sperm death after fertilization on conventional ivf can be a good prediction of ivf outcomes

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DESIGN: Retrospective study. MATERIALS AND METHODS: All fresh, non-donor in conventional IVF without ICSI cycles performed from January 2012 to December 2013 (n¼838) were included in this study. The patients were divided into two groups: Study group (A group): A total of 115 patients underwent subnormal sperm count (R5 and <20 106/mL). Control group (B group): A total of 723 patients underwent normal sperm count R20 106/mL). Both groups became normal count after sperm treatment. RESULTS: Patient characteristics were similar between two groups. In each group, age (A group: 35.33.6; B group: 35.74.0 P¼0.73), basal FSH (7.42.6; 7.33.7mIU/ml P¼0.76), duration of rFSH (9.02.3; 9.13.2day P¼0.81), and endometrial thickness (10.12.0; 10.12.1mm P¼0.41) did not show significant differences. There were no significant dif- ferences between two groups in the number of MII oocytes (10.86.6; 10.16.2 P¼0.93), fertilization rate (85.2%; 88.8% p¼0.64), pregnancy rate (57.4%; 54.8% P¼0.85), and ongoing pregnancy rate (49.6%; 49.2% P¼0.70). CONCLUSION: Subnormal sperm group could obtain the same fertiliza- tion rate and pregnancy rate as those of normal group in conventional IVF without ICSI. Generally, ICSI is carried out for subnormal sperm because the possibility of fertilization may decline. But the safety of ICSI has not been proved yet and according to our results, ICSI was not necessarily needed in case of subnormal sperm. Therefore, it seems that conventional IVF without ICSI is sufficient as a fertilization method for subnormal sperm. P-189 Tuesday, October 21, 2014 MEN WITH A COMPLETE ABSENCE OF NORMAL FORMS (0%) ON STRICT MORPHOLOGY EXHIBIT HIGH RATES OF SUCCESS WITHOUT IN VITRO FERTILIZATION. J. R. Kovac, a R. P. Smith, b M. Cajipe, c J. Scovell, c R. Ramasamy, c J. Dupree, c G. Langille, c D. J. Lamb, c L. I. Lipshultz. c a Urology of Indiana, Carmel, IN; b University of Virginia, Charlottesville, VA; c Baylor College of Medicine, Houston, TX. OBJECTIVE: In couples with infertility, an abnormal strict morphology of 0% normal forms (NF) is often used as a criterion to proceed rapidly to in- vitro fertilization (IVF). A paucity of data exists examining reproductive suc- cess for these couples outside of IVF. We investigated the outcomes of men with 0% NF to determine success of natural conception (NC) & intra-uterine insemination (IUI) compared to IVF. DESIGN: Cohort study on patients from a high volume, tertiary, infertility clinic from 2010-13. MATERIALS AND METHODS: A total of 24 men with 0% NF (strict Kruger criteria) were identified with 18 randomly selected men >4% NF used as controls. Patient charts were reviewed & men contacted & administered an IRB-approved telephone questionnaire to ascertain outcomes. RESULTS: Average age of men & spouses in the 0% NF & control cohorts was not significantly different. Men with 0% NF revealed normal levels of LH, FSH, testosterone & estradiol. As expected, FSH was significantly higher in men with 0% NF (p¼0.04). Semen analysis demonstrated similar volumes in men with 0% NF compared to controls while forward progression (20.1 vs. 2.40.1, p¼0.0003), density (71.4 vs. 507, p<0.0001) & total motile counts (71.8 vs. 599, p<0.0001) were different. Of those men with 0% NF, 42% (n¼10/24) achieved pregnancy. A total of 29% (n¼7/24) did not require IVF (NC¼25%, n¼6/24; IUI¼4.2%, n¼1/24). IVF was used in 12.5% (n¼3/24). Controls achieved pregnancy on 78% of occasions (n¼14/18) of which 72% (n¼13/18) did not require IVF (NC¼67%, n¼12/18; IUI¼5.5%, n¼1/18). IVF was used in 22% of control men (n¼4/ 18). Only one case was for primary infertility (5.5%) while 3 were for sec- ondary infertility following a natural birth (17%). In men with 0% NF & the first pregnancy was conceived without IVF (n¼7), 100% had success on subsequent occasions (n¼7/7). In control men with sequential births where the first pregnancy was conceived without IVF (n¼6), 50% (n¼3/6) went on to require IVF with subsequent pregnancies. CONCLUSION: Men with 0% NF conceived without IVF in 29% of cases. Control men had success 72% of the time. Strict morphology provides