Myo-inositol in health and disease: its impact on semen parameters and male fertility.

BACKGROUND: Myo-inositol (cis-1,2,3,5-trans-4,6-cyclohexanehexol; MI) is the most prominent of nine inositol stereoisomers. MI, its phosphate derivatives, and associated lipids are widely found in vegetables and animal tissues and are known to participate in numerous biological processes. OBJECTIVES: To perform a review analysis on MI presence, functions, and impact in male fertility. MATERIALS AND METHODS: A thorough search of listed publications in PubMed on MI and its derivatives was done. RESULTS: Published information was found and compiled on MI identification, natural dietary sources and absorption, biosynthesis, concentrations, as well as MI as its derivatives (PI, PIP, GPI, IPG) roles in several human tissues and body fluids in health and disease. A section was focused on MI presence, biosynthesis, and functions in the mammalian male genital tract and in spermatozoa, and summarized reports describing the impact of in vivo and in vitro MI supplementation on human semen quality and fertility. Studies reported a discrete improvement in sperm motility in fresh and frozen-thawed semen, and a better sperm performance in natural and assisted fertility. DISCUSSION AND CONCLUSION: MI was reported as an effective supplement for sperm quality. In any case, several study designs lack appropriate controls or data analysis to confirm the relevance of the findings. While promising, larger prospective randomized controlled studies will be required to confirm the positive effect of MI supplementation in male infertility management. Moreover, further investigations are encouraged to unravel MI roles in sperm physiology and the underlying molecular mechanisms.

Fibroblast growth factor 2 (FGF2) is present in human spermatozoa and is related with sperm motility. The use of recombinant FGF2 to improve motile sperm recovery.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate several functions of somatic cells. In a previous work, we reported FGFR expression in human spermatozoa and their involvement in motility. This study aimed to evaluate the presence and localization of fibroblast growth factor 2 (FGF2) in human spermatozoa, to determine the relationship of FGF2 levels with conventional semen parameters and to assess the effect of recombinant FGF2 (rFGF2) on sperm recovery in a selection procedure. Western immunoblotting analysis using an antibody against FGF2 revealed an 18-kDa band in sperm protein extracts. The protein was immunolocalized in the sperm flagellum and acrosomal region, as well as in all germ cells. Sperm FGF2 levels, assessed by flow cytometry, showed a positive (p < 0.05) correlation with sperm concentration, motility, total sperm number and total motile cells per ejaculate. Moreover, samples with abnormal motility depicted diminished (p < 0.01) FGF2 levels compared to those with normal motility. Spermatozoa exposed to rFGF2 bound the protein, exhibited higher (p < 0.05) total and motile sperm recoveries, and increased (p < 0.01) kinematic parameters after the swim-up. Findings herein presented lead to consider sperm FGF2 level as a potential marker of sperm quality, and rFGF2 as a supplement for improving sperm recovery in selection techniques.

Glass wool filtration of bull cryopreserved semen: a rapid and effective method to obtain a high percentage of functional sperm.

Frozen-thawed bull sperm are widely used in assisted reproductive technologies, but cryopreservation negatively affects semen quality. Several sperm selection techniques have been developed to separate motile sperm from non-motile cells. The aim of the present study was to evaluate the effectiveness of the glass wool column filtration to select functional sperm from frozen-thawed bull semen samples. Frozen semen from six Holstein bulls was thawed and filtered through a glass wool column, followed by assessment of routine and functional sperm parameters. In a set of experiments, sperm aliquots were also processed by swim up to compare both selection methods. Samples recovered in the glass wool filtrate had high percentages of viable (94 ± 3%, mean ± SD), progressively motile (89 ± 4%), acrosome-intact (98 ± 1%), and non-capacitated (80 ± 10%) sperm; these values were higher (P < 0.05) than those obtained after performing the swim up procedure. Moreover, the glass wool filtration yielded 67 ± 19% motile cells, in comparison with 18 ± 8% obtained with swim up (P < 0.05), calculated as the concentration of progressively motile cells selected relative to their concentration in the sample before the selection procedure. Glass wool-filtered sperm were able to undergo capacitation-related events, based on the increase in the percentage of cells classified as capacitated by CTC staining (B-pattern) after incubation with heparin (50 ± 5%) in comparison with control conditions with no heparin (17 ± 4%) or heparin + glucose (16 ± 2%; P < 0.05). Moreover, they underwent acrosomal exocytosis in response to pharmacologic (calcium ionophore A23187 and lysophosphatidylcholine) and physiological (follicular fluid) stimuli, and they fertilized in vitro matured cumulus-oocyte complexes and denuded oocytes (two-cell embryos: 72 ± 4% and 52 ± 6%, respectively). We conclude that glass wool filtration is a low-cost, simple, and highly effective procedure to select functionally competent sperm for reproductive technologies in the bull, which may be useful for other domestic and farm animals, as well as for endangered species.

