Lack of impact of semen quality on fertilization in assisted conception.

BACKGROUND: Defective semen quality is one of the commonest causes of infertility. The diagnosis of male fertility depends upon a descriptive evaluation of human semen, however a normal semen analysis does not necessarily indicate satisfactory fertility potential. AIMS: (i) to examine the semen quality of patients undergoing treatment by assisted conception, (ii) to explore relationships between semen quality and treatment outcomes, and (iii) to look at inter-laboratory variation in the assessment of semen quality. METHODS: Semen quality in patients undergoing assisted conception treatment between 2001 and 2004 was reviewed. Data on female age, egg numbers and fertilization outcomes was obtained by case note review. RESULTS: The thresholds used to direct patients towards IVF or ICSI treatment were comparable with the normal values promulgated by WHO, with the exception of morphology. Semen quality was not predictive of fertilization rates. When the results of independent measurements of the same sample were compared, there was diagnostic disagreement in between 10%-29% of samples. CONCLUSIONS: The conventional criteria of semen quality are used to determine treatment strategy for couples undergoing assisted conception but are not reflected in fertilization rates, emphasising the limited utility of the conventional criteria of semen quality in the assessment of sperm function. There remains significant inter-laboratory variation in the results of semen analysis.

Distress and concerns in couples referred to a specialist infertility clinic.

OBJECTIVES: The aims of this study were to examine emotional distress and infertility-related concerns in male and female members of couples referred to a specialist infertility clinic and to determine changes in these over time. METHODS: A prospective cohort study with a 6-month follow-up. Emotional distress was measured using the Hospital Anxiety and Depression Scale, and concerns by a specially designed questionnaire. RESULTS: The response rate achieved was 38%. At baseline, 25.7% of women and 8.9% of men had scores of greater than 10 on the Hospital Anxiety and Depression Scale (HADS) Anxiety subscale, and 2.7% of women and 1.8% of men had scores of greater than 10 on the HADS Depression subscale. At 6-month follow-up the HADS scores were substantially unchanged. Females reported a significantly greater infertility-related concerns regarding life satisfaction, sexuality, self-blame, self-esteem and avoidance of friends compared with males. CONCLUSIONS: The prevalence of emotional disorder identified was low. There were gender differences in the nature of the specific concerns reported. The degree of distress and concerns did not change significantly over time. There are a minority of patients, mainly females, with clinically significant distress and infertility-related concerns amongst patients attending infertility clinics who deserve psychological attention.

Investigation of suppression of the hypothalamic-pituitary-gonadal axis to restore spermatogenesis in azoospermic men treated for childhood cancer.

BACKGROUND: Does suppression of the hypothalamic-pituitary-gonadal (HPG) axis restore spermatogenesis in men rendered azoospermic following treatment of childhood cancer? METHODS: Seven men with azoospermia secondary to treatment for childhood cancer, median age (range), 22.2 (18-25.3) years, aged 10.4 (4.4-13.3) years at original diagnosis, participated. Each subject underwent semen analysis and testicular biopsy, followed by treatment with medroxyprogesterone acetate (MPA), 300 mg i.m. repeated after 12 weeks, with 800 mg testosterone pellets s.c. on day 1 to suppress the HPG axis. Hormone and semen analysis was performed every 6 weeks for 48 weeks. A second testicular biopsy was performed at week 48. RESULTS: Before HPG axis suppression, mean +/- SEM plasma LH was 9.0 +/- 1.8 U/l, testosterone 17.9 +/- 1.5 nmol/l and FSH 22.4 +/- 4.4 U/l. Median (range) venous plasma and seminal plasma inhibin B levels were 10.0 (7.8-35) and 11.2 (7.8-770) ng/l respectively. During HPG suppression, FSH and LH levels were undetectable for > or =12 weeks followed by a gradual return to pretreatment concentrations by 48 weeks. All men remained azoospermic at study completion and complete absence of germ cells on biopsies was demonstrated by immunocytochemistry for all specimens pre- and post-HPG axis suppression. CONCLUSIONS: HPG axis suppression with MPA-testosterone for > or =12 weeks did not restore spermatogenesis in azoospermic men treated with gonadotoxic radiotherapy and chemotherapy for childhood cancer.

