Sperm toxicity testing on lubricant gels: should we be recommending 'fertility-friendly' specialist products?

Couples trying to conceive or providing samples for Assisted Reproductive Technologies (ART) are advised against the use of lubricant-gels due to the risk of sperm-toxicity. However, gels now exist which are specifically formulated to help couples conceive but without consensus on their toxicity relative to non-specialist products. This study tested gels recently introduced as 'sperm friendly' (FertilSafe Plus, Fertile Check) alongside established lubricants intended for pleasure only using a recently published toxicity testing regime. Computer Assisted Sperm Analysis (CASA) was performed at 1 and 2 h on donor sperm (n = 12) pre-incubated with each gel (10% v/v) and controls. All gels led to a significant loss of motility/velocity at 1 and 2 h (p < 0.01), with the most significant loss from the 2 Durex pleasure products (11% and 15%, vs 47% progression) at 60 min, although these performed better than saliva (used as negative control). Incubation with FertilSafePlus led to the smallest loss of motility (24% vs 47%) at 1 h. Saliva and products designed for lubrication only exhibited the most negative effect on motility and those marketed as 'sperm safe' could be considered the best performers. Whether these affects are due to direct toxicity or are indirect due to other factors such as viscosity, pH or osmolality remains uncertain.

Is pre-freeze sperm preparation more advantageous than post-freeze?

Human sperm cryopreservation is characterised to this day by sub-optimal success rates. Interestingly, a traditional approach to improving post-thaw outcome has been to integrate standard sperm preparation techniques into freezing protocols as a means of selecting sperm with the highest fertilisation potential prior to insemination. However, no consensus has been reached yet regarding the optimal timing (before or after freezing) of this selection step. Following analysis of a total of 20 human semen samples, which were divided into two aliquots prepared by density gradient centrifugation either before or after freezing, this study demonstrated higher post-thaw total (P < 0.0001), progressively motile (P = 0.005) and vital (P < 0.0001) sperm counts for frozen-prepared semen samples. The present study suggests that direct insemination with frozen-prepared sperm with minimal intervening post-thaw processing might be a more advantageous approach to current clinical practices, particularly for donor and patient intrauterine insemination programmes. Further research into cryopreservation-induced coiled sperm tail morphology is also warranted. LAY SUMMARY: Freezing and storing of sperm in liquid nitrogen ('sperm cryopreservation') is the current method of choice for preserving the fertility of a wide scope of men. Nevertheless, sub-optimal sperm survival is still associated with traditional cryopreservation methods, namely 'slow freezing', and may affect fertility treatment success rates. Interestingly, a widely applied approach for selecting high-quality sperm before treatment has been to incorporate 'sperm preparation' techniques, such as density gradient centrifugation, in slow freezing protocols. There is, however, an ongoing debate regarding which is the optimal timing of this selection step: before or after freezing. In this study, we collected 20 human semen samples which were divided into two portions and subjected to density gradient centrifugation either before or after freezing. Post-thaw semen analyses demonstrated significantly improved sperm counts (P < 0.05) when density gradient centrifugation was performed before freezing, thus suggesting this approach to be more advantageous for current clinical practices.

Is azoospermia the appropriate standard for post-vasectomy semen analysis? Or an unachievable goal of best practice laboratory guidelines.

The increasingly stringent laboratory-approach to diagnosing azoospermia for post-vasectomy semen analysis (PVSA) continues to be at odds with the simpler approach desired by clinicians. This study describes the analysis of 10 years of PVSA and discusses the outcome in relation to risk, cost and assesses whether more stringent procedures are required. PVSA was performed on 4788 patients initially using a 2-test strategy (16 and 20 weeks post-surgery), moving to 1 test during 2013-2014. Azoospermia was confirmed by the analysis of 10 µl of semen followed by 10 µl of centrifuged pellet. In total, there were 9260 tests with a median of 1.93 tests/patient and 18.7 weeks to clearance. Surgical failure occurred in 1.75%, falling to 1.1% between 2011 and 2016. There were no cases of unwanted pregnancy, recanalization or complaints although misdiagnosis was detected in 1 case as a result of failure to confirm patient identification. Azoospermia performed according to World Health Organization (WHO) guidelines is sufficiently robust to confirm success/failure of vasectomy. With uncertainty surrounding the diagnosis, efforts to improve detection of occasional non-motile sperm are futile, cost more and fail to reduce risk of inappropriate clearance. Misdiagnosis is more likely from patient identification error and mitigation may include reverting to the safety net of a 2-test strategy.

Independent and combined effects of diethylhexyl phthalate and polychlorinated biphenyl 153 on sperm quality in the human and dog.

A temporal decline in human and dog sperm quality is thought to reflect a common environmental aetiology. This may reflect direct effects of seminal chemicals on sperm function and quality. Here we report the effects of diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153) on DNA fragmentation and motility in human and dog sperm. Human and dog semen was collected from registered donors (n = 9) and from stud dogs (n = 11) and incubated with PCB153 and DEHP, independently and combined, at 0x, 2x, 10x and 100x dog testis concentrations. A total of 16 treatments reflected a 4 × 4 factorial experimental design. Although exposure to DEHP and/or PCB153 alone increased DNA fragmentation and decreased motility, the scale of dose-related effects varied with the presence and relative concentrations of each chemical (DEHP.PCB interaction for: DNA fragmentation; human p < 0.001, dog p < 0.001; Motility; human p < 0.001, dog p < 0.05). In both human and dog sperm, progressive motility negatively correlated with DNA fragmentation regardless of chemical presence (Human: P < 0.0001, r = -0.36; dog P < 0.0001, r = -0.29). We conclude that DEHP and PCB153, at known tissue concentrations, induce similar effects on human and dog sperm supporting the contention of the dog as a sentinel species for human exposure.

CASA in the medical laboratory: CASA in diagnostic andrology and assisted conception.

CASA has been used in reproductive medicine and pathology laboratories for over 25 years, yet the 'fertility industry' generally remains sceptical and has avoided automation, despite clear weaknesses in manual semen analysis. Early implementers had difficulty in validating CASA-Mot instruments against recommended manual methods (haemocytometer) due to the interference of seminal debris and non-sperm cells, which also affects the accuracy of grading motility. Both the inability to provide accurate sperm counts and a lack of consensus as to the value of sperm kinematic parameters appear to have continued to have a negative effect on CASA-Mot's reputation. One positive interpretation from earlier work is that at least one or more measures of sperm velocity adds clinical value to the semen analysis, and these are clearly more objective than any manual motility analysis. Moreover, recent CASA-Mot systems offer simple solutions to earlier problems in eliminating artefacts and have been successfully validated for sperm concentration; as a result, they should be viewed with more confidence in relation to motility grading. Sperm morphology and DNA testing both require an evidence-based consensus and a well-validated (reliable, reproducible) assay to be developed before automation of either can be of real clinical benefit.

Safe Storage of Gametes and Embryos: No Time for Complacency.

Recent serious untoward incidents in the field of assisted reproduction have once again highlighted the need for vigilance and in particular improved risk management in relation to the cryopreservation of gametes and embryos. Despite increasing levels of regulation and the requirement to adhere to total quality management practices, catastrophic incidents such as the death of an employee or the loss of a freezer full of patient embryos or sperm continue to occur sufficiently frequently for the industry to be concerned and highlight the need to make practices considerably safer. Potential losses through litigation could be considerable if the gamete or embryo bank is found to be negligent and fails to provide the necessary resources and implement recognizable control measures, which may include suitable facilities with adequate ventilation and oxygen monitoring, competent staff, and an appropriate level of well-maintained equipment; round-the-clock emergency procedures, early warning systems to deal with a failing vessel; and contingency to mitigate losses within a failing vessel and validated procedures which are embedded into the organization which lead to a high-quality end product, for example, frozen embryo while permitting a safe system of work. Since cross-contamination incidents have occurred in other disciplines such as blood cryostorage, which have led to the transmission of viral disease, the biological safety of the end product also requires careful consideration despite the fact that no similar incident appears to have come to light in the field of reproductive medicine. Implementation of risk reduction measures may include screening for blood borne viruses and possibly bacterial infection, careful selection of appropriate packaging plastics, and consideration as to whether packaging is suitable for immersion in liquid nitrogen. If not, vapor phase storage may be considered as additional mitigation. In the light of recent events, centers should place risk management of their cryofacilities and service high on the agenda and ensure that it becomes integral to future objective setting and business planning.

