Deregulation of sperm nuclear protamine ratio (P1/P2) has been shown to correlate with male factor infertility in humans, but the cause of this abnormal protein expression has yet to be identified. Recent studies have shown that there is little genetic variability in the coding regions of either of the protamine gene sequences. However, these studies did not investigate the 5' or 3' non-coding regions of these genes for mutations that might account for changes in the transcriptional or translational regulation of the protamines. In an effort to determine if genetic variation in these non-coding regions may account for aberrant protamine expression, we have sequenced the 5' and 3' untranslated regions (UTRs) of both protamine 1 (P1) and protamine 2 (P2) genes in a population of infertile men with protamine deregulation, men presenting for infertility work-up with normal protamine ratios, and a population of unrelated, fertile men from the Utah Genetic Reference Project (UGRP). This analysis has identified 14 single nucleotide polymorphisms (SNPs), of which 13 were novel SNPs in the UTRs of P1 and P2, and verified the existence of a variable length repeat (VLR), GAn, in the P2 5' region. The SNP frequencies and VLR allelic frequencies did not achieve statistical significance between the populations, however, one of the SNPs identified in the 3' UTR of protamine 2 was found at a low frequency in the abnormal protamine patients, but was completely absent in men with verified normal protamine ratio and donors of known fertility. In conclusion, a number of SNPs have been reported in the protamine genes and the untranslated regions, however, these gene variants do not appear to be responsible for protamine deficiency. Hence, the underlying cause for aberrant protamine expression may possibly be due to abnormalities in candidate spermatogenic transcriptional/translational regulators, post-translational modifiers, or as-of-yet unidentified factors affecting the testicular environment.
Seminal vesicle cysts with ipsilateral renal agenesis is rare. When the patient is symptomatic, surgical treatment may be necessary. However, the seminal vesicle is difficult to access surgically, and current transurethral or open surgical approaches have inherent shortcomings. The laparoscopic techniques developed in the last decade may overcome the difficulties in the surgical treatment of seminal vesicle pathology. In this study we report a patient diagnosed with left seminal vesicle cyst and ipsilateral renal agenesis who was managed successfully through the laparoscopic approach. The patient was a 41-year-old who suffered from perineal pain and intermittent hemospermia for 20 years. Ultrasonography and computerized tomography, CT, indicated a cyst of the left seminal vesicle and an absent left kidney. The total laparoscopic operation time was 90 minutes and the estimated blood loss was 80 ml. With a follow-up of 13 months, the patient had total relief of his preoperative symptoms without complication.
A Tris-citric acid-glucose (TCG) diluent containing low concentrations (6%) of egg yolk, and a TCG extender containing lactose (without egg yolk), were compared for use in the cryopreservation of Spanish ibex (Capra pyrenaica) epididymal spermatozoa. To optimize the collection of epididymal spermatozoa, two spermatozoa recovery methods were tested: i) by using small cuts in the cauda epididymides and ii) by the application of air pressure (from a syringe) inside the vas deferens. The percentage of viable spermatozoa recovered was lower (P < 0.05) with the air pressure method. No significant differences were seen in the efficacy of the two diluents as determined by percentage viability of thawed sperm, membrane integrity (as determined by the hypo-osmotic swelling test), or acrosome integrity. The use of the TCG-lactose medium strongly reduced sperm motility (P < 0.001). The sperm samples that had been diluted with TCG-6% egg yolk extender showed a greater incidence (P < 0.05) of morphological abnormalities. TCG-lactose alone, does not well preserve motility when cryopreserving Spanish ibex epididymal spermatozoa.
Testicular microlithiasis (TM) is an unusual ultrasonographic manifestation in testicular parenchyma. Limited information is available about TM in Taiwanese men. We performed a retrospective analysis to investigate the characteristics of TM and its association with testicular cancer and infertility in Taiwan. Male patients who had received scrotal ultrasonography because of scrotal symptoms or infertility between January 2000 and December 2003 were recruited. The incidence of TM was 7.6%. Both testicular microlithiasis and testicular cancer occurred chiefly in the third decade. Patients with TM exhibit a higher chance of testicular cancer (6% vs. 0.9%). No local field effect between TM and testicular cancer was observed. Testicular microlithiasis severity is not positively correlated with sperm quality and sterility. Forty-eight patients (32%) were available at follow-up. No patient developed a testicular tumor or elevated tumor markers (AFP, beta-hCG) during follow-up. We suggest monthly self-examination, annual scrotal ultrasonography and tumor markers screening between the age of 20 and 30 years of patients with TM.
