Detection of a novel mutation in the testis-specific antizyme-3 gene
2
stages. Genes of interest included, DAZ, TSPY, Zona pellucida binding protein gene, Transition protein 1, Protamine 2, neural polypyrimidine tract binding protein gene, bromodomain (testis specific) and testis specific ankyrin motif-containing protein genes, and Synaptojanin. The analysis of each biopsy with respect to these spermatogenic stage-specific genes con- firmed the gene signatures findings and, in the vast majority of cases, correlated with testis biopsy histology. However, careful analysis of gene expression data did suggest that more advanced spermatogenic stages were present in biopsies classified with early maturation arrest phenotype. Conclusions: Based on the use of cDNA microarrays, it is possible to generate genetic signatures or “portraits” for individual testis biopsies as an alternative to routine histology. In general, such signatures correlate with standard morphologic patterns, but in some cases they suggest that more advanced spermatogenic stages exist than can be currently defined by morphology. These findings may have important implications in sperm detection for ICSI. Supported by: NIH (RAR); Lance Armstrong Foundation, California Urology Foundation (PJT, RAR). Monday, October 14, 2002 3:00 P.M. O-83 Sperm motility in men with spinal cord injury (SCI) is enhanced by inactivating cytokines in the seminal plasma. Josh M. Randall, Sarmistha Basu, Teodoro C. Aballa, Charles M. Lynne, Nancy L. Brackett. Univ of Miami Sch of Medicine, Miami, FL. Objective: Men with SCI have a unique sperm profile characterized by normal to high sperm concentrations and abnormally low sperm motility. The cause of this condition is unknown, but may be related to abnormalities in the seminal plasma. Their semen contains elevated concentrations of activated T cells (J Androl, in press) which are known to secrete cytotoxic cytokines. The cytokines TNF, IL1, and IL6 are elevated in semen of SCI men (unpublished data). To determine if elevated cytokines may be con- tributing to low sperm motility in men with SCI, the present study used monoclonal antibodies (mAb) to block cytokine activity in fresh semen. The resulting effect on sperm motility was determined. Design: In a within subjects design, sperm motility of each subject’s fresh untreated semen was compared to that of the same semen specimen treated with mAb to neutralize cytokine activity. Materials/Methods: Nine men with SCI participated in the study. The mean age (range 21 to 43 years) was 32.7 2.3 years ( standard error of the mean, SEM). The mean years post injury (range 3 to 32) was 11.9 3.1, and the level of injury was C5/6 in 2 subjects, T1 to T7 in 3 subjects, and T8 to T12 in 4 subjects. Each subject had undergone at least four ejacula- tions spaced 4 to 8 weeks apart prior to semen collection for this study. Specimens were collected by penile vibratory stimulation or electroejacu- lation. Each specimen was separated into eight 100 l aliquots. To these aliquots, mAb to TNF (A), IL1 (B), and IL6 (C) were added singly and in all possible combinations, so that the seven treatment groups were: A, B, C, AB, AC, BC, ABC. One aliquot remained untreated as a control. After a 1 hour incubation at room temperature, sperm motility was analyzed in each preparation. Sperm motility in untreated versus treated preparations was compared by analysis of variance. Results: Mean sperm motility increased in all treatment groups, however, the only group that reached statistical significance was the group receiving mAb against all three cytokines (ABC) (see table below). Group Untreated A B C AB AC BC ABC % motility 25.9 4.5 30.4 5.9 34.1 7.5 36.4 8.1 34.3 8.5 35.2 8.5 38.3 9.5 46.2 5.9 Signifi- cance NA NS NS NS NS NS NS p 0.02 % motility mean percent of motile sperm SEM. NS not significant. NA not applicable. Conclusions: Neutralization of specific cytokines enhances sperm motil- ity in semen of men with SCI. To the best of our knowledge this is the first intervention that significantly improves sperm motility in this group of severely impaired patients. Supported by: Grants to NLB from the Miami Project to Cure Paralysis and the State of Florida Specific Appropriations. Monday, October 14, 2002 3:45 P.M. O-84 Increased incidence of triplet repeat diseases expanded alleles in azoospermic men: A new concern for ICSI? Maria Rosa Maduro, Rob- erto Casella, Alex G. Smith, Dolores J. Lamb. Baylor Coll of Medicine, Houston, TX; Clin of Urology, Univ Hosp, Basel, Switzerland. Objective: Triplet repeat diseases (TRDs) are autosomal dominant neu- rodegenerative disorders, associated with abnormal expansion of polymor- phic CAG/CTG, CGG/CCG or GAA/TTC motifs embedded within coding and non-coding regions of specific genes. A feature of TRDs is anticipation, whereby