Session 23: Paramedical Laboratory

Su-ming Hsu

Session 23: Paramedical Laboratory

Human Reproduction, 2010

i35 Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010 O-087 Maternal death from ectopic pregnancy and miscarriage J. Neilson 1 1 University of Liverpool, School of Reproductive & Developmental Medicine, Liverpool, United Kingdom One of the three health-related UN Millennium Development Goals (MDGs) seeks to see a 75% reduction in maternal mortality worldwide by the year 2015. This is the MDG that is most off-target, and it is unlikely to be achieved. Progress in reducing maternal mortality can only be demonstrated by accurate population measurements – which can be extremely challenging in low-income countries – which is where the large majority of maternal deaths occur. The UK has had a continuous audit of maternal deaths since 1952. Identiﬁcation of deaths has become very accurate by linking death certiﬁcation with birth certiﬁcation, nationally. The audit is ‘conﬁdential’ in that assessment is made without knowledge of the names of the patients or clinicians or of hospitals. A similar model has been adopted elsewhere e.g. South Africa. Reports are published at 3 yearly intervals and generic messages in recent reports have included the importance of social exclusion and some ethnicities, the growing problem of obesity and its signiﬁcance to maternal mortality, and the signiﬁ- cance of suicide. This presentation will review trends in early pregnancy mater- nal deaths since the early 1950s, and focus on recent important ﬁndings. Deaths from ectopic pregnancy are more common than from miscarriage or from ter- mination of pregnancy. A major concern has been the failure to suspect ectopic pregnancy in women with atypical clinical presentations, notably diarrhoea and vomiting. Deaths from sepsis may be an increasing problem, including deaths after miscarriage. The symptoms and signs of serious, pregnancy-related sepsis may be non-speciﬁc and deterioration can be extremely rapid. There is a need to reinforce the importance of basic clinical observations and encourage the use of monitoring tools such as the Modiﬁed Early Obstetric Warning Score (MEOWS). SELECTED ORAL COMMUNICATION SESSION SESSION 23: PARAMEDICAL LABORATORY Monday 28 June 2010 17:00 - 18:00 O-088 Relationship between DNA damage and sperm head birefringence C.G. Petersen 1 , L.D. Vagnini 2 , C.M. Junta 2 , A.L. Mauri 3 , F.C. Massaro 3 , L.F.I. Silva 4 , J. Ricci 3 , M. Cavagna 5 , A. Pontes 6 , R.L.R. Barufﬁ 3 , J.B.A. Oliveira 1 , J.G. Franco Jr. 1 1 Centre for Human Reproduction Prof. Franco Jr /Paulista Centre for Di- agnosis Research and Training / Department of Gynecology and Obstetrics Botucatu Medical School São Paulo State University - UNESP, Research Unit, Ribeirao Preto, Brazil 2 Paulista Centre for Diagnosis Research and Training, Research Unit, Ribei- rao Preto, Brazil 3 Centre for Human Reproduction Prof. Franco Jr / Paulista Centre for Diag- nosis Research and Training, Research Unit, Ribeirao Preto, Brazil 4 Centre for Human Reproduction Prof. Franco Jr / Paulista Centre for Diag- nosis Research and Training / Postgraduate Fellow Department of Gynecol- ogy and Obstetrics Botucatu Medical School Sao Paulo State University – UNESP, Research Unit, Ribeirao Preto 5 Centre for Human Reproduction Prof. Franco Jr, Research Unit, Ribeirao Preto, Brazil 6 Department of Gynecology and Obstetrics Botucatu Medical School São Paulo State University - UNESP, Research Unit, Botucatu, Brazil Introduction: Sperm head birefringence (SHBF) selection has been proposed to improve ICSI outcomes. Published studies have demonstrated that injection of spermatozoa with partial SHBF improves the development of viable em- bryos and rates of implantation and pregnancy in ICSI cycles. On the other hand, successful assisted reproduction techniques partly depend on the inherent integrity of sperm DNA, since high DNA fragmentation has been associated with increased rates of spontaneous abortion. The objective of this study was to evaluate the relationship between pattern human SHBF and DNA damage. a given period of treatment, we and