- Email: [email protected]
Unfolded protein response is a key regulator of oxygen dependant trophoblast differentiation
P-178 Tuesday, October 23, 2012 DYNAMIC ANALYSIS OF THE RELATIONSHIP BETWEEN THE TIMING OF SYNGAMY AND HUMAN EMBRYONIC DEVELOPMENT USING TIME-LAPSE CINEMATOGRAPHY. M. Ueda, K. Iwata, A. Imajo, K. Yumoto, Y. Iba, Y. Mio. Reproductive Centre, Mio fertility clinic, Yonago, Tottori, Japan. OBJECTIVE: In 2011, the Istanbul consensus workshop proposed that the assessment of syngamy (24h post-insemination) is a very sensitive predictor for embryonic quality. The value of this technique as an independent predictor of embryonic outcome is still unclear. In this study, we analyzed the relationship between the timing of syngamy and human embryonic development using time-lapse cinematography (TLC). DESIGN: Time-lapse study. MATERIALS AND METHODS: We have developed a TLC system, which is described elsewhere. The culture was maintained at an optimal conditions. After IVF or ICSI procedures, donated oocytes (n¼203) were analyzed by TLC. In IVF, the cumulus cells were removed at 1h post insemination and the oocytes were analyzed for TLC. In ICSI, TLC was commenced with the oocytes after the procedure. RESULTS: A total of 203 oocytes were examined and 128 were normally fertilized embryos (IVF:50, ICSI:78) and the rest were abnormally fertilized (n¼13), undetectable (n¼35) and unfertilized (n¼27). In normally fertilized embryos, the times required from the extrusion of 2nd polar body to syngamy during IVF and ICSI were 21.23.6h and 20.83.6h, respectively. The time required for syngamy in good quality embryos (GQE) was 20.03.3h, which was significantly less than 22.14.3h in poor quality embryos (PQE)(P<0.05). In addition, the time required for syngamy in abnormally fertilized embryos was 25.16.3h which was delayed compared to normally fertilized embryos. CONCLUSION: Although there was no difference in the time required for syngamy by the procedure of insemination (IVF or ICSI), the time required for syngamy in GQE was significantly shorter than that in PQE. This suggests that syngamy timing would be a useful parameter to assess the quality of human embryos. Furthermore, the time required for syngamy between normally and abnormally fertilized embryos was significantly different, suggesting that we could distinguish the normally fertilized from abnormal embryos by assessment of timing of syngamy.
phology of blastocysts carrying errors affecting smaller chromosomes was similar to that of euploid embryos. This is in agreement with the fact that such aneuploid conceptions are capable of implanting and sometimes lead to clinical pregnancies and/or miscarriages. Such associations were not observed at the cleavage stage.
P-180 Tuesday, October 23, 2012 DOES SEMEN CRYOPRESERVATION HAVE INFLUENCE ON EMBRYO KINETICS? L. Muela Garcıa,a M. Martınez Morales,a B. Gadea Navaro,a I. Perez Cano,a M. Mu~noz Cantero,a M. Meseguer Escriva.b aIVI Alicante, Alicante, Spain; bIVI Valencia, Valencia, Spain. OBJECTIVE: To determine whether the semen cryopreservation has an influence on embryo kinetics from IVF and ICSI treatments by the use of a digital analysis system EmbryoscopeÒ(UnisenseFertilitech,Denmark). DESIGN: Retrospective study of 110 cycles from our ovum.donation.program Oocyte mean/patient¼13.12. MATERIALS AND METHODS: Embryos (n837) were classified in four groups: FrozenSemenforIVF(n98),FreshSemenIVF(n284),FrozenSemenICSI(n209) andFreshSemenICSI(n246).The following parameters were evaluated: Pronuclear breakdown PNBD, cleavage time T2, division time to 3cells T3, to 4cells T4, 5cells T5, 6cells T6, 7cells T7, 8cells T8, Morula M and blastocyst B formation. Statistical analyses were carried out by the test ANOVA. * Significant differences P<0,05. RESULTS: TABLE 1. and Table 2. Division FreshSemen Timing(h) IVF PNBD T2 T3 T4 T5 T6 T7 T8 M B
