Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline.

The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline.

Azoospermia: is sample centrifugation indicated? A national survey of practice and the Oxford experience.

OBJECTIVE: Some men with azoospermia on general laboratory testing have low quantities of sperm in the ejaculate that can only be identified through sample centrifugation and careful examination of the pellet droplets (extended sperm preparation [ESP]). Such sperm can be used for IVF-ICSI as an alternative to either surgical sperm retrieval (SSR) or donor sperm. The aims of the present study were to: 1) assess UK IVF clinic practice with regard to ESP in men with azoospermia; and 2) to analyze the outcome of ESP and SSR in azoospermic men attending the Oxford Fertility Unit. DESIGN: National survey of all 70 IVF units plus chart review. SETTING: Assisted conception unit. PATIENT(S): One hundred twenty-two azoospermic men referred to the Oxford Fertility Unit. MAIN OUTCOME MEASURE(S): Proportions of UK IVF clinics performing ESP for azoospermia. Proportions of azoospermic men in Oxford with sperm identified at ESP and, if necessary, SSR. Relationship between serum FSH and outcome. RESULT(S): In part 1 of the study, 55 (79%) of the 70 UK IVF clinics returned completed questionnaires. Fifty clinics (91%) routinely performed ESP for men with azoospermia on general laboratory testing, four clinics (7%) proceeded straight to SSR without prior ESP, and one clinic varied in their approach. When clinics were asked whether they used serum FSH levels when considering whether to proceed to SSR 28 (51%) did, 9 (16%) did not, and 18 (33%) varied in their approach. The value placed on testicular volume similarly varied. Part 2 of the study included 122 men referred to the Oxford Fertility Unit with azoospermia on general laboratory testing. Eighty-seven men underwent ESP. Motile sperm was found, cryopreserved, and later used during IVF-ICSI treatment in 19 men (22%). Eighty-one men underwent SSR (after either a negative ESP or declining ESP). Viable sperm was found in 66 men undergoing SSR (81%). There was a statistically significant relationship between serum FSH and the chance of retrieving sperm with SSR (P=0.002) but not with ESP. CONCLUSION(S): The majority (91%) of IVF clinics in the UK routinely perform ESP in men with azoospermia on general testing. Only half routinely used serum FSH levels as predictors of SSR outcome. The value of ESP is confirmed by our findings in Oxford. Twenty-two percent of men with azoospermia on general laboratory testing had sufficient sperm found at ESP to proceed to IVF-ICSI without resorting to the use of either SSR or donor sperm. Serum FSH levels were not related to the chance of finding sperm during ESP but were related to the outcome of SSR. Our results suggest that ESP should be considered for all men with azoospermia and no apparent obstruction.