Sperm DNA damage in potentially fertile homozygous beta-thalassaemia patients with iron overload.

BACKGROUND: To test the hypothesis that human sperm DNA could sustain iron-induced oxidative damage and reduce its fertilizing ability, we studied patients with homozygous beta-thalassaemia major (HbTh) as a model of iron overload. METHODS: Sperm from six thalassaemic patients and five age-matched controls were assessed by the sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Semen parameters, endocrine markers of testicular function, iron profiles and the presence of organ dysfunction were also determined. RESULTS: All patients with HbTh were iron overloaded (median ferritin: 2251 microg/l) and had evidence of spontaneous spermatogenesis. Thalassaemic patients had more sperm DNA damage than the controls (P < 0.01). The sperm DNA damage by SCSA and TUNEL were positively correlated (P < 0.05). Sperm motility and TUNEL results were negatively correlated (P < 0.05), while the age of onset of chelation and sperm DNA damage were positively associated with both SCSA (R(2) = 0.80, P = 0.016) and TUNEL data (R(2) = 0.67, P < 0.044). No other biochemical or clinical data were associated with sperm DNA damage. CONCLUSIONS: The increase in sperm DNA damage and the negative correlation between sperm motility and DNA damage suggest that iron overload in HbTh predisposes sperm to oxidative injury. This finding has important implications in assisted reproductive procedures such as ICSI where there is increased risk of transmitting defective DNA to the offspring.

Sperm morphological defects related to environment, lifestyle and medical history of 1001 male partners of pregnant women from four European cities.

BACKGROUND: Recently, differences in semen quality have been found among the partners of pregnant women from four European cities: Turku, Copenhagen, Edinburgh and Paris. METHODS: During this study, slides from the four centres were subjected to a centralized assessment of sperm morphology. The percentages of sperm defects were recorded using a multiple-entry classification enabling the calculation of the multiple anomalies index (MAI), which is the mean number of anomalies per abnormal sperm. The relationships between various sperm abnormalities and self-reported data on medical history, lifestyle and occupational factors were examined. RESULTS: Significant differences in the MAI and most of the sperm defects were found between the four cities, and more abnormalities were found in spring than in winter. An increase in some sperm abnormalities was related to medical treatment of the mother during pregnancy, higher birthweight and previous treatment for cryptorchidism. Significant variations of several sperm defects were related to stress, weekly working time, occupational posture and metal welding, suggesting directions for further exposure studies. CONCLUSION: The present study indicated that the detailed assessment of sperm abnormalities is a useful biomarker of the effect of various external factors which may qualitatively affect human spermatogenesis.

Effect of a phytoestrogen food supplement on reproductive health in normal males.

Animal studies and human intervention trials have demonstrated the cancer chemopreventive properties of plant phytoestrogens, and phytoestrogen supplements are now widely available 'over-the-counter'. However, consumption of phytoestrogen-rich diets can cause impaired fertility and reproductive tract disorders in some animals and the apparent decline in human sperm quality over recent decades may be related to increased exposure to environmental endocrine disruptors. The present study determines the effects of a short-term phytoestrogen supplement on semen quality and serum sex steroid and gonadotrophin levels in human males. Healthy volunteers took a supplement containing 40 mg of isoflavones daily for 2 months and donated blood and semen samples monthly for 2 months before and 4 months after supplementation. Semen samples were analysed for ejaculate volume, sperm concentration, total sperm count, motility and morphology. Blood samples were analysed for sex hormone and gonadotrophin levels and phytoestrogen concentrations, and testicular volume was measured using an orchidometer. The phytoestrogen supplement increased plasma genistein and daidzein concentrations to approx. 1 microM and 0.5 microM respectively; yet, there was no observable effect on endocrine measurements, testicular volume or semen parameters over the study period. This is the first study to examine the effects of a phytoestrogen supplement on reproductive health in males. We conclude that the phytoestrogen dose consumed had no effect on semen quality.