impor- tant information about the process of spermatogenesis but should not be used to predict fertilization potential. In men with severe teratozoospermia & where maternal age allows, alternative modalities may be considered prior to immediate IVF. Supported by: JRK is Supported by a Male Reproductive Health Research (MHRH) Career Development Physician Scientist Award (K12) (HD073917- 01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program to DJL. P-190 Tuesday, October 21, 2014 THE EFFECTS OF SPERM CONCENTRATION ON IN VITRO EM- BRYO DEVELOPMENT. B. Tilley, a G. Navarrete, a S. Chantilis, a,b K. Lee, a,b M. Thomas, a,b R. Gada, a,b S. Purcell, a M. Meintjes. a,c a Dallas Fertility Center, Dallas, TX; b Dallas-Ft. Worth Fertility Associates, Dallas, TX; c Frisco Institute for Reproductive Medicine, Frisco, TX. OBJECTIVE: To determine if decreased sperm concentration used in con- ventional insemination would affect normal and abnormal fertilization rate or subsequent in vitro embryo development. DESIGN: Prospective sibling oocyte study. MATERIALS AND METHODS: Patients were< 41 years old or using donor oocytes, had R 8 oocytes retrieved, <2 previous cycles, and used fresh ejaculated sperm. Oocytes were divided equally into four different concen- trations of motile sperm: (A) 0.15x10 ^ 6/mL, (B) 0.10x10 ^ 6/mL, (C) 0.075x10 ^ 6/mL, and (D) 0.05x10 ^ 6/mL. Odd numbers of oocytes went to the greatest sperm concentration first. Sperm was added 6 hours post retrieval and fertilization assessed 18 h later. Fertilization took place in 200ul drops of P-1 + 5mg/mL HSA (Irvine) and embryos were cultured in G1/G2 (Vitrolife) + 10% SSS (Irvine) all under oil in humidified 6%CO2 and 5%O2. Normal and abnormal fertilization, day 5 blastocyst formation per 2PN, number of freeze quality blastocysts per 2PN, and number transferred per 2PN were analyzed by ANOVA. Differences between treatments were assessed using Fisher’s LSD. Odds of obtaining low fertilization, defined as <50% 2PN, were calculated using logistic regression. RESULTS: A total of 533 oocytes were retrieved from 25 patients. Overall normal and abnormal fertilization rates were 73.7 and 14.3%; respectively. Overall, implantation and clinical pregnancy rates were 57.5% and 62.5%; respectively, with an average number of embryos transferred of 1.7. The odds of insemination resulting in low fertilization were not increased by decreasing sperm concentration. Treatment group means SE are shown in the table below. No significant differences between treatment groups were observed for any variable. Treatment Oocytes 2PN Abnormal Blastocyts ET Cryo A 144 74.85.0 16.44.4 48.56.4 18.44.9 13.44.4 B 137 79.35.0 10.53.1 52.26.5 11.43.2 25.35.6 C 130 68.55.1 16.13.9 41.73.9 11.54.7 13.34.9 D 122 72.35.7 14.25.7 43.45.7 11.94.8 17.34.2 CONCLUSION: Decreasing sperm concentration to only 50,000 motile sperm/mL for conventional insemination does not significantly affect normal or abnormal fertilization. However, decreased sperm concentration did not result in any improvement in embryo development either. P-191 Tuesday, October 21, 2014 SPERM DEATH AFTER FERTILIZATION ON CONVENTIONAL IVF CAN BE A GOOD PREDICTION OF IVF OUTCOMES. S. J. Kwak, I. H. Park, K. H. Lee, S. G. Kim, H. G. Sun, Y. Y. Kim, J. H. Lee, J. Y. Park. Infertility Lab., Mamapapa&- Baby Obstetrics and Gynecology, Ulsan, Republic of Korea. OBJECTIVE: Live and motile sperm is usually observed after fertilization in the culture dish, but interestingly dead and immotile sperm was sometimes observed. However, the association of sperm death after fertilization in the culture dish and IVF outcomes have not yet reported. In this study, we inves- tigated relationship association of IVF outcomes and sperm death in the cul- ture dish after fertilization on conventional IVF. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: We analyzed 472 cycles from patients aged under 40 years undergoing conventional IVF from January 2012 to December 2013. After fertilization, the patients were classified depending on sperm condition in the culture dish into two groups : group A (n¼435): high percentage of the living sperm with hyperactivated motility, group B e202 ASRM Abstracts Vol. 102, No. 3, Supplement, September 2014

