Mathew J. Tomlinson

Reduced senescence and retained nuclear DNA integrity in human spermatozoa prepared by density gradient centrifugation.

AbstractPurpose: To investigate whether removal of extraneous cells and immotile spermatozoa from a sperm preparation by density gradient centrifugation could help to maintain normal spermatozoa in a viable state and retain their deoxyribonucleic acid integrity. Methods: Sperm motility was assessed on a daily basis in aliquots of neat semen, extended semen, and spermatozoa prepared on a PureSperm® density gradient. At the same time, aliquots of each sperm sample were preserved for TUNEL assay and nick translation. Results: Spermatozoa prepared using density gradient centrifugation survived three times as long as spermatozoa in neat semen or in extended semen. Both deoxyribonucleic acid integrity and sperm motility were retained in the gradient preparations. Conclusions: Preparing spermatozoa by density gradient centrifugation is advantageous in prolonging sperm survival and maintaining deoxyribonucleic acid integrity, presumably by removing sources of reactive oxygen species. Stored spermatozoa could be used for a second attempt at fertilization if oocyte immaturity was suspected.

Validation of a novel computer-assisted sperm analysis (CASA) system using multitarget-tracking algorithms

OBJECTIVE To determine the accuracy and precision of a novel computer-assisted sperm analysis (CASA) system by comparison with existing recommended manual methods. DESIGN Prospective study using comparative measurements of sperm concentration and motility on latex beads and immotile and motile sperm. SETTING Tertiary referral fertility center with strong academic links. PATIENT(S) Sperm donors and male partners of couples attending for fertility investigations. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Achievement of Accubead target value for high and low concentration suspensions. Repeatability as demonstrated by coefficients of variation and intraclass correlation coefficients. Correlation and limits of agreement between CASA and manual methods. RESULT(S) The CASA measurements of latex beads and sperm concentrations demonstrated a high level of accuracy and repeatability. Repeated Accubead measurements attained the required target value (mean difference from target of 2.61% and 3.71% for high- and low-concentration suspensions, respectively) and were highly reproducible. Limits of agreement analysis suggested that manual and CASA counts compared directly could be deemed to be interchangeable. Manual and CASA motility measurements were highly correlated for grades a, b, and d but could not be deemed to be interchangeable, and manual motility estimates were consistently higher for motile sperm. CONCLUSION(S) The novel CASA system was able to provide semen quality measurements for sperm concentration and motility measurements which were at least as reliable as current manual methods.

Sperm quality and its relationship to natural and assisted conception: British Fertility Society Guidelines for practice

Abstract Reports on the influence of semen parameters on natural or assisted pregnancy are contradictory, suggesting that the many confounding variables which contribute to outcome have not been taken into account. However, it is possible to derive some consensus for both natural and assisted conception by focussing on studies which use WHO-recommended semen analysis on relatively large populations, applying appropriate statistics and accounting for ‘female factors’. The concentration of progressively motile sperm has consistently been shown to be the most predictive factor with regard to outcome. Around 64% of studies suggest that a reasonable chance of success with artificial insemination requires at least 5 × 106 motile sperm and this is supported by the WHOs revised reference range for natural conception. Sperm morphology remains controversial, with a lack of standardisation across centres, the adoption of ever-stricter scoring criteria and changing reference values. Antisperm antibodies do not appear to influence outcome independently of sperm motility and agglutination. Sperm DNA damage appears to be related to sperm quality, embryo development and pregnancy loss, yet there remains no consensus on the best testing procedures, clinical reference values and how patients with an adverse result should be managed. In conclusion, laboratories should continue to focus on reducing the uncertainty and improving the quality of their basic semen analysis.

Monitoring fertility (semen analysis) by cancer survivors who banked sperm prior to cancer treatment

