Spermatozoa Of Infertile Men Research Articles

Testicular‐sparing microsurgery for suspected testicular masses

To describe a microsurgical technique for removing suspected testicular masses with sparing of the testicular parenchyma, and to describe case studies. Six men were referred with testicular lesions (3-6 mm) detected on ultrasonography (US); in one, the lesion was palpable. US showed hypoechoic lesions and in two cases were mixed hypoechoic and anechoic. In these men, the testicular lesion was identified by US before surgery, giving three-dimensional coordinates to facilitate intraoperative recognition. A traditional inguinal incision was used and the funiculus clamped subinguinally without opening the canal. The testicle was isolated after sectioning the gubernaculum testis. In a separate operative field, an equatorial incision of the albuginea was made in a plane orthogonal to the major axis of the testicle, sparing the subtunical vasa. The parenchymal lobuli were dislodged and the seminiferous tubules dissociated, the nodule identified and completely removed, together with approximately 1 mm of surrounding healthy tissue. This technique can also be used for microsurgical testicular sperm extraction (MicroTESE), to retrieve sperm in infertile men. In two infertile men MicroTESE was also performed. Histology revealed one case each of seminoma, Leydig-cell tumour, Leydig cell hyperplasia, atrophy, normality in the incidental forms, and complicated cysts of the albuginea. In the follow-up for infertility reasons, no scarring was observable on the tunica albuginea in the men who had conservative therapy. One year later the patient with seminoma was free of disease. The increasingly frequent detection of benign testicular lesions, particularly in infertile men, calls for a surgical approach that must be as conservative as possible for the testicular parenchyma. We think that microsurgery should be the first-line technique in small suspected testicular lesions in infertile men.

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.

Incidence of Fas positivity and deoxyribonucleic acid double-stranded breaks in human ejaculated sperm

Objective To determine the incidence of Fas positivity and DNA double-strand breaks (DSB) as indicators of early- and late-stage apoptosis in ejaculated sperm. Design Fas positivity was assessed by flow cytometry and DSB by the neutral Comet assay. Setting Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. Patient(s) and intervention(s) Forty-five infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies. Main outcome measure(s) Percentage Fas-positive cells, percentage DNA fragmentation, olive tail moment. Result(s) The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozospermic semen. No Fas positivity was observed in fertile mens' sperm. Deoxyribonucleic acid fragmentation (DSB) was greater in infertile than in fertile men's sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage. Conclusion(s) Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis.

Preimplantation genetic diagnosis by fluorescence in situ hybridization: clinical possibilities and pitfalls.

Preimplantation genetic diagnosis using the fluorescence in situ hybridization technique (FISH) is being used widely to prevent the transmission of sex-linked diseases, to screen for translocations, and for aneuploidy screening in specific in vitro fertilization (IVF) patient groups, along with FISH analysis of spermatozoa in infertile men. In this study, we aim to critically analyze our clinical results in patients at risk of transmitting sex-linked diseases (n = 55), in carriers of translocations (n = 43), in women who have recurrent miscarriage (two or more miscarriages) (n = 128), recurrent IVF failure (three or more failed IVF attempts) (n = 47), and patients of advanced maternal age (37 years old or older) (n = 79). The use of the FISH technique in carriers of sex-linked diseases and translocation patients prevents transmission of these conditions and provides good IVF outcome. In patients with recurrent miscarriage, implantation failure, and advanced maternal age, a high incidence of embryos with abnormal chromosomes 13,16,18,21,22, X, and Y was observed (range 69-75%), as expected. In those three groups of patients, the selection of euploid embryos for transfer resulted in good pregnancy rates with a low incidence of miscarriage. Limitations and pitfalls of this technique are also discussed.

Genes and male infertility: what can go wrong?

Genes and male infertility: what can go wrong?