Neural cadherin is expressed in human gametes and participates in sperm-oocyte interaction events.

Neural cadherin (N-cadherin) is a transmembrane glycoprotein involved in calcium-dependent cell-cell adhesion and signalling events. The previous evidence shows N-cadherin expression in the human gonads and gametes; however, N-cadherin subcellular localization in human spermatozoa and oocytes, and its involvement in fertilization remain to be characterized. In this study, expression of N-cadherin in human spermatozoa and testis was confirmed by RT-PCR and protein forms were identified using Western immunoblotting. N-cadherin localization in testicular and ejaculated spermatozoa, in cells that had undergone capacitation and acrosomal exocytosis, as well as in oocytes was assessed using immunocytochemistry. Participation of the adhesion protein in fertilization was evaluated using the HemiZona Assay (HZA) and the zona pellucida (ZP)-free hamster oocyte sperm penetration assay (SPA). Both the N-cadherin transcript and the mature protein form (135 kDa) were found in spermatozoa and testis. The protein was mainly immunolocalized in the acrosomal region of testicular, non-capacitated and capacitated spermatozoa, and was found in the equatorial segment after acrosomal exocytosis. N-cadherin was also detected in oocytes, in conjunction with beta-catenin, a member of the adhesion complex. Sperm incubation with anti N-cadherin antibodies did not affect their ability to interact with homologous ZP in the HZA; by contrast, presence of the antibodies in the SPA led to a significant (p < 0.01) reduction in the percentage of penetrated oocytes. In conjunction, results indicate that N-cadherin is a sperm protein of testicular origin localized in cellular regions involved in gamete interaction. N-cadherin would not participate in sperm-ZP interaction, but it would have a role in sperm-oolemma adhesion/fusion events.

Expression of epithelial cadherin in the human male reproductive tract and gametes and evidence of its participation in fertilization.

Epithelial cadherin (E-cadherin) has been involved in several calcium-dependent cell-cell adhesion events; however, its participation in gamete interaction has not been fully investigated. Our results have demonstrated expression of E-cadherin mRNA in the human male reproductive tract showing higher levels in the caput, corpus and cauda epididymis than in the testis. The mature 122 kDa E-cadherin was detected in epididymal protein extracts and was localized in the epithelial cells from the three epididymal regions. Moreover, the 86 kDa E-cadherin ectodomain was found in cauda epididymal and seminal plasma. Western immunoblotting of human sperm protein extracts allowed the identification of four E-cadherin forms (122, 105, 97 and 86 kDa). The protein was localized in the acrosomal region of intact spermatozoa, remained associated with the head of acrosome-reacted cells and was also detected on the oocyte surface. A similar localization was determined for other proteins of the adhesion complex (beta-catenin and actin). Spermatozoa incubated with anti-E-cadherin antibodies showed impaired binding to homologous zona pellucida (ZP); in addition, presence of these antibodies inhibited the penetration of human spermatozoa to ZP-free hamster oocytes. The results presented here describe the expression of E-cadherin in the male reproductive tract and gametes and strongly suggest its involvement in adhesion events during human fertilization. The identification of proteins involved in gamete interaction will contribute to the understanding of the molecular basis of fertilization and help in the diagnosis and treatment of infertility.