Regional differences in semen quality in Europe.

Recent reports have indicated a decrease in semen quality of men in some countries, and suggested regional differences. A study was undertaken of semen samples from 1082 fertile men from four European cities (Copenhagen, Denmark; Paris, France; Edinburgh, Scotland; and Turku, Finland). Semen analysis was standardized, inter-laboratory differences in assessment of sperm concentration were evaluated, and morphology assessment centralized. Lowest sperm concentrations and total counts were detected for Danish men, followed by French and Scottish men. Finnish men had the highest sperm counts. Men from Edinburgh had the highest proportion of motile spermatozoa, followed by men from Turku, Copenhagen and Paris. Only the differences between Paris/Edinburgh and Paris/Turku were statistically significant (P < 0.003 and P < 0.002 respectively). No significant differences in morphology were detected. A general seasonal variation in sperm concentration (summer 70% of winter) and total sperm count (summer 72% of winter) was detected. Semen quality of a 'standardized' man (30 years old, fertile, ejaculation abstinence of 96 h) were estimated. Typically, sperm concentrations (x 10(6)/ml) for winter/summer were: Turku 132/93; Edinburgh 119/84; Paris 103/73; and Copenhagen 98/69. These differences in semen quality may indicate different environmental exposures or lifestyle changes in the four populations. However, it remains to be seen whether such changes can account for these differences. These data may also serve as a reference point for future studies on time trends in semen quality in Europe.

Differential expression of oestrogen receptor alpha and beta proteins in the testes and male reproductive system of human and non-human primates.

The role(s) oestrogens play in male adult reproductive function remains uncertain. We have used antibodies specific for oestrogen receptor- alpha (ERalpha) and - beta (ERbeta) to investigate their distribution within the male. In testes from adult human, macaque and marmoset, ERbeta protein was detected in Sertoli cells, Leydig cells and peritubular myoid cells. In germ cells, the intensity of immunostaining for ERbeta was variable between species. Immunoexpression in preleptotene, leptotene and zygotene spermatocytes was low/absent in all species. Elongated spermatids were consistently immunonegative. No ERalpha immunoexpression was detected in testes. ERbeta was detected in epithelial and stromal cell nuclei throughout the male reproductive system [efferent ductules (ED), epididymis, vas deferens, seminal vesicles] and in the bladder. ERalpha was detected in non-ciliated epithelial cells in the ED, but rarely in epithelial and basal cells within the epididymis. Epithelial cells from seminal vesicles and bladder were immunonegative for ERalpha. Expression of ERalpha in stromal cells was rare in the ED, epididymis and bladder but more frequent in seminal vesicles. Expression of ERalpha, and long and short forms of ERbeta, was confirmed by Western blotting. The widespread expression of ERbeta suggests that it is the primary target for modulation of tissue function via oestrogenic ligands in the male reproductive system.

Male reproductive health: cause for concern?

A substantial body of evidence has accumulated in recent years that human semen quality may be deteriorating. This has been associated with evidence of other changes in male reproductive health, including increases in congenital malformations and testicular cancer in humans, and similar problems in wildlife. Unfortunately, the evidence remains inconclusive. It has been suggested that these changes may be due to environmental xeno-oestrogens acting during development. Although there is now a large quantity of data indicating that this is a plausible hypothesis, evidence of causality, rather than association, remains to be provided. The potential importance of these changes for human health is considerable and urgent research is required to clarify the situation.

Impact of a deep saturation dive on semen quality.

The demonstration dive 'Aurora' has provided an opportunity to study the impact of extreme hyperbaric conditions on male fertility. This operation involved a 33-day diving programme during which divers were exposed to a maximum pressure of 4.6 Mega Pascals (Mpa) for 7 days. At days - 4, + 27, + 34, + 82 and + 263 relative to the initiation of the dive, semen samples were analysed to determine the quality of spermatogenesis and the functional competence of the spermatozoa. A dramatic fall in semen quality was observed in association with the dive and by day + 82 the potential fertility of the men was seriously compromised as evidenced by oligoasthenoteratozoospermic semen profiles and the poor fertilizing potential of the spermatozoa. These studies indicate, for the first time, that the severe hyperbaric conditions associated with deep saturation dives have a profound effect on male reproductive function.