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Page 1: Sperm death after fertilization on conventional IVF can be a good prediction of IVF outcomes

DESIGN: Retrospective study.MATERIALS AND METHODS: All fresh, non-donor in conventional

IVF without ICSI cycles performed from January 2012 to December 2013(n¼838) were included in this study. The patients were divided into twogroups: Study group (A group): A total of 115 patients underwent subnormalsperm count (R5 and <20 � 106/mL). Control group (B group): A total of723 patients underwent normal sperm count R20 � 106/mL). Both groupsbecame normal count after sperm treatment.

RESULTS: Patient characteristics were similar between two groups. Ineach group, age (A group: 35.3�3.6; B group: 35.7�4.0 P¼0.73), basalFSH (7.4�2.6; 7.3�3.7mIU/ml P¼0.76), duration of rFSH (9.0�2.3;9.1�3.2day P¼0.81), and endometrial thickness (10.1�2.0; 10.1�2.1mmP¼0.41) did not show significant differences. There were no significant dif-ferences between two groups in the number of MII oocytes (10.8�6.6;10.1�6.2 P¼0.93), fertilization rate (85.2%; 88.8% p¼0.64), pregnancyrate (57.4%; 54.8% P¼0.85), and ongoing pregnancy rate (49.6%; 49.2%P¼0.70).

CONCLUSION: Subnormal sperm group could obtain the same fertiliza-tion rate and pregnancy rate as those of normal group in conventional IVFwithout ICSI. Generally, ICSI is carried out for subnormal sperm becausethe possibility of fertilization may decline. But the safety of ICSI has notbeen proved yet and according to our results, ICSI was not necessarilyneeded in case of subnormal sperm. Therefore, it seems that conventionalIVF without ICSI is sufficient as a fertilization method for subnormalsperm.

P-189 Tuesday, October 21, 2014

MEN WITH A COMPLETE ABSENCE OF NORMAL FORMS (0%)ON STRICTMORPHOLOGY EXHIBIT HIGH RATES OF SUCCESSWITHOUT IN VITRO FERTILIZATION. J. R. Kovac,a R. P. Smith,b

M. Cajipe,c J. Scovell,c R. Ramasamy,c J. Dupree,c G. Langille,c

D. J. Lamb,c L. I. Lipshultz.c aUrology of Indiana, Carmel, IN; bUniversityof Virginia, Charlottesville, VA; cBaylor College of Medicine, Houston, TX.

OBJECTIVE: In couples with infertility, an abnormal strict morphology of0% normal forms (NF) is often used as a criterion to proceed rapidly to in-vitro fertilization (IVF). A paucity of data exists examining reproductive suc-cess for these couples outside of IVF. We investigated the outcomes of menwith 0% NF to determine success of natural conception (NC) & intra-uterineinsemination (IUI) compared to IVF.

DESIGN: Cohort study on patients from a high volume, tertiary, infertilityclinic from 2010-13.

