P-356

Apoptosis in human ejaculated abnormal sperm cells. Ziskind Genia,Toubi Elias, Tal Joseph, Kessel Aharon, Ohel Gonen, Ilan Calderon. BnaiZion Medical Ctr, Haifa, Israel.

Introduction: Despite effort, little information is available on the biolog-ical significance of apoptosis in human spermatozoa or its possible role inmale infertility.

Objectives: 1) To determine whether apoptosis can be measured inejaculated spermatozoa by flow cytometry using the annexin V assay. 2) Tocompare apoptosis levels in the sperm population of infertile asthenotera-tozoospermic men with that of normal-fertile donors.

Materials and methods: The study group included ten asthenoteratozo-ospermic men who underwent ICSI. The control group consisted of tennormal-fertile donors. Sperm samples were assessed according to WHOstandards. Ejaculates from infertile men and fertile donors were examined30 min. and 4 hrs. after collection. The percentage of apoptosis in bothgroups was examined with annexin V staining (translocation of membranephosphatidylserine-PS).

Results: Apoptosis was measured in spermatozoa by flow cytometryusing the annexin V assay. Expression of PS on the outer leaflet of the cellmembrane was observed in all patients of the two groups studied. 3) In thecontrol group, the mean percentage of apoptotic sperm cells after 30minutes and 4 hours was 18.4% (� 3.9) and 20.9% (� 5.8) compared to32.6% (� 6.9) and 42.9% (� 10.6) in the study group (P�0.001). In thecontrol group, the mean percentage of dead sperm after 30 minutes and 4hours was 20.6% (� 3.7) and 22.6% (� 4.3), compared to 33.8% (� 8) and48.6% (� 11) in the study group (P�0.001).

Conclusions: 1) Spermatozoa from infertile asthenoteratozoospermic menand normal- fertile donors display translocation of membrane PS as diag-nosed by the annexin V assay. 2) Ejaculated spermatozoa from infertileasthenoteratozoospermic men had significantly higher percentage of apo-ptotic and dead cells compared to normal-fertile donors. 3) The annexin Vassay is valiable in identifing sperm population of poor functional compe-tence.

P-357

Anti-mullerian hormone as a spermatogenesis marker in non-obstruc-tive azoospermia? Emmanuelle Thibault, S. Mirallie, Y. Al Hussein, M.Jean, O. Bouchot, P. Barriere. Hopital Mere et Enfant, CHU, Nantes,France.

Introduction: Before surgical retrieval in non-obstructive azoospermia,presence of spermatozoa remains difficult to evaluate.

The Anti-Mullerian Hormone (AMH or MIS: Mullerian Inhibiting Sub-stance) is a glycoprotein specifically produced by Sertoli cells and involvedin regression of Mullerian ducts in male embryos.

In male adults, the role of AMH is still unknown. Since AMH valuesseem to be correlated with persistent spermatogenesis ( Fusijawa et al. 2002Hum. Reprod. Vol 17,968-970), this property could be used to evaluate theprobability to recover spermatozoa in testicular biopsies intended for non-obstructive azoospermic patients (Fenichel et al. 1999 Hum. Reprod. Vol14, 2020-2024).

Materials and Methods: Seminal AMH have been evaluated by Enzyme-Linked Immuno-Assay in seminal plasma (Beckman Coulter, Marseille.France) in 14 fertile donors (Group I), in 24 non- obstructive azoospermicpatients for whom a testicular biopsy has been performed (Group II) and in6 obstructive azoospermic patients (Group III).

Results:

Group II values have been compared with results of testicular biopsies :15 values were above the threshold of sensitivity (0.7 pmol/L) with 10correlated to positive biopsy (positive predictive value: 66.7%)and 9 valueswere found undetectable with 7 correlated to negative biopsy (negative

predictive value: 77.8%). As reported, sensibility and specificity of AMHlevels are respectively 83.3% and 58.3%.

Discussion and conclusion: AMH, in association with other parameterssuch as testicular volume, FSH, Inhibin B, Ymicrodeletions could allow toimprove predictive value of male assessment in non-obstructive azoosper-mia.These datas are actually confirmed by current increase of the serie wichcould be published in next monthes.

P-358

Effect of exogenous �-nicotinamide adenine dinucleotide phosphatereduced (NADPH) on Sperm DNA integrity. Tamer M. Said, Rakesh K.Sharma, Suresh C. Sikka, Anthony J. Thomas Jr., Ashok Agarwal. Cleve-land Clin Fdn, Cleveland, OH; Tulane Univ Medical Sch, New Orleans, LA.

Objective: Human spermatozoa have been shown to generate reactiveoxygen species (ROS) via oxidation of endogenous NADPH that is involvedin sperm capacitation. However, excessive ROS generation is also known tocause sperm DNA damage. The objective of our study was to detect the abilityof mature and immature spermatozoa to generate ROS in response to exoge-nous NADPH and to measure the extent of the subsequent DNA damage.

Design: Prospective-controlled study.Materials and Methods: Semen samples were collected from five donors

with normal standard semen parameters. Samples were divided into matureand immature fractions using Isolate double density gradient (90%, 47%).Each fraction was further subdivided into 2 subsets; the first was dividedinto 3 aliquots and incubated with 5 mM NADPH for 0, 3 and 24 hours. Thesecond subset was similarly divided and incubated without NADPH to serveas control. Sperm DNA damage and ROS levels were assessed usingterminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick endlabeling (TUNEL)-coupled flow cytometry and chemiluminescence assay (x106 counted photons per minute) using lucigenin as a probe.

Results: The median and interquartile values (25thand 75th percentiles) ofDNA damage are shown in the table. Significantly higher DNA damage (P�0.05) was seen in both NADPH treated sample fractions compared tocontrols only after 24 hours incubation. Levels of ROS in the maturefraction (90%) were significantly higher than controls (16.4 � 9.6 vs.2.36 � 3.8, P � 0.032) only after 24 hours of incubation, while in theimmature fraction (47%) ROS levels were significantly higher at both 3(1.45 � 1.15 vs. 0.4 � 0.3, P � 0.032) and 24 hours (18.38 � 13.46 vs.3.2 � 6.03, P � 0.032). DNA fragmented sperm showed a positive corre-lation with ROS levels (r � 0.54, P � 0.002) in samples incubated withNADPH for 24 hours.

*P value �0.05 considered significant in samples compared to controls.†P value �0.05 considered significant after incubation for 24 hours com-pared to 0 and 3 hours.

Conclusions: Our results indicate that NADPH may play a role in spermROS production and DNA fragmentation. ROS generation is triggeredearlier in the immature fraction but DNA damage takes longer time todevelop.

Supported by: Funds from The Cleveland Clinic Foundation.

P-359

Human sperm cryopreservation using sucrose as a cryoprotectantshows a promising future. Amjad Hossain, Nazrul Islam, Anthony Ca-ruso, Amos Madanes. The Midwest Fertility Ctr, Downers Grove, IL.

Objective: Glycerol in combination with egg yolk is by far the mostwidely used and successful cryoprotectant for human sperm. The cryopre-served sperm need to be separated from both glycerol and yolk before theycan be used in any in vitro fertilization procedure. Thus, the glycerol methodrequires rigorous post thaw washing which may lead to disappointing yield

FERTILITY & STERILITY� S239

particularly from severly oligozoospermic samples. The sucrose is known asa nonpenetrating cryoprotectant and is used in embryo freezing and thaw-ing. In this study we explored the feasibility of utilizing sucrose in spermcryopreservation.

Design: Sucrose was used in different concentrations to determine it’scryoprotecting capability and to simplify freeze-thaw techniques.

Materials and Methods: The surplus washed sperm (with � 95% motility)from IVF patient was used in the experiment. The concentration wasadjusted to 40 M and 5 M per ml in mHTF medium to represent normo-zoospermic and oligozoospermic samples, respectively. Three ul sperm ofeither concentrations was added to 50 ul of 500, 400, 300, 200, and 100 mMsucrose, and incubated for 5 min. Droplets of 10 ul size of each treatmentwere made on a 60 mm size dish lid (Falcon 1007) and immediately plungedinto liquid nitrogen. The dish was brought out of liquid nitrogen and thawedat either ambient temperature or at 37 C. Once thawing was complete (�5min), mHTF medium was added to each droplet so that the final sucroseconcentration become 5, 50, or 100 mM. The droplets were immediatelycovered with oil and incubated at 37 C. The sperm motility in the abovestated conditions were evaluated before freezing as well as 30 min andovernight post thaw incubation.

Results: The pre-freeze motility, by 5 min incubation in 500, 400, 300,200, and 100 mM sucrose was 0, 6, 20, 20, and 70%, respectively. The postthaw motility (10-15%) was documented only in 300, 200 and 100 mMconditions. Those survived exhibited forward progression with high motilitygrade ( grade 3-4). Motility (1-3 per field) was also observed in the samplesafter 20 hrs (overnight). The post thaw motility pattern was identical10-15%) in normozoospermic and oligozoospermic samples. Differenttypes of hypo-osmotic swellings (a-g types) were exhibited by the post thawsperm in all treatment conditions. Thawing temperetures (ambient or 37 C)did not influence the post thaw yield. The final concentration of sucrose inpost thaw samples varied from 5 to 100 mM, and did not show anycorrelation the with post thaw motility.

Conclusion: This pilot study indicates that sucrose alone can be used insperm cryopreservation at 100-300 mM concentration range. The apparentlow post thaw viability in sucrose technique (� 15%) hopefully could beimproved by adjusting and/ or modifying the protocol. A further study of thesucrose technique on actual semen sample is now in progress in ourlaboratory.

P-360

Effects of oxidative stress on human sperm activation. Edna E. Tirado,Rajendra Sharma, Dennis E. Sawyer, Yogesh C. Awasthi, David B. Brown.The Univ Texas Med Branch, Galveston, TX; The Univ of Newcastle,Newcastle, Australia.

Objective: In previous studies, the human sperm activation assay (HSAA)has been used to demonstrate that individuals who produce sperm withabnormal HSAA scores do not have successful ART attempts at pregnancy(Brown and Nagamani, Yale J Biol Med 65: 29, 1992; Brown et al., FertSteril 64: 612, 1995). An abnormal HSAA response is possibly a result ofsperm DNA/chromatin that has been damaged by oxidative stress and/orexposure to reproductive toxicant(s). In this study, we will determine ifsperm exposed to increasing concentrations of reactive oxygen species(ROS; hydrogen peroxide, H2O2) for 15 and 60 min, will damage the spermDNA/chromatin such that the treated sperm responds abnormally in theHSAA.

Design: H2O2 exposure time-dose response study.Materials and Methods: Semen was collected from a fertile male who had

previously been shown to produce sperm that responded normally in theHSAA. Sperm were isolated from the seminal plasma as described in theabove references. The sperm pellet was resuspended in phosphate bufferedsaline (PBS), pH � 7.4, and divided into 4 aliquots, then incubated at 37°Cin PBS with H2O2 concentrations of 0 �M, 10 �M, 50 �M, and 100 �M.Aliquots were taken at 15 and 60 min, each aliquot divided into 2 equalportions. The sperm from one portion was isolated and permeabilized, thenassayed in the HSAA as described in the above references. The other portionwas centrifuged and the supernatant was collected and the pellet wassonicated. Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) con-centrations in the supernatants and sonicated sperm samples were deter-mined by spectrophotometry.

Results: The HSAA 15 min time point showed an 8% increase in fullydecondensed sperm over that observed for the control in sperm treated for

60 min in 50 �M H2O2, and for 15 and 60 min in 100 �M H2O2. The lipidperoxidation measurements (MDAHNE) for the sonicated sperm samplesare shown in Table 1. There was no detectable increase in fully decondensedsperm over that observed for the control in the sperm exposed to 10 �M and50 �M H2O2 for 15 min, or for sperm exposed to 10 �M H2O2 for 60 min.At the HSAA 3 hour time point, for all exposure times and concentrations(with the exception of the sperm exposed to 100�M H2O2 for 60 min),100% of the sperm had recondensed. The sperm that had been exposed tothe highest concentration of H2O2 for the longest time had only 48% of thesperm recondense.

Conclusion: The preliminary data from this study indicate that humansperm exposed to high concentrations of H2O2 have a definite increase inlipid peroxidation and decondensation rates. The sperm exposed to 50 �MH2O2 (15 min and 60 min) and 100 �M H2O2 (15 min) recondensedcompletely despite an extensive increase in lipid peroxidation. Interestingly,the sperm exposed to 100 �M H2O2 for 60 min, that had maximal lipidperoxidation, had a dramatic decrease in the number of sperm that recon-densed. Further experiments will be performed to establish the significanceand clinical relevance of this data.

Supported by: CONACYT Mexico, Grant 90735.

P-361

Immunolocalization of Fertility-Associated Antigen (FAA) on bovine,equine, and human sperm. George R. Dawson, Tod C. McCauley, JaniceN. Oyarzo, Joseph S. Grossman, Sheldon H. F. Marks, Roy L. Ax. TheUniv of Arizona, Dept of Animal Science, Tucson, AZ; TMI Lab and TheUniv of Arizona, Dept of Animal Science, Tucson, AZ; The Univ ofArizona, Tucson, AZ; TMI Lab and Arizona Andrology Lab and Cryobank,Tucson, AZ; The Univ of Arizona, Dept of Animal Science and TMI Lab,Tucson, AZ.

Objective: Seminal heparin-binding proteins (HBP) mediate heparin in-duced capacitation of bovine sperm in vitro. One bovine HBP produced bythe accessory sex glands has homology to human deoxyribonuclease(DNase) I-like protein, and was “coined” fertility-associated antigen (FAA).Western blot detection of FAA on bovine sperm is associated with a 17%increase in fertility in natural and A.I. settings. The objective of thisexperiment was to localize FAA on bovine, equine and human ejaculatedspermatozoa, and human epididymal spermatozoa.

Design: A comparative analysis of immunolocalization of a fertilityrelated bovine sperm protein in bovine, equine and human sperm.

Materials and Methods: A 22 kDa recombinant FAA fragment wascloned, expressed and used as antigen to produce rabbit immune sera.Indirect immunofluorescence of sperm was performed using anti-recombi-nant FAA antisera. Bovine (n�6), equine (n�3), and human (n�4) semenwas centrifuged and washed to remove seminal plasma. Discard samples ofhuman cauda epididymal sperm were utilized following vasovasostomy.Spermatozoa were permeabilized in ethanol, mounted on slides and air-dried. Slides were washed in PBS, nonspecific binding sites were blockedwith 10% normal goat serum, and primary antibodies were added (1: 500)and incubated for 2 h at R.T.. After 3 washes in PBS, slides were incubatedwith FITC-conjugated goat anti-rabbit secondary antibodies (1: 160) for 45min. Preimmune sera and secondary antibodies alone served as controls.Slides were washed in PBS and anti-fade mounting medium was applied.Slides were analyzed with a Leica Diaplan microscope equipped for epi-fluorescence and images were captured with linked AlphaImager software.In addition, bull and stallion sperm were solubilized in Lammeli buffer,proteins separated by SDS-PAGE and Western blotting was performed withthe FAA antisera (1: 1000) following standard protocols.

Results: Immunoreactive FAA was detected in the acrosome directlyunderlying the plasma membrane of bull spermatozoa. Interestingly, adistinctly different pattern of immunolocalization was observed on humansperm. FAA localized specifically to the equatorial segment of ejaculatedhuman sperm. FAA was not detected on stallion sperm. Fluorescence wasundetectable in cauda epididymal human sperm, therefore the FAA epitope

S240 Abstracts Vol. 80, Suppl. 3, September 2003