Alus and B1s are short interspersed repeat elements (SINEs) derived from the 7SL RNA gene. Alus and B1s exist in the cytoplasm as non-coding RNA indicating that they are actively transcribed, but their function, if any, is unknown. Transcription of individual SINEs is a prerequisite for retroposition, but it is also possible that individual Alu and B1 elements have some cellular functions. Previous studies suggest that transcription of Alu elements depends on the presence of an RNA polymerase-III bipartite promoter and the poly-A tail. Sequencing of small RNAs has demonstrated that the members of the Y and S subfamily are expressed. We analyzed almost one million Alu sequences longer than 200 nucleotides for the presence of RNA polymerase-III bipartite promoter sequences. More than half contained a promoter indicating some potential for expression. We searched 7.7 million human EST sequences in dbEST for the presence of Alu noncoding RNAs and found evidence for the expression of 452. Analysis of mouse spermatogenic dbEST libraries revealed an apparent relationship between the level of differentiation and the level of B1-related sequences in the EST library.
The existence of a complex population of mRNAs in human sperm is well documented but their role is not completely elucidated. Evidence for a latent transcriptional capacity and/or a potential translation de novo in mature spermatozoa from fertile men has been provided and is helpful in understanding the final steps of sperm maturation (capacitation and/or the acrosome reaction). Spermatogenesis is controlled by gonadotrophins and testosterone, their effects are modulated by locally-produced factors that include estrogens derived from the irreversible transformation of androgens by aromatase. The data demonstrating an additional source of estrogens in rat germ cells along with several studies showing a decreased sperm motility in men deficient in aromatase has led to the further explanation of the expression of aromatase in ejaculated spermatozoa from fertile men. A significant decrease in the amount of aromatase transcripts in the immotile sperm fraction was recorded. In addition, the levels of transcripts encoding for proteins involved in either nuclear condensation protamines 1 and 2 (Prm1 and Prm2) or in capacitation endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide sythase (nNOS) and c-myc were compared in low and high motile sperm prepared from the same sample. No significant change in the ratio of c-myc/Prm2 between the two populations of spermatozoa was observed. Conversely the amount of Prm1 mRNA was significantly higher in the low motile fraction than in the high motile fraction; in most of the high motile sperm samples analyzed, eNOS and nNOS transcripts were undetectable, whereas they were observed in low motile sperm. Moreover, a partial or complete disappearance of c-myc transcripts was observed after capacitation. Analysis of the mRNA profile in human ejaculated sperm could be helpful either as a diagnostic tool to evaluate the male gamete quality and/or as a prognostic value for fertilization and embryo development.
Administration of testosterone undecanoate (TU) over 12 months to men with sexual dysfunction and signs of the metabolic syndrome, restored their plasma testosterone (T) levels to the mid-range of reference values. This had a beneficial effect on their sexual functioning as evidenced by an improvement of their scores on the International Index of Erectile Function. The scores on the Aging Male Symptoms score, AMS, were also improved. Most impressive were the improvements in the parameters of the metabolic syndrome; they all improved and appeared largely correlated (i.e., decline in waist circumference with declines of plasma cholesterol and LDL and increase in plasma HDL). Sex hormone binding globulin, SHBG, may be considered as an indicator of the severity of the metabolic syndrome; levels of SHBG initially fell, probably as a result of rising plasma T levels. But over the last six months of the observation period when plasma T rose further, there was a significant increase in plasma SHBG which may be interpreted to indicate an improvement of the metabolic syndrome. Blood pressure improved slightly but significantly. in this cohort of elderly men (54-76 years; median 64 years) there were no safety concerns over a one year period of T administration. Prostate specific antigen, PSA, levels remained stable; the International Prostate Symptoms Score, IPSS, improved slightly. Liver functions and plasma glucose remained stable. Hemoglogin and hematocrit values increased significantly but remained within reference values.