MATERIALS AND METHODS: A total of 24 men with 0% NF(strict Kruger criteria) were identified with 18 randomly selected men>4% NF used as controls. Patient charts were reviewed & men contacted& administered an IRB-approved telephone questionnaire to ascertainoutcomes.

RESULTS: Average age of men & spouses in the 0%NF& control cohortswas not significantly different. Men with 0% NF revealed normal levels ofLH, FSH, testosterone & estradiol. As expected, FSH was significantlyhigher in men with 0% NF (p¼0.04). Semen analysis demonstrated similarvolumes in men with 0%NF compared to controls while forward progression(2�0.1 vs. 2.4�0.1, p¼0.0003), density (7�1.4 vs. 50�7, p<0.0001) & totalmotile counts (7�1.8 vs. 59�9, p<0.0001) were different. Of those men with0%NF, 42% (n¼10/24) achieved pregnancy. A total of 29% (n¼7/24) did notrequire IVF (NC¼25%, n¼6/24; IUI¼4.2%, n¼1/24). IVF was used in12.5% (n¼3/24). Controls achieved pregnancy on 78% of occasions(n¼14/18) of which 72% (n¼13/18) did not require IVF (NC¼67%,n¼12/18; IUI¼5.5%, n¼1/18). IVF was used in 22% of control men (n¼4/18). Only one case was for primary infertility (5.5%) while 3 were for sec-ondary infertility following a natural birth (17%). In men with 0% NF &the first pregnancy was conceived without IVF (n¼7), 100% had successon subsequent occasions (n¼7/7). In control men with sequential birthswhere the first pregnancy was conceived without IVF (n¼6), 50% (n¼3/6)went on to require IVF with subsequent pregnancies.

CONCLUSION:Menwith 0%NF conceivedwithout IVF in 29% of cases.Control men had success 72% of the time. Strict morphology provides impor-tant information about the process of spermatogenesis but should not be usedto predict fertilization potential. In men with severe teratozoospermia &where maternal age allows, alternative modalities may be considered priorto immediate IVF.

Supported by: JRK is Supported by a Male Reproductive Health Research(MHRH)Career Development Physician Scientist Award (K12) (HD073917-

e202 ASRM Abstracts

01) from the Eunice Kennedy Shriver National Institute of Child Health andHuman Development (NICHD) Program to DJL.

P-190 Tuesday, October 21, 2014

THE EFFECTS OF SPERM CONCENTRATION ON IN VITRO EM-BRYO DEVELOPMENT. B. Tilley,a G. Navarrete,a S. Chantilis,a,b

K. Lee,a,b M. Thomas,a,b R. Gada,a,b S. Purcell,a M. Meintjes.a,c aDallasFertility Center, Dallas, TX; bDallas-Ft. Worth Fertility Associates, Dallas,TX; cFrisco Institute for Reproductive Medicine, Frisco, TX.

OBJECTIVE: To determine if decreased sperm concentration used in con-ventional inseminationwould affect normal and abnormal fertilization rate orsubsequent in vitro embryo development.DESIGN: Prospective sibling oocyte study.MATERIALS AND METHODS: Patients were< 41 years old or using

donor oocytes, hadR 8 oocytes retrieved,<2 previous cycles, and used freshejaculated sperm. Oocytes were divided equally into four different concen-trations of motile sperm: (A) 0.15x10̂6/mL, (B) 0.10x10̂6/mL, (C)0.075x10̂6/mL, and (D) 0.05x10̂6/mL. Odd numbers of oocytes went tothe greatest sperm concentration first. Spermwas added 6 hours post retrievaland fertilization assessed 18 h later. Fertilization took place in 200ul drops ofP-1 + 5mg/mLHSA (Irvine) and embryos were cultured in G1/G2 (Vitrolife)+ 10% SSS (Irvine) all under oil in humidified 6%CO2 and 5%O2. Normaland abnormal fertilization, day 5 blastocyst formation per 2PN, number offreeze quality blastocysts per 2PN, and number transferred per 2PN wereanalyzed by ANOVA. Differences between treatments were assessed usingFisher’s LSD. Odds of obtaining low fertilization, defined as <50% 2PN,were calculated using logistic regression.RESULTS: A total of 533 oocytes were retrieved from 25 patients. Overall