Sperm chromosome aneuploidy analysis in a man with globozoospermia

Objective To determine the frequencies of chromosome aneuploidy and diploidy in sperm from a male with globozoospermia. Design Assessment of sperm chromosome aneuploidy and diploidy frequencies by multicolor fluorescence in situ hybridization (FISH) analysis. Setting University research laboratory. Patient(s) An infertile patient with round-headed sperm (globozoospermia). Intervention(s) Sperm samples were obtained by masturbation for cytogenetic analysis. Main outcome measure(s) Aneuploidy and diploidy frequencies were assessed by multicolor FISH analysis for chromosomes 1, 15, 21, X, and Y and compared with those of five control donors. Result(s) A minimum of 10,000 sperm was analyzed per chromosome probe, for a total of 30,145 sperm. There was a statistically significantly increased frequency of XY disomy in the man with globozoospermia compared with the case in normal donors. The frequency of aneuploidy for chromosomes 1, 15, 21, XX, and YY was not statistically significantly increased. The frequency of diploidy was also not statistically significantly different. Conclusion(s) A previous report demonstrated an increased frequency of chromosome 15 aneuploidy in sperm of a globozoospermia patient who fathered a trisomy 15 conceptus. No other report has studied the frequency of chromosome 15 aneuploidy in these infertile men. Our study does not demonstrate an increased risk for chromosome 15 aneuploidy associated with globozoospermia. However, an elevated frequency of XY disomy was discovered, which is the most common type of chromosome abnormality observed in sperm of infertile men.

Aneuploidy in spermatozoa of infertile men with teratozoospermia.

Recent studies have shown that aneuploidy in spermatozoa of infertile men with poor semen quality is increased. The purpose of this study was to determine whether poor sperm morphology is associated with the incidence of spermatozoa with numerical chromosome abnormalities. Semen samples from 20 infertile teratozoospermic men were studied using multicolour fluorescence in situ hybridization (FISH). Men were divided into four groups according to the proportion of normal sperm morphology: infertile men with <10% (group A, n=7), 10-19% (group B, n=6), and 20-29% (group C, n=7) of morphologically normal spermatozoa, and controls (group D, n=5) with > or =30% normal forms. Two hybridizations were performed. All the samples were analysed using probes for chromosomes 1 and 7 and, in addition, in group A and in controls with normal semen parameters probes for chromosomes X, Y and 18 were also used. Ten thousand spermatozoa were scored per hybridization. Severely teratozoospermic men (<10% normal forms) had significantly higher frequency of disomy 7, 18, YY, XY and diploidy in their spermatozoa when compared with controls. The results suggest that poor sperm morphology is associated with numerical chromosome abnormalities of spermatozoa. Severely teratozoospermic men may be at an increased risk of producing aneuploid offspring.

Prevalence of human herpesvirus types 1–8 in the semen of infertility patients and correlation with semen parameters

The herpesvirus group consists of eight DNA viruses: herpes simplex virus types 1 and 2 (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus (HHV) types 6, 7, and 8. They have also been detected in genital secretions, but reported prevalence data vary widely, especially for HSV, with a prevalence range from 3.1% (HSV-2; done by polymerase chain reaction [PCR]) in men with genital HSV-2 infection to 46% (HSV-1 and HSV-2; in situ hybridization) in spermatozoa of infertile men. The presence of HSV and CMV has been found to be associated with altered semen parameters.

Aneuploidy in human spermatozoa: FISH analysis in men with constitutional chromosomal abnormalities, and in infertile men.

Reproductive difficulties are associated intimately with cytogenetic abnormalities. This article reviews multicolour fluorescence in situ hybridization studies on spermatozoa from men with constitutional chromosomal abnormalities and the consequences for spermatozoa, and on chromosomal abnormalities in the spermatozoa of infertile men who have normal somatic karyotypes. In 47,XYY men, the frequencies of 24,XY and 24,YY spermatozoa appear to be < or = 1%. Klinefelter (47,XXY) and mosaic Klinefelter patients had sperm aneuploidy frequencies of 2-25% and 1.5-7.0%, respectively. Robertsonian translocation carriers had 3-27% spermatozoa unbalanced for the chromosomes involved in the translocation, with a possible modest interchromosomal effect, but none of the increased frequencies of chromosomal disomy approached 1%. The frequency of chromosomally unbalanced spermatozoa in reciprocal translocations averages 50%, is strongly dependent on the chromosomes involved in the individual translocation, and may be slightly increased as a result of a small interchromosomal effect. Infertile men with a normal karyotype and low sperm concentration or certain types of morphologically abnormal spermatozoa have a significantly increased risk of producing aneuploid spermatozoa, particularly for the sex chromosomes. An increased risk of sperm aneuploidy was not observed in infertile men with poor sperm motility or in those with a normal karyotype and normal semen parameters.