DNA integrity in human spermatozoa: relationships with semen quality.

The literature contains conflicting evidence regarding the existence of DNA damage in spermatozoa from infertile male patients. To examine this phenomenon, we have studied ejaculated spermatozoa from normozoospermic semen donors and from a group of the unselected male partners of couples attending an infertility clinic for initial investigation. Classical semen analysis according to World Health Organization (WHO) guidelines was undertaken with computer-assisted sperm analysis (CASA). Spermatozoa were prepared by sequential washing and centrifugation and were analyzed for DNA fragmentation using three assays: 1) a single-cell gel electrophoresis (comet) assay, 2) in situ nick translation with prior chemical decondensation (ISNT-decondensed), and 3) in situ nick translation without prior chemical decondensation (ISNT-condensed). In addition, reactive oxygen species (ROS) generation by spermatozoa was measured, and seminal plasma was analyzed for its total reactive antioxidant potential (TRAP). When the donor and patient groups were compared, the latter had lower levels of semen quality and higher levels of DNA damage, which was particularly apparent using the comet assay. Highly significant negative correlations were observed between DNA fragmentation, detected by all three assays, and semen quality, particularly sperm concentration. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage. We conclude that a significant proportion of infertile men have elevated levels of DNA damage in their ejaculated spermatozoa.

Male infertility and intracytoplasmic sperm injection (ICSI).

Micro-assisted fertilization in the form of intracytoplasmic sperm injection (ICSI) has truly revolutionised the treatment options for couples with impaired semen quality, and those with both obstructive and non-obstructive azoospermia. In general, the major issues which relate to the success of ICSI are those related to the success of conventional IVF, and the high multiple pregnancy rate remains a major cause for concern. There is growing evidence that the short-term health of ICSI offspring is relatively unremarkable, but our growing understanding of the genetic basis of much of male subfertility and of the impaired genomic integrity which characterises the oligozoospermic phenotype indicate a cautious approach to the longer term health of ICSI offspring.

Successful in-vitro fertilization pregnancy with spermatozoa from a patient with Kartagener's syndrome: case report.

This paper reports on the successful treatment by in-vitro fertilization (IVF) of a couple in whom the male partner had Kartagener's syndrome. His spermatozoa were severely asthenozoospermic with deficient dynein arms and disordered microtubular configuration. On computer-assisted sperm analysis (CASA) motile spermatozoa displayed straight non-progressive motility with minimal amplitude of lateral head displacement and none were hyperactivated. This is the first case report in which spermatozoa with axonemal disruption in a man with immotile cilia syndrome (ICS) have been shown to be able to penetrate the zona pellucida and fertilize oocytes. IVF may be a suitable treatment for certain variants of ICS.

Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa.

Reactive oxygen metabolites are known to disrupt sperm-oocyte fusion, sperm movement, and DNA integrity; however, the relative sensitivities of these elements to oxidative stress are unknown. In this study these factors were assessed in human spermatozoa exposed to increasing levels of oxidative stress achieved through the stimulation of endogenous oxidant generation with NADPH or direct exposure to hydrogen peroxide. At low levels of oxidative stress, DNA fragmentation was significantly reduced while the rates of sperm-oocyte fusion were significantly enhanced. As the level of oxidative stress increased, the spermatozoa exhibited significantly elevated levels of DNA damage (p < 0.001) and yet continued to express an enhanced capacity for sperm-oocyte fusion. At the highest levels of oxidative stress, extremely high rates of DNA fragmentation were observed but the spermatozoa exhibited a parallel loss in their capacities for movement and oocyte fusion. These studies emphasize how redox mechanisms can either enhance or disrupt the functional and genomic integrity of human spermatozoa depending on the intensity of the oxidative stimulus. Because these qualities are affected at different rates, spermatozoa exhibiting significant DNA damage are still capable of fertilizing the oocyte. These results may have long-term implications for the safety of assisted conception procedures in cases associated with oxidative stress.

Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection.

We present the first evidence that genetically damaged human spermatozoa are able to form normal pronuclei in oocytes after intracytoplasmic sperm injection (ICSI). The role of reactive oxygen species (ROS) as a cause of chromatin and DNA damage is well recognized. The same class of molecule can be found in the semen of males with severe infertility, who remained infertile until the advent of ICSI. In this study we have investigated the role of ROS in the induction of chromatin damage, DNA strand breakage and the subsequent ability of spermatozoa to decondense and form pronuclei after ICSI. Spermatozoa from normozoospermic men participating in our research programme were exposed to oxidizing environments created by co-incubation with hydrogen peroxide, reduced nicotinamide adenine dinucleotide phosphate (NADPH) or activated white cells. The subsequent ability of the spermatozoa to decondense in vitro was examined using sequential incubations in EDTA, dithiothreitol and sodium dodecyl sulphate, and the amounts of DNA strand breakage were assessed using an in-situ nick translation protocol. Finally, cells exposed to hydrogen peroxide, NADPH and activated leukocytes were microinjected into hamster oocytes, and their ability to decondense and form normal pronuclei was determined. The results indicate that human sperm chromatin becomes cross-linked under conditions of oxidative stress and exhibits increased DNA strand breakage, yet the rate of pronucleus formation is no different from that of untreated control cells. The ability of genetically damaged spermatozoa to achieve normal fertilization following ICSI has implications for the practice of this form of assisted conception therapy.

Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants.

Exposure of human spermatozoa to nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the dose dependent generation of reactive oxygen species (ROS) which, at a critical level of intensity, induced lipid peroxidation, DNA damage and a dramatic decline of sperm motility. This system was then used as a model for screening the ability of different antioxidants to combat oxidative stress created through the excessive intracellular generation of toxic oxygen products of metabolism. A variety of antioxidants that has previously been shown to be protective against extracellularly derived oxidants (e.g. superoxide dismutase, catalase, vitamin E, hypotaurine) were ineffective in this system. Albumin, however, could provide complete protection against NADPH induced oxidative stress via mechanisms that did not involve the suppression of the lipid peroxidation cascade but rather the inactivation of lipid peroxides generated during this process. Albumin did not protect against DNA damage induced by NADPH but was extremely effective at preventing DNA fragmentation arising from the suppression of glutathione peroxidase activity with mercaptosuccinate. These studies emphasize that the design of clinically effective antioxidant treatments will depend, critically, upon the source of the oxidative stress. For cases involving excessive intracellular ROS generation, albumin appears to be an important means of neutralizing lipid peroxide-mediated damage to the sperm plasma membrane and DNA.

Epidemiology and aetiology of male infertility.

Infertility is a common problem, affecting perhaps one couple in six, the majority of whom now seek medical care. Although diagnostic problems make it difficult to establish the extent of the male partner's contribution with certainty, a number of studies suggest that male problems represent the commonest single defined cause of infertility. The World Health Organization has proposed a scheme for the diagnostic classification of male infertility, based upon a standardized approach to clinical assessment and to the assessment of semen quality. Some of these classifications are now controversial, and many are descriptive, rather than aetiological. Increasingly, the importance of occupation, environmental and particularly genetic factors in the causation of male infertility is being recognized.

Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma.