P-179 Tuesday, October 23, 2012 THE EFFECT OF ANEUPLOIDY ON EMBRYO MORPHOLOGY AND PREIMPLANTATION DEVELOPMENT FROM THE CLEAVAGE TO THE BLASTOCYST STAGE. S. Jaroudi,a S. Alfarawati,a,b M. Poli,b D. Wells,a,b E. Fragouli.a,b aReprogenetics UK, Oxford, Oxfordshire, United Kingdom; bNuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, Oxfordshire, United Kingdom. OBJECTIVE: Aneuploidy might have an adverse effect on various embryo characteristics including morphology. This study involved a detailed investigation of the impact of aneuploidy on embryo morphology at different developmental stages. The copy numbers of all 24 chromosomes were evaluated. DESIGN: Chromosome screening and data analysis. MATERIALS AND METHODS: 1256 embryos generated from 229 patients (average maternal age 39 years) were investigated. Embryo morphology was assessed and recorded on days 3 and/or 5-6 postfertilization and chromosome analysis was carried out using microarray comparative genomic hybridisation (aCGH). RESULTS: At the cleavage stage aneuploidy was common even in better grade embryos (80% affected; 27% with 1 error, 23% with 2-3 errors, 30% with R4 errors). The majority (51%) of the best quality blastocysts were euploid. Of the good quality aneuploid blastocysts, most (34%) had 1 error, whilst only 2% had R4 errors. 19% of these blastocysts were affected by aneuploidies compatible with a clinical pregnancy (trisomy 13, 16, 18, 21, 22, X0, or XXY), whereas only 7% carried errors affecting larger chromosomes (1-12). 59% of cleavage stage embryos and 52% of blastocysts were females and 41% and 49% were males. However, amongst the fastest growing cleavage-stage embryos (R10 cells) 72% were female, while the opposite was noted for rapidly developing blastocysts (grade 5 or 6; 55% males). CONCLUSION: A weak association was observed between aneuploidy and morphology at the blastocyst stage, but not earlier in development. At the cleavage stage complex abnormalities were observed in all grades, whereas this was only seen in ‘early’ blastocysts (P<0.0001). The mor-
S164
ASRM Abstracts
*
Frozen SemenIVF
P
24,780,18 25,250,28 0,19 28,140,79 28,340,39 0,88 37,690,45 38,090,87 0,66 40,650,34 41,620,56 0,14 51,580,50 51,711,03 0,9 54,360,44 55,640,88 0,17 57,34 0,50 59,410,91 0,05 58,960,47 59,97 0,96 0,32 88,960,64 90,66 1,06 0,17 101,650,65 105,041,21 0,01*
Fresh SemenICSI
Frozen SemenICSI
24,910,20* 24,210,23* 27,87 0,27* 26,870,27* 38,51 0,46 37,620,58 40,95 0,43 40,170,44 50,970,73 50,060,81 54,66 0,54 53,210,72 57,13 0,50 55,080,66 59,60 0,49 56,710,61 90,58 0,86 90,080,89 104,580,74 104,090,84
P 0,02* 0,01* 0,22 0,21 0,41 0,10 0,01* 0,00* 0,69 0,66
Significant differences P<0,05
In the IVFgroup, kinetics were not significantly different from fresh and frozen semen except in blastocyst formation. Those embryos from fresh semen developed faster than those with frozen semen. However, for the ICSIgroup, significant differences were found for PNBD,T2,T7andT8. CONCLUSION: The use of the EmbryoscopeÒ allows us to study continuously the division timing thanks to the time lapse technology, which permits us the selection of those embryos with highest implantation potential. The results obtained showed similar division timing for the parameters studied, so the embryo development is not affected by semen cryopreservation. Supported by: IVI.
