D. Stewart Irvine

Fertility preservation for young patients with cancer: who is at risk and what can be offered?

Estimates suggest that by 2010, one in 715 people in the UK will have survived cancer during childhood. With increasing numbers of children cured, attention has focused on their quality of life. We discuss the causes of impaired fertility after cancer treatment in young people, and outline which patients are at risk and how their gonadal function should be assessed. With the report of a livebirth after orthotopic transplantation of cryopreserved ovarian tissue and the continued development of intracytoplasmic sperm injection for men with poor sperm quality, we assess established and experimental strategies to protect or restore fertility, and discuss the ethical and legal issues that arise.

Protective effect of antioxidants on the impairment of sperm motility by activated polymorphonuclear leukocytes

OBJECTIVE To test the ability of antioxidants to reduce the loss of sperm motility caused by reactive oxygen species generated by polymorphonuclear leukocytes (PML). DESIGN Standardized preparations of leukocyte-contaminated semen were created by suspending known concentrations of purified spermatozoa and PML in seminal plasma. After the stimulation of reactive oxygen species generation with phorbol ester, the spermatozoa were washed and incubated in culture medium before an analysis of their movement. The ability of antioxidants to counteract the free radical-induced loss of sperm motility observed under these circumstances was assessed. SETTING An institutional research laboratory. PARTICIPANTS The semen was obtained from normal volunteers. INTERVENTIONS The following were tested: vitamins C and E, dimethylsulfoxide, catalase, hypotaurine, N-acetylcysteine, and reduced glutathione. MAIN OUTCOME MEASURES Reactive oxygen species generation was monitored by luminol-dependent chemiluminescence. Sperm motility was assessed manually and by computer-aided semen analysis. RESULTS Consistent impairment of sperm motility and average path velocity was observed in the presence of activated PML. This effect was reduced by the concomitant presence of glutathione, N-acetylcysteine, hypotaurine, and catalase. CONCLUSION Antioxidants can protect against the damaging effect of leukocyte-derived reactive oxygen species on sperm movement and may be of clinical value in assisted conception procedures.

Semen quality and spermatozoal DNA integrity in survivors of childhood cancer: a case-control study.

BACKGROUND Treatment of childhood cancer can result in impaired spermatogenesis. Intracytoplasmic sperm injection (ICSI), however, can enable men to achieve fatherhood, and has focused attention on gamete integrity in men with oligozoospermia. Our aim was to assess testicular function in survivors of childhood cancer. METHODS We assessed testicular function in 33 survivors of childhood cancer and 66 age-matched controls. The median age at diagnosis and at the start of the trial was 10.0 years (range 2.2-16.9) and 21.9 years (16.5-35.2), respectively. We assessed pubertal staging, measured plasma sex steroid hormone concentrations, and analysed semen quality, including spermatozoal DNA integrity. FINDINGS Ten (30%) individuals were azoospermic and six (18%) oligozoospermic (sperm concentration, 20 x 10(6)/mL). Sperm concentration was significantly lower in the non-azoospermic group than in controls (median 37.1 x 10(6)/mL, IQR 19.7 x 10(6) to 89.9 x 10(6), vs 90.7 x 10(6)/mL, 50.5 x 10(6) to 121.5 x 10(6); p=0.002). In the non-azoospermic cancer survivor group, inhibin B concentrations were lower than in controls (mean 153.3 ng/L, SEM 17.8, vs 223.7 ng/L, 8.8; p,0.001), and FSH concentrations were higher (6.6 U/L, 0.9, vs 3.2 U/L, 0.2; p,0.001). Only 11 (33%) survivors of childhood cancer had normal semen quality. There was no significant difference in sperm DNA integrity between the non-azoospermic and control groups (9%, 5-13, vs 11%, 7-16; p=0.06). INTERPRETATION Sperm concentration is reduced after treatment for cancer. However, the sperm produced seems to carry as much healthy DNA as those produced by the healthy population, suggesting that assisted conception can be considered as a treatment option for these men.

Predicting the fertilizing potential of human sperm suspensions in vitro: importance of sperm morphology and leukocyte contamination

OBJECTIVE To determine the relationships between sperm function tests and fertilization of human oocytes in vitro. DESIGN Analysis of infertile patients undergoing IVF therapy. SETTING Diagnostic Andrology Laboratory and Assisted Conception Service. PATIENTS Forty-one couples who underwent IVF-ET therapy were studied. INTERVENTIONS None. MAIN OUTCOME MEASURES The ability of human spermatozoa to achieve fertilization in vitro was examined in relation to numerous criteria of semen quality, including the conventional semen profile, the computer-aided assessment of sperm movement, ionophore-induced acrosome reaction, acridine orange staining, sperm morphology, and chemiluminescent signals induced by phorbol ester and N-formyl-methionyl-leucyl-phenylalanine (FMLP). RESULTS Significant correlations were observed between fertilization rates and several attributes of the sperm preparations, including elements of sperm function (acrosome reaction), movement (percentage motile, hyperactivation, the amplitude of lateral sperm head displacement), morphology (normal morphology, midpiece defects, multiple anomalies index), nuclear normality (acridine orange staining), and reactive oxygen species generation (chemiluminescence induced by phorbol ester and FMLP). In a stepwise multiple regression analysis, an accurate prediction of fertilization rates was obtained using a multiple regression equation incorporating six variables of which sperm morphology and FMLP-induced chemiluminescence were the most informative. CONCLUSIONS A set of criteria have been identified that accurately predict the fertilizing potential of human sperm suspensions in vitro and that place particular emphasis on sperm morphology and the degree of leukocyte contamination.

