Role of sperm chromatin abnormalities and DNA damage in male infertility

Sperm DNA integrity is essential for the accurate transmission of genetic information. It has a highly compact and complex structure and is capable of decondensation-features that must be present in order for a spermatozoon to be considered fertile. Any form of sperm chromatin abnormalities or DNA damage may result in male infertility. In support of this conclusion, it was reported that in-vivo fecundity decreases progressively when > 30% of the spermatozoa are identified as having DNA damage. Several methods are used to assess sperm chromatin/DNA, which is considered an independent measure of sperm quality that may yield better diagnostic and prognostic approaches than standard sperm parameters (concentration, motility and morphology). The clinical significance of this assessment lies in its association not only with natural conception rates, but also with assisted reproduction success rates. Also, it has a serious impact on the offspring and is highly prognostic in the assessment of fertility in cancer patients. Therefore, screening for sperm DNA damage may provide useful information in cases of male idiopathic infertility and in those men pursuing assisted reproduction. Treatment should include methods for prevention of sperm DNA damage.     

The pathophysiology of varicoceles in the light of current molecular and genetic information

Varicoceles are a common cause of male infertility, but despite data being obtained from animal models and human studies the pathophysiology remains unclear. Recently, molecular and genetic information has been reported on men with varicoceles which may shed new light onto the causes of decreased semen parameters and poor sperm function. Here, a number of studies are reviewed in an attempt to develop a working hypothesis for the relationship of varicoceles and infertility. New studies on testicular tissue of men with varicoceles have demonstrated increased apoptosis among developing germ cells, which may be the cause of oligospermia. Other studies with semen have shown increased levels of reactive oxygen species (ROS) in association with poor sperm motility. Recent studies of morphologically abnormal spermatozoa have demonstrated disruption of the sperm head actin by cadmium, a cation reported to be present in high concentrations among some men with varicoceles. Finally, microdeletions of the alpha-1 subunit of the sperm calcium channels in a proportion of men with varicoceles suggests a genetic defect leading to abnormal acrosomal function. The intent of this review was to explain the pathophysiology of varicoceles, and the findings seem to support a 'co-factor' hypothesis. In order for varicoceles to be associated with infertility, they exist as 'co-factors' along with other molecular/genetic problems.     

Globozoospermia revisited

Globozoospermia is a rare (incidence <0.1%) but severe disorder in male infertility. Total globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa lacking an acrosome. It is still unclear whether patients whose ejaculate contains both normal and globozoospermic cells (partial globozoospermia) suffer from a variation of the same syndrome. Apart from the fact that affected males suffer from reduced fertility or even infertility, no other physical characteristics can be associated with the syndrome. ICSI is a treatment option for these patients, although low fertilization rates after ICSI show a reduced ability to activate the oocyte. In globozoospermic cells, the use of acrosome markers has demonstrated an absent or severely malformed acrosome. Chromatin compaction appears to be disturbed but is not consistently over- or undercondensed. In some cases, an increased number of cells with DNA fragmentation have been observed. The analysis of the cytogenetic composition revealed an increased aneuploidy rate in some cases. Nonetheless, no increased number of spontaneous abortions or congenital defects has been reported in pregnancies conceived after ICSI. The pathogenesis of globozoospermia most probably originates in spermiogenesis, more specifically in acrosome formation and sperm head elongation. In several knockout mouse models, a phenotype similar to that in humans was found. Together with the occurrence of affected siblings, these findings indicate a genetic origin, which makes globozoospermia a good candidate for genetic analysis. More research is needed to elucidate the pathogenesis of human globozoospermia to further understand globozoospermia as well as (abnormalities in) spermiogenesis and spermatogenesis in general.     

TEST-yolk media and sperm quality

Media prepared with egg yolk and two buffers, TES and Tris,called TEST-yolk have been shown to have beneficial effectson the survivability, fertilizing capacity and storage potentialof human spermatozoa. Egg yolk lipoproteins are the criticalcompounds for the beneficial effects, with a synergistic effectdue to the TES-Tris buffer component TEST-yolk media have beenused in the sperm penetration assay, the hemizona assay, spermpreparation for clinical in-vitro fertilization, artificialinsemination with homologous spermatozoa, cryopreservation,sperm samples with a positive antisperm antibody test, and preparationfor techniques requiring capacitated spermatozoa. Few harmfulconsequences of TEST-yolk have been reported, although controlledtrials are required to evaluate therapeutic effects in the treatmentof male infertility.     