STUDY QUESTION What medical and psychological variables predict why men with banked sperm do not return for semen analysis after their cancer treatment has ended? SUMMARY ANSWER Men who decline the offer of semen analysis are less likely to have reported adverse side effects during cancer treatment, and have a more negative experience of banking sperm and a more negative attitude towards disposal of their stored semen than those who attend. WHAT IS KNOWN ALREADY Previous authors have noted that male cancer survivors seem reluctant to have their fertility tested after their treatment has ended. Moreover, the utilization rates of banked sperm are very low (<10%) and the majority of samples are kept for many years without being used. STUDY DESIGN, SIZE AND DURATION A cross-sectional study of 499 cancer survivors who were sent a questionnaire about their views on sperm banking, fertility and post-treatment semen analysis between April 2008 and December 2010. PARTICIPANTS AND SETTING Men (aged 18-55 years) who had banked sperm in Sheffield and Nottingham (UK) prior to gonadotoxic treatment for cancer more than 5 years previously. MAIN RESULTS AND THE ROLE OF CHANCE Completed questionnaires were received from 193 men (38.7% response rate) whose samples had been banked for 9.18 ± 3.70 years (range = 4.94-26.21) and whose current age was 35.08 ± 7.08 years (range = 21.58-54.34; mean ± SD). One-third (35.8%) had never attended for semen analysis. In multivariate analysis, the odds of not attending for semen analysis were significantly greater among men who did not experience adverse treatment side effects [odds ratio (OR) = 5.72, 95% confidence interval (CI) = 2.10-15.56], who reported a more negative experience of banking sperm (OR = 1.82, 95% CI = 1.17-2.82) and a more negative attitude to disposal of their stored semen (OR = 1.56, 95% CI = 1.01-2.42). LIMITATIONS AND REASONS FOR CAUTION Only 38.7% of those eligible agreed to take part. We do not know the characteristics of men who declined to take part, if they agreed to attend semen analysis without completing the questionnaire or whether they had chosen to have semen analysis performed elsewhere (e.g. private sector). Some of the measures used (e.g. experience of banking sperm) relied on mens recall of events many years previously. WIDER IMPLICATIONS OF THE FINDINGS New strategies are required to encourage these men to engage with fertility monitoring programmes if sperm banks are to be used cost-effectively and men are to be given appropriate fertility advice. STUDY FUNDING AND COMPETING INTERESTS This paper was supported by funding from Cancer Research-UK to C.E., A.A.P. and R.R. (C481/A8141). The views expressed are those of the authors. No competing interests declared.

Sperm donor recruitment within an NHS fertility service since the removal of anonymity

The marked decline in the number of sperm donors recruited in the UK has been largely attributed to changes in regulations and in particular those related to the removal of anonymity. After a 5-year period of inactivity, the sperm donor bank in Nottingham was provided with limited resources to try and recruit donors who were willing to be identified on the HFEA register. Marketing was sporadic and at first low cost and the enquiry rate only increased significantly when the centres website became operational and higher cost advertising was used. Over a 4-year period, a total of 151 enquiries gave rise to 14 useable donors at a cost of approximately £5,500 each. Donor sperm was generally of high quality having been density gradient prepared prior to cryopreservation and provided an overall ongoing pregnancy rate of 21.6% and 45.6% by IUI and IVF, respectively. The overall exercise demonstrated that identifiable donors were coming forward but in lower numbers compared to those observed before 2005. At current treatment prices, centres should be aware that recouping the costs of donor recruitment and processing may be difficult and that the cost of both donor sperm and donor insemination are likely to rise significantly.

The history of sperm cryopreservation

While there have been several relatively recent comprehensive reviews of mammalian sperm cryopreservation (Watson, 2001; Leibo, 2004), this chapter is not meant to be a review of the literature of mammalian sperm cryopreservation. Instead, we intend to provide an understanding of the history, current status, and potential future direction of mammalian sperm cryobiology. There are many reasons why sperm cryopreservation is important, including: (1) maintenance of genetic diversity in domestic and wild species populations (Wildt, 1992; Critser & Russell, 2000); (2) facilitating the distribution of “genetically superior” domestic species lines; (3) treatment of iatrogenic infertility (Kuczynski et al., 2001; Ranganathan et al., 2002; Tash et al., 2003; Agarwal et al., 2004; Nalesnik et al., 2004); and (4) genetic banking of genetically modified animal models of human health and disease (Critser & Russell, 2000; Knight & Abbott, 2002). While actual references to sperm cryobiology and cryopreservation date as far back as the 1600s (Sherman, 1964), it was not until the development of artificial insemination (AI) in the late 1950s and early 1960s, when the dairy industry needed longer-term storage methods for bull sperm, that sperm cryopreservation became a major area of scientific investigation. Polge et al. (1949) made a pivotal discovery that showed that the use of glycerol (a permeating solute) could provide protection to cells at low temperatures. This is often cited as the defining moment in the establishment of modern sperm cryobiology. However, there is actually a very important body of literature which predates it, some of which established the scientific basis upon which their work was interpreted and further developed (Lovelock, 1953a; Lovelock & Polge, 1954; Menz & Luyet, 1965; Luyet, 1966; MacKenzie & Luyet, 1967a; 1967b; Luyet & Rapatz, 1970; Luyet, 1971; Rasmussen & Luyet, 1972; Rapatz & Luyet, 1973), and some of which was parallel literature that was not incorporated into the main pathway of sperm cryobiology mainly because it was published in Russian (Katkov, 2005). Based upon this scientific history, the development of “successful” mammalian sperm cryopresveration methods was established. It is critical to realize, however, that even today only a very few mammalian species’ sperm can be effectively cryopreserved. Even in those cases, the “success,” as measured by postthaw motility, routinely is 50 percent or less than that of the prefreeze motility. Successful cryopreservation varies highly among species, individuals within species, and even within ejaculates of individuals, which is largely attributed to the differences in biophysical characteristics among cell types (Gao et al., 1997; Thurston et al., 2002; Walters et al., 2005). Chapter

Working Party on Sperm Donation Services in the UK: report and recommendations.

