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recurrent abortion was common had a higher incidence of sperm chromo-somal abnormalities. Men with compromised semen parameters presentedwith higher levels of gonosomal disomy and meiotic arrest. In group B, theabsence of a chromosomal sperm defect, at least for the probes utilized,indicated that this was not the sole responsible factor for the repeatedimplantation failure. In all, this data suggests that a sperm aneuploidy assaymay be useful adjunct in the screening of infertile men undergoing ART.

Supported by: Institutional.

MALE REPRODUCTION AND UROLOGY

Wednesday, October 25, 20063:00 pm

O-246

IDEAL CULTURE TIME FOR IMPROVEMENT IN SPERM MO-

TILITY FROM TESTICULAR ASPIRATES OF AZOOSPERMIC

MEN. D. S. Morris, D. E. Davis, R. L. Dunn, T. G. Schuster, D. A. Ohl,G. D. Smith. Univ of Michigan, Ann Arbor, MI.

OBJECTIVE: Motility of testicular-derived spermatozoa reflects viabilityand predicts success with intracytoplasmic sperm injection. Although im-provements in motility are seen when incubated in media for extendedperiods, no guidelines suggest duration and culture usage for optimalimprovement in motility. We sought to characterize improvement in motil-ity for sperm isolated by testicular aspiration by incubation in differentmedia over time.

DESIGN: Prospective, side-by-side comparative laboratory analysis.MATERIALS AND METHODS: Diagnostic and therapeutic fine needle

testicular aspirations were performed on 47 azoospermic men. The numberof non-motile and motile sperm per 20 high-powered fields (HPF) wereassessed at initial collection and following 24 or 48 hours of incubation ineither processing media (PM human tubal fluid HEPES buffer gentamicin human serum albumin; Irvine Scientific) or Hams F10 albumin (F10; Irvine Scientific). Using the number of motile sperm and thetotal number of sperm, the motility was calculated as a percentage. A mixedregression model controlling for side, media, and baseline motility wascreated to analyze the change in motility between 24h and 48h.

RESULTS: Initial motility was 1.1 3.7% (mean SD). Motilityimproved in PM media to 8.7 17.6% at 24h and 13.7 27.6% at 48h.Likewise, motility improved in F-10 media to 11.4 21.3% at 24h and16.3 29.2% at 48h. The improvement in motility from 24 to 48 hours wassignificant (p0.027). The mean change from 24h to 48h was 4.8 12.7sperm/20 HPF. A descriptive analysis showed that 45% of samples in-creased from 24h to 48h while 21% declined and 34% were unchanged.These results were confirmed when limiting analysis to samples withnon-zero motility at 24h, i.e. those with motility at 24h continued to showsignificant improvement at 48h. There was no significant difference be-tween PM and F10 media for improvement in motility between 24 and 48hours (p0.45).

CONCLUSION: Incubation in PM or F10 media improves sperm motilitywith significant improvement noted between 24 and 48 hours. These resultssuggest the ideal timing of oocyte retrieval for ICSI may correlate with 48hour sperm incubation. Additionally, the finding of decreased motility insome specimens from 24 to 48 hours raises the potential for offeringdiagnostic aspiration and culture prior to ICSI to individualize the labora-tory management of testis tissue.

Supported by: None.

Wednesday, October 25, 20063:15 pm

O-247

SELF-ASSEMBLY OF CABYR BY OLIGOMERIZATION AND ITS

BINDING WITH ROPPORIN AND AKAP3 IN HUMAN SPERM

FIBROUS SHEATH. Y. Li, W. He, A. Mandal, Y. Kim, J. Herr. Centerfor Research in Contraceptive and Reproductive Health, Dept of CellBiology, Univ of Virginia, Charlottesville, VA.

OBJECTIVE: Calcium-binding tyrosine phosphorylation-regulated pro-tein (CABYR) is a highly polymorphic, calcium-binding tyrosine, as well as

serine/threonine, phosphorylated fibrous sheath protein involved in capac-itation. Six splice variants of human CABYR have been reported, involvingtwo coding regions, CR-A and CR-B. A putative domain homologous to theRII dimerization and AKAP-binding domains of PKA at the N-terminus ofCRA suggests that CABYR may self-assemble and bind to AKAPs, how-ever, no direct evidence has yet confirmed this speculation and no bindingpartners of CABYR have been reported. The objective of this study was totest the hypotheses that 1) CABYR variants oligomerize and 2) CABYRbinds other sperm proteins.

DESIGN: To identify CABYR oligomers, reduced and non-reducedsperm extracts were compared by diagonal 2-D gel electrophoresis and

immunoblotted with sera to CABYR variants. Immunoprecipitates withimmune and pre-immune sera to CABYR variants were compared by 2-Dgel analysis and unique spots microsequenced to identify binding proteins.

MATERIALS AND METHODS: Celis or CSH extracts of swim-upsperm were separated by diagonal or blue native/SDS-PAGE. Immunoblot-ting employed rat anti-sera and mouse mAbs to rec-CABYR-coding regionA or B. Roche IP buffer [soluble proteins] or a dialyzed Celis extract[insoluble proteins] were immunoprecipitated with the above antibodies,analyzed on 2D gel, unique spots were cored and MALDI microsequenced.

RESULTS: The most abundant CABYR isoforms in sperm containedonly CRA, and included the 86KDa, calcium binding form [Dev. Biol.286:46-56]. CABYR variants containing both CRA and CRB, or CRB-onlywere identified. The predominant heterodimer at 130KDa in non-reducingconditions was composed of a 86KDa CRA variant and a 50KDa CRA andCRB-containing variant. A principal oligomer of 200-220KDa in non-reducing conditions dissociated into 130KDa86KDa or 86KDa50KDa

isoforms upon reduction. This indicated that the 200KDa complex is atrimer composed of two 86KDa and one 50KDa variant or an oligomercontaining 86KDa, 50KDa variants and another protein. Additional highermolecular weight oligomers were also noted in non-reducing and nativeconditions. These data support the model that CABYR forms heterodimersand trimers that further oligomerize in the assembly of the supermolecularstructure of the fibrous sheath. AKAP3 was coimmunoprecipitated withCABYR using anti-CRA polyclonal antibody, and conversely, CABYR wascoimmunoprecipitated with AKAP3 using anti-AKAP3 polyclonal anti-body. The coimmunoprecipitated CABYR contained not only the CRA-onlyvariant but also some CRB-containing variants. The CRA-only variant wasalso coimmunoprecipitated with CRB-containing variants by anti-CRBpolyclonal antibody. This confirmed the former results that differentCABYR variants participate in the assembly of the supermolecular structureof fibrous sheath by oligomerization. Co-IP and mass spectrometry showedthat ropporin, another RII-like domain containing protein, also coimmuno-

precipitated with CABYR, indicating ropporin is a CABYR binding partner.This is the first demonstration that CABYR binds with another RII-likedomain containing protein.

CONCLUSION: CABYR variants form oligomers and complex withropporin as well as the scaffolding protein AKAP3 in the fibrous sheath.

Supported by: D43 TW/HD 000654 from the Fogarty International Cen-ter, NIH.

Wednesday, October 25, 20063:30 pm

O-248

REPEAT TESTICULAR SPERM EXTRACTION (TESE): OUT-

COME, CLINICAL CORRELATES AND COMPLICATIONS OF

DIFFERENT INTERVALS BETWEEN FIRST AND SUBSEQUENTTESE. H. M. El-Fakahany, A. Nisbet, S. C. Honig. Dermatology, STDs&Andrology Deaprtment, Al-Minya Faculty of Medicine, Al-Minya, Egypt;Urology Dept, Univ of Connecticut School of Medicine, Farmington, CT;Urology Center, New Haven, CT.

OBJECTIVE: The introduction of ICSI with testicular sperm extraction(TESE) has changed the treatment options for azoospermic men whether itis obstructive (OA) or non-obstructive (NOA). While in OA, sperm can besurgically retrieved in 100% of cases, in NOA, sperm can be retrieved inonly 50% (Tournaye et al, 1997), as these patients may have only small fociof active spermatogenesis (Amer et al, 1999). Few studies had been done toevaluate repeated testicular sperm extraction. The objective of our study isto assess whether the intervals between repeated TESE procedures for NOApatients affect the outcome and/or the complications.

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DESIGN: Retrospective chart review of patients with NOA from a singlemale reproductive surgeon.

MATERIALS AND METHODS: Retrospective chart review was per-formed on all patients undergoing TESE/ICSI over a 7-year period. Patientswere included if they had evidence of non-obstructive azoospermia docu-mented with several centrifuged semen analysis. Patients were excluded iftestis histology and/or TESE findings were consistent with normal spermat-ogenesis. 107 patients charts were reviewed and all patients who weresubjected to at least two TESE procedures in our clinic were included. 21patients met the inclusion criteria and clinical data, surgical outcome,intervals between procedures, and complications were analyzed. All patients

underwent open TESE without microdissection.RESULTS: Of the 21 patients, 13 had two TESE, 7 patients had three

TESE, and 1 patient had 4 TESE procedures. Interval between TESEprocedures ranged from two months to ten years. 90.5% (19/21 pts) of ptshad sperm seen on the initial TESE. Of patients with a successful firstTESE, 89% (17/19 pts) had a successful second TESE. Of pts undergoinga third TESE, 86% (6/7pts) had sperm retrieved and 100% of patientsundergoing 4 TESE had sperm retrieved (1/1). There was no correlationbetween success of repeated TESE and testis size or FSH levels. Intervalsbetween procedures did not affect the outcome of the second TESE if thefirst TESE was successful. When using one-year or less interval betweenprocedures with the first TESE being successful, the success rate of secondTESE was 92.3% (one year) and 83.3% (one year) respectively. Of ptsundergoing repeat TESE less than 6 months from previous, all retrievals(5/5 pts) were successful. Post-operative complications such as hematomaor prolonged pain were rare and no difference was seen with differing

intervals.CONCLUSION: Repeat TESE is a safe and highly efficacious procedure

in patients with NOA. Different intervals between procedures appear not toaffect sperm retrieval rates if the first procedure was successful. Even shortintervals of less than 6 months between TESE show high success rates

Supported by: None.

Wednesday, October 25, 20063:45 pm

O-249

THE RISK OF INTRACYTOPLASMIC SPERM/SPERMATID IN-

JECTION FOR NON-MOSAIC KLINEFELTER SYNDROME PA-

TIENTS. A. Tanaka, I. Tanaka, M. Nagayoshi, S. Awata, N. Himeno, H.

Kusunoki. Saint Mother Hospital, Kitakyusyu, Japan.

OBJECTIVE: Previously published results of sperm analysis using mul-ticolor FISH in XXY indicated the presence, in most cases, of an increasein the proportion of abnormal XY and XX spermatozoa in XXY males. Ithas been reported that the sex chromosome abnormality incidence rate forICSI children increased when Klinefelter syndrome patient sperm had beenused. We conducted this study to investigate the mechanism of germ-cellmosaics.

DESIGN: Chromosomal FISH analysis of spermatogenic cells found innon-mosaic Klinefelter syndrome patients.

MATERIALS AND METHODS: This study dealt with 35 men who hadpreviously been diagnosed as having non-mosaic Klinefelter syndrome. In5 patients, spermatozoa were found, however these were not examinable byFISH analysis due to their immotility and/or head deformation therefore weperformed FISH analysis on these 5 patients spermatogenic cells. Four