Intracytoplasmic sperm injection (ICSI) in 2006: evidence and evolution

The introduction of intracytoplasmic sperm injection (ICSI)in 1992 has dramatically changed the management of severe maleinfertility. In severe male infertility, live birth rates withICSI are superior to those with other non-donor treatments.In non-male infertility, however, pregnancy rates are not betterwith ICSI than with in vitro fertilization (IVF). With obstructiveor non-obstructive azoospermia, reasonable pregnancy rates arenow possible with ICSI after recovery of sperm from the testesfollowed by ICSI. Genetic counselling is indicated for severemale infertility, whether or not ICSI is considered. ICSI isindicated in preimplantation genetic diagnosis (PGD) to avoidcontamination by extraneous DNA in the case of PCR-based testingand to increase the number of embryos available for testing.In turn, PGD may be indicated in pregnancies that are at highrisk of aneuploidy because of genetic factors associated withazoospermia. As with IVF, not all couples succeed, but 2% ofcouples with failed ICSI cycles will conceive without treatment.ICSI outcome studies indicate that there is a significant increasein prematurity, low birthweight, and perinatal mortality associatedwith single and multiple births, similar to the outcomes ofconventional IVF. However, as evidenced in long-term follow-upstudies, the higher rates of urogenital abnormalities and increaseduse of healthcare may be associated with paternal characteristics.     

Knockout mouse models of sperm flagellum anomalies

To date, 21 knockout mouse models are known to bear specific anomalies of the sperm flagellum structures leading to motility disorders. In addition, genes responsible for flagellar defects of two well-known spontaneous mutant mice have recently been identified. These models reveal genetic factors, which are required for the proper assembly of the axoneme, the annulus, the mitochondrial sheath and the fibrous sheath. Many of these genetic factors follow unexpected cellular pathways to act on sperm flagellum morphogenesis. These mouse models may bear anomalies which are restricted to the spermatozoa or display more complex phenotypes that often include neuropathies and/or cilia-related diseases. In human, several structural disorders of the sperm flagellum found in brothers or consanguineous men probably have a genetic origin, but the genes involved have not yet been identified. The mutant mice we present in this review are invaluable models, which can be used to identify potential candidate genes for infertile men with specific sperm flagellum anomalies.     

Function of aquaporins in female and male reproductive systems

The flow of water and some other small molecules across cell membranes is important in many of the processes underlying reproduction. The fluid movement is strongly associated with the presence of aquaporins (AQPs) in the female and male reproductive systems. It has been suggested that AQPs mediate water movement into the antral follicle and play important roles in follicle development. AQPs are known to be involved in the early stage of spermatogenesis, in the secretion of tubule liquid and in the concentration and storage of spermatozoa. Fluid reabsorption in some regions of the male reproductive tract is under steroid hormone control and could be mediated by various AQPs. Also AQPs take part in the processes of fertilization, blastocyst formation (as the pathway for transtrophoectodermal water movement during cavitation) and implantation. Alterations in the expression and function or regulation of AQPs have already been demonstrated in disorders of the male reproductive system, such as abnormal sperm motility, the abnormal epididymis and infertility seen in cystic fibrosis, and varicocele. This article extensively reviews the distribution of AQPs in mammalian reproductive tissues and discusses their possible physiological and pathophysiological roles.     

Sperm function assays and their predictive value for fertilization outcome in IVF therapy: a meta-analysis

The prevalence of male infertility and the availability of new, highly successful therapeutic options make the testing of sperm functional competence mandatory. An objective, outcome-based examination of the validity of the currently available assays was performed based upon the results obtained from 2906 subjects evaluated in 34 prospectively designed, controlled studies. The aim was carried out through a meta-analytical approach that examined the predictive value of four categories of sperm functional assays: computer-aided sperm motion analysis (CASA); induced-acrosome reaction testing; sperm penetration assay (SPA); and sperm-zona pellucida binding assays for IVF outcome. Results demonstrated a high predictive power of the sperm-zona pellucida binding and the induced-acrosome reaction assays for fertilization outcome. On the other hand, the findings indicated a poor clinical value of the SPA as predictor of fertilization and a real need for standardization and further investigation of the potential clinical utility of CASA systems. This analysis points out to limitations of the current tests and the need for standardization of methodologies and provides objective evidence on which clinical management and future research can be based.     

Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection

In this study we investigated whether morphology and chromatinanomalies in human spermatozoa can influence fertilization afterintracytoplasmic sperm injection (ICSI). We examined unfertilizedoocytes, using the fluorochrome Hoechst 33342, to determinewhether a relationship exists between failure of fertilizationand sperm chromatin quality. Sperm chromatin packaging qualitywas assessed using the chromomydn A3 (CMA3) fluorochrome, andthe presence of DNA damage in spermatozoa, using in-situ nicktranslation. Normal males present sperm parameters with a normalmorphology of >20%, CMA3 fluorescence of <30% and exhibitendogenous nicks in <10% of their spermatozoa. When patientswere separated according to these values no difference was observedin their fertilization rates after ICSL When the unfertilizedICSI oocytes were examined, we found that patients with CMA3fluorescence of <30% and nicks in <10% of their spermatozoahad only 17.5 and 21.6% respectively of their unfertilized oocytescontaining spermatozoa that remained condensed. In contrast,patients with higher CMA3 and nick values had a significantlyhigher number, 412 and 48.9%, of their unfertilized oocytescontaining condensed spermatozoa. Sperm morphology did not showany such pattern. The percentage of spermatozoa which had initiateddecondensation in unfertilized oocytes was not influenced bymorphology, CMA3 fluorescence or nicks. In light of these resultswe postulate that poor chromatin packaging and/or damaged DNAmay contribute to failure of sperm decondensation after ICSIand result in failure of fertilization.     

Oxidative stress and male infertility--a clinical perspective

Oxidative stress occurs when the production of potentially destructivereactive oxygen species (ROS) exceeds the bodies own naturalantioxidant defenses, resulting in cellular damage. Oxidativestress is a common pathology seen in approximately half of allinfertile men. ROS, defined as including oxygen ions, free radicalsand peroxides are generated by sperm and seminal leukocyteswithin semen and produce infertility by two key mechanisms.First, they damage the sperm membrane, decreasing sperm motilityand its ability to fuse with the oocyte. Second, ROS can alterthe sperm DNA, resulting in the passage of defective paternalDNA on to the conceptus. This review will provide an overviewof oxidative biochemistry related to sperm health and will identifywhich men are most at risk of oxidative infertility. Finally,the review will outline methods available for diagnosing oxidativestress and the various treatments available.     

Sperm banking and assisted reproduction treatment for couples following cancer treatment of the male partner

In recent years, the survival of young males suffering from cancer has been improved. Development of new techniques such as IVF and intracytoplasmic sperm injection enables even low quality spermatozoa to be used successfully. It is possible therefore to preserve fertility potential of cancer patients before embarking on adjuvant chemotherapy and radiotherapy. Recognizing the importance of protecting the fertility potential of these young males, we present our recommendations for sperm cryopreservation based on the 11 year experience of Bourn Hall and the British Joint Council for Clinical Oncology consultation report. This paper discusses the options available for patients who recover from cancer to become fathers. In many cases patients are concerned about possible abnormalities and teratogenic risks to their future children who have been conceived naturally or by fertility treatment. The data available in the literature may reassure the medical community that there is no such increased risk. However, due to the relatively small number of children born after such treatment, a long-term follow-up is required. There is an ongoing debate regarding the justification for the programme due to the small number of patients who make use of their banked spermatozoa. The authors believe in the importance of protecting the fertility potential of cancer patients, enabling them to father their genetic children in the future while fighting their illness.     

Antisperm immunity in natural and assisted reproduction

Research conducted in the last 40 years has provided evidence that antisperm antibodies (ASA) can impair the fertilizing capacity of human spermatozoa. It is established that ASA can be present at different sites, can act against different antigens and can impair fertility in various ways. In fact, in the past it was amply demonstrated that ASA can act negatively on sperm motility and on cervical mucus penetration. In recent years, owing to the improvement and spreading of IVF techniques, it has been possible to demonstrate the effect of antibody-bound spermatozoa at the level of in-vitro gamete interaction. The literature demonstrates that the various previously used treatments for immunological infertility, i.e. medical therapy, intrauterine insemination with husband's spermatozoa (AIH) and IVF, usually had poor success. The primary choice of treatment in immunological infertility, especially in the most severe cases and when the sperm head is involved, is ICSI. ASA evaluation in all couples who undergo the various techniques of insemination or IVF is imperative.     