normal and abnormal fertilization rates were 73.7 and 14.3%; respectively.Overall, implantation and clinical pregnancy rates were 57.5% and 62.5%;respectively, with an average number of embryos transferred of 1.7. Theodds of insemination resulting in low fertilization were not increased bydecreasing sperm concentration. Treatment group means � SE are shownin the table below. No significant differences between treatment groupswere observed for any variable.

Treatment Oocytes 2PN Abnormal Blastocyts ET Cryo

Vol. 10

2, No. 3, Suppleme nt, Septe

A

144 7 4.8�5.0 16.4�4.4 48.5�6.4 1 8.4�4.9 1 3.4�4.4 B 137 7 9.3�5.0 10.5�3.1 52.2�6.5 1 1.4�3.2 2 5.3�5.6 C 130 6 8.5�5.1 16.1�3.9 41.7�3.9 1 1.5�4.7 1 3.3�4.9 D 122 7 2.3�5.7 14.2�5.7 43.4�5.7 1 1.9�4.8 1 7.3�4.2

CONCLUSION: Decreasing sperm concentration to only 50,000 motilesperm/mL for conventional insemination does not significantly affect normalor abnormal fertilization. However, decreased sperm concentration did notresult in any improvement in embryo development either.

P-191 Tuesday, October 21, 2014

SPERM DEATH AFTER FERTILIZATION ON CONVENTIONALIVF CAN BE A GOOD PREDICTION OF IVFOUTCOMES. S. J. Kwak, I. H. Park, K. H. Lee, S. G. Kim,H. G. Sun, Y. Y. Kim, J. H. Lee, J. Y. Park. Infertility Lab., Mamapapa&-Baby Obstetrics and Gynecology, Ulsan, Republic of Korea.

OBJECTIVE: Live and motile sperm is usually observed after fertilizationin the culture dish, but interestingly dead and immotile spermwas sometimesobserved. However, the association of sperm death after fertilization in theculture dish and IVF outcomes have not yet reported. In this study, we inves-tigated relationship association of IVF outcomes and sperm death in the cul-ture dish after fertilization on conventional IVF.DESIGN: Retrospective cohort study.MATERIALS AND METHODS: We analyzed 472 cycles from patients

aged under 40 years undergoing conventional IVF from January 2012 toDecember 2013. After fertilization, the patients were classified dependingon sperm condition in the culture dish into two groups : group A (n¼435):high percentage of the living sperm with hyperactivated motility, group B

mber 2014

Page 2: Sperm death after fertilization on conventional IVF can be a good prediction of IVF outcomes

(n¼37): more than 80% of immotile with dead sperm in the culture dish. Wecompared IVF outcomes between two groups.

RESULTS: Therewere no differences in patients age (group A; 35.12�2.9vs. group B; 35.16�3.1, p¼0.94), number of oocytes (9.2�5.4 vs. 7.8�5.5,p¼0.13) and sperm counts (53 x 10̂6/mL�26.62 vs. 50.13 x 10̂6/mL�34,p¼0.53). And also fertilization rate did not differ between two groups(72.8% vs. 69.6%, p¼0.32). However, the clinical pregnancy rate in groupB was significantly higher than group A (56.3% vs. 75.6%, p¼0.02).

CONCLUSION: In this study, dead sperm group showed significantlyhigher pregnancy rate compared with living sperm group. We suppose thathigh percentage of dead sperm means good quality sperms burn out thelimited amount of nutrients in the culture dish. Therefore, high percentageof sperm death after fertilization on conventional IVF could be a good pre-diction of IVF outcome.

P-192 Tuesday, October 21, 2014

REPRODUCTIVEOUTCOMEOFOOCYTE ACTIVATION BY CAL-CIUM IONOPHORE A23187 FOLLOWING INTRACYTOPLASMICSPERM INJECTION (ICSI) IN PATIENTS WITH PREVIOUS COM-PLETE FERTILIZATION FAILURE. T. Caballero, P. J. Buzzi,M. P. Zappacosta, A. Valcarcel, F. Lorenzo. IFER Instituto de Ginecolog�ıay Fertilidad, Buenos Aires, Argentina.

OBJECTIVE: Fertilization failure after ICSI may be due to the inability ofthe spermatozoa to trigger oocyte activation which, is characterized by a risein intracellular calcium concentration. Treatment with calcium ionophoremay increase the free intracellular calcium, thereby mimicking physiologicalcell-signaling mechanisms resulting in oocyte activation.The aim of thisstudy was to evaluate the impact on reproductive outcome of oocyte activa-tion (OA) by calcium ionophore (A23187) in patients with complete fertiliza-tion failure following ICSI during previous In Vitro Fertilization (IVF)cycles.

DESIGN: Prospective cohort study.MATERIALS AND METHODS: Between January 2011 and December

2013 30 couples who had prior insuccessful ICSI treatments due to com-plete fertilization failure accepted to enter this study. Following ICSI 217injected oocytes were activated with calcium ionophore A23187 for twentyminutes. Embryo transfer was performed on day 3. Fertilization and topquality embryos rate, implantation, clinical pregnancy and birth rateswere analyzed.

RESULTS: Oocyte retrieval in 30 patients resulted in 288 cumulus-oocytecomplexes. After denudation, 217 (84.3%) were mature and, consequently,available for ICSI. Fertilization rate was 63.1% (137/217), cleavage ratewas 92% (126/137). A mean of 2.9 Day 3 embryos were transferred to 26 pa-tients. Four couples had no embryos to transfer. Clinical pregnancy rate was38.5% (10/26), miscarriage rate was 20% (2/10). Nine healthy babies wereborn (one twin pregnancy).

CONCLUSION: The clinical use of ionophores in assisted reproduction isstill limited by insufficient knowledge about their potential toxic effect on oo-cytes and embryos. However, healthy children already born seem not to beaffected by ionophore treatment in our study. Assisted oocyte activationwith calcium ionophore may become a reasonable and efficient treatmentin cases of very low or complete fertilization failure. More prospectivecontrolled studies should be performed to confirm these results.

EMBRYO BIOLOGY

P-193 Tuesday, October 21, 2014

ACCURATE QUANTIFICATION OF SPECIFIC PROTEINS OF IN-TEREST IN SINGLE HUMAN BLASTOCOELS USING TARGETEDMASS SPECTROMETRY. M. Poli,a,b A. Ori,c S. Jaroudi,d T. Child,a,b

M. Beck,c D. Wells.a,d aUniversity of Oxford, Oxford, United Kingdom;bOxford Fertility Unit, Oxford, United Kingdom; cEuropean MolecularBiology Laboratory, Heidelberg, Germany; dReprogenetics UK, Oxford,United Kingdom.

OBJECTIVE: In this study we investigate the feasibility of detecting andquantifying specific protein targets secreted within single human blastocoelsusing Targeted Mass Spectrometry (MS).

DESIGN: Prospective Cohort Study.MATERIALS AND METHODS: Blastocoel fluid was removed from 80

Day5/6 fully expanded blastocysts donated to research. The samples were

FERTILITY & STERILITY�

batched in groups of 20, trypsin digested and analyzed by tandem MS. Inde-pendent confirmation of the embryonic origin of the identified proteins wasachieved via transcriptomic analysis of 18 blastocysts using a combination ofRNA amplification and microarray analysis. Initial experiments allowedcompilation of a catalogue of detectable blastocoel proteins, after whichwe selected 26 peptides of interest and designed related Single ReactionMonitoring (SRM) Assays. Subsequently, the SRM Assays and TargetedMS were applied to the analysis of individual blastocoel samples. Homolo-gous synthetic peptides were spiked-in to allow absolute quantification ofthe targets. Finally, variations in protein profiles across all samples wereexamined.RESULTS: Analysis of combined tandemMS runs identified 287 proteins

that exist within the human blastocoel (false discovery rate of <1%). Com-parison of transcriptomic and proteomic data suggests that>80% of proteinsfound within blastocoel fluid are derived from genes actively transcribed atthe blastocyst stage, confirming the high specificity of tandem MS. UsingTargeted MS, we succeeded in measuring the concentration of specific pro-teins in single blastocoels. Interestingly, we observed that the ratio between‘‘housekeeping’’ and modulated proteins can vary drastically across samplessuggesting significant differences between embryos in terms of molecularphenotype.CONCLUSION: This study confirms the feasibility of accurately

measuring individual peptides released by the human blastocyst into the blas-tocoel cavity using TargetedMS. This strategy allows the analysis of proteinsof embryonic origin only, avoiding background noise from medium compo-nents. Not only has this work provided a catalogue of biologically interestingproteins secreted by embryos nearing the time of implantation, but the dataproduced is likely to be of relevance to the phenotype of the embryo andmay therefore have clinical value. The use of protein concentration ratiosas embryo viability/implantation markers is currently under investigation.Supported by: Institutional funding.

P-194 Tuesday, October 21, 2014

ORC4 PLAYS A ROLE IN POLAR BODY EXTRUSION IN THEMOUSE OOCYTE AND ZYGOTE. H. Nguyen, M. Ko,M. A. Ortega, J. Marh, W. S. Ward. Institute for Biogenesis Research -John A Burns School of Medicine, Honolulu, HI.

OBJECTIVE: Six proteins, ORC1-6, make up the origin recognition com-plex (ORC) that prepares DNA replication origins for licensing. In somaticcells, the ORC1-6 proteins bind to an unlicensed origin to form a complex,but in mammalian cells four proteins ORC2-5 bind to many origins firstand then ORC1 bind to this complex. The complex recruits Cdc6 and Cdt1and allow licensing to occur by loading Mcm2-7. We tested if ORC1-6 pro-teins in the mouse oocyte and zygote behave the similarity to the licensingpathways in somatic cells.DESIGN: At different stages of meiosis and the first cell division cycle,

embryos were stained for one of five ORC proteins, and MCM7 which asso-ciates with chromosomes during cell cycle as a positive control. We used 10oocytes/group for each experiment.MATERIALS AND METHODS: Immunocytochemistry (ICC) was used

to detect ORC1-6 proteins, and cells were analyzed by confocal microscopy.RESULTS: We found that ORC1, 3, 5 and 6 all localized to the area be-

tween the separating maternal chromosomes at anaphase II after fertilization,as we had previously reported for ORC2. ORC1, 3 and 5 were not detected inzygotic G1. ORC6 was found in the nucleolar periphery of the pronuclei andpolar body nucleus at zygotic G1. Interestingly, in both meiotic divisions,ORC4 surrounds the set of chromosomes that will eventually be discardedin the polar bodies, but not the chromosomes that segregate into the oocyte.ORC4 localizes around the future polar body chromosomes as sphere-likestructure in anaphase in both meiotic divisions as the two chromosomesets migrate apart. In zygote G1, ORC4 surrounds the nuclei of 1st and2sd polar bodies, but is absent from the paternal or maternal pronuclei.The absence of ORC4 in the pronuclei was surprising because ORC4 isrequired for DNA replication in somatic cells. Moreover, we did findORC2 in zygotic pronuclei, as expected. At mitosis, the ORC4 was absentfrom the chromosomes at metaphase, but appeared on both sets of separatingchromosomes at telophase. At this point, the ORC4 in the polar body alsomigrated into the nuclei.CONCLUSION: We are currently conducting additional experiments to

determine the functional roles of ORC4 in polar body formation. Our resultssuggest that ORC4 localization can be used to help identify the DNA strandsthat destined to be expelled in the polar body. They also suggest that ORC4 is

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