different sides of each testis were biopsied. Biopsied tissues were washed inRBC lysing buffer for hemolysis and cut into pieces using hand-heldscissors at 4oC in D-PBS containing 0.125% collagenase (Type IA-S) and0.01% DNase. This mixture was incubated in a shaky water bath at 32.5oCfor 20 min, after which the supernatant and cells were removed andresuspended in D-PBS containing 0.125% collagenase (Type IA-S) and0.01% DNase for further incubation in a shaky water bath for 10 min. Thespermatogenic cell suspension was washed three times in PBS (Ca,Mg free), centrifuged at 280g for 10 min. Spermatogonia, primaryspermatocyte and spermatid are selected respectively following their pecu-liar cellular characteristics and then fixed in the droplet on slide (methanol: acetic acid : water 3:1:4 ).After fixation, the slides were rinsed withD-PBS, then with Milli Q water, and finally dehydrated. Double target FISHwas performed using directly labeled DNA probes specific for chromo-somes X and Y (Vysis, CEP DNA probe). The probe mixture was added to

the slide under a coverslip and the nuclear DNA and probe DNA weresimultaneously denatured for 5 min at 75oC. The slides were incubated in achamber (Hybrite, Vysis) at 42 oC for 120 minutes to allow hybridization,and then counterstained in DAPI.

RESULTS: The percentage of XY/XXY in the spermatogonia was 69.1%(159/230), and 30.9% (71/230) respectively. All primary spermatocyteswere XY, all spermatid chromosomes were haploid, and both X and Y-chromosomes appeared almost equal in frequency (X: Y 48.3%: 51.7%).FISH Analysis for Each Spermatogenic Cells Stage in Non-mosaicKlinefelter Syndrome Patients.

CONCLUSION: Our data indicates that Klinefelter syndrome males whoproduced spermatozoa in any numbers were XY / XXY mosaics and thatXXY cells were unable to enter meiosis but normal spermatogenesis wouldoccur in the XY line. Although the mechanism of diploid spermatozoaproduction is still unknown, the risk of intracytoplasmic sperm or spermatidinjection into oocytes may be lower than previously expected.

Supported by: None.

Wednesday, October 25, 20064:00 pm

O-250

THE PROTECTIVE EFFECTS OF A HEAT SHOCK RESPONSE

AMONG INFERTILE MEN WITH VARICOCELES. S. H. Benoff,I. R. Hurley, L. Yuan, H. Xu, J. L. Marmar. The Feinstein Institute forMedical Research, Manhasset, NY; Robert Wood Johnson Medical Schoolat Camden, Camden, NJ.

OBJECTIVE: Varicoceles cause treatable male infertility, but theirpathophysiology remains unclear. Currently, reflux from varicoceles isbelieved to raise testes temperature in association with androgen depletion,elevated nitric oxide levels and oxidative stress. Increased germ cell apo-ptosis occurs, leading to abnormal spermatogenesis and decreased spermdensity. Elevated testes cadmium (Cd) acts independently, correlating withincreased Fas ligand (FasL), germ cell apoptosis and low sperm density. Inanimal models, Cd elevates nitric oxide. Heat shock proteins (e.g. HSP70-1)may protect testis from high temperature, but heat shock response has notbeen investigated. The goal of this study was to determine if heat shockresponse antagonized other factors noted in testis biopsies from men with

varicoceles.DESIGN: Testis biopsies were sorted into 2 groups ( heat-inducible

HSP70-1 mRNA) to determine whether expression of Leydig cell cyto-chrome P450 side chain cleavage enzyme [SCC], FasL, inducible nitricoxide synthase (iNOS), endothelial NOS (eNOS), germ cell apoptosis andsperm density correlated with heat shock response.

MATERIALS AND METHODS: Testes were biopsied by ultrasonically-guided single-stick percutaneous aspiration at varicocelectomy (17 leftvaricocele; 28 bilateral varicoceles) or at sperm retrieval for ICSI (4 priorvasectomy/normal spermatogenesis controls) and formalin fixed. HSP70-1mRNA was assessed by RT-PCR against constitutively expressed HSP70-HOM control. Cd was determined by atomic absorption. Apoptosis wasquantified by TUNEL in paraffin sections and iNOS, eNOS, SCC, and FasLexpression were assessed by 2 blinded observers scoring antibody stainingon a scale of 0-4. Data was analyzed using SigmaStat v.3.0 and t-tests (t) or

S106 Abstracts Vol. 86, Suppl 2, September 2006