VARICOCELECTOMY IMPROVES INTRAUTERINE INSEMINATION SUCCESS RATES IN MEN WITH VARICOCELE

We determined whether varicocele treatment before intrauterine insemination significantly affects intrauterine insemination success rates. A total of 58 infertile couples, of whom the women had normal evaluations and men had abnormal semen analyses and a history of varicocele, were included in this study. They were identified after reviewing the charts of all women undergoing intrauterine insemination for male factor infertility at our center. Of the men 24 participated in 63 intrauterine insemination cycles without varicocele treatment, while in the remaining 34 varicocele was treated before a total of 101 intrauterine insemination cycles. Variables associated with pregnancy or live birth were analyzed using repeat measures logistic regression with generalized estimating equation techniques. An initial stepwise generalized estimating equation was performed without including varicocele treatment status. Subsequently varicocele treatment status and the significant associated factors were included in analysis. The semen characteristics of untreated and treated varicocele groups were compared with repeat measures analysis of variance. On pre-wash semen analysis patients with untreated varicocele had significantly higher mean motility plus or minus standard error than patients whose varicoceles were treated (48.6% +/- 2.3% versus 38.1% +/- 1.8%, p = 0.02). However, no statistically significant difference was noted in the mean post-wash total motile sperm count in the treated and untreated groups (7.2 +/- 1.0 versus 14.8 +/- 2.6, p = 0.1). Despite these findings the pregnancy and live birth rates per cycle were significantly higher in patients in whom varicocele was treated than in those without varicocele treatment (11.8% versus 6.3%, p = 0.04 and 11.8% versus 1.6%, p = 0.007, respectively). Varicocele treatment may not improve semen characteristics in all men but it appears to improve pregnancy and live birth rates among couples undergoing intrauterine insemination for male factor infertility. A functional factor not measured on routine semen analysis may affect pregnancy rates in this setting. Men should be screened for varicocele before intrauterine insemination is initiated for male factor infertility.

Absence of deleted in azoospermia ( DAZ) genes in spermatozoa of infertile men with somatic DAZ deletions

Objective: To determine the presence or absence of the deleted in azoospermia ( DAZ) gene clusters in the Y-bearing spermatozoa in semen of severely oligozoospermic men or in testicular biopsy samples of azoospermic men with somatic DAZ deletions. Design: Prospective study. Setting: Academic hospital. Patient(s): Nineteen patients attending our clinics for therapeutic intracytoplasmic injection of sperm. Intervention(s): Peripheral blood lymphocytes were used to obtain somatic DNA for analysis using the polymerase chain reaction. Analysis of chromosomes X and Y and the detection of the DAZ gene clusters were carried out with the fluorescence in situ hybridization technique in spermatozoa remaining after intracytoplasmic sperm injection. Main Outcome Measure(s): Presence or absence of the DAZ gene clusters in matched somatic DNA and Y-bearing spermatozoa. Result(s): Seven patients appeared to have a somatic DAZ deletion. Three-color fluorescence in situ hybridization showed that all Y-bearing spermatozoa examined from these men carried the same deletion. Conclusion(s): The DAZ deletions present in the seven men would all have been transmitted if they had fathered sons through artificial fertilization techniques using the sperm cells examined in this study.

Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4.

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.

Glutathione and hypotaurine in vitro: effects on human sperm motility, DNA integrity and production of reactive oxygen species.

Sperm DNA integrity is of paramount importance for the accurate conveyance of genetic material. DNA damage may be a major contributory factor in male infertility as DNA from sperm of infertile men has been found to be more susceptible to induced DNA damage in vitro than DNA from fertile men. Reactive oxygen species (ROS) are a significant source of DNA damage and human sperm are extremely sensitive to ROS attack due to their high content of polyunsaturated fatty acids and lack of capacity for DNA repair. Seminal plasma, which contains a wealth of antioxidants, provides sperm with crucial protection against oxidative insult. However, during preparation for use in assisted conception techniques, sperm are separated from seminal plasma and deprived of that essential protection. The aim of this study was to determine the effects of supplementation with glutathione and hypotaurine during sperm preparation on subsequent sperm motility, DNA integrity, induced DNA damage and ROS generation. Semen samples (n = 45) were divided into aliquots and prepared by Percoll density centrifugation (95.0-47.5%) using medium which had been supplemented with these antioxidants to a number of different concentrations all within physiological levels. Control aliquots were included which had no glutathione or hypotaurine added. Sperm motility was determined using computer-assisted semen analysis. DNA damage was induced using H(2)O(2) and DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay, while ROS generation was measured using chemiluminescence. Addition of glutathione and hypotaurine, either singly or in combination, to sperm preparation medium had no significant effect on sperm progressive motility or baseline DNA integrity. Despite this, sperm were still afforded significant protection against H(2)O(2)-induced damage and ROS generation.