Uncertainty associated with assessing semen volume: are volumetric and gravimetric methods that different?

The World Health Organization laboratory manual for the examination of human semen suggests that an indirect measurement of semen volume by weighing (gravimetric method) is more accurate than a direct measure using a serological pipette. A series of experiments were performed to determine the level of discrepancy between the two methods using pipettes and a balance which had been calibrated to a traceable standard. The median weights of 1.0ml and 5.0ml of semen were 1.03 g (range 1.02-1.05 g) and 5.11 g (range 4.95-5.16 g), respectively, suggesting a density for semen between 1.03g and 1.04 g/ml. When the containers were re-weighed after the removal of 5.0 ml semen using a serological pipette, the mean residual loss was 0.12 ml (120 µl) or 0.12 g (median 100 µl, range 70-300 µl). Direct comparison of the volumetric and gravimetric methods in a total of 40 samples showed a mean difference of 0.25ml (median 0.32 ± 0.67ml) representing an error of 8.5%. Residual semen left in the container by weight was on average 0.11 g (median 0.10 g, range 0.05-0.19 g). Assuming a density of 1 g/ml then the average error between volumetric and gravimetric methods was approximately 8% (p < 0.001). If, however, the WHO value for density is assumed (1.04 g/ml) then the difference is reduced to 4.2%. At least 2.4-3.5% of this difference is also explained by the residual semen remaining in the container. This study suggests that by assuming the density of semen as 1 g/ml, there is significant uncertainty associated with the average gravimetric measurement of semen volume. Laboratories may therefore prefer to provide in-house quality assurance data in order to be satisfied that 'estimating' semen volume is 'fit for purpose' as opposed to assuming a lower uncertainty associated with the WHO recommended method.

Replacing IUI with IVF for initial treatment of unexplained infertility: why this NICE recommendation is cause for concern.

The latest guidelines from the National Institute for Health and Care Excellence (NICE) for assisted conception recommend that people experiencing unexplained infertility should no longer be offered stimulated intra-uterine insemination (IUI) as a first-line treatment, but rather be directed towards IVF or alternatively be left to expectant management. NICE has acknowledged that the cited evidence leading to this decision was not sufficiently robust. As such, we are concerned that accordance with these new NICE guidelines may result in people with no identifiable cause of their infertility being prematurely referred for IVF treatment. Since IVF constitutes a more invasive and expensive treatment process, which also represents an additional and unnecessary cost pressure to the National Health Service, there is a longstanding need for a robust clinical trial to resolve the uncertainty as to whether one treatment is more appropriate than another. Until such data is available, we suggest that provision of stimulated IUI, in centres achieving a satisfactory live birth rate, represents a significant cost-saving to those commissioning fertility services, with lower risks to people treated.

Standards in reporting cryopreserved donor sperm characteristics: should they be reported post-thaw or post-wash?

An increase in the reliance on imported donor samples has been the consequence of a continued shortage of UK donors. Disputes can arise between suppliers and purchasers if the sperm quality is not as expected, yet there appears to be no requirement for the standardization of methods for sperm processing or analysis. Following analysis of 102 donor intrauterine insemination cycles, this study demonstrates that the motile sperm concentration is significantly (P < 0.05) reduced after the necessary removal of cryoprotectant before insemination. Suppliers of donor spermatozoa should therefore provide information on standards used for sperm assessment and whether analysis is performed before or after washing in order that purchasers are better informed about the quality of the end product they are committed to buying.

How strict should specimen acceptance or rejection criteria be for diagnostic semen analysis? An opinion.