the disease severity increases and the age of onset decreases from one generation to the next due to genomic instability in meiotic cells. Some infertile patients present genomic instability. Several TRDs, like Kennedy Disease (KD) and Myotonic Dystrophy (MD) are characterized by de- creased or abolished reproductive fitness; for others, this association has not been investigated, although animal models suggest this possibility. The objective of this study is to determine whether severe idiopathic male infertility patients exhibit meiotic instability with an increased incidence of TRD abnormal alleles. Design: A prospective controlled study of TRDs-associated loci repeat lengths in the somatic and germline tissues of non-obstructive azoospermic men. Men with obstructive azoospermia but normal histology served as controls. Statistical analysis was performed by a Cochran-Mantel-Haenszel test of differences between the severe infertile patients diagnosis (Sertoli cell only, maturation arrest, hypospermatogenesis) and the controls. Materials/Methods: DNA was extracted from paired blood and testis samples of 41 azoospermic patients and 20 controls and amplified by polymerase chain reaction for 6 loci associated with TRDs: KD, MD, Huntington Disease (HD) and Spinocerebellar Ataxia 1, 2 and 6 (SCA-1, SCA-2 and SCA-6, respectively). Triplet repeat lengths were determined by fragment analysis using an ABI 310 Analyzer and GeneScan software. Results: A significant increased incidence of total instability (94%) was observed between the paired blood and testis samples of Sertoli cell only (SCO) patients as compared to controls (p 0.08). When instability was present, repeat expansions were almost always observed in the patients testis, relative to their blood. Importantly, triplet repeat lengths above the minimum necessary to develop the TRD were observed in the somatic tissue of 5.3% of the SCO patients, but not the control group for the MD locus. Moreover, at the MD and SCA-2 loci, repeat lengths large enough to cause TRDs were observed in the testicular tissue of 15.8% and 5.3% of the SCO patients, as compared to 0% of the controls. Conclusions: SCO patients present a higher than normal incidence of genomic instability, with a propensity to repeat expansion in the testis at several TRDs loci. MD-associated alleles are present at a higher than normal frequency in the SCO patients. Thus, male infertility may be an early symptom of MD. A higher than expected percentage of patients seeking infertility evaluation may be mild-MD symptomatic carriers. A high per- centage of expanded MD and SCA-2 alleles are present in the testis of SCO men. As all patients were candidates for testicular sperm extraction and ICSI, there is concern that the offspring conceived may be at increased risk for MD and SCA-2. Supported by: Supported in part by NIH P01HD 36289 – 01 (to DJL), Swiss National Science Foundation (to RC), Roche Research Foundation (to RC and DJL). Monday, October 14, 2002 4:00 P.M. O-85 Detection of a novel mutation in the testis-specific antizyme-3 gene. Greg Christensen, Ivaylo Ivanov, John Atkins, Peter Schlegel, Anna Mielnik, Douglas Carrell. Univ of Utah Sch of Medicine, Salt Lake City, UT; Cornell Univ Sch of Medicine, New York, NY. Objective: Mutations in both the Y-linked and autosomal genes are known causes for male infertility in animals and humans. Ornithine decar- boxylase and spermine synthase are two genes in the polyamine biosynthe- sis pathway that produce male infertility in mice when knocked-out or S32 Abstracts Vol. 78, No. 3, Suppl. 1, September 2002
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Transcript of Detection of a novel mutation in the testis-specific antizyme-3 gene
stages. Genes of interest included, DAZ, TSPY, Zona pellucida bindingprotein gene, Transition protein 1, Protamine 2, neural polypyrimidine tractbinding protein gene, bromodomain (testis specific) and testis specificankyrin motif-containing protein genes, and Synaptojanin. The analysis ofeach biopsy with respect to these spermatogenic stage-specific genes con-firmed the gene signatures findings and, in the vast majority of cases,correlated with testis biopsy histology. However, careful analysis of geneexpression data did suggest that more advanced spermatogenic stages werepresent in biopsies classified with early maturation arrest phenotype.
Conclusions: Based on the use of cDNA microarrays, it is possible togenerate genetic signatures or “portraits” for individual testis biopsies as analternative to routine histology. In general, such signatures correlate withstandard morphologic patterns, but in some cases they suggest that moreadvanced spermatogenic stages exist than can be currently defined bymorphology. These findings may have important implications in spermdetection for ICSI.
Supported by: NIH (RAR); Lance Armstrong Foundation, CaliforniaUrology Foundation (PJT, RAR).
Monday, October 14, 20023:00 P.M.
O-83
Sperm motility in men with spinal cord injury (SCI) is enhanced byinactivating cytokines in the seminal plasma. Josh M. Randall, SarmisthaBasu, Teodoro C. Aballa, Charles M. Lynne, Nancy L. Brackett. Univ ofMiami Sch of Medicine, Miami, FL.
Objective: Men with SCI have a unique sperm profile characterized bynormal to high sperm concentrations and abnormally low sperm motility.The cause of this condition is unknown, but may be related to abnormalitiesin the seminal plasma. Their semen contains elevated concentrations ofactivated T cells (J Androl, in press) which are known to secrete cytotoxiccytokines. The cytokines TNF, IL1, and IL6 are elevated in semen of SCImen (unpublished data). To determine if elevated cytokines may be con-tributing to low sperm motility in men with SCI, the present study usedmonoclonal antibodies (mAb) to block cytokine activity in fresh semen. Theresulting effect on sperm motility was determined.
Design: In a within subjects design, sperm motility of each subject’s freshuntreated semen was compared to that of the same semen specimen treatedwith mAb to neutralize cytokine activity.
Materials/Methods: Nine men with SCI participated in the study. Themean age (range 21 to 43 years) was 32.7 � 2.3 years (� standard error ofthe mean, SEM). The mean years post injury (range 3 to 32) was 11.9 � 3.1,and the level of injury was C5/6 in 2 subjects, T1 to T7 in 3 subjects, andT8 to T12 in 4 subjects. Each subject had undergone at least four ejacula-tions spaced 4 to 8 weeks apart prior to semen collection for this study.Specimens were collected by penile vibratory stimulation or electroejacu-lation. Each specimen was separated into eight 100 l aliquots. To thesealiquots, mAb to TNF (A), IL1 (B), and IL6 (C) were added singly and inall possible combinations, so that the seven treatment groups were: A, B, C,A�B, A�C, B�C, A�B�C. One aliquot remained untreated as a control.After a 1 hour incubation at room temperature, sperm motility was analyzedin each preparation. Sperm motility in untreated versus treated preparationswas compared by analysis of variance.
Results: Mean sperm motility increased in all treatment groups, however,the only group that reached statistical significance was the group receivingmAb against all three cytokines (A�B�C) (see table below).
Group Untreated A B C A�B A�C B�C A�B�C
% motility 25.9 � 4.5 30.4 � 5.9 34.1 � 7.5 36.4 � 8.1 34.3 � 8.5 35.2 � 8.5 38.3 � 9.5 46.2 � 5.9
Signifi-cance
NA NS NS NS NS NS NS p� 0.02
% motility � mean percent of motile sperm � SEM. NS � not significant. NA � not applicable.
Conclusions: Neutralization of specific cytokines enhances sperm motil-ity in semen of men with SCI. To the best of our knowledge this is the firstintervention that significantly improves sperm motility in this group ofseverely impaired patients.
Supported by: Grants to NLB from the Miami Project to Cure Paralysisand the State of Florida Specific Appropriations.
Monday, October 14, 20023:45 P.M.
O-84
Increased incidence of triplet repeat diseases expanded alleles inazoospermic men: A new concern for ICSI? Maria Rosa Maduro, Rob-erto Casella, Alex G. Smith, Dolores J. Lamb. Baylor Coll of Medicine,Houston, TX; Clin of Urology, Univ Hosp, Basel, Switzerland.
Objective: Triplet repeat diseases (TRDs) are autosomal dominant neu-rodegenerative disorders, associated with abnormal expansion of polymor-phic CAG/CTG, CGG/CCG or GAA/TTC motifs embedded within codingand non-coding regions of specific genes. A feature of TRDs is anticipation,whereby the disease severity increases and the age of onset decreases fromone generation to the next due to genomic instability in meiotic cells. Someinfertile patients present genomic instability. Several TRDs, like KennedyDisease (KD) and Myotonic Dystrophy (MD) are characterized by de-creased or abolished reproductive fitness; for others, this association has notbeen investigated, although animal models suggest this possibility. Theobjective of this study is to determine whether severe idiopathic maleinfertility patients exhibit meiotic instability with an increased incidence ofTRD abnormal alleles.
