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.

Deletion of the entire AZFc locus on the human Y chromosome leads to male infertility. The functional roles of the individual gene families mapped to AZFc are, however, still poorly understood, since the analysis of the region is complicated by its repeated structure. We have therefore used single-nucleotide variants (SNVs) across approximately 3 Mb of the AZFc sequence to identify 17 AZFc haplotypes and have examined them for deletion of individual AZFc gene copies. We found five individuals who lacked SNVs from a large segment of DNA containing the DAZ3/DAZ4 and BPY2.2/BPY2.3 gene doublets in distal AZFc. Southern blot analyses showed that the lack of these SNVs was due to deletion of the underlying DNA segment. Typing 118 binary Y markers showed that all five individuals belonged to Y haplogroup N, and 15 of 15 independently ascertained men in haplogroup N carried a similar deletion. Haplogroup N is known to be common and widespread in Europe and Asia, and there is no indication of reduced fertility in men with this Y chromosome. We therefore conclude that a common variant of the human Y chromosome lacks the DAZ3/DAZ4 and BPY2.2/BPY2.3 doublets in distal AZFc and thus that these genes cannot be required for male fertility; the gene content of the AZFc locus is likely to be genetically redundant. Furthermore, the observed deletions cannot be derived from the GenBank reference sequence by a single recombination event; an origin by homologous recombination from such a sequence organization must be preceded by an inversion event. These data confirm the expectation that the human Y chromosome sequence and gene complement may differ substantially between individuals and more variations are to be expected in different Y chromosomal haplogroups.

Deletions of the DAZ gene family in distal Yq11 are always associated with deletions of the azoospermia factor c (AZFc) region, which we now estimate extends to 4.94 Mb. Because more Y gene families are located in this chromosomal region, and are expressed like the DAZ gene family only in the male germ line, the testicular pathology associated with complete AZFc deletions cannot predict the functional contribution of the DAZ gene family to human spermatogenesis. We therefore established a DAZ gene copy specific deletion analysis based on the DAZ-BAC sequences in GenBank. It includes the deletion analysis of eight DAZ-DNA PCR markers [six DAZ-single nucleotide varients (SNVs) and two DAZ-sequence tag sites (STS)] selected from the 5' to the 3'end of each DAZ gene and a deletion analysis of the gene copy specific EcoRV and TaqI restriction fragments identified in the internal repetitive DAZ gene regions (DYS1 locus). With these diagnostic tools, 63 DNA samples from men with idiopathic oligozoospermia and 107 DNA samples from men with proven fertility were analysed for the presence of the complete DAZ gene locus, encompassing the four DAZ gene copies. In five oligozoospermic patients, we found a DAZ-SNV/STS and DYS1/EcoRV and TaqI fragment deletion pattern indicative for deletion of the DAZ1 and DAZ2 gene copies; one of these deletions could be identified as a 'de-novo' deletion because it was absent in the DAZ locus of the patient's father. The same DAZ deletions were not found in any of the 107 fertile control samples. We therefore conclude that the deletion of the DAZ1/DAZ2 gene doublet in five out of our 63 oligozoospermic patients (8%) is responsible for the patients' reduced sperm numbers. It is most likely caused by intrachromosomal recombination events between two long repetitive sequence blocks (AZFc-Rep1) flanking the DAZ gene structures.

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 man's 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.

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. Non-pulmonary 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 ten deletions, we have identified an additional thirty eight novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, twenty 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 whereas four familial cases with three showing 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 gene regulation. 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.

We have developed a rapid screening protocol for deletion analysis of the complete AZFa sequence (i.e. 792 kb) on the Y chromosome of patients with idiopathic Sertoli-cell-only (SCO) syndrome. This Y deletion was mapped earlier in proximal Yq11 and first found in the Y chromosome of the SCO patient JOLAR, now designated as the AZFa reference patient. We now show that similar AZFa deletions occur with a frequency of 9% in the SCO patient group. In two multiplex polymerase chain reaction experiments, deletions of the complete AZFa sequence were identified by a typical deletion pattern of four new sequence-tagged sites (STS): AZFa-prox1, positive; AZFa-prox2, negative; AZFa-dist1, negative; AZFa-dist2, positive. The STS were established in the proximal and distal neighbourhoods of the two retroviral sequence blocks (HERV15yq1 and HERV15yq2) which encompass the break-point sites for AZFa deletions of the human Y chromosome. We have found deletions of the complete AZFa sequence always associated with a uniform SCO pattern on testicular biopsies. Patients with other testicular histologies as described in the literature and in this paper have only partial AZFa deletions. The current AZFa screening protocols can therefore be improved by analysing the extension of AZFa deletions. This may provide a valuable prognostic tool for infertility clinics performing testicular sperm extraction, as it would enable the exclusion of AZFa patients with a complete SCO syndrome.

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Deletions of the AZFc region in Yq11.2, which include the DAZ gene family, are responsible for most cases of male infertility and were associated with severe oligozoospermia and also with a variable testicular pathology. To uncover the functional contribution of DAZ to human spermatogenesis, a DAZ gene copy-specific deletion analysis was previously established and showed that DAZ1/DAZ2 deletions associate with oligozoospermia. In this study we applied the same screening method to 50 control fertile males and 91 non-obstructive azoospermic males, 39 with Sertoli cell-only syndrome (SCOS) and 52 with meiotic arrest (MA). Samples were also screened with 24 sequence-tagged sites to the different AZF regions, including 114 control fertile males. After biopsy (testicular sperm extraction, TESE), residual spermiogenesis was found in 57.7% MA and 30.8% SCOS cases (incomplete syndromes). DAZ1/DAZ2 deletions were associated with the testicular phenotype of residual spermiogenesis as they were only found in two patients (8%) with incomplete MA. Differences between incomplete (23.3%) and complete (4.5%) MA cases regarding AZFc and DAZ1/DAZ2 deletion frequencies, and between incomplete (58.3%) and complete (11.1%) SCOS cases for AZFc deletions, suggest that incomplete syndromes might represent an aggravation of the oligozoospermic phenotype. As successful TESE was achieved in 87.5% of MA cases with AZFc and DAZ1/DAZ2 deletions and in 58.3% of SCOS cases with AZFc deletions, the present results also suggest that these molecular markers might be used for the establishment of a prognosis before TESE.