Molecular determinants of invasion in endometrial cancer.

Endometrial carcinoma is the most common gynaecological malignancy in the western world and the most frequent among infiltrating tumours of the female genital tract. Despite the characterisation of molecular events associated with the development of endometrial carcinoma, those associated with the early steps of infiltration and invasion in endometrial cancer are less known. Deep myometrial invasion correlates with more undifferentiated tumours, lymph-vascular invasion, node affectation and decreased global survival. In this review we present an overview of the molecular pathology of myometrial infiltration that defines the initial steps of invasion in endometrial cancer. Down-regulation of E-cadherin as a main player of epithelial to mesenchymal transition, as well as modifications on other molecules involved in cell-cell contacts, render cells with a migratory phenotype. In addition, altered signalling pathways and transcription factors associate with myometrial invasion, histologic grade and metastasis.

Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation.

BACKGROUND: Human semen is composed of a heterogeneous population of sperm with varying degrees of structural and functional differentiation and normality, which result in subpopulations of different quality. METHODS: Using a discontinuous Percoll gradient, we separated three subsets of sperm [(45%; L45), (65%; L65) and (90%; L90) fractions] from normozoospermic human semen samples from healthy donors and proceeded to characterize their morphology, motility and hyperactivation, as well as their ability to undergo tyrosine phosphorylation under capacitating conditions. RESULTS: As expected, sperm isolated from the lowest density layer (L45) showed the poorest quality, displaying the smallest percentage of morphologically normal and motile sperm. During a capacitating incubation, this subset of cells also showed deficient capacity to undergo hyperactivation and protein tyrosine phosphorylation. Conversely, sperm isolated from the other layers (L65 and L90) showed a time-dependent progressive increment in tyrosine phosphorylation, establishing statistically significant differences with sperm from L45. The tyrosine phosphorylation deficiency of L45 sperm could be overcome when sperm from that fraction were stimulated with activators of the cAMP-dependent kinase (PKA) pathway (dbcAMP + pentoxifylline), pointing to the sperm's plasma membrane as the main site of such deficiency. CONCLUSIONS: Poor quality sperm isolated from a Percoll gradient display an intrinsic tyrosine phosphorylation deficiency, possibly caused by a plasma membrane defect, which is associated with their inability to undergo normal capacitation and, ultimately, acquire optimal fertilizing potential.

Expression of human proacrosin in Escherichia coli and binding to zona pellucida.

Proacrosin is a multifunctional protein present in the sperm acrosome. This study characterizes the expression of human proacrosin in bacteria and assesses zona pellucida binding activity. The cDNA encoding human proacrosin was subcloned in pGEX-3X and pET-22b vectors. In the pGEX system, expression of the full-length fusion protein was not detected. In the pET system, an expression product with an apparent molecular size similar to that expected for the proenzyme (Rec-40, 42-44 kDa) was recognized by a monoclonal antibody to human acrosin, AcrC5F10. A 32-34-kDa protein (Rec-30), not recognized by AcrC5F10 on Western blots, was the major expression product. Proteins of 21 (Rec-20) and 18 (Rec-10) kDa were recovered as insoluble expression products as were Rec-40 and Rec-30, and truncated products from the C terminus were detected in the soluble fraction. Rec-40 and Rec-30 coexisted at any culture time tested. Immune serum raised against Rec-30 (AntiRec-30) stained the acrosomal region of permeabilized human spermatozoa and recognized the recombinant proteins and proacrosin from human sperm extracts. Amino acid sequence analysis indicated that Rec-30, Rec-20, and Rec-10 are N-terminal fragments of proacrosin. The recombinant proteins Rec-40, -30, -20, and -10 were found to interact with homologous (125)I-zona pellucida glycoproteins.

The relationship between critical evaluation of sperm morphology and the TYB-optimized zona free hamster oocyte sperm penetration assay.