P-181 Tuesday, October 23, 2012 UNFOLDED PROTEIN RESPONSE IS A KEY REGULATOR OF OXYGEN DEPENDANT TROPHOBLAST DIFFERENTIATION. J. C. Robins, L. A. Underhill, B. Bhagavath, S. A. Carson. Obstetrics and Gynecology, Women and Infants Hospital/Warren Alpert School of Medicine at Brown University, Providence, RI. OBJECTIVE: Low O2 tension promotes invasive trophoblast differentiation. However, the mechanisms regulating this are unclear. The hypoxia inducible factor 1a (HIF1a) is critical for differentiation. The unfolded protein response system (UPR) is as an important O2 regulator in other cells. The objectives of this study are 1) to determine the O2 dose-dependant expression of HIF1a and GRP78 (UPR specific protein), 2) to determine if UPR is activated during O2 independent differentiation, and 3) to determine if UPR activation will affect trophoblast differentiation.
Vol. 98, No. 3, Supplement, September 2012
DESIGN: Basic science. MATERIALS AND METHODS: Mouse trophoblast stem cells (TSCs) were cultured in low O2 (2%, 5%, 10%), or 20% O2 tension for 72h. Protein was purified and assayed by Western Blot for HIF1a and GRP78. In a parallel experiment, TSCs were placed in differentiation media for 72h and assayed for HIF1a and GRP78 protein. Finally, TSCs were differentiated in the presence or absence of thapsigargin, a UPR inducer. Differentiation was determined by measuring pathway specific gene expression with qRT-PCR. RESULTS: HIF1a protein is measurable at all O2 tensions. There is a significant increase in HIF1a protein at 3h in the low O2 treated cells when compared to 20%; however, there are no significant differences in the amount of HIF1a among low O2 concentrations. GRP78 protein was significantly induced by low O2 at 24h in a dose dependant fashion. Differentiation induced the expression of GRP78 protein independent of O2 tension. Induction of UPR results in a 3-fold increase of Pl2 expression, a marker of invasive trophoblast differentiation. CONCLUSION: HIF1a is not tightly regulated by O2 tension in trophoblast cells. Therefore, while required, HIF1a is not the regulator of O2 dependant differentiation. Our data demonstrate that UPR is activated by both low O2 and differentiation in trophoblasts and activation leads to invasive trophoblast differentiation. Therefore, UPR is key in determining the pathway of O2 regulated differentiation. Supported by: NIGMS 8 P20 GM103537-10.
P-182 Tuesday, October 23, 2012 DEVELOPMENTAL REGULATION OF APOPTOSIS PHENOMENA DURING EARLY EMBRYONIC DEVELOPMENT IN MOUSE AND HUMAN SPECIES: ROLES OF Bcl2 FAMILY GENES. I. Boumela,a S. Assou,a D. Haouzi,a H. Dechaud,b S. Hamamah.a,b aIRB-Inserm U1040, Montpellier, France; bDepartement de Biologie de la Reproduction, CHRU Montpellier, Montpellier, France. OBJECTIVE: To compare the expression profile of anti (Bcl2, Mcl1, Bclx, Bcl2l10) and pro-apoptotic (Bax, Bcl2l13, Bik, Bim, Noxa) Bcl2 family genes during early development in mouse and human species. DESIGN: Human mature MII oocytes, day 3 (D3, 6-8 cells) and D5 (blastocyst) embryos were included after informed consent of the patients and IRB approval. Mouse MII oocytes, D1.5 (2 cells), D2.5 (6-8 cells) and D3.5 embryos (blastocyst) were collected from mice under in vivo conditions. The dynamic of Bcl2 family gene expression before and after embryonic genome activation was determined. MATERIALS AND METHODS: Total RNA was extracted from single samples and gene expression was analyzed by qRT-PCR. RESULTS: Most of the Bcl2 family genes exhibited similar expression patterns during human and mouse early embryonic development. Thus, Bcl2l10 followed a maternal profile with high levels in human (x5, P<0,05) and mouse (x334, P<0,05) oocytes and a rapid decrease after fertilization. Mcl1, Bik and Noxa genes were expressed after embryonic genome activation occurring on D3 in humans (x245, x1661, x608 respectively; P<0,05) and D1.5 in mice (x31, x119, x3 respectively; P<0,05). In addition, Bcl-x and Bcl2l13 were constituvely expressed throughout early embryonic development. However, two members were differentially expressed between the two species. Indeed, while human Bim and Bax genes appear to be exclusively embryonic, with expression only in D3 (x48 for Bim, P<0,05) and/or D5 embryos (x9, x28 for Bim and Bax respectively, P<0,05), their murine homologs were detected at all developmental stages, including MII oocytes. CONCLUSION: Our data reveal that the expression of Bcl2 family genes during early embryonic development is conserved between both human and mouse species. Interestingly, some differences may exist for certain genes including Bax, which is a key regulator of female fertility in mice, and thus should be considered when extrapolating experimental data from mice to humans. Supported by: Ferring, Genevrier, MSD and Vitrolife companies. P-183 Tuesday, October 23, 2012 COMPARISON AMONG DIFFERENT HUMAN EMBRYO MORPHOLOGICAL CLASSIFICATIONS REGARDING PREGNANCY PROGNOSTIC IN IN VITRO FERTILIZATION (IVF) CYCLES. G. M. Gindri, C. L. V. Werneck, A. S. Aguiar, M. C. E. C. Martins, P. G. Sa, M. C. A. Cardoso. Laboratory, Vida Centro de Fertilidade da Rede D’Or, Rio de Janeiro, RJ, Brazil.
