Victor Chow

Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal

Male factor infertility has been associated with abnormal DNA methylation at imprinted genes. Little information is available on the status of imprinting in the sperm of men with azoospermia, including the association between aberrant imprinting and obstructive azoospermia (OA) or non-OA (NOA). Analysis of DNA methylation at imprinted genes in the sperm of men undergoing vasectomy reversal would aid determination of whether aberrant imprinting is associated with obstruction. Testicular sperm was retrieved from testicular biopsies obtained from men with azoospermia (N=18), including OA (N=10), NOA (N=5), and unknown pathology (N=3), and from men undergoing vasectomy reversal (N=17). Sperm was also obtained from proven fertile men (N=9). DNA methylation was investigated at multiple CpG sites within the differentially methylated regions (DMRs) of three imprinted genes, H19, IG-GTL2 and MEST, using bisulphite sequencing. Unique clones representative of single cells were analyzed. We found a significant decrease in DNA methylation at the H19 DMR in testicular sperm of azoospermic men compared with proven fertile men. The decrease was also significant between OA and proven fertile men, and between men undergoing vasectomy reversal and proven fertile men, suggesting that aberrant DNA methylation may be associated with obstruction. Changes in DNA methylation at IG-GTL2 and MEST DMRs among groups were not significant. Our data suggest that imprinting abnormalities may be associated with obstruction and may occur in response to changes in testicular environment and not only spermatogenesis failure, as previously reported. Methylation at the H19 DMR was particularly prone to modification in testicular sperm.

Silencing of unpaired meiotic chromosomes and altered recombination patterns in an azoospermic carrier of a t(8;13) reciprocal translocation

BACKGROUND Male carriers of structural chromosomal abnormalities provide a useful model for studying the effects of impaired synapsis on human meioses and male fertility. METHODS We used immunofluorescent techniques to examine recombination (MLH1), synapsis (SYCP3/SYCP1) and transcriptional inactivation (BRCA1/gammaH2AX/RNA polymerase II) of meiotic chromosomes in an azoospermic carrier of a t(8;13) reciprocal translocation. Two biopsies were performed 1 year apart and on different testes. RESULTS Global recombination rates differed between the two biopsies. Although global recombination rates were not altered when compared with control men, recombination frequencies were reduced specifically on the rearranged chromosomes. Asynapsed quadrivalents were observed in 90% and 87% of pachytene nuclei from the first and second biopsies, respectively, and were frequently associated with the sex chromosomes. BRCA1 and gammaH2AX, two proteins implicated in meiotic sex chromosome inactivation, localized along asynapsed regions regardless of whether or not they were associated with the sex chromosomes. Immunostaining for RNA polymerase II provided further evidence that unsynapsed regions are silenced during human meiosis. CONCLUSIONS The fidelity of synapsis is a critical factor in determining the outcome of gametogenesis in humans, as the transcriptional inactivation of asynapsed regions may silence meiotic genes, leading to meiotic arrest and infertility.

CUA Guideline: The workup of azoospermic males.

A committee was established at the request of the CUA to determine guidelines for the investigation and management of azoospermia. Members of the committee, all of whom have special expertise in the investigation and management of male infertility, were chosen from different communities across Canada. The members represent different practices in different communities.

45,X/46,XY mixed gonadal dysgenesis: A case of successful sperm extraction

Infertility is common among couples, about one third of cases are the result of solely male factors, and rarely abnormalities of genetic karyotypes are the root cause. Individuals with a 45X,/46,XY mosaiscism are rare in the literature and very few have fertile potential. We discuss a case of a 27-year-old male with known mixed gonadal dysgenesis, 50:50 split mosaiscism of 45,X/46,XY, presenting for evaluation of 1.5 year history of infertility. He demonstrated low volume non-obstructive azoospermia. Upon testicular biopsy, spermatozoa were extracted. These sperm were subjected to aneuploidy studies demonstrating 95.95% euploidy. The sperm were further assessed and placed in cryopreservation after being deemed sufficient for potential intracytoplasmic sperm injection. This is a unique case of viable sperm in a man with mixed gonadal dysgenesis, 45,X/46,XY mosaiscism.