The aim of this study was to prospectively investigate the spermatozoa ultrastructure in relation to the results of in vitro fertilization - embryo transer (IVF-ET).Forty-nine consecutive couples admitted for IVF-ET were prospectively evaluated for electron microscopic spermatozoa morphology and the outcome of IVF-ET. Thirty-four couples revealed successful fertilization, defined as presence of two pronuclei 14-16 hours after spermatozoa administration, while the remaining 15 formed the failure group. Spermatozoa fixed with 2.5% glutaraldehyd and embedded in Spurr's resin were analyzed with JAM 100 S transmission electron microscope (TEM) for the following ultrastructure abnormalities: head deformity, cytoplasmic residues, chromatin condensation failures, acrosomal alterations, neck defects, mid-piece defects, principal piece and end-piece defects and immature forms.Successful IVF-ET couples revealed a significantly higher percentage of normal spermatozoa utrastructure (32.0 +/- 13.1% versus 17.1 +/- 13.4%, p < 0.001). Failed IVF-ET couples represented a significantly higher percentage of chromatin condensation failures (9.8 +/- 5.1% versus 5.7 +/- 5.3%, p < 0.05) and tail defects (16.7 +/- 11.5% versus 7.2 +/- 7.2%, p < 0.001). A positive correlation between normal ultrastructure spermatozoa percentage and fertilized oocytes percentage was found (r = 0.35, p < 0.05).Our data suggest that spermatozoa TEM findings correlate with IVF-ET results. Ultrastructural estimation of spermatozoa can improve the diagnosis of male fertility and may explain some reasons of failure in assisted reproduction methods. We consider systematic TEM spermatozoa examination in cases with failed IVF-ET prior to intracytoplasmic sperm injection (ICSI).
The Sperm Quality Analyzer (SQA) IIB, a member of the SQA-II family of machines which uses the scatter of light by sperm as an indicator of sperm motility, was systematically evaluated as a means of analyzing objectively the motility of porcine epididymal sperm. The sperm motility (%) and the Sperm Motility Index (SMI) are calculated by the machine using pre-programmed algorithms designed for human sperm. The machine performed well and was able to detect changes in sperm motility under experimental conditions. However, two major limitations of this machine were identified, (i) the readings obtained were influenced by the concentration of the sperm suspension despite the actual sperm motility remaining constant, and (ii) the machine was unable to differentiate between progressive and non-progressive motility. It should therefore be recognized that (a) the sperm concentration must be kept constant in studies in vitro if differences between treatment groups are to be identified, and (b) the inability to separate progressive motility from that of total motility will restrict the usefulness of this and similar machines to studies monitoring changes in total motility alone.
cAMP responsive element modulator (CREM) activator isoforms are involved in mammalian spermatogenesis and spermiogenesis. CREM proteins are highly expressed in postmeiotic germ cells of rodents and primates. Homozygous CREM inactivated mice exhibit round spermatid maturation arrest. The lack of CREM expression at both the mRNA and protein levels is associated with spermatid maturation arrest in infertile patients. Using real-time RT-PCR, we have examined the levels of CREM activator isoform mRNAs: CREMθ1, CREMθ2 and CREMt2 + Ex-γ in gametogenic and interstitial cell fractions from normal human testis, in homogenized tissue samples from spermatogenic arrest and from testicular tumors. We have shown for the first time the presence of CREM activator isoform containing exon γ (CREMτ2 + Exγ) in normal human spermatogenesis. Among the three CREM isoforms, CREMθ1 was expressed in its highest level in the male gonads. In comparison, CREMθ2 mRNA was significantly less suggesting that the P3 promoter is much more active in human testis than the P4 promoter. Minimal-nill levels of mRNA for either of the CREM activator isoforms were detected in lymphocytes or in gonadal tissues from patients with SCOS (Sertoli Cell Only Syndrome). This data underlines the significance of CREMθ1 isoform in the regulation of transcription during post-meiotic germ cell differentiation.