Protamines and male infertility

Protamines are the major nuclear sperm proteins. The human sperm nucleus contains two types of protamine: protamine 1 (P1) encoded by a single-copy gene and the family of protamine 2 (P2) proteins (P2, P3 and P4), all also encoded by a single gene that is transcribed and translated into a precursor protein. The protamines were discovered more than a century ago, but their function is not yet fully understood. In fact, different hypotheses have been proposed: condensation of the sperm nucleus into a compact hydrodynamic shape, protection of the genetic message delivered by the spermatozoa, involvement in the processes maintaining the integrity and repair of DNA during or after the nucleohistone-nucleoprotamine transition and involvement in the epigenetic imprinting of the spermatozoa. Protamines are also one of the most variable proteins found in nature, with data supporting a positive Darwinian selection. Changes in the expression of P1 and P2 protamines have been found to be associated with infertility in man. Mutations in the protamine genes have also been found in some infertile patients. Transgenic mice defective in the expression of protamines also present several structural defects in the sperm nucleus and have variable degrees of infertility. There is also evidence that altered levels of protamines may result in an increased susceptibility to injury in the spermatozoan DNA causing infertility or poor outcomes in assisted reproduction. The present work reviews the articles published to date on the relationship between protamines and infertility.     

The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility

Current treatments of subfertile couples are usually empiric, as the true cause of subfertility often remains unknown. Therefore, we outline the role of nutritional and biochemical factors in reproduction and subfertility. A literature search was performed using MEDLINE, Science Direct and bibliographies of published work with both positive and negative results. The studies showed that folate has a role in spermatogenesis. In female reproduction, folate is also important for oocyte quality and maturation, implantation, placentation, fetal growth and organ development. Zinc has also been implicated in testicular development, sperm maturation and testosterone synthesis. In females, zinc plays a role in sexual development, ovulation and the menstrual cycle. Both folate and zinc have antioxidant properties that counteract reactive oxygen species (ROS). Thiols, such as glutathione, balance the levels of ROS produced by spermatozoa and influence DNA compaction and the stability and motility of spermatozoa. Oocyte maturation, ovulation, luteolysis and follicle atresia are also affected by ROS. After fertilization, glutathione is important for sperm nucleus decondensation and pronucleus formation. Folate, zinc, ROS and thiols affect apoptosis, which is important for sperm release, regulation of follicle atresia, degeneration of the corpus luteum and endometrial shedding. Therefore, the concentrations of these nutrients may have substantial effects on reproduction. In conclusion, nutritional and biochemical factors affect biological processes in male and female reproduction. Further research should identify pathways that may lead to improvements in care and treatment of subfertility.     

Regulation of sperm function by reactive oxygen species

Sperm capacitation can be increased by the addition of reactive oxygen species (ROS) and decreased by antioxidants. Broadly consistent results have been achieved with a wide variety of methods and across different species. Exposure to ROS increases protein tyrosine phosphorylation consequent on an increase in cAMP and activation of tyrosine kinase and inhibition of tyrosine phosphatase. The measurement of ROS production by sperm is complicated by contamination of suspensions by leukocytes, laying many studies open to doubt. In human sperm the observation that extracellular NADPH could support superoxide production detected with the chemiluminescent probe lucigenin and had physiological effects similar to hydrogen peroxide led to the suggestion that they contained NADPH oxidase activity to generate ROS to support capacitation. However, the realization that lucigenin can signal superoxide artefactually, combined with failure to detect superoxide production using spin trapping techniques or to detect NADPH oxidase components in mature sperm, and confirmation of old reports that NADPH solution contains substantial amounts of hydrogen peroxide due to autoxidation, have undermined this hypothesis. Although the presence of significant NADPH oxidase activity in mature human sperm now seems less likely, other observations continue to suggest that they can make ROS in some way. There is stronger evidence that animal sperm can make ROS although these may be mainly of mitochondrial origin.     

Smoking and reproduction: gene damage to human gametes and embryos

Assisted conception is a useful methodology for detecting disturbances in clinical outcome, meiotic maturation, and genetic integrity of human gametes. Germinal cells are vulnerable to genetic damage from smoking, but can repair damage during meiosis. In ejaculated spermatozoa, repair capacity declines drastically. Smoking alters the meiotic spindle of oocytes and spermatozoa, leading to chromosome errors which affect reproductive outcomes. Smoking is associated with reduced numbers of retrieved oocytes, leading to early age of menopause. Oocyte elimination occurs preferentially during meiosis I, a period sensitive to genetic damage. Smoking inhibits embryo fragmentation; inhibition may confer survival advantage to embryos genetically altered. Smoking is associated with low sperm quality, but clinical effects are not recognized. Cadmium (a heavy metal), nicotine (a toxic alkaloid), and its metabolite cotinine, are detectable in gonadal tissues and fluids in association with smoking. Cotinine incorporates into ovarian granulosa-lutein cells, compromising the developmental potential of follicles. Benzo[a]pyrene is a carcinogenic polycyclic aromatic hydrocarbon resulting from cigarette combustion. Its reactive metabolite binds covalently to DNA, forming adducts. Smoking-related adducts were detectable in ovarian granulosa-lutein cells, oocytes, spermatozoa and preimplantation embryos. Transmission of altered DNA from smoking by spermatozoa was demonstrated in preimplantation embryos and in association with increased risk of childhood cancer.     