Design: A prospective controlled study of TRDs-associated loci repeatlengths in the somatic and germline tissues of non-obstructive azoospermicmen. Men with obstructive azoospermia but normal histology served ascontrols. Statistical analysis was performed by a Cochran-Mantel-Haenszeltest of differences between the severe infertile patients diagnosis (Sertolicell only, maturation arrest, hypospermatogenesis) and the controls.
Materials/Methods: DNA was extracted from paired blood and testissamples of 41 azoospermic patients and 20 controls and amplified bypolymerase chain reaction for 6 loci associated with TRDs: KD, MD,Huntington Disease (HD) and Spinocerebellar Ataxia 1, 2 and 6 (SCA-1,SCA-2 and SCA-6, respectively). Triplet repeat lengths were determined byfragment analysis using an ABI 310 Analyzer and GeneScan software.
Results: A significant increased incidence of total instability (94%) wasobserved between the paired blood and testis samples of Sertoli cell only(SCO) patients as compared to controls (p �0.08). When instability waspresent, repeat expansions were almost always observed in the patientstestis, relative to their blood. Importantly, triplet repeat lengths above theminimum necessary to develop the TRD were observed in the somatic tissueof 5.3% of the SCO patients, but not the control group for the MD locus.Moreover, at the MD and SCA-2 loci, repeat lengths large enough to causeTRDs were observed in the testicular tissue of 15.8% and 5.3% of the SCOpatients, as compared to 0% of the controls.
Conclusions: SCO patients present a higher than normal incidence ofgenomic instability, with a propensity to repeat expansion in the testis atseveral TRDs loci. MD-associated alleles are present at a higher than normalfrequency in the SCO patients. Thus, male infertility may be an earlysymptom of MD. A higher than expected percentage of patients seekinginfertility evaluation may be mild-MD symptomatic carriers. A high per-centage of expanded MD and SCA-2 alleles are present in the testis of SCOmen. As all patients were candidates for testicular sperm extraction andICSI, there is concern that the offspring conceived may be at increased riskfor MD and SCA-2.
Supported by: Supported in part by NIH P01HD 36289–01 (to DJL),Swiss National Science Foundation (to RC), Roche Research Foundation(to RC and DJL).
Monday, October 14, 20024:00 P.M.
O-85
Detection of a novel mutation in the testis-specific antizyme-3 gene.Greg Christensen, Ivaylo Ivanov, John Atkins, Peter Schlegel, AnnaMielnik, Douglas Carrell. Univ of Utah Sch of Medicine, Salt Lake City,UT; Cornell Univ Sch of Medicine, New York, NY.
Objective: Mutations in both the Y-linked and autosomal genes areknown causes for male infertility in animals and humans. Ornithine decar-boxylase and spermine synthase are two genes in the polyamine biosynthe-sis pathway that produce male infertility in mice when knocked-out or
S32 Abstracts Vol. 78, No. 3, Suppl. 1, September 2002
altered. The purpose of this study was to evaluate another polyamineregulator, the testis-specific Antizyme-3 gene, for mutations in a group of200 azoospermic or severely oligozoospermic males.
Design: A prospective study in which the DNA from consenting, severelyoligozoospermic (less than 5 M/ml) or azoospermic patients was evaluatedby single strand conformation polymorphism (SSCP) analysis and directsequencing for Antizyme-3 mutations. Individuals who had been diagnosedwith any condition or treatment connected with infertility (eg., cysticfibrosis, Klinefelters, varicocele, chemotherapy, etc.) were not included.
Materials/Methods: A total of 250 general population controls and 200azoospermic or severely oligozoospermic males were screened for alter-ations of the Antizyme-3 gene. Five sets of PCR primers were designed tospan each of the five exons and promoter region of the gene. PCR reactionswere optimized to give clean, ample samples. Products from the PCRreactions were used in an SSCP analysis to detect potential polymorphismsor mutations and followed up with direct sequencing when an alteration wasdetected.
Results: No alterations were found in the 250 control individuals. TheSSCP analysis detected a single azoospermic patient from the 200 screenedwith an alteration in exon five. Direct sequencing of the exon confirmed amutation converting C to T and resulting in a change of Pro164 to Ser. Thepatient had a wild type allele in addition to the mutant allele and wasdetermined to be heterozygous for the Antizyme-3 locus.
Conclusions: The results indicate that a mutation of the Antizyme-3 geneis the likely cause for infertility in one of our study patients. To the best ofour knowledge, this is the first report of a non-endocrine, autosomal muta-tion responsible for human male infertility. These data also suggest thatmutations in the Antizyme-3 gene are rare, and not a common cause ofinfertility in humans. Many genes have been identified from animal modelsas male infertility candidates and global screening of these genes should beconducted to determine which are clinically relevant.
Supported by: N/A
Monday, October 14, 20024:15 P.M.
O-86
Male infertility on the internet. Michael P. Zahalsky, Stephanie K. Lam,Chistopher Diblasio, Harris M. Nagler. Beth Israel Medical Ctr, New York,NY.
Objective: The Internet has become a resource for couples searching forinformation on male infertility. This study analyzes the quality of theinformation on male infertility available to the layperson on the Internet.
Design: A prospective review of male infertility data on the Internet.Materials/Methods: The phrase “Male Infertility” was entered into five
Internet search engines: Yahoo, Google, Excite, MSN, and AOL. The first20 web pages within each search engine were analyzed by a panel (3physicians and one non-healthcare professional. Each web page was clas-sified according to the following characteristics: site ownership, targetaudience, and presence of information on varicoceles. The target audiencewas identified as healthcare professionals, laypersons, or both. When websites presented information regarding varicoceles, the attitude towards sur-gical treatment was evaluated. Reviewers assessed whether a web site wasprimarily advertising or informational. The credibility and quality of websites was assessed by a grading scale of six parameters published by theAmerican Medical Association (AMA) and by the Health On the NetFoundation (HON) Code of Conduct for medical and health web pages. Outof a total score of 6, web sites scoring 3 or less were considered to have poorcredibility.
Results: A total of 250,340 web pages were generated. One hundredpages were reviewed. 73 sites were considered relevant, 27 were excludedbased on our exclusion criteria. Yahoo returned the highest number ofrelevant web pages (100%) while AOL returned the lowest (55%). Theownership of the web sites are summarized in the table. Physicians/health-care professionals and commercial enterprises owned 69.8% of web pages.Only two web sites were owned by a medical society, while none wereowned by a government agency. 94.6% of web pages were targeted to thelayperson. 72.6% of web pages were deemed to be primarily advertise-ments. Only 17.8% listed clear references and sources for the informationprovided. 67.1% did not provide when their information had been updated.58.9% of the web pages had a poor credibility score of equal or less than 3.
Most of the pages (68.5%) posted information provided by qualified healthprofessionals or clearly stated if it was not given by a healthcare profes-sional. Only 3 web pages (4.1%) had received Health on the Net (HON)certification. No links to the AMA, American College of Obstetrics andGynecology, or American Urological Association web pages were listed onany of the search engine results.
Characteristics of the 73 relevant Web Pages.
Ownership Number Percentage
Nonprofit organization 3 4.1%Academic institution 11 15.1%Government Agency 0 0.0%Medical Society/Association 2 2.7%MD or Healthcare Professional 29 39.7%Commercial enterprise 22 30.1%Health Web site 4 5.5%Lay person 2 2.7%
Conclusions: There are no clear guidelines to monitor web pages, and thislack of regulation can lead users to inaccurate or incomplete information.Medical associations need to assume a more active role in the process ofcertifying health-related web sites and should provide more information andadequate references to guide the layperson searching the Internet.
Supported by: Male Infertility Research Fund. Department of Urology.I.W. Decamp Foundation.
SOCIETY FOR REPRODUCTIVE ENDOCRINOLOGYAND INFERTILITY
Monday, October 14, 20022:00 P.M.
O-87
Serum mullerian-inhibiting substance is a reliable predictor of theovarian follicular status. Renato Fanchin, Joelle Taieb, Luca Maria Scho-nauer, Ghada Hosny, Carlo Torrisi, Rene Frydman. Hosp Antoine Beclere,Clamart, France.
Objective: Mullerian-inhibiting substance (MIS) is a glycoprotein hor-mone that belongs to the transforming growth factor beta superfamily andis expressed by granulosa cells in adult women. It is possible that MIS playsan autocrine role on the physiology of FSH-sensitive follicles and itssecretion may reflect the activity of early antral follicles during the luteal-follicular transition.The present investigation was designed to characterizethe possible relationship between the number of early antral follicles andserum MIS levels and to weigh this relationship against that between earlyantral follicles and other putative markers of ovarian functioning andfertility potential on cycle day 3.
Design: Prospective study.
FERTILITY & STERILITY� S33