To investigate seminal antioxidant systems and their correlation with hormonal pattern in varicocele patients, we studied 33 varicocele (VAR) patients (12 oligozoospermic, 21 normozoospermic) and 34 non-VARs (10 idiopathic oligozoospermic, 24 normozoospermic). Non-enzymatic total antioxidant capacity (TAC) was measured using H(2)O(2)-metamyoglobin, which generates the radical form, spectroscopically detectable, of the chromogen 2,2',-azinobis-3-ethylbenzothiazoline-6-sulphonate (ABTS): time till appearance (Lag) of ABTS*(-) signifies antioxidant concentration. Lag was significantly longer in VARs than controls suggesting ineffective utilization of antioxidants. A significant direct correlation (r = 0.65, p < 0.01) of Lag with sperm count was observed in non-VARs, while in VARs it was inverted, as well as with hematic FSH levels. A multivariate analysis including FSH, Lag, progressive spermatozoa, oligozoospermia and varicocele indicated a strong inverse correlation between FSH and motility (r(2) = 0.31, p > F = 0.0007), not modified by Lag (r(2) = 0.31, p > F = 0.002). Their inverse correlation with Lag may suggest that higher FSH levels, improving sperm antioxidant efficiency, counterbalance varicocele-induced dyspermia.
The purpose of our study was to evaluate the effect of age and sperm DNA integrity on sperm survival. Semen samples from fifty six unselected patients undergoing infertility evaluation were assessed in terms of standard semen parameters, DNA integrity, and sperm survival after 6-24 h of incubation. Prolonged incubation of density gradient selected sperm adversely effects sperm survival in older patients and patients with extensive sperm DNA damage. Immediate preparation of such samples prior to use for assisted reproductive technology (ART) may overcome the negative effect of extended incubation.
A very rare case of successful pregnancy was reported using testicular sperm from a severely infertile man given the treatment of hCG in advance. Prior to treatment, no sperm were present in the ejaculate and testicle. hCG was administrated to the patient for spermatogenesis. After a period of hCG treatment, a few immature sperm were observed in the minced testicular specimen. A successful pregnancy was established by ICSI using the sperm. Treatment by administration of hCG to generate sperm followed by ICSI for the azoospermia patients was effective and practicable.
Upon assuming the position of Editor-in-Chief for Archives of Andrology, now Archives of Andrology: Journal of Reproductive Systems, I would like to welcome the new members of the Editorial Board who share the vision that is driving the evolution of this journal. Our mission is to facilitate the next significant advance in reproductive biology by encouraging the use of systems approaches to enable the translation of research and knowledge from the bench to the bedside. Since the completion of the human and other species’ genomes, biology has been faced with the challenge of connecting seemingly isolated elements from the now data-rich environment. Problems created by the scope of samples and wealth of data, like management and appropriate analysis, are common among many fields. Yet most fields remain apart recreating the same solution even though when coalesced, novel solutions and practical applications would result. For example, one can envisage that by using complex data sets that range from a simple morphology and family history to gene chip based analysis, new global-based gene diagnostics yielding patient specific ‘‘designer’’ treatments could evolve. Spurred on by these advances and their inherent potential, reproductive science is emerging from a descriptive field to one that uses a systems-wide approach as a means to integrate outwardly unconnected elements. One of the goals that can now be realized is to understand the mechanism that mediates development of the ‘‘ultimate’’ stem cells, i.e., the sperm and the egg, to that of complex mature adult systems. Reproductive Biology now requires a central coordinating forum to serve as a catalyst that embraces a systemswide approach. Archives of Andrology: Journal of Reproductive Systems will rise to this challenge by facilitating discussion and dissemination of the results of novel implementations directed to reproductive systems. These advances can be communicated as Research Articles, Communications, Applications Notes (e.g. protocols), Clinical Corner (e.g. case reports), Review Articles, Hypotheses, and Letters to the Editor. The journal will serve both the basic and medical sciences highlighting the use of systems approaches, including genomic, cellular, proteomic, bioinformatic, molecular, and biochemical, to address fundamental questions in reproductive biology, reproductive medicine, and translational research. Archives of Andrology: Journal of Reproductive Systems will reach out to the fields of medical molecular biology, genomics, proteomics, and bioinformatics, while showcasing the development of analytical tools including data mining and management information systems. Areas of interest include: male factor fertility and infertility encompassing its genetic, biochemical, cellular, morphological, ultra-structural, physiological, Archives of Andrology: Journal of Reproductive Systems, 53:107–108, 2007 Copyright # Informa Healthcare ISSN: 0148-5016 print=1521-0375 online DOI: 10.1080/01485010701283674
Hormonal responses were assessed in men with prostate cancer (T2-4, Nx, Mx) who were randomized to receive either a single injection of goserelin 3.6 mg or leuprolide 3.75 mg. Testosterone increased over the first week, with a significantly higher mean rate of change of total testosterone (day 3) and free testosterone (day 3 and 7) with leuprolide. Following in initial rise in luteinizing hormone (LH), the rate of decrease in LH levels was significantly greater with goserelin by day 28. There are significant differences in endrocrine response to goserelin and leuprolide in the 4 weeks following administration.