Medical laboratory accreditation (previously by Clinical Pathology Accreditation UK Ltd and now by the United Kingdom Accreditation Service) has been integral to improving standards and service quality in the UK. With the recent introduction of the ISO15189 standard, all laboratories offering a clinical diagnostic service are required to demonstrate further improvement, with more emphasis on validation and assessment of the uncertainty levels associated with testing. This applies not only to 'bench testing', but also to the evidence-base for all pre-analytical and post-analytical procedures. To reduce the risk of external influences on andrology test results, semen sample rejection criteria were developed, including confirmation of patient identity, a strict time limit from sample production to testing, the use of toxicity-tested containers, a prescribed sexual abstinence and a need for complete sample collection. However, such criteria were originally developed by the World Health Organization in order to standardise analysis rather than reject testing outright, and should therefore be implemented with caution. Rejecting samples with normal semen parameters because they fail to meet some of the criteria as outlined above would be a waste of resources and adversely affect user (the person who requested or provided the sample) satisfaction. This document evaluates the evidence base underlying commonly used criteria for specimen rejection and suggests how they may be applied more pragmatically in order to improve efficiency and reduce the waste of resources.

How do men in the United Kingdom decide to dispose of banked sperm following cancer treatment?

Current policy in the UK recommends that men bank sperm prior to cancer treatment, but very few return to use it for reproductive purposes or agree to elective disposal even when their fertility recovers and their families are complete. We assessed the demographic, medical and psychological variables that influence the decision to dispose by contacting men (n = 499) who banked sperm more than five years previously, and asked them to complete questionnaires about their views on sperm banking, fertility and disposal. From 193 responses (38.7% response rate), 19 men (9.8%) requested disposal within four months of completing the questionnaire. Compared with men who wanted their sperm to remain in storage, they were significantly more confident that their fertility had recovered (OR = 1.78, 95% CI = 1.05-3.03, p = 0.034), saw fertility monitoring (semen analysis) as less important (OR = 0.61, 95% CI = 0.39-0.94, p = 0.026), held more positive attitudes to disposal (OR = 5.71, 95% CI = 2.89-11.27, p < 0.001), were more likely to have experienced adverse treatment side-effects (OR = 4.37, CI = 1.61-11.85, p = 0.004) and had less desire for children in the future (OR = 0.41, 95% CI = 0.26-0.64, p < 0.001). Information about men's reasons to dispose of banked sperm may be helpful in devising new strategies to encourage men to engage with sperm banking clinics and make timely decisions about the fate of their samples.

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

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 WHO's 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.

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.

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 centre's 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.

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.

Risks associated with cryopreservation: a survey of assisted conception units in the UK and Ireland.

A number of recent, high-profile incidents involving loss or damage to cryopreserved material held in IVF units or sperm storage centres have highlighted the need for centres to carefully review their cryostorage practice and take action. Critical disasters involving lost or damaged patient material, although high profile, are still thought to be rare, and there has been concern that we should ensure that any response is proportionate to risk. However, as no regulators, manufacturers or similar professional disciplines have collected information in the long term, our knowledge of the true incidence of such adverse events is extremely poor. Recognizing the need for some solid data, the UK Association of Clinical Embryologists (ACE) conducted a survey on the subject, at its joint meeting with the Association of Irish Clinical Embryologists (ICE) (January, 2006). Questions were asked in relation to the risk of: injury to personnel; and potential loss of and potential damage to stored material. The number of serious and not so serious adverse events/situations relating to both staff and sample safety are discussed in detail. The incidence of problems was certainly higher than we had imagined; the lack of general training and awareness amongst our staff is a serious cause for concern, and appears to leave the industry vulnerable. Moreover, the survey highlighted the need for a coordinated approach to the collection of more detailed information both prospectively and retrospectively. Regulators, manufacturers, allied professional bodies and, more importantly, centres should be encouraged to share both recent and historic data relating to adverse events, in order that accurate risk assessments can be made in future.

Managing risk associated with cryopreservation.

Patients who consent to the frozen storage of sperm or embryos quite rightly expect the storing centre to do everything reasonably possible to keep them in optimum conditions. Both the process of cryopreservation and the cryofacility are loaded with risk, from patient/sample processing, through to the eventual utilization or disposal of specimens. The risk management process should focus on minimizing losses, including staff injury, premature warming of cells and tissues, mistaken identity, and transmission of infection. Early warning and monitoring systems should be in place for quality assurance and to prevent incidents involving cryovessels turning critical. Centres must ensure that every reasonable practical measure that can be put in place is done so, and that resourcing of the service adequately reflects the liability it represents.