The objective of this study was to analyse the relationship between the percentage of spermatozoa in semen with normal morphology, assessed using the Tygerberg criteria, and sperm fertilizing ability assessed using the TYB-optimized zona free hamster oocyte sperm penetration assay (TYB-optimized SPA), to evaluate the predictive value of strict morphology on outcome of the SPA. In a retrospective study, 56 samples were analysed. In addition to routine semen parameters, the percentage of spermatozoa with normal morphology (A forms) and the average number of penetrations per oocyte (Sperm Capacitation Index) was evaluated in all cases. Using a multiple linear regression analysis with all semen parameters, sperm morphology was the best predictor (p = 0.001) of the SPA score. The agreement between the percentage of A forms and the Sperm Capacitation Index beyond chance (kappa coefficient) was 0.5842. Twenty-two specimens had abnormal SPA scores, with 21 exhibiting abnormal sperm morphology (Sensitivity = 96%). The remaining 34 samples had normal Sperm Capacitation Index values; of these, 23 had normal sperm morphology in semen (Specificity = 68%). The positive predictive value was 96%, and the negative predictive value was 66%. All semen samples from control donors had normal semen parameters and Sperm Capacitation Index values. In conclusion, the percentage of spermatozoa with normal morphology assessed using Tygerberg criteria (> 14% A forms) are predictive of the results in the TYB-optimized SPA. However, sperm morphology appears to be a better predictor when it is normal than when it is abnormal.

Strontium supports human sperm capacitation but not follicular fluid-induced acrosome reaction.

The ability of strontium (Sr(2+)) to replace calcium (Ca(2+)) in maintaining human sperm function has still not been completely characterized. In the present study, acrosome reaction (AR) inducibility in response to human follicular fluid (hFF) was compared in spermatozoa incubated in either Ca(2+)- or Sr(2+)-containing media. Other events related to sperm capacitation, such as protein tyrosine phosphorylation and hyperactivation as well as zona pellucida (ZP) recognition under both conditions, were also analyzed. Spermatozoa incubated overnight in the presence of Sr(2+) were unable to undergo the AR when exposed to hFF. Nevertheless, when spermatozoa were incubated under this condition and then transferred to medium with Ca(2+), sperm response to hFF was similar to that of cells incubated throughout in the presence of Ca(2+). The sperm protein tyrosine phosphorylation patterns and the percentages of sperm motility and hyperactivation were similar after incubation in Ca(2+)- or Sr(2+)-containing media. Under both conditions, the same binding capacity to homologous ZP was observed. Similar results were obtained when EGTA was added in order to chelate traces of Ca(2+) present in Sr(2+) medium. From these results, it can be concluded that Sr(2+) can replace Ca(2+) in supporting capacitation-related events and ZP binding, but not hFF-induced AR of human spermatozoa.

Papanicolaou and Kruger assessment of sperm morphology: thresholds and agreement.

The current World Health Organization guidelines (1992) suggest that the presence of > or = 30% normal sperm forms (i.e. PAP criteria) is consistent with normal semen quality. Critical evaluation of sperm morphology (CE; Kruger classification) has shown an excellent correlation with human in vitro fertilization. Utilizing Kruger criteria, > 14% normal sperm forms has been proposed as indicative of normal semen quality. We have performed a retrospective analysis on 261 individuals to assess the agreement between PAP and Kruger criteria for normal sperm morphology (NSM). When the threshold for NSM by PAP was set at 30%, a significant agreement was found between the percentage normal forms of both criteria (Kappa coefficient = 0.37; p < 0.001). Sixty-seven (92%) of the 73 men found to have abnormal sperm morphology by PAP had abnormal semen by Kruger classification. When the threshold for NSM by PAP was established at 50%, the Kappa coefficient was 0.48 (p < 0.001). Sixty of the 72 samples (83%) classified as normal by PAP staining were normal by Kruger criteria. Interestingly, when NSM by PAP was between 30 and 50%, the specimen was just as likely to have normal or abnormal sperm morphology by Kruger (40 vs. 60%, respectively). These results strongly suggest that a high or low percentage of NSM by PAP is in agreement with the Kruger classification. The excellent agreement of Kruger and WHO criteria at the extremes (< 30% and > 50%) may obviate the need for Kruger assessment. However, when WHO morphology is between 30 and 50%, the addition of Kruger evaluation may provide meaningful information to help better diagnose a patient and plan his treatment.