The extragenomic action of progesterone on human spermatozoa is influenced by redox regulated changes in tyrosine phosphorylation during capacitation

Capacitation had no effect on the ability of progesterone to elicit a rapid calcium transient in the acrosomal domain of human spermatozoa but had a marked influence of the ability of this steroid to induce a biological response. The development of this responsiveness to progesterone appeared to be redox regulated in that it was promoted by the stimulation of reactive oxygen species generation and inhibited by the presence of antioxidants, including catalase and membrane permeant thiols. The ability of redox conditions to influence the biological responsiveness of human spermatozoa did not involve changes in the dynamics of the calcium transients induced by progesterone but was causally linked with clear differences in tyrosine phosphorylation. We conclude that the ability of human spermatozoa to respond to the calcium transients induced by progesterone depends on a background of phosphotyrosine expression that can be profoundly influenced by the redox status of the spermatozoa during capacitation.

Superoxide dismutase in human sperm suspensions: Relationship with cellular composition, oxidative stress, and sperm function

Sensitive techniques have been developed for monitoring superoxide dismutase (SOD) activities in human sperm preparations. In contradiction to the protective role normally assigned to SOD, populations of defective spermatozoa recovered from the low density region of Percoll gradients were found to have three times more SOD than functionally competent preparations pelleting in high density Percoll. SOD activity was negatively correlated with the movement characteristics of human spermatozoa and their capacity for oocyte fusion, and positively associated with the induction of peroxidative damage. SOD activity was also highly correlated with other markers of the cytoplasmic space, creatine kinase (CK), and glucose-6-phosphate dehydrogenase (G-6-PDH). We conclude that while SOD may play a physiological role in maintaining a balance between O2.- and H2O2, high levels of this enzyme are associated with impaired sperm function because (a) the human spermatozoon is highly susceptible to the cytotoxic effects of H2O2, (b) O2.- is an important mediator of normal sperm function, and (c) high SOD activities reflect errors in spermatogenesis associated with germ cell exfoliation and the retention of excess residual cytoplasm by the spermatozoa.

Male Infertility and DNA Damage in Doppel Knockout and Prion Protein/Doppel Double-Knockout Mice

The prion protein (PrP) and Doppel (Dpl) have many structural and biochemical properties in common, leading to the suggestion that the lack of an obvious phenotype in PrP-deficient mice maybe because of compensation by Dpl. To test this hypothesis and also investigate the function of Dpl we have generated Prnd(-/-) and Prnp(-/-)/Prnd(-/-) mouse lines. Both develop normally and display an identical male sterility phenotype that differs from that reported for another Prnd(-/-) mouse line. Sperm from both our mutant lines were present at normal concentrations, had normal motility, and no morphological abnormalities. Despite only rarely fertilizing oocytes in vivo, because of an inability to perform the acrosome reaction, mutant sperm were capable of fertilization in vitro, albeit at reduced rates compared to wild type. Elevated levels of oxidative DNA damage were found in both types of mutant sperm and resulting embryos failed at an early stage. Therefore we found no evidence that Dpl compensates for the loss of PrP function in mutant mouse lines, but it does have an important anti-oxidant function necessary for sperm integrity and male fertility.

The value of adenosine triphosphate (ATP) measurements in assessing the fertilizing ability of human spermatozoa

A study was undertaken to examine the relationship between adenosine triphosphate (ATP) levels and the fertilizing capacity of human spermatozoa in vitro and in vivo. The concentration of ATP in semen was found to be positively correlated with the ability of sperm to fuse with zona-free hamster oocytes. However, it was also demonstrated that a large part of this relationship depends upon the relationship between semen ATP concentrations and sperm number. Measurements of ATP levels in cryostored ejaculates used in an artificial insemination by donor program revealed that such measurements were not able to distinguish fertile from infertile ejaculates. However, among fertile donors, ATP levels did seem to contribute useful information on relative fertility. It is concluded that ATP measurement has a limited role in the laboratory evaluation of sperm function.

Field trial of a diluent for the transportation of human semen at ambient temperatures

OBJECTIVE To examine the ability of a citrate-yolk buffer extender to preserve human semen samples at ambient temperatures over a 25- to 30-hour period. DESIGN Human semen samples were diluted 1:1 with citrate-yolk buffer or homologous seminal plasma and transported at ambient temperature between two distant locations (London to Edinburgh, United Kingdom). Various criteria of semen quality then were assessed before and after 25 to 30 hours storage and transportation in these diluents. SETTING An institutional research laboratory and a private fertility clinic. PATIENT(S) Samples were provided by 21 donors of unknown fertility and 7 asthenozoospermic patients. INTERVENTION(S) The diluent used to preserve human semen comprised an egg yolk buffer supplemented with fructose and citrate. MAIN OUTCOME MEASURE(S) Aspects of semen quality assessed included movement, hyaluronate penetration, viability, acrosome reaction, and reactive oxygen species generation. RESULT(S) The deterioration of semen quality at ambient temperatures could be prevented by the presence of citrate-yolk buffer, permitting the accurate analysis of oxidative stress and human sperm function, 25 to 30 hours postejaculation. CONCLUSION(S) Citrate-yolk buffer offers considerable promise as a medium for the ambient temperature storage and transportation of human semen.