our patients are becoming liberated from the tyranny of current treatment paradigms. We are at last acknowledging the harm ovarian stimulation does to the embryo, the endometrium in which it must implant, and the woman undergoing treatment who must endure this assault on her physical, psychological and, too often, ﬁnancial health. In this debate we will address these disadvantages, and explore the potential beneﬁts of end- ing our love affair with ovarian stimulation for IVF, and embarking on a new relationship with nature. O-085 Con H. Fatemi 1 1 UZ Brussel, Centre for Reproductive Medicine, Brussels, Belgium The two major complications observed in modern IVF are the occurrence of ovarian hyperstimulation syndrome (OHSS) and multiple pregnancies. In the United States of America and some European countries the multiple pregnancy rates after IVF have been reported to be more than 36%. Multiple pregnancies are the cause of preterm deliveries with low birthweight causing admission to neonatal unit care in more than 63% of those patients. Moreover, Low birth weight is associated with increased adult incidence of cardiovascular disease and associated disorders such as hypertension and diabetes mellitus. To avoid the mentioned complications, two approaches have been proposed: IVF in natural cycle or single stimulated GnRH antagonist cycle triggered by an agonist followed by single embryo transfer (SET) of cryo preserved embryos in natural cycle (FET). By performing IVF in natural cycle, hCG has to be administered for ﬁnal oocyte maturation and oocyte retrieval. Otherwise, the oocyte retrieval can not be planned on an adequate way. However, by the administration of hCG, one can no longer speak about a natural cycle, but an “unstimulated” cycle. More- over, hCG administrated in a natural cycle, seems to have a negative impact on the endometrium and the outcome. About a decade ago, GnRH antagonist protocols for the prevention of a premature LH surge were introduced, allowing ﬁnal oocyte maturation to be triggered with a single bolus of a GnRH agonist (GnRHa). GnRHa is as effective as hCG for the induction of ovulation and apart from the LH surge a FSH surge is also induced. GnRHa triggering of ﬁnal oocyte maturation does posses advantages over hCG triggering in terms of an almost complete elimination of OHSS and the retrieval of more mature oocytes. However, a poor clinical outcome is present, when GnRHa is used to trigger ﬁnal oocyte maturation in IVF/ICSI antagonist protocols, due to an uncorrect- able luteal phase deﬁciency. Since the impact of agonist trigger seems to be on the endometrium and not the oocyte/embryo quality, it has been suggested to cryopreserve the embryos and transfer in consecutive natural cycles. Vitriﬁcation is a cryopreservation technique that leads to a glass-like solidi- ﬁcation, with rapid cooling of cells or tissues. vitriﬁcation of human zygotes at the pronuclear stage is a successful and reliable method with favorable out- comes and has been recommended as a routine technique for cryopreservation of human embryos. Moreover, Limited evidence is available regarding the optimal preparation of the endometrium for implantation in FET cycles. Recently, it was demon- strated that for patients with a regular cycle, FET in a spontaneous natural cycle has high implantation and ongoing pregnancy rates. In conclusion, by performing a single stimulation in a GnRH antagonist cycle triggered by a GnRH agonist followed by Cryo-thawed SET in consec- utive natural cycles, the two major complications of COH for IVF could be eliminated almost completely without jeopardizing the outcome. INVITED SESSION SESSION 22: PREVENTION OF MATERNAL DEATH IN EARLY PREGNANCY Monday 28 June 2010 17:00 - 18:00 O-086 Pregnancy and thromboembolism M. Greaves 1 1 University of Aberdeen, Head of School of Medicine & Dentistry, Aberdeen, United Kingdom Downloaded from https://academic.oup.com/humrep/article-abstract/25/suppl_1/i35/590625 by guest on 06 June 2020

i36 Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010 effects of vitriﬁcation at two extremes of pre-implantation embryo development; two-cