O-289 Tuesday, October 15, 2013 05:00 PM

OUTCOMES OF PREWASHED VERSUS UNWASHED DONORSPERMSAMPLESWHENUSEDFOR INTRAUTERINE INSEMINA-TION (IUI). C. R. Juneau, S. R. Kassira, J. W. Edmonds, M. R. McLean,J. F. McLaren. Obstetrics and Gynecology, University of Alabama at Bir-mingham, Birmingham, AL.

OBJECTIVE: Donor sperm is commercially available in unwashed (ICI)and prewashed (IUI-ready) preparations. There is limited data regardingthe efficacy of on site processed ICI versus IUI-ready samples when usedfor intrauterine inseminations. Our hypothesis was that IUI-ready donorspecimens provide a higher total motile sperm count (TMSC) at the timeof IUI and a higher subsequent pregnancy rate.

DESIGN: This retrospective cohort study included all patients who under-went donor IUI from 2005-2012 at an academic REI practice.

MATERIALS AND METHODS: All patients aged 19-42 years who un-derwent donor IUI were identified and data abstracted on type of donor spermsample, age, indication, use of fertilitymedications, and other covariates. Theprimary outcome, TMSC at the time of IUI, was collected, as were the sec-ondary outcomes of chemical and clinical pregnancy rates and live birth rate.Parametric and non-parametric measures were used to analyze outcomes.

RESULTS: Of the total 353 donor IUI cycles, 71 (20%) were IUI-readysamples and 282 (80%) were ICI samples. Indication for therapy was malefactor (34%), same sex (39%) and single female (26%). The majority of cy-cles were performed with oral (73%) or injectable (14%) ovulation agents.The median TMSC was not significantly different between the IUI-ready(18x10^6) and ICI (15x10^6) preparations (p¼0.08). The incidence of aTMSC <10x10^6 was 11% for IUI-ready samples and 20% for ICIsamples (p¼0.09). The OR for a TMSC <10x10^6 was 0.51 (p¼0.10,95%CI: 0.23-1.13) for IUI-ready vs ICI samples. No difference in biochem-ical, clinical, or live birth rates were seen between the two groups.

CONCLUSION: IUI-ready donor specimens had a higher TMSC andlower incidence of TMSC <10x10^6 compared to ICI samples, althoughthe difference was not statistically significant in our study population. Itdoes not appear that IUI-ready specimens provide a significant benefit overICI specimens in centers where ICI samples can be processed post-thawfor IUI procedures.

O-290 Tuesday, October 15, 2013 05:15 PM

ENHANCEMENTOF SPERMKINEMATIC PERFORMANCEWITHB VITAMIN SUPPLEMENTATION. J. E. Adams,a M. A. Delaney,a

W. P. Gibbons,a G.M. Grunert,b E. Kovanci,a W.-S. A.Wun.b aReproductiveEndocrinology& Infertility, Baylor College ofMedicine, Houston, TX; bFer-tility Specialists of Houston, Houston, TX.

OBJECTIVE: B vitamins play an important role in mitochondrial func-tion, cellular metabolism, and the prevention of oxidative stress, whichmay benefit sperm function. Our objective was to investigate the effect of in-dividual B vitamin supplementation at varying concentrations on standardsperm kinematical parameters in gradient washed human sperm sampleswith normal or mildly abnormal baseline parameters.

DESIGN: Prospective cohort study.MATERIALS ANDMETHODS: Discarded pooled semen without identi-

fiable information was used for the study with exempt status IRB approval.Each sample was processed through a 40% and 80% isolate density gradient,washed, and divided. Thiamine (B1), Riboflavin (B2), Inositol (B3), andBiotin (B7) were individually added to the sperm samples at concentrationsof 10-10 M to 10-6M. Sample counts of at least 800 spermatozoa per slide foreach preparation and the control were then analyzed with the HamiltonThorne Computer Aided Semen Analyzer (CASA) for kinematical parame-ters at 1, 2, 4, and 20 hours. CASA analysis of motility, hyperactivation (HA),% rapid motility, curve linear velocity (VCL), amplitude of lateral headdisplacement (ALH), and linearity (LIN) were recorded. A mixed effectmodel was used for statistical analysis.

RESULTS: Overall, there was a significant improvement (p<0.05) inmotility at 20 hours with all 4 vitamins. HAwas also significantly increased(p<0.05) with B1 and B2 at the 2-4 hour time points. Significant increases inVCL and ALH, and decreases in LIN were noted in relation to HA. No toxiceffects were observed.

CONCLUSION: Supplementation of washed sperm with B vitaminsimproves long term motility and HA at roughly physiologic concentrationsof 10-9M to 10-7M, which may benefit use in intrauterine insemination(IUI)/ART. We plan a clinical trial of B vitamin enhanced semen preparationfor IUI.