FERTILITY & STERILITYÒ
OBJECTIVE: Determine which of the three morphological embryo classifications: Veeck, Ziebe S et al. and ASEBIR, indicates a better prognostic of pregnancy. DESIGN: A retrospective study from IVF cycles from August 2010 through December 2011 which included 2078 cleaved embryos from 303 cycles in 288 different patients. Only transferred embryos on D2 and D3 have been considered. Blastocysts and embryos from thawing techniques were excluded. MATERIALS AND METHODS: Embryos were evaluated at the inverted microscope at 42 and 66 h (+/-2 h) post-insemination and all data are stored for analyses. For association measure among the three different classifications the x2 test has been used, when associating each one with implantation success, and the Spearman correlation coefficient to measure association between classifications. RESULTS: The success of implantation does significantly associate with the ASEBIR classification (x23¼9,701; P¼0,0212) and does not significantly associate with Ziebe S. et al (x24¼7,0789; P¼0,06943) nor with Veeck (x24¼5,7409; P¼0,2193) classifications. The positive bhCG does significantly associate with the ASEBIR classification (x23¼8,1537; P¼0,04294) and does not significantly associate with Ziebe S. et al (x24¼6,3375; P¼0,0963) nor with Veeck (x24¼5,0742; P¼0,2798) classifications. The association two by two demonstrated that they are highly correlated to each other (ASEBIR x Ziebe S. et al¼0,867; ASEBIR x Veeck¼0,293; Ziebe S. et al x Veeck¼0,308). CONCLUSION: The results show that only ASEBIR classification is significantly associated with positive bhCG and implantation success. This classification more faithfully describes pregnancy prognostics and could be considered the best one. It was shown that the parameters evaluated by this classification are in fact important to embryo development. Although all classifications have been highly associated, we see a greater association between ASEBIR and Ziebe S, et al.
