sought to investigate the chances of misjudging sperm morphology due toasymmetrical abnormalities or variable head positioning during fixation.

DESIGN: Prospective, randomized, double-blind study.MATERIALS AND METHODS: Standard morphology slides were pre-

pared using TestSimplets� and scored according to the 2010 WHO criteria.An aliquot of semen was added to polyvinylpyrrolidone, placed on a slide,analyzed at 600x, where a single motile spermatozoon was targeted and aten-second video was recorded. This was repeated for five spermatozoaand ten subsequent still images were obtained for each sperm at differentrotational plane. These 50 images were randomly displayed to a team of an-drologists who were asked to assess the presence or absence of sperm headirregularities. Inter-observer reliability was assessed by the intra-class corre-lation coefficient (ICC) using a two-way random effects model.

RESULTS: The semen sample used for the study had a volume of 4.5mL,concentration of 28million/mL, motility of 46%, and 2% normal morphology.Seven andrologists participated in the study. Despite the inter-observer concor-dance of the sperm images, there was overt discordance with respect to theangle of observation on whether the spermatozoon was normal or abnormal.This indicated that the various images of the same spermatozoon were scoreddifferently according to the presented position. From this we can extrapolatethe poor reliability of the standard bidimensional method particularly whenasymmetric abnormalities are present. The concordance of inter-observeragreement (ICC) ¼ 0.53 (95% CI¼0.21, 0.91) (P<0.0001) demonstratingpoor reliability of the morphological assessments of the same spermatozoon.

CONCLUSION: These results underscore the innate limitations and thehighly subjective nature of standard 2-D sperm staining and assessment.By keeping a spermatozoon in a fluid environment, our analysis is not subjectto staining and air-drying artifacts. Because spermatozoa are not sphericalbut have flattened surfaces, this observation highlights the chances offalse-negative interpretation with conventional staining.

O-293 Wednesday, October 22, 2014 12:15 PM

MULTILAYER DENSITY GRADIENT AS A USEFUL TECHNIQUEFOR SPERM SEX SORTING. P. Nicotra, H. Uriondo, E. Barrios,S. Papier, G. Fiszbajn, F. Nodar, C. Alvarez Sedo. CEGYR - Geneticsand Reproductive Medicine, Capital Federal, Buenos Aires, Argentina.

OBJECTIVE: Several studies have been carried out for sperm cell sortingin order to allow gender selection. Most of them were performed using flowcitometry considering the differences between spermDNA staining (X or Y).However, flow citometry technologies are expensive and it can have a cost-effectiveness impact over clinical treatments. Kaneko et al., (1983) have de-mostrated that performing a multilayer density gradient could improve spermgender selection in humans. Thus, the objective of this study was to describeour results on sperm gender selection based on centrifugation densitygradient without altering sperm characteristics.

DESIGN: Prospective cohort study.MATERIALS AND METHODS: This study included the analysis of 18

semen samples from men who underwent ART procedures. The inclusioncriteria were: age <45 y/o, sperm concentration> 5million/mL, and normalhormonal levels. Samples with severe teratozoospermia were excluded.Seminal parameters: volume, concentration, progressive motility andmorphology were taken into account for the analysis. All samples were pro-cessed by centrifugation density gradient consisting in four layers (90%,60%, 45%, and 20%). Fractions were collected after centrigutation for 25 mi-nutes at 300g. An aliquot from each fraction were fixed and spread on a slideglass, for subsequent sperm chromosomal analysis using FISH technique (18,X and Y). DNA fragmentation was evaluated by TUNNEL assay. Further-

Initial sperm parameters

N¼18 X�SD

Age 38.5�4.5Volume 3.1�0.9Concentration(millions/mL) 63.9�31.2Progressive motility 47.5�13.4Morphology 10.1�5.9Vitality 74.5�8.5DNA fragmentation 27.6�8.5Acrosome reaction 14.5�3.8X rate 50.5%�1.2%Y rate 49.5%�1.5%

e100 ASRM Abstracts

more, acrosomal reaction was assessed by immunocytochemistry using aspecific anti-acrosine antibody.RESULTS: Sperm parameters and variables evaluated are shown in table

1. When the pellet was analyzed, the proportion of X-bearing sperm was71%�3.1%, DNA fragmentation: 17.8�5.1% and acrosome reaction was:8.7�1.6%. When the gradient became lighter the proportion of Y-bearingsperm was: 76%�3.8%, DNA fragmentation: 16.1�7.2% and acrosome re-action was: 10.6�2.4%.

CONCLUSION: Multiple centrifugation density gradient allows the sep-aration of sperm bearing different sexual chromosomes. This is a preliminarystudy that needs more population to be completely to be sure about the effi-cacy of this method. In a future, this tool could be applied for gender selectionin ART in cases of prevention of X-linked diseases.

O-294 Wednesday, October 22, 2014 12:30 PM

EFFECT OF SPERM MATURITY AND ASSISTED OOCYTE ACTI-VATION ON MORPHOKINETICS OF EARLY HUMANEMBRYOS. T. Takeuchi,a Y. Mori,a Y. Nakajo,a,b N. Aono,a

T. Okuda,a K. Kyono.a,b aKyono ART Clinic Takanawa, Minatoku, Tokyo,Japan; bKyono ART Clinic, Sendai, Miyagi, Japan.

OBJECTIVE: ICSI with testicular sperm extraction (TESE) is a feasibletherapeutic option for azoospermic patients, however, testicular sperm haslower fertilizability owing to its immaturity. Assisted oocyte activation(AOA) is often used to treat activation failure after ICSI. The objective ofthis study was to assess the effect of sperm immaturity and induced oocyteactivation on early embryonic events by Time-lapse monitoring (TM).DESIGN: Comparative morphological assessment of ICSI zygotes.MATERIALS AND METHODS: A total of 74 patients comprised of 3

groups, 57 with ejaculated sperm (EJ), 10 with AOA, and 7 with TESE. Num-ber of zygotes in each group was 196, 48, and 65, respectively. For AOA, oo-cytes were briefly exposed to Ca ionophore after ICSI. Inseminated oocyteswere individually cultured in a TM system (EmbryoscopeTM). Images wereacquired every 10 min for up to 144 hrs. Zygotes were categorized as eitherthose which developed to blastocysts (BL), or those which did not (non-BL).Time points of each morphokinetic event, such as 2nd polar body extrusion(PBII), pronuclear (PN) appearance/disappearance, embryonic cell divisions,and intervals between cell cleavages were analyzed.RESULTS: Blastulation rates for EJ, AOA, and TESEwere 54.1, 41.7, and

44.6%, respectively, and all comparable. After transferring 59 EJ embryos 13(22.0%) implanted (IMP). In EJ, the time point for the 2-cell stage wasshorter in BL than in non-BL (P < 0.01), while the interval between the 2-and 3-cell stage was longer in BL (P < 0.01). Among replaced embryos,IMP zygotes developed to the 8-cell stage faster than their non-IMP (55.0� 6 vs. 61.1 � 8 hrs, P < 0.01). In both AOA and TESE, time points forPN disappearance, the 2-, and 4-cell stage were shorter in BL than in non-BL (P < 0.05), while intervals between the 2- and 3-cell stage in AOA,and between the 4- and 5-cell stage in TESE were longer in BL than innon-BL (P < 0.05). Overall, in comparison to EJ zygotes, TESE extrudedPBII more slowly (P< 0.05), while AOA developed faster to the 4-cell stage(P < 0.05).CONCLUSION: Faster and more synchronous cleavage was associated

with higher developmental potential of zygotes irrespective of sperm originand activation methods. Although the blastocyst formation rate was similar,sperm immaturity appeared responsible for slower first cleavage, and chem-ically induced activation accelerated early cleavage.

O-295 Wednesday, October 22, 2014 12:45 PM

NEXT GENERATION BISULFITE SEQUENCING REVEALSCONSISTENT POPULATION-WIDE REGIONAL SPERM DNAMETHYLATION ALTERATIONS WITH AGE. T. G. Jenkins,a

K. I. Aston,a C. Pfluger,b B. R. Cairns,b D. T. Carrell.a aAndrology andIVF Laboratories, University of Utah, Salt Lake City, UT; bDepartment ofOncological Sciences, University of Utah, Salt Lake City, UT.

OBJECTIVE: To confirm, and further describe DNA methylation alter-ations that are common in sperm as a result of aging. Our objective was tobetter understand the cell population dynamics of DNA methylation alter-ations associated with aging.DESIGN: Samples from our general population tissue bank were selected

based on age alone for a comparison of ‘‘young’’ and ‘‘aged’’ sperm DNAmethylation profiles. A total of 19 aged (>45 years of age) and 47 young(<25 years of age) samples were selected for this analysis.

Vol. 102, No. 3, Supplement, September 2014