Before the advent of intracytoplasmic sperm injection (ICSI) semen preparation techniques focused on the need to sustain the fertilizing potential of the spermatozoa particularly by reducing oxidative stress. However, for severely oligozoospermic patients treated by ICSI, sperm preparation protocols are used which aim to maximize sperm recovery rather than sperm function. In this study we have examined the impact of different sperm preparation techniques on oxidative stress, sperm motion and DNA integrity. Reactive oxygen species (ROS) generation was monitored using luminol-dependent chemiluminescence, seminal antioxidant activity was assessed using a total reactive antioxidant potential (TRAP) assay while sperm motility and DNA damage were evaluated using computer assisted semen analysis and in-situ nick translation respectively. The results demonstrate a significant increase in the levels of ROS generated by samples prepared by swim-up from a washed pellet compared with spermatozoa isolated directly from seminal plasma. This oxidative stress was associated with a highly significant increase in the level of DNA damage sustained by the spermatozoa while the quality of sperm motility remained largely unchanged. These results suggest that if repeated centifugation protocols are to be used to prepare spermatozoa, strategies should be developed for minimizing collateral DNA damage.

Evaluation of a spectrophotometric assay for the measurement of malondialdehyde and 4-hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function.

A spectrophotometric assay for the measurement of malondialdehyde and 4 hydroxyalkenals (MA + 4HA) has been evaluated for the detection of sperm pathologies involving oxidative stress. In order to make sensitive measurements of MA + 4HA on human spermatozoa, the stimulation of a lipid peroxidation cascade with a ferrous ion promoter was found to be necessary. The optimal configuration for the promoter was defined (0.64 mM FeSO4 + 20 mM ascorbate for 2 h in Ca2+ and Mg2 free Hanks' balanced salt solution) and the assay used in a series of studies to elucidate the functional significance of MA + 4HA determinations. Such measurements were found to give highly significant correlations (p < 0.001) with the loss of motility induced by oxidative stress created either with a xanthine oxidase, free radical generating system or by prolonged incubation under aerobic conditions. Experiments involving the stimulation and suppression of lipid peroxide release from human sperm suspensions, in concert with a bioassay for cytotoxicity, confirmed the strength and causative nature of these associations. Measurements of lipid peroxidation potential in highly purified, leucocyte-free sperm suspensions revealed the presence of inverse correlations with the motility of the spermatozoa, their viability, their competence for sperm-oocyte fusion and, most significantly, the quality of sperm movement in the original semen samples. Similar negative correlations were observed between sperm function and phorbol ester-stimulated reactive oxygen species generation but, unlike the MA + 4HA determinations, these relationships were obfuscated by the presence of leucocytes. We conclude that the measurement of MA + 4HA in human spermatozoa provides important information on the underlying quality of spermatogenesis and should be of value in the clinical diagnosis of infertility involving oxidative stress and the selection of patients for antioxidant therapy.

Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis.

In men, inhibin B is the circulating isoform involved in the regulation of follicle stimulating hormone (FSH) secretion. Within the testis, inhibin B may have a role in Sertoli and germ cell interactions, thus secretion into seminal plasma may reflect seminiferous tubule function. Using specific immunoassays, inhibin B was present in seminal plasma in fertile men (n = 105) and in unselected men attending an infertility clinic (n = 174) with a wide range in concentration from undetectable (<15 pg/ml) up to 54,100 pg/ml (geometric mean 280 pg/ml). There was a highly significant correlation between seminal plasma inhibin B concentration and sperm concentration (r = 0.46, P < 0.001), but no correlation with percentages of spermatozoa with progressive motility or normal morphology. Inhibin A and isoforms containing pro and alphaC immunoreactivity were not detectable. In post-vasectomy seminal plasma samples (18 of 20) inhibin B was undetectable, indicating that the testis is the predominant source. In unselected men attending an infertility clinic, inhibin B was undetectable in 17% (present in remainder; maximum concentration 26,200 pg/ml; mean 263 pg/ml), with a highly significant correlation between seminal plasma inhibin B and sperm concentration (r = 0.55, P < 0.0001). In men with oligo/ azoospermia (sperm concentration <20 x 10(6)/ml), seminal plasma inhibin B concentrations were lower in those with elevated plasma FSH concentrations (mean values 42 and 205 pg/ml, P < 0.05). Inhibin alpha and betaB subunits were localized predominantly in Sertoli and Leydig cells, using immunohistochemistry. We conclude that inhibin B of testicular origin is present in normal human seminal plasma, but with a very wide range in concentration, and may reflect the functional state of the seminiferous epithelium.