Mark Hamilton* (Chair) (Chair, British Fertility Society (BFS): Clinician) Allan Pacey (Secretary, BFS: Andrologist) Mathew Tomlinson (Chair, Association Biomedical Andrologists: Andrologist) Daniel Brison (Scientist Representative BFS Committee: Embryologist) Lawrence Shaw (Clinician, Private Sector) Cathy Turner (BFS Nurse Representative, London) Laura Witjens (Chair, National Gamete Donation Trust (NGDT)) Pip Morris (Donor Recruitment Manager, (NGDT)) Clare Brown (Chair, Infertility Network UK (INUK)) Olivia Montuschi (Donor Conception Network (DCN)) Joanne Adams (Donor Recruitment Manager, Manchester, Andrologist) Brian Lieberman (Clinician, NHS & Private sector) Jennifer Speirs (BFS Counsellor Representative, Edinburgh)

Uncertainty of measurement and clinical value of semen analysis: has standardisation through professional guidelines helped or hindered progress?

This article suggests that diagnostic semen analysis has no more clinical value today than it had 25–30 years ago, and both the confusion surrounding its evidence base (in terms of relationship with conception) and the low level of confidence in the clinical setting is attributable to an associated high level of ‘uncertainty’. Consideration of the concept of measurement uncertainty is mandatory for medical laboratories applying for the ISO15189 standard. It is evident that the entire semen analysis process is prone to error every step from specimen collection to the reporting of results and serves to compound uncertainty associated with diagnosis or prognosis. Perceived adherence to published guidelines for the assessment of sperm concentration, motility and morphology does not guarantee a reliable and reproducible test result. Moreover, the high level of uncertainty associated with manual sperm motility and morphology can be attributed to subjectivity and lack a traceable standard. This article describes where and why uncertainty exists and suggests that semen analysis will continue to be of limited value until it is more adequately considered and addressed. Although professional guidelines for good practice have provided the foundations for testing procedures for many years, the risk in following rather prescriptive guidance to the letter is that unless they are based on an overwhelmingly firm evidence base, the quality of semen analysis will remain poor and the progress towards the development of more innovative methods for investigating male infertility will be slow.

Review and follow-up of patients using a regional sperm cryopreservation service: ensuring that resources are targeted to those patients most in need

Are all patients undergoing chemotherapy for long‐term sperm banking at risk of permanent sterility? Male fertility is generally lower in men with cancer and all patient groups are at risk of azoospermia. Careful management is required to ensure that samples are not stored for excessively long periods should they not be required. A retrospective analysis of 1688 patient records and prospective recall of patients for semen testing were perfomed. Pre‐therapy fertility was compared with a group of pre‐vasectomy patients as a comparator. Those who fail to bank spermatozoa, rates of disposal of samples and the utilization in assisted reproduction were also examined. Sperm quality was poorest in testicular cancer (TC) patients followed by those with Hodgkins lymphoma (HL) prior to treatment. Post‐therapy data were available in 376 patients (42%). Sperm number was lowest (and azoospermia highest at 77%) in patients with HL treated with regimens other than adriamycin, bleomycin, vinblastine and dacarbazine (ABVD). Non‐HL NHL and leukaemic patients had similarly high rates of azoospermia at 46 and 55%. HL patients treated with ABVD (11%) and TC patients (9.7%) had the lowest rates of azoospermia. Azoospermia was seen in every treatment group except for TC patients receiving carboplatin. Only 45 patients used their samples in ART (4.5%) in 10 years. Little is known about the fertility status of the patients not coming forward for follow‐up testing, those conceiving naturally, those with no intention of conceiving and some which may have psychological reasons for not attending. In conclusion, virtually all patients undergoing chemotherapy are potentially at risk of temporary or permanent infertility. However, as uptake and utilization of stored material remain low, sperm banks should be carefully managed to ensure that resources are targeted to the patients most in need.

Is your andrology service up to scratch

The quality of the end product from andrology services continues to lack consistency and in some cases fails to meet the needs of the end users (patients or clinicians). Results of external quality assessment (EQA) schemes continue to show unacceptably wide variation for the results of a single specimen. Some laboratories are able to show that the results of semen analyses relate to both natural and assisted pregnancy and are therefore useful in the management of the infertile couple, whereas others claim that their value is limited to the identification of severe male factor infertility. With wide variation in standardisation of methodology, levels of staff training and quality assurance, it is entirely understandable that such discrepancies persist. The following article proposes that Quality Assurance (QA) is derived from standardisation of methods and implementation of good practice for the entire analytical process, i.e. from the collection and delivery of the specimen, through analysis and processing, to the eventual reporting and interpretation of the result to the clinician. Without appropriate QA, the value of diagnostic testing will remain limited and will vary according to the individual or individual laboratory performing the test.