Response of routine semen analysis and critical assessment of sperm morphology by Kruger classification to therapeutic varicocelectomy.

PURPOSE: We studied the effect of varicocelectomy on Kruger morphology and semen parameters. MATERIALS AND METHODS: A total of 33 subfertile men diagnosed with varicoceles was evaluated 3 months before, and 3 to 4 and 6 to 8 months after varicocelectomy. Evaluation involved routine semen analysis and sperm morphology using Kruger classification. RESULTS: Significant improvement in sperm concentration and count was found after varicocelectomy (sperm count preoperatively 117.1 +/- 29, 3 to 4 months postoperatively 162.5 +/- 41 and 6 to 8 months postoperatively 139.8 +/- 25 million sperm, p = 0.0095). Using Kruger classification, evaluation of sperm morphology revealed overall significant increase in percentage of normal A forms at 3 to 4 and 6 to 8 months after surgery (from 9.8 +/- 5.8% A forms, 13.6 +/- 7.7% A forms, and 14.5 +/- 7.5% A forms, respectively, p = 0.0002, normal greater than 14%). Twelve of the 26 patients (46%) with abnormal sperm morphology preoperatively and greater than 4% A forms reached normal levels 3 months postoperatively. Six months after surgery only 6 patients maintained normal values and 3 of the initial 14 nonresponders became normal (9 of 26, 36%). Three patients with severe teratozoospermia (less than 4% A forms) showed improvement in sperm morphology. Four patients with normal sperm morphology preoperatively were not affected by varicocelectomy. CONCLUSIONS: Surgical correction of varicocele was associated with significant improvement in sperm morphology evaluated using Kruger classification. Concentration and count improved after varicocelectomy. Changes were observed as early as 3 months after surgery.

Male immunologic infertility: sperm performance on in vitro fertilization.

OBJECTIVE: To analyze sperm performance in a group of patients with male immunologic infertility treated with IVF-ET. DESIGN: Retrospective clinical study. SETTING: Patients attending a private IVF clinic. PATIENT(S): The study group comprised seven men with significant levels of surface-bound antisperm antibodies treated in nine IVF cycles. The control group comprised nine couples with female tubal infertility and no indication of male factor infertility treated on the same cycle. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fertilization rate, early embryonic development, implantation, and clinical pregnancy rate (PR). RESULT(S): Forty-six (44.2%) of 104 inseminated oocytes were fertilized in the study group compared with 65 (84.4%) of 77 in the control group, which was a significant difference. Surface-bound antisperm antibodies significantly inhibited early embryonic cleavage in the study group (13 [28.3%] of 46 embryos with at least 3 blastomeres) compared with the control group (41 [63.1%] of 65 embryos, with at least 3 blastomeres). The percentage of good-quality embryos (grades 1 and 2) was similar in the study and control groups (71.7% and 78.5%, respectively). The percentage of poor-quality embryos (grade 4 and two pronuclei) was higher in the study group compared with the control group (13.9% versus 9.2%, respectively); however, the difference was not significant. The implantation rate and clinical PR were lower in the study group (3% and 11%, respectively) compared with the control group (9.5% and 44%, respectively), but the difference was not statistically significant. CONCLUSION(S): The fertilization rate and early embryonic cleavage of human embryos was found to be reduced significantly in patients with high levels of surface-bound antisperm antibodies. Moreover, embryonic quality and the PR may be compromised by the presence of significant levels of surface-bound antisperm antibodies.

Urine contamination of seminal fluid after transurethral resection of the ejaculatory ducts.