P-184 Tuesday, October 23, 2012 IDENTIFICATION OF DISPLACED ENDOMETRIAL GLANDS AND EMBRYONIC DUCT REMNANTS IN FEMALE FOETAL REPRODUCTIVE TRACT: POSSIBLE PATHOGENETIC ROLE IN ENDOMETRIOTIC AND PELVIC NEOPLASTIC PROCESSES. J. Bouquet de la Joliniere,a J. M. Ayoubi,b L. Gianaroli,a J. B. Dubuisson,a A. Feki,a J. Gogusev.c aGynecology, HFR Fribourg H^opital cantonal, Fribourg, Switzerland; bGynecology, Foch Hospital, Suresnes, France; cInserm U 567, Hopital Necker, Paris, France. OBJECTIVE: The goal of our study was to define an embyological determinism of endometriosis and adenomyosis. DESIGN: Recent findings strongly promoted the hypothesis that common pelvic gynaecological diseases including endometriosis and ovarian neoplasia may develop de novo from ectopic endometrial-like glands and/or embryonic epithelial remnants. To verify the frequency, the anatomical localization and the phenotype of misplaced endometrial tissue along the fetal female reproductive tract, histological and immunohistochemical analyses of uteri, fallopian tubes, and uterosacral ligaments were performed. MATERIALS AND METHODS: Reproductive organs were collected from seven female fetuses at autopsy, five of them from gestational ages between 18 and 26 weeks and two fetuses with gestational ages of 33 and 36 weeks deceased of placental anomalies. Serial sections from areas containing ectopic glands and embryonic duct residues were analyzed by histological and immunohistochemical procedures. RESULTS: Numerous ectopic endometrial glands and stroma were detected in the myometrium in two foetuses with low levels of expression of estrogen alpha (ER-a) and progesterone receptors (PR). The embryonic ducts were localized in the uterine broad and ovarian ligaments and under the fallopian tube serosa in six foetuses. Low levels of steroid receptors expression were found in the embryonic residues, whereas CEA and Ca 125 were not detected. The embryonic residues stromal component strongly expressed the CD 10 and vimentin proteins. CONCLUSION: The anatomical and the immunohistochemical features of the ectopic organoid structures identified in foetal female reproductive tract suggest that endometriotic as well as neoplastic disease in adult women may develop on the basis of misplaced endometrial glands and/or embryonic cell remnants.
S165
phology of blastocysts carrying errors affecting smaller chromosomes was similar to that of euploid embryos. This is in agreement with the fact that such aneuploid conceptions are capable of implanting and sometimes lead to clinical pregnancies and/or miscarriages. Such associations were not observed at the cleavage stage.
P-180 Tuesday, October 23, 2012 DOES SEMEN CRYOPRESERVATION HAVE INFLUENCE ON EMBRYO KINETICS? L. Muela Garcıa,a M. Martınez Morales,a B. Gadea Navaro,a I. Perez Cano,a M. Mu~noz Cantero,a M. Meseguer Escriva.b aIVI Alicante, Alicante, Spain; bIVI Valencia, Valencia, Spain. OBJECTIVE: To determine whether the semen cryopreservation has an influence on embryo kinetics from IVF and ICSI treatments by the use of a digital analysis system EmbryoscopeÒ(UnisenseFertilitech,Denmark). DESIGN: Retrospective study of 110 cycles from our ovum.donation.program Oocyte mean/patient¼13.12. MATERIALS AND METHODS: Embryos (n837) were classified in four groups: FrozenSemenforIVF(n98),FreshSemenIVF(n284),FrozenSemenICSI(n209) andFreshSemenICSI(n246).The following parameters were evaluated: Pronuclear breakdown PNBD, cleavage time T2, division time to 3cells T3, to 4cells T4, 5cells T5, 6cells T6, 7cells T7, 8cells T8, Morula M and blastocyst B formation. Statistical analyses were carried out by the test ANOVA. * Significant differences P<0,05. RESULTS: TABLE 1. and Table 2. Division FreshSemen Timing(h) IVF PNBD T2 T3 T4 T5 T6 T7 T8 M B