This study tested a method for maintaining human spermatozoa without freezing for subsequent use in intracytoplasmic sperm injection (ICSI). We demonstrated that human sperm stored in electrolyte-free solution maintain their motility and viability for at least 4 and 6 weeks, respectively. We also have shown that preserved spermatozoa are fully functional in ICSI. Sperm chromosome analysis after injection of human sperm into mouse oocytes revealed that two weeks of storage does not negatively affect sperm DNA integrity. A mouse model was used to analyze the ability of preserved sperm to participate in normal embryogenesis. Mouse sperm preserved in electrolyte-free solution in a similar manner as human sperm maintained motility for up to 3 weeks. When mouse spermatozoa stored for 1 week were injected into the oocytes by ICSI, they yielded normal blastoctysts and normal viable fetuses. The results of the study bear significance for human assisted reproduction technologies (ART) and provide clinicians and infertile patients with a new method that can simplify sperm preparation for ICSI, assisting men who are unable to provide semen on the day of assisted fertilization.
The aim of this study was to assess the frequency of AZF microdeletions in peripheral leukocytes and testicular cells in Chinese men with idiopathic infertility. Expression in testicular cells was also determined. In this study, we screened 62 idiopathic infertile patients, in whom karyotype, sperm count and hormonal parameters were evaluated. Genomic DNA was extracted from the peripheral leukocytes. Molecular analysis was performed by two multiplex polymerase chain reactions (PCR) using a set of eight sequence tagged sites (STS) from 3 different regions of the Y chromosome. Total cellular RNA was extracted from the testicular tissue using a Trizol-method. Reverse Transcription (RT) reactions were performed to synthesize cDNA. Amplification of DFFRY, RBM and DAZ genes was performed to analyze their expression in testicular cells. in this cohort, we found 12 submicroscopic deletions (12/62, 19.4%). Nine patients (9/33, 27.2%) were detected in the azoospermic group and three (3/29, 10.3%) in the severe oligozoospermic group. RT-PCR analysis from testicular cells gave normal amplifications for SRY and DFFRY mRNA in 62 idiopathic patients; two patients were negative for RBM expression; no RBM and DAZ were detected for a case; 12 patients had no expression in the AZFc region involving the DAZ gene. Of 12 cases, three patients with normal PCR analysis of DAZ gene on genomic DNA showed no RT-PCR amplification for DAZ mRNA. The use of RT-PCR of specific spermatid expressed genes in conjunction with examining microdeletions using peripheral leukocytes is suggested to avoid the transmission of the Y chromosomal microdeletions from a father to a son via testicular sperm aspiration (TESE), intracytoplasmic sperm injection (ICSI)
The purpose of this study was to examine the effect of sperm preparation by density gradient on the intra-individual variation in sperm motility. Patients presenting for density gradient (DG) sperm preparation were analyzed retrospectively. Patients who had more than one preparation were included. The variation within each patient was studied using the coefficient of variation (CV = standard deviation/mean x 100). Density gradient preparation resulted in a reduction in the CV of sperm motility (CV motility before DG: 19.8 +/- 15.82% vs. CV motility after DG: 15.9 +/- 17.97%, p < 0.001). However, CV of sperm concentration (44.2 +/- 26-51%) and CV progressively motile sperm (49.2 +/- 28.48%) remained very high after DG. This variability should be reflected in counseling patients undergoing intrauterine insemination.