FERTILITY & STERILITY�

O-291 Tuesday, October 15, 2013 05:30 PM

PRELIMINARY RESULTS OF A NEW METHOD OF VITRIFICA-TION OF SPERMATOZOA. D. Pab�on, M. Maseguer, D. Castell�o,P. Romero, A. Cobo, M. J. De los Santos. IVF Lab, IVI-Valencia, Valencia,Spain.

OBJECTIVE: Sperm vitrification appears as alternative method forimproving conventional freezing. New sperm vitrification studies have eval-uated the ability of sucrose to protect against damage sperm physicochemicalproduced by freezing. Therefore, we developed and evaluated a new methodof vitrification, using a combination of 0,25 M trehalose-sucrose (V) andcompared the results using a conventional methods for sperm cryopreserva-tion(CC).DESIGN: A prospective non-randomized study to compared the efficacy

of spermatozoa vitrification protocol respect to the conventional freezingprotocol.MATERIALS AND METHODS: The sperm vitrification solution was

0,25M trehalose-sucrose and plunge in liquid nitrogen directly in microdrop-lets of 10-20ml. A total of 25 semen samples were vitrified using the newmethod (V) and 25 samples were frozen by the conventional method contain-ing glycerol (CC). The survival rates and sperm mobility rates were calcu-lated before and after cryopreservation. Statistical analysis was performedusing ANOVA test and Chi square, p<0.02 were considered statistically sig-nificant.RESULTS: The survival rate was significantly higher when spermatozoa

were vitrified with 0,25 M trehalose-sucrose, compared with the survival us-ing the conventional method of freezing, V ¼ 77,60% (CI95%73,30-81,90)vs CC¼ 40,84% (CI95% 33,77-47.92).The progressive motility parameterswere significantly higher respect the conventional freezing (V ¼ 76,56%� 10,34 vs CC ¼ 47,86 � 12,77). (P< 0,02). The sperm vitality beforeand after vitrification was (88.92 � 6.24 vs 75.76 � 7.8) respectively.CONCLUSION: Our preliminary results show that this new vitrification

method can improve survival after warning sperm. A study designed to assessthe fertilizing capacity of sperm in ICSI and embryo quality is being evalu-ated to compare the two methods of sperm cryopreservation in parallel.

O-292 Tuesday, October 15, 2013 05:45 PM

DISTINCT EXPRESSION OF SPERMATOGENIC MARKERS INTESTICULAR BIOPSIES FROM AZOOSPERMIC PA-TIENTS. M. Huleihel,a M. Azab,a S. Kleiman,b R. Hauser,b

H. Yavetz,b E. Lunenfeld.c aMicrobiology, Immunology and Genetics,Ben-Gurion University of the Negev, Beer-Sheva, Israel; bInstitute for theStudy of Fertility, Lis Maternity Hospital, Tel Aviv, Israel; cDep. OB/GYNand IVF Unit, Soroka Medical Center, Beer-Sheva, Israel.

OBJECTIVE: To evaluate the expression of premeiotic, meiotic and post-meiotic cell markers in testicular biopsies of azoospermic patients.DESIGN: Biopsies from patients with histology of normal spermatogen-

esis, Sertoli only syndrome or incomplete maturation arrest were examined.MATERIALS AND METHODS: Testicular biopsies were defined ac-

cording to their histology as normal (n¼11), Sertoli Cell Only Syndromewith no sperm retrieved in TESE (SCOS; n¼13) and mixed atrophy withsperm retrieved (MA; n¼12). The study was performed after writteninformed consents obtained from the patients. The expression levels ofthe premeiotic (OCT4, GFR-a1, SALL4, CD9, a-6-INTEGRIN, C-KIT),meiotic (CREM-1), and postmeiotic (PROTAMINE) markers were exam-ined in the biopsies by real time PCR analysis (qPCR), using specificprimers for each marker.RESULTS: All patients with normal spermatogenesis expressed all the

premeiotic, meiotic and postmeiotic markers. Most of the patients (>84%)with SCOS expressed GFR-a1, SALL4, CD9 and a-6-INTEGRIN whichare known to be premiotic markers. However, only 54% expressed OCT4(premiotic marker). None of them expressed CREM-1(meiotic marker) andonly one expressed PROTAMINE (post meiotic marker). The expressionlevels of OCT4, GFR-a1, SALL4, and C-KIT were significantly lowercompared to normal (p<0.004). Most of the patients (>74%) with MA ex-pressed all the premeiotic and meiotic markers, but only 58% expressedPROTAMINE (post meiotic marker). Only C-KIT expression levels weresignificantly lower compared to normal (p<0.007).CONCLUSION: Our results clearly show the detection of different stages

of the spermatogenic cascade by markers expression and enlarge our under-standing of the spermatogenic process.

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