P-179 Tuesday, October 23, 2012 THE EFFECT OF ANEUPLOIDY ON EMBRYO MORPHOLOGY AND PREIMPLANTATION DEVELOPMENT FROM THE CLEAVAGE TO THE BLASTOCYST STAGE. S. Jaroudi,a S. Alfarawati,a,b M. Poli,b D. Wells,a,b E. Fragouli.a,b aReprogenetics UK, Oxford, Oxfordshire, United Kingdom; bNuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, Oxfordshire, United Kingdom. OBJECTIVE: Aneuploidy might have an adverse effect on various embryo characteristics including morphology. This study involved a detailed investigation of the impact of aneuploidy on embryo morphology at different developmental stages. The copy numbers of all 24 chromosomes were evaluated. DESIGN: Chromosome screening and data analysis. MATERIALS AND METHODS: 1256 embryos generated from 229 patients (average maternal age 39 years) were investigated. Embryo morphology was assessed and recorded on days 3 and/or 5-6 postfertilization and chromosome analysis was carried out using microarray comparative genomic hybridisation (aCGH). RESULTS: At the cleavage stage aneuploidy was common even in better grade embryos (80% affected; 27% with 1 error, 23% with 2-3 errors, 30% with R4 errors). The majority (51%) of the best quality blastocysts were euploid. Of the good quality aneuploid blastocysts, most (34%) had 1 error, whilst only 2% had R4 errors. 19% of these blastocysts were affected by aneuploidies compatible with a clinical pregnancy (trisomy 13, 16, 18, 21, 22, X0, or XXY), whereas only 7% carried errors affecting larger chromosomes (1-12). 59% of cleavage stage embryos and 52% of blastocysts were females and 41% and 49% were males. However, amongst the fastest growing cleavage-stage embryos (R10 cells) 72% were female, while the opposite was noted for rapidly developing blastocysts (grade 5 or 6; 55% males). CONCLUSION: A weak association was observed between aneuploidy and morphology at the blastocyst stage, but not earlier in development. At the cleavage stage complex abnormalities were observed in all grades, whereas this was only seen in ‘early’ blastocysts (P<0.0001). The mor-
S164
ASRM Abstracts
*
Frozen SemenIVF
P
24,780,18 25,250,28 0,19 28,140,79 28,340,39 0,88 37,690,45 38,090,87 0,66 40,650,34 41,620,56 0,14 51,580,50 51,711,03 0,9 54,360,44 55,640,88 0,17 57,34 0,50 59,410,91 0,05 58,960,47 59,97 0,96 0,32 88,960,64 90,66 1,06 0,17 101,650,65 105,041,21 0,01*
Fresh SemenICSI
Frozen SemenICSI
24,910,20* 24,210,23* 27,87 0,27* 26,870,27* 38,51 0,46 37,620,58 40,95 0,43 40,170,44 50,970,73 50,060,81 54,66 0,54 53,210,72 57,13 0,50 55,080,66 59,60 0,49 56,710,61 90,58 0,86 90,080,89 104,580,74 104,090,84
P 0,02* 0,01* 0,22 0,21 0,41 0,10 0,01* 0,00* 0,69 0,66
Significant differences P<0,05
In the IVFgroup, kinetics were not significantly different from fresh and frozen semen except in blastocyst formation. Those embryos from fresh semen developed faster than those with frozen semen. However, for the ICSIgroup, significant differences were found for PNBD,T2,T7andT8. CONCLUSION: The use of the EmbryoscopeÒ allows us to study continuously the division timing thanks to the time lapse technology, which permits us the selection of those embryos with highest implantation potential. The results obtained showed similar division timing for the parameters studied, so the embryo development is not affected by semen cryopreservation. Supported by: IVI.
P-181 Tuesday, October 23, 2012 UNFOLDED PROTEIN RESPONSE IS A KEY REGULATOR OF OXYGEN DEPENDANT TROPHOBLAST DIFFERENTIATION. J. C. Robins, L. A. Underhill, B. Bhagavath, S. A. Carson. Obstetrics and Gynecology, Women and Infants Hospital/Warren Alpert School of Medicine at Brown University, Providence, RI. OBJECTIVE: Low O2 tension promotes invasive trophoblast differentiation. However, the mechanisms regulating this are unclear. The hypoxia inducible factor 1a (HIF1a) is critical for differentiation. The unfolded protein response system (UPR) is as an important O2 regulator in other cells. The objectives of this study are 1) to determine the O2 dose-dependant expression of HIF1a and GRP78 (UPR specific protein), 2) to determine if UPR is activated during O2 independent differentiation, and 3) to determine if UPR activation will affect trophoblast differentiation.