Spermatoceles are benign cystic dilations of the epididymis. Despite their relatively common occurrence, it is not clear why or when men want this lesion treated. We present a single institution series of men undergoing spermatocelectomy. We describe the clinical characteristics of men with these lesions and hypothesize that men with spermatoceles seek intervention when the lesion approximates the size of a testicle. The characteristics of 24 men who sought excision of symptomatic spermatoceles were reviewed. Specific characteristics included subject age, duration of diagnosis, symptom type, and symptom duration. Spermatoceles were characterized by size, sidedness, and associated findings. Simple descriptive statistics were used for analysis. The mean age of men seeking spermatocelectomy was 46 years. Most men (58%) sought surgery due to a combination of pain and sensation of mass. The mean duration of symptoms was 48 months. At the time of excision, the average size of spermatoceles was 4.2 cm in greatest diameter, and most (71%) were right sided. Men who experienced pain as an isolated symptom were younger by ∼ 10 years compared to those who experienced mass.Men in this series appear to tolerate spermatoceles for a relatively long period of time. Once they seek excision, spermatoceles have grown to roughly the size of a normal testicle and men are bothered both by pain and mass symptoms.
François Cuzin Inserm, U636, F-06108, Nice, France and Université de NiceSophia Antipolis, Laboratoire de Génétique du Développement Normal et Pathologique, Nice, France The question addressed in the title is raised from the observations of two groups. The presence of RNA in human spermatozoa [reviewed by Ostermeier et al. 2004; Krawetz 2005; Miller et al. 2005] and the observation of paramutation in an animal model [Rassoulzadegan et al. 2006] led us to conclude that the hereditary transmission of this epigenetic modification is associated with the transfer of RNA molecules accumulated in the sperm head. The tantalizing question is whether paramutation occurs in humans. It is, however, only in the animal model that it is possible to examine this epigenetic determination. The same genetic analysis is not possible in human since unambiguous identification of subtle modifications of gene expression requires a homogenous genetic background and serial crosses between siblings. Failing these requirements, multiple explanations are always possible for apparently non Mendelian inheritance. However, once the phenomenon has been recognized in the animal model, then consequences and correlates can be searched in the normal and pathological human conditions. To that purpose, results and hypotheses generated by the mouse studies have to be made available to the extremely difficult task of analyzing the scarce clinical genetic data. The possible function of sperm RNA in humans remains highly speculative. Analysis of paramutation in the mouse provides a new frame for hypotheses on its possible role in hereditary transmission and thus a role for sperm RNA. Excellent reviews on paramutation in plants are available [reviewed by Chandler and Stam 2004], including a comparison of plant and mouse [Chandler 2007]. The aim of this summary is to propose to the reader a working hypothesis on the possibility of gene controls exerted by RNA transferred from sperm to the embryo. History of spermatozoal RNA starts with the pioneer work performed on human sperm [reviewed in Krawetz 2005]. Its possible role(s) remain a matter of speculation [reviewed by Ostermeier et al. 2004; Miller et al. 2005]. However we have recently reported the hereditary transmission of an epigenetic change in expression of the mouse Kit gene [Rassoulzadegan et al. 2006]. Hereditary epigenetic variation was observed in plants by Brink in 1956 and has since been extensively studied as paramutation. In the mouse, the initial observation was a dramatic departure from the expected Mendelian distribution in the progeny of crosses between heterozygotes carrying the wild type Kit allele and the null allele Kit . Most of the progeny genotyped as wild type Kitþ=þ homozygotes derived from crosses between Kit tm1Alf=þ while heterozygotes maintained a large degree of variation. Received 09 March 2007; accepted 29 May 2007.