Andrology: Assessment of human sperm functional changes after in-vitro coincubation with cells retrieved from the human female reproductive tract

Human spermatozoa must undergo functional changes prior to fertilization;however, the site of this physiological event is still unclear.To evaluate the influence of the female reproductive tract onsperm fertilizing capacity, fertile sperm samples were coincubatedwith endometrial, oviductal, granulosa and cumulus cells, follicularfluid and maternal serum. Sperm penetration into the zona-freehamster ova and motion parameters were measured daily for 72h. Compared to control samples, endometrial and oviductal cellcultures did not alter sperm fertilizing capacity or their movementcharacteristics. Sperm coincubated with follicular fluid, granulosaor cumulus cells exhibited a significantly higher ability topenetrate zona-free hamster ova for up to 48 h. Sperm motilityincreased at 4 h in the presence of follicular fluid and serum.At 24 h sperm velocity and amplitude of lateral head displacementsignificantly declined in sperm samples exposed to serum, andvelocity also declined in follicular fluid and with coincubationusing ovarian follicle cells. Sperm motility and velocity decreasedat 48 h in the presence of serum, follicular fluid, cumulusor granulosa cells. Our findings may suggest that specific secretoryfactors produced in the human pre-ovulatory ovarian follicleenhance human sperm fertilizing capacity.     

Consequences on offspring of abnormal function in ageing gametes

The present review aims to analyse (i) the molecular, biochemical and cellular changes that accompany oocyte and sperm ageing in any of the internal or external environments where they can reside, and (ii) the consequences of the abnormal function in ageing gametes on pre- and post-implantation development and later life of offspring. This review also aims to propose and discuss cellular/molecular mechanisms framed within the 'free radical theory of ageing'. It appears that the ageing of gametes prior to fertilization may affect many molecular, biochemical and cellular pathways that may jeopardize not only pre- and post-implantation embryo/fetal development but also later life of offspring. Consequences of gamete ageing range from decreased vigour (with the concomitant decrease in intelligence, reproductive fitness and longevity) of apparently normal-looking offspring to severe congenital, epigenetic and/or genetic anomalies. All these effects may be easily prevented by efficient diffusion of both the potential risks of gamete ageing and the steps that should be taken by couples wishing to achieve pregnancy to guarantee a correct maturational synchronization of gametes at fertilization. Although in-vitro antioxidant therapy appears to protect from or retard the ageing process of gametes, it may not assure the total absence of negative effects on the resulting offspring.     

The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans

BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.     

Exploiting common targets in human fertilization and HIV infection: development of novel contraceptive microbicides

The continued high rates of unintended pregnancies and the unrelentless expansion of the acquired immune deficiency syndrome (AIDS) epidemic, especially in less developed countries, warrant the development of novel strategies to help individuals avoid these risks. Dually active compounds displaying contraceptive and microbicidal anti-human immunodeficiency virus (anti-HIV) properties constitute one such strategy. Sharing the same anatomical and functional context, sperm fertilization and genital infection by HIV offer an opportunity for simultaneous intervention. Some of the molecules and mechanisms used by sperm to fertilize the oocyte are similar, if not identical, to those used by HIV while infecting host cells. An example of common structures is the lipid membrane surrounding the spermatozoon and the HIV core. Disruption of its architecture by surface-active compounds exerts both spermicidal and virucidal activity. A more specific alteration of lipid rafts [membrane microdomains enriched in cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins] by beta-cyclodextrins also results in similar effects. During fertilization and infection, both sperm and HIV interact with their target cell receptors through chemical charges, hydrophobic forces and carbohydrate recognition. Anionic polymers such as cellulose sulphate and polystyrene sulphonate (PSS) inhibit sperm and HIV cell binding. Because some of the molecules involved in this interaction, e.g. heparin sulphate proteoglycan, are also used by other pathogens to infect their target tissues, polyanions exert broad antimicrobial activity as well. During fertilization and infection, sperm and HIV, as well as other microbes, use signal transduction molecules and mechanisms such as adenyl cyclase/cyclic adenosine monophosphate (cAMP)-dependent kinase, calcium and tyrosine phosphorylation, whose inhibition has been shown to impair sperm function and HIV replication. These commonalities at the level of sperm and HIV structure, cell binding and fusion processes, and signalling pathways therefore provide the biological framework to develop bifunctional inhibitors with both antimicrobial and contraceptive properties.