Vol. 98, No. 3, Supplement, September 2012
DESIGN: Basic science. MATERIALS AND METHODS: Mouse trophoblast stem cells (TSCs) were cultured in low O2 (2%, 5%, 10%), or 20% O2 tension for 72h. Protein was purified and assayed by Western Blot for HIF1a and GRP78. In a parallel experiment, TSCs were placed in differentiation media for 72h and assayed for HIF1a and GRP78 protein. Finally, TSCs were differentiated in the presence or absence of thapsigargin, a UPR inducer. Differentiation was determined by measuring pathway specific gene expression with qRT-PCR. RESULTS: HIF1a protein is measurable at all O2 tensions. There is a significant increase in HIF1a protein at 3h in the low O2 treated cells when compared to 20%; however, there are no significant differences in the amount of HIF1a among low O2 concentrations. GRP78 protein was significantly induced by low O2 at 24h in a dose dependant fashion. Differentiation induced the expression of GRP78 protein independent of O2 tension. Induction of UPR results in a 3-fold increase of Pl2 expression, a marker of invasive trophoblast differentiation. CONCLUSION: HIF1a is not tightly regulated by O2 tension in trophoblast cells. Therefore, while required, HIF1a is not the regulator of O2 dependant differentiation. Our data demonstrate that UPR is activated by both low O2 and differentiation in trophoblasts and activation leads to invasive trophoblast differentiation. Therefore, UPR is key in determining the pathway of O2 regulated differentiation. Supported by: NIGMS 8 P20 GM103537-10.
P-182 Tuesday, October 23, 2012 DEVELOPMENTAL REGULATION OF APOPTOSIS PHENOMENA DURING EARLY EMBRYONIC DEVELOPMENT IN MOUSE AND HUMAN SPECIES: ROLES OF Bcl2 FAMILY GENES. I. Boumela,a S. Assou,a D. Haouzi,a H. Dechaud,b S. Hamamah.a,b aIRB-Inserm U1040, Montpellier, France; bDepartement de Biologie de la Reproduction, CHRU Montpellier, Montpellier, France. OBJECTIVE: To compare the expression profile of anti (Bcl2, Mcl1, Bclx, Bcl2l10) and pro-apoptotic (Bax, Bcl2l13, Bik, Bim, Noxa) Bcl2 family genes during early development in mouse and human species. DESIGN: Human mature MII oocytes, day 3 (D3, 6-8 cells) and D5 (blastocyst) embryos were included after informed consent of the patients and IRB approval. Mouse MII oocytes, D1.5 (2 cells), D2.5 (6-8 cells) and D3.5 embryos (blastocyst) were collected from mice under in vivo conditions. The dynamic of Bcl2 family gene expression before and after embryonic genome activation was determined. MATERIALS AND METHODS: Total RNA was extracted from single samples and gene expression was analyzed by qRT-PCR. RESULTS: Most of the Bcl2 family genes exhibited similar expression patterns during human and mouse early embryonic development. Thus, Bcl2l10 followed a maternal profile with high levels in human (x5, P<0,05) and mouse (x334, P<0,05) oocytes and a rapid decrease after fertilization. Mcl1, Bik and Noxa genes were expressed after embryonic genome activation occurring on D3 in humans (x245, x1661, x608 respectively; P<0,05) and D1.5 in mice (x31, x119, x3 respectively; P<0,05). In addition, Bcl-x and Bcl2l13 were constituvely expressed throughout early embryonic development. However, two members were differentially expressed between the two species. Indeed, while human Bim and Bax genes appear to be exclusively embryonic, with expression only in D3 (x48 for Bim, P<0,05) and/or D5 embryos (x9, x28 for Bim and Bax respectively, P<0,05), their murine homologs were detected at all developmental stages, including MII oocytes. CONCLUSION: Our data reveal that the expression of Bcl2 family genes during early embryonic development is conserved between both human and mouse species. Interestingly, some differences may exist for certain genes including Bax, which is a key regulator of female fertility in mice, and thus should be considered when extrapolating experimental data from mice to humans. Supported by: Ferring, Genevrier, MSD and Vitrolife companies. P-183 Tuesday, October 23, 2012 COMPARISON AMONG DIFFERENT HUMAN EMBRYO MORPHOLOGICAL CLASSIFICATIONS REGARDING PREGNANCY PROGNOSTIC IN IN VITRO FERTILIZATION (IVF) CYCLES. G. M. Gindri, C. L. V. Werneck, A. S. Aguiar, M. C. E. C. Martins, P. G. Sa, M. C. A. Cardoso. Laboratory, Vida Centro de Fertilidade da Rede D’Or, Rio de Janeiro, RJ, Brazil.