Transurethral resection of ejaculatory duct obstruction has assumed a significant role in the treatment of infertile men. The potential impact of disruption of the ejaculatory duct apparatus after transurethral resection has not been studied. The seminal plasma of patients was evaluated after transurethral resection of the ejaculatory ducts by determining creatinine levels as a measure of urine contamination of semen. Analysis of semen parameters was retrospectively performed on preoperative and postoperative samples in 8 subfertile men diagnosed with ejaculatory duct obstruction treated by transurethral resection. These were not 8 consecutive patients but rather individuals from a larger series who had seminal plasma frozen preoperatively and postoperatively. A significant increase in seminal plasma creatinine levels postoperatively was detected in 7 of 8 patients evaluated. In patients who were requested to produce 2 specimens within 1 hour high levels of creatinine were found in both ejaculates, although creatinine levels were lower in the second ejaculate. The patient who postoperatively had low levels of creatinine in seminal plasma demonstrated an improvement in sperm concentration and morphology, and his wife became pregnant. Transurethral resection of the ejaculatory ducts results in marked improvement in some semen parameters. However, the impact of urine contamination in semen after transurethral resection of the ejaculatory ducts must be assessed in the management of patients who present with ejaculatory duct obstruction.

Cystic fibrosis and congenital agenesis of the vas deferens, antisperm antibodies and CF-genotype.

Antisperm antibodies are formed as a result of vasal and epididymal obstruction. Fourteen males of different ages (pre-, peri- and post-pubertal) with bilateral congenital vasal agenesis and epididymal obstruction secondary to cystic fibrosis (CF), and seven men with congenital bilateral aplasia of the vas deferens (CBAVD) were evaluated with regard to both the presence and levels of serum antisperm antibodies, and the CF-genotype. While IgA and IgG were not detected among pre- and peri-pubertal CF patients, 4 out of 10 (40%) exhibited IgM binding to sperm tail-tip. Post-pubertal CF patients showed high antisperm antibody (ASA) levels in 3 of the 4 males (75%) evaluated for the three isotypes assayed. ASA were found in 5 of 7 CBAVD patients (71%); IgG (n = 3) and IgM (n = 4) were found to be the predominant isotypes bound to sperm tail-tip. CF-genotype analysis revealed two pre-pubertal patients with the DeltaF508/DeltaF508 CF-genotype and a positive ASA response, thus suggesting an earlier or more severe blockage. In addition, the two CBAVD patients found to have a ?/? CF-genotype on the initial screening did not have ASA. The altered antigenicity of sperm associated with initiation of spermatogenesis appears to modify the antisperm antibody isotypes. Further studies on a larger number of patients may allow for a better understanding of the ASA response, as well as a better understanding of a possible phenotype/genotype association between the CF-genotype and the immunologic response.

Molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin.

In the present study, molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin is described. The full-length cDNA encoding human proacrosin was utilized to recover a 17-kb human genomic clone which was sequenced without further subcloning. The nucleotide sequences of the exons agree with the sequence of the cDNA reported previously. More than 500 bases of the promoter region were sequenced and found to be highly GC rich but devoid of an identifiable TATA box. These findings are generally consistent with a recently published report [Keime, S., Adham, I. M. & Engel, W. (1990) Eur. J. Biochem. 190, 195-200]. However, further sequence analysis revealed discrepancies between our clone and that previously reported. Sequencing of the first intron showed similarity with the published data for 54 bases of the 5' region, beginning with the donor splice site, and for 114 bases at the 3' end. However, 500 bases sequenced distal to the initial 54 bases at the 5' end of intron 1 showed no similarity with the published sequence. In addition, the boundaries of intron 3 differed such that a cytosine residue previously reported to be in exon 3 was found to be the first base of exon 4. Detailed studies were undertaken to confirm that our clone constitutes the authentic sequence of human proacrosin. Cloning and characterization of the human proacrosin gene may allow for informative studies of its regulation, and for a more detailed examination of its role in fertilization.