In vitro inhibition of superoxide anion production and superoxide dismutase activity by zinc in human spermatozoa.

The in vitro effect of zinc on superoxide anion (O2-) generation and on SOD-like activity in spermatozoa of infertile men was investigated. The formation of superoxide anion was stimulated by NADPH and the level of superoxide anion was measured by the reduction of ferricytochrome c. Both Percoll-isolated (n = 14) and washed spermatozoa (n = 14) exposed to 1 mmol/L zinc (60 min, 37 degrees C), released less (p < 0.002 and p < 0.04, respectively) superoxide anions than did zinc-untreated spermatozoa. These results implicate a possible role for zinc as a scavenger of excessive superoxide anions produced by defective spermatozoa in semen after ejaculation. Additionally, zinc was found to dose-dependently inhibit superoxide dismutase (SOD)-like activity of spermatozoa in vitro. The inhibition of SOD-like activity by an equal concentration of zinc (1 mmol/L) was less pronounced in oligospermic (p < 0.002; n = 16) and asthenozoospermic (p < 0.0005; n = 20) than in normozoospermic samples (p < 0.0001; n = 20). This differential ability of zinc to inhibit SOD-like activity may be relevant to the physiological function of spermatozoa in fertilization. The evidence that zinc may elicit an inhibition of both superoxide anion production and SOD-like activity in human spermatozoa, indicate the existence of novel, zinc-related mechanism(s) involved in the oxidative events occurring after ejaculation, with a possible modulatory effect on germ cell function.

Fertilization antigen-1 removes antisperm autoantibodies from spermatozoa of infertile men and results in increased rates of acrosome reaction

Objective: To determine if fertilization antigen (FA)-1 will remove autoantibodies from the surface of sperm cells of immunoinfertile men by immune adsorption and permit an increased acrosome reaction (AR). Design: Prospective analytic study. Setting: University medical center. Patient(s): Men from 18 infertile couples with autoantibodies present on their spermatozoa. Intervention(s): Sperm samples after processing were examined for antibody binding and AR before and after adsorption with control medium or FA-1. Main Outcome Measure(s): Sperm-bound antibody was assessed by the immunobead assay (immunoglobulin [Ig] A and IgG) and the AR by induction with ionophore A23187. Result(s): Adsorption with FA-1 compared with control medium increased immunobead-free swimming sperm an average of 50% and 76% for IgA and IgG antisperm antibodies, respectively, with 78% and 100% of the 18 semen specimens increasing significantly. The AR rate increased an average of 10.3% compared with control medium and showed improvement in 78% of the sperm samples after FA-1 adsorption. Conclusion(s): The FA-1 sperm antigen appears to significantly free sperm cells coated with autoantibodies in the semen of most infertile men examined. Reducing sperm-bound antibodies that inhibited the AR allowed the sperm cells to undergo successful AR induction by calcium ionophore.

Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men

Sperm samples from infertile men with oligozoospermia or teratozoospermia were studied by multicolour fluorescence in-situ hybridization (FISH) using DNA probes for chromosomes 13 and 21. A total of 90 809 sperm nuclei from nine infertile men and 182 799 sperm nuclei from 18 control donors were analysed. There was a highly significant increase in the frequency of spermatozoa disomic for chromosome 13 in infertile patients (0.28%) compared to control donors (0.13%) (two-tailed Z statistic P < 0.0001) and for chromosome 21 (0.48% in infertile men versus 0.37% in controls, P < 0.0001). Also there was a significantly increased frequency of diploid spermatozoa in infertile men (0.85%) compared to control donors (0.66%) (P < 0.0001). Our previous studies on these same infertile patients demonstrated increased frequencies of sperm disomy for chromosomes 1 and XY. This suggests that infertile men, who are prime candidates for intracytoplasmic sperm injection, may be at a very small increased risk of aneuploid offspring.

Defective function of membrane potential (psi) on sperm of infertile men.

Membrane potential across the plasma membrane (psi) of human sperm was determined by measuring the accumulation of triphenylmethylphosphonium (TPMP+). Washed sperm of fertile or infertile men were suspended, incubated in low-K+ and high-Na+ or high-K+ and low-Na+ medium, and allowed to take up the cation TPMP+ to a steady state (20 min at 37 degrees C). Under these conditions a psi of -75 +/- 6 mV was observed in sperm cells of fertile men, while in spermatozoa of idiopathic and asthenozoospermic infertile men, the psi was of 35 +/- 5 mV and 32 +/- 5 mV, respectively. Upon depolarization of the sperm membrane by K+, the transport system Na(+)-K+ in spermatozoa of idiopathic infertile men decreased 53 +/- 3.5%, while that in sperm cells asthenozoospermic men decreased 57 +/- 3% with respect to spermatozoa of fertile men. The results of these studies indicate their possible application to the diagnosis of male infertility.

Abnormal expression of protein 4.1 in spermatozoa of infertile men with teratospermia

Molecular defects responsible for morphologically abnormal spermatozoa (teratospermia) associated with male sterility are largely unknown. We report defective expression of protein 4.1, a cytoskeletal protein initially recognised in red cells. In some patients with severely amorphous sperm heads, protein 4.1 had an abnormal sub-cellular localisation (tail instead of head) and appeared as high-molecular-weight isoforms, especially a 135 kDa species instead of the 82 kDa isoform seen in fertile sperm. Because the 135 kDa isoform is characteristic of immature germ cells from testis, its presence in teratospermia suggests profoundly disturbed sperm differentiation.

Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe teratozoospermia

To evaluate the acrosome reaction and its prerequisite, a calcium influx, in spermatozoa of infertile men with a high incidence of abnormal sperm forms. Prospective, controlled study. Academic tertiary assisted reproduction center. Patients (n = 14) were allocated in the study after semen evaluation showed teratozoospermia (< 14% normal sperm forms) as diagnosed by strict criteria. After swim-up separation of the motile fraction, acrosome reactions were evaluated using Pisum sativum agglutinin (both spontaneously and exogenously induced with P and the calcium ionophore A23187, both at 10 microM); the intracellular-free [Ca2+]i was assessed by the fluorescent fura-2 indicator (basal and after P). Patients did not show the typical P-induced wave of [Ca2+]i that was observed in controls but rather a blunted response, no response at all, or abnormal basal [Ca2+]i levels. The percent of basal acrosome reaction was significantly lower for patients versus controls postswim-up, and at 1 hour and 3 hours. Furthermore, there was a significant difference in the response of acrosome reaction to P both at 1 hour and 3 hours, with patients showing almost no response at all. However, patients' acrosome reaction response to the calcium ionophore was similar to those of fertile men. Infertile patients with a high incidence of abnormal sperm forms as diagnosed by strict criteria have a low incidence of spontaneous acrosome reaction and a diminished P-stimulated acrosome reaction, whereas the nonspecific response to a calcium ionophore is conserved. Parallel abnormalities of [Ca2+]i were observed in patients, suggesting that these sperm populations may have a defective nongenomic P sperm receptor and/or abnormalities of other membrane transduction systems.

Sperm nuclear instability and staining with aniline blue: abnormal persistence of histones in spermatozoa in infertile men.

During mammalian spermiogenesis, replacement of the somatic histones by basic proteins, the protamines, allows normal sperm nuclear condensation. In this study we have evaluated the degree of chromatin compaction in spermatozoa from 191 infertile subjects, affected by different testicular disorders, compare with that in 50 fertile sperm donors (controls). In infertile men, there was a higher percentage of unstable spermatozoa after incubation with sodium dodecyl sulphate (SDS) and of stained spermatozoa after staining with aniline blue (P less than 0.001 vs. controls). Furthermore, a positive linear correlation was found between SDS-unstable spermatozoa and stained spermatozoa (P less than 0.001), suggesting that sperm instability was related to a defect in histone-replacement by sperm-specific nucleoproteins, protamines. When the patients were considered according to pathology, high sperm nuclear instability and a high percentage of stained spermatozoa were detected in groups affected by varicocele, idiopathic infertility and in patients with a history of unilateral cryptorchidism. In the latter group the same alterations were observed even when the cryptorchid testis had been removed during surgery. In the group with a past history of mumps orchitis these parameters did not show any difference when compared with controls.