FERTILITY & STERILITYÒ
OBJECTIVE: Determine which of the three morphological embryo classifications: Veeck, Ziebe S et al. and ASEBIR, indicates a better prognostic of pregnancy. DESIGN: A retrospective study from IVF cycles from August 2010 through December 2011 which included 2078 cleaved embryos from 303 cycles in 288 different patients. Only transferred embryos on D2 and D3 have been considered. Blastocysts and embryos from thawing techniques were excluded. MATERIALS AND METHODS: Embryos were evaluated at the inverted microscope at 42 and 66 h (+/-2 h) post-insemination and all data are stored for analyses. For association measure among the three different classifications the x2 test has been used, when associating each one with implantation success, and the Spearman correlation coefficient to measure association between classifications. RESULTS: The success of implantation does significantly associate with the ASEBIR classification (x23¼9,701; P¼0,0212) and does not significantly associate with Ziebe S. et al (x24¼7,0789; P¼0,06943) nor with Veeck (x24¼5,7409; P¼0,2193) classifications. The positive bhCG does significantly associate with the ASEBIR classification (x23¼8,1537; P¼0,04294) and does not significantly associate with Ziebe S. et al (x24¼6,3375; P¼0,0963) nor with Veeck (x24¼5,0742; P¼0,2798) classifications. The association two by two demonstrated that they are highly correlated to each other (ASEBIR x Ziebe S. et al¼0,867; ASEBIR x Veeck¼0,293; Ziebe S. et al x Veeck¼0,308). CONCLUSION: The results show that only ASEBIR classification is significantly associated with positive bhCG and implantation success. This classification more faithfully describes pregnancy prognostics and could be considered the best one. It was shown that the parameters evaluated by this classification are in fact important to embryo development. Although all classifications have been highly associated, we see a greater association between ASEBIR and Ziebe S, et al.
P-184 Tuesday, October 23, 2012 IDENTIFICATION OF DISPLACED ENDOMETRIAL GLANDS AND EMBRYONIC DUCT REMNANTS IN FEMALE FOETAL REPRODUCTIVE TRACT: POSSIBLE PATHOGENETIC ROLE IN ENDOMETRIOTIC AND PELVIC NEOPLASTIC PROCESSES. J. Bouquet de la Joliniere,a J. M. Ayoubi,b L. Gianaroli,a J. B. Dubuisson,a A. Feki,a J. Gogusev.c aGynecology, HFR Fribourg H^opital cantonal, Fribourg, Switzerland; bGynecology, Foch Hospital, Suresnes, France; cInserm U 567, Hopital Necker, Paris, France. OBJECTIVE: The goal of our study was to define an embyological determinism of endometriosis and adenomyosis. DESIGN: Recent findings strongly promoted the hypothesis that common pelvic gynaecological diseases including endometriosis and ovarian neoplasia may develop de novo from ectopic endometrial-like glands and/or embryonic epithelial remnants. To verify the frequency, the anatomical localization and the phenotype of misplaced endometrial tissue along the fetal female reproductive tract, histological and immunohistochemical analyses of uteri, fallopian tubes, and uterosacral ligaments were performed. MATERIALS AND METHODS: Reproductive organs were collected from seven female fetuses at autopsy, five of them from gestational ages between 18 and 26 weeks and two fetuses with gestational ages of 33 and 36 weeks deceased of placental anomalies. Serial sections from areas containing ectopic glands and embryonic duct residues were analyzed by histological and immunohistochemical procedures. RESULTS: Numerous ectopic endometrial glands and stroma were detected in the myometrium in two foetuses with low levels of expression of estrogen alpha (ER-a) and progesterone receptors (PR). The embryonic ducts were localized in the uterine broad and ovarian ligaments and under the fallopian tube serosa in six foetuses. Low levels of steroid receptors expression were found in the embryonic residues, whereas CEA and Ca 125 were not detected. The embryonic residues stromal component strongly expressed the CD 10 and vimentin proteins. CONCLUSION: The anatomical and the immunohistochemical features of the ectopic organoid structures identified in foetal female reproductive tract suggest that endometriotic as well as neoplastic disease in adult women may develop on the basis of misplaced endometrial glands and/or embryonic cell remnants.
S165