Genetic characterization of two 46,XX males without gonadal ambiguities

PurposeTo evaluate hypotheses which explain phenotypic variability in sex determining region Y positive 46,XX males. We investigate two 46,XX males without gonadal ambiguities.MethodsCytogenetic and molecular analyses were used to identify the presence of Y chromosome material and to map the translocation breakpoint. Finally, the pattern of X chromosome inactivation was studied using the methylation assay at the androgen receptor locus.ResultsThe presence of Y chromosome material, including the sex determining region Y gene, was demonstrated in both men. However, the amount of translocated Y chromosome material differed between the patients. Different X chromosome inactivation patterns were found in the patients; random in one patient and non-random in the other.ConclusionsWe found a lack of association between phenotype and X chromosome inactivation pattern. Our cytogenetic and molecular analyses show support for the position effect hypothesis explaining the phenotypic variability in XX males.

Meiotic recombination, synapsis, meiotic inactivation and sperm aneuploidy in a chromosome 1 inversion carrier.

Disrupted meiotic behaviour of inversion carriers may be responsible for suboptimal sperm parameters in these carriers. This study investigated meiotic recombination, synapsis, transcriptional silencing and chromosome segregation effects in a pericentric inv(1) carrier. Recombination (MLH1), synapsis (SYCP1, SYCP3) and transcriptional inactivation (γH2AX, BRCA1) were examined by fluorescence immunostaining. Chromosome specific rates of recombination were determined by fluorescence in-situ hybridization. Furthermore, testicular sperm was examined for aneuploidy and segregation of the inv(1). Our findings showed that global recombination rates were similar to controls. Recombination on the inv(1) and the sex chromosomes were reduced. The inv(1) associated with the XY body in 43.4% of cells, in which XY recombination was disproportionately absent, and 94.3% of cells displayed asynapsed regions which displayed meiotic silencing regardless of their association with the XY body. Furthermore, a low frequency of chromosomal imbalance was observed in spermatozoa (3.4%). Our results suggest that certain inversion carriers may display unimpaired global recombination and impaired recombination on the involved and the sex chromosomes during meiosis. Asynapsis or inversion-loop formation in the inverted region may be responsible for impaired spermatogenesis and may prevent sperm-chromosome imbalance.

Meiotic and sperm aneuploidy studies in three carriers of Robertsonian translocations and small supernumerary marker chromosomes

OBJECTIVE To study the meiotic behaviour of one carrier of a small supernumerary marker chromosome (sSMC): 47,XY,+mar; one carrier of a Robertsonian translocation (ROB): 45,XY,rob(13;21) (q10;q10); and one carrier of both a sSMC and a ROB: 46,XY,rob(13;21) (q11.1;q11.1),+mar. DESIGN Case-control study. SETTING University-affiliated research center and hospital. PATIENT(S) Subfertile men with ROB and sSMC. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The chromosomal origin of the sSMC was assessed by multiplex fluorescence in situ hybridization. The segregation of the ROB and sSMC in sperm and possible interchromosomal effects were examined by fluorescence in situ hybridization. Synapsis, meiotic recombination, and meiotic inactivation were investigated in ejaculate spermatocytes of the 47,XY,+mar and 45,XY,rob(13;21) carriers using immunostaining. RESULT(S) In the 47,XY,+mar and 46,XY,rob(13;21),+mar carriers, the sSMC was found in 13.5% and 11.5 % of sperm, respectively. Analysis of meiotic segregation of chromosome 13 and 21 showed that 91.2% of sperm were normal/balanced in the 46,XY,rob(13;21),+mar case, whereas 88.4% of sperm were normal/balanced in the 45,XY,rob(13;21) case. Interchromosomal effects involving the sex chromosomes were found in both sSMC carriers. Both 47,XY,+mar and 45,XY,rob(13;21) carriers showed decreased global recombination, impaired synapsis, and an association of abnormal chromosomes with the XY body. CONCLUSION(S) Carriers of marker chromosomes produce sperm with markers at frequencies lower than theoretically expected. Carriers of ROB and sSMC showed decreased recombination, impaired synapsis, and association of abnormal chromosomes with the XY body, which may contribute to an interchromosomal effect. Using immunofluorescence techniques to analyze ejaculate-derived spermatocytes from subfertile men provides a novel technique for examining meiosis without the need for a testicular biopsy.

Altered Crossover Distribution and Frequency in Spermatocytes of Infertile Men with Azoospermia

During meiosis, homologous chromosomes pair to facilitate the exchange of DNA at crossover sites along the chromosomes. The frequency and distribution of crossover formation are tightly regulated to ensure the proper progression of meiosis. Using immunofluorescence techniques, our group and others have studied the meiotic proteins in spermatocytes of infertile men, showing that this population displays a reduced frequency of crossovers compared to fertile men. An insufficient number of crossovers is thought to promote chromosome missegregation, in which case the faulty cell may face meiotic arrest or contribute to the production of aneuploid sperm. Increasing evidence in model organisms has suggested that the distribution of crossovers may also be important for proper chromosome segregation. In normal males, crossovers are shown to be rare near centromeres and telomeres, while frequent in subtelomeric regions. Our study aims to characterize the crossover distribution in infertile men with non-obstructive (NOA) and obstructive azoospermia (OA) along chromosomes 13, 18 and 21. Eight of the 16 NOA men and five of the 21 OA men in our study displayed reduced crossover frequency compared to control fertile men. Seven NOA men and nine OA men showed altered crossover distributions on at least one of the chromosome arms studied compared to controls. We found that although both NOA and OA men displayed altered crossover distributions, NOA men may be at a higher risk of suffering both altered crossover frequencies and distributions compared to OA men. Our data also suggests that infertile men display an increase in crossover formation in regions where they are normally inhibited, specifically near centromeres and telomeres. Finally, we demonstrated a decrease in crossovers near subtelomeres, as well as increased average crossover distance to telomeres in infertile men. As telomere-guided mechanisms are speculated to play a role in crossover formation in subtelomeres, future studies linking crossover distribution with telomere integrity and sperm aneuploidy may provide new insight into the mechanisms underlying male infertility.

Evaluation of DNA methylation at imprinted DMRs in the spermatozoa of oligozoospermic men in association with MTHFR C677T genotype.

Altered DNA methylation has been previously identified in the spermatozoa of infertile men; however, the origins of these errors are poorly understood. DNA methylation is an epigenetic modification which is thought to play a fundamental role in male germline development. DNA methylation reactions rely on the cellular availability of methyl donors, which are primarily products of folate metabolism, where a key enzyme is methylenetetrahydrofolate reductase (MTHFR). The MTHFR C677T single nucleotide polymorphism (SNP) reduces enzyme activity and may potentially alter DNA methylation processes during germline development. The objective of this study was to determine whether altered DNA methylation in spermatozoa is associated with the MTHFR C677T SNP. DNA methylation was evaluated at the H19, IG‐GTL2, and MEST imprinted differentially methylated regions in the spermatozoa of 53 men – 44 oligozoospermic men and nine fertile men with normal sperm parameters via bisulfite sequencing of sperm clones. The 44 infertile men were stratified by severity of oligozoospermia – three normal (>15 million spermatozoa/mL), eight moderate (5–15 million spermatozoa/mL), 23 severe (1–5 million spermatozoa/mL), and 10 very severe (<1 million spermatozoa/mL). MTHFR C677T SNP genotyping was conducted in a subset of 44 peripheral blood samples via restriction fragment length polymorphism. A total of three men – severe oligozoospermic and CT genotype – were found to be altered, which is defined as having ≥50% of their clones altered, where an altered clone was in turn defined as ≥50% of CpGs with incorrect DNA methylation patterns. The incidence of three altered men within the CT subgroup, however, was not significantly higher than the incidence in the CC subgroup. Taken together, altered DNA methylation in spermatozoa was not significantly associated with the MTHFR C677T SNP; however, there was a trend for higher incidence of alterations among severe oligozoospermic infertile men with CT genotypes.

Meiotic behaviour and sperm aneuploidy in an infertile man with a mosaic 45,X/46,XY karyotype

The meiotic behaviour of the germ cells in 45,X/46,XY men has not been extensively studied. This study investigated the meiotic events and sperm aneuploidy in an azoospermic man with a 45,X/46,XY (50/50) mosaic karyotype to better understand the fate of the 45,X cells and the production of chromosomally abnormal spermatozoa. Combining immunofluorescence techniques and fluorescence in-situ hybridization, meiotic recombination, synapsis, meiotic sex chromosome inactivation (MSCI) and configuration were analysed, as well as sperm aneuploidy in the patient and 10 normal, fertile men. Despite the 50:50 somatic mosaicism in the patient, 25% of pachytene cells analysed were 45,X. Furthermore, 63% of pachytene cells were 46,XY with paired sex chromosomes, and 12% were 46,XY with unpaired sex chromosomes, which displayed abnormal MCSI patterns. Although the patients testicular spermatozoa showed increased aneuploidy, the majority were of normal constitution. The X:Y sperm ratio was significantly increased compared with the controls (P < 0.001), which may indicate that some 45,X cells gave rise to X-bearing spermatozoa. The findings provide insight into the fate of 45,X/46,XY cells in meiosis, supporting the hypothesis that stringent checkpoints ensure the favourable production of spermatozoa with normal chromosomal constitution despite an individuals abnormal karyotype.