression in imprinted genes particularly those related to fetal or placental growth. Quantitative Real Time PCR was performed to evaluate gene expressions patterns of the four mentioned genes in spontaneous miscarriages or fetal deaths from 38 women. Expression levels of PHLDA2 gene were upregulated in the first trimester pregnancy cases and all four imprinted genes studied were upregulated in the second trimester of pregnancy cases comparing with controls. In third trimester PEG10 was downregulated in fetal samples group. This is the first study presenting data from human imprinted genes expression in spontaneous miscarriages or fetal deaths cases from the three trimesters of pregnancy.

Identification of new breakpoints in AZFb and AZFc

Microdeletions in AZFa, AZFb and AZFc regions lead to different patterns of male infertility, from severe oligozoospermia to non-obstructive azoospermia. Intrachromosomal homologous recombination mechanisms were already identified in patients with simultaneous microdeletions in the AZFb and AZFc regions. Ten patients with atypical AZFb and AZFc deletion patterns were studied. The definition of those microdeletions and the fine characterization of the respective breakpoints were performed using sequence tagged sites/single nucleotide variants-PCR and DNA sequencing. Y-chromosome haplogroups were determined to establish a putative association with the patterns obtained. Seven deletion patterns were identified, P5/terminal (30%; 3/10), P5/P1 distal (20%; 2/10), IR4/distal-P2, IR2/proximal-P1, IR4/distal-P1, P4/terminal and complete AZFb/c deletion (10%; 1/10). Breakpoint sequence analysis suggests that only in one patient the P5/P1 distal deletion pattern was due to a homologous recombination mechanism. Sequence alignment of the other deletion patterns suggest that they have resulted from non-homologous recombination mechanisms.

Cytological and Expression Studies and Quantitative Analysis of the Temporal and Stage-Specific Effects of Follicle-Stimulating Hormone and Testosterone During Cocultures of the Normal Human Seminiferous Epithelium

Abstract In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.

AZFb microdeletions and oligozoospermia—which mechanisms?

OBJECTIVE To characterize the deletion patterns and its breakpoints in oligozoospermic patients presenting AZFb and AZFc microdeletions and to understand the recombination mechanisms underlying these microdeletions. DESIGN Case report. SETTING Genetics Department of Faculty of Medicine of Porto, Porto, Portugal. PATIENT(S) Two men with severe oligozoospermia and two men with nonobstructive azoospermia identified as having different AZFb+c deletion patterns via Y chromosome microdeletion analysis. INTERVENTION(S) Definition of microdeletions and the fine characterization of the respective breakpoints by sequence-tagged sites (STS) polymerase chain reaction (PCR) and single-nucleotide variant (SNV) PCR. MAIN OUTCOME MEASURE(S) Study of the fine structure of the Y-chromosome and discussion of the putative mechanisms involved in each microdeletion pattern. RESULT(S) From the four patients studied, three deletion patterns were identified: IR4/distal-P2 (25%; 1 of 4), P5/proximal-P1 (50%; 2 of 4), and P5/distal-P1 (25%; 1 of 4). Although severe oligozoospermia is normally associated with AZFc, a complete AZFb deletion was found in one case. CONCLUSION(S) Analysis of these patients has revealed a new putative region that may be involved in spermatogenesis conservation.

Prognostic factors for successful testicle spermatid recover

Men with non-obstructive azoospermia exhibit different histopathologic syndromes in the testicle biopsy, varying from aplasia, Sertoli cell only syndrome, maturation arrest and hypoplasia. The genetic basis of these syndromes is discussed. We present the diagnostic testicle biopsies performed on 160 consecutive non-obstructive azoospermic patients, and these results were correlated with the findings after multiple bilateral treatment testicle biopsy. Each syndrome had to be reevaluated as for the presence of at least one focus of spermatogenesis up to the primary spermatocyte, round spermatid, elongating spermatid, elongated spermatid, or spermatozoa stages. The clinical outcome using donor sperm-IVF, spermatid or sperm intracytoplasmic injection is thereafter presented. A new prognosis based on the findings of this large clinical series coupled to results obtained with Y chromosome molecular screening is offered. Alternative treatments to donor sperm for men without spermatids are discussed.

Y-chromosome microdeletions in nonobstructive azoospermia and severe oligozoospermia.

The aim of the present work was to present the outcomes of the patients with Y-chromosome microdeletions treated by intracytoplasmic sperm injection (ICSI), either using fresh (TESE) or frozen-thawed (TESE-C) testicular sperm and ejaculated sperm (EJAC). The originality of this work resides in the comparisons between the different types of Y-microdeletions (AZFa, AZFb, and AZFc) and treatments, with detailed demographic, stimulation, embryological, clinical, and newborn (NB) outcomes. Of 125 patients with Y-microdeletions, 33 patients presented severe oligozoospermia (18 performed ICSI with ejaculated sperm) and 92 secretory azoospermia (65 went for TESE with 40 having successful sperm retrieval and performed ICSI). There were 51 TESE treatment cycles and 43 TESE-C treatment cycles, with a birth of 19 NB (2 in AZFa/TESE-C, 12 in AZFc/TESE, and 5 in AZFc/TESE-C). Of the 29 EJAC cycles, there was a birth of 8 NB (in AZFc). In TESE and EJAC cycles, there were no significant differences in embryological and clinical parameters. In TESE-C cycles, there was a significant lower oocyte maturity rate, embryo cleavage rate and mean number of embryos transferred in AZFb, and a higher mean number of oocytes and lower fertilization rate in AZFc. In conclusion, although patients with AZFc microdeletions presented a high testicular sperm recovery rate and acceptable clinical outcomes, cases with AZFa and AZFb microdeletions presented a poor prognosis. Due to the reported heredity of microdeletions, patients should be informed about the infertile consequences on NB and the possibility of using preimplantation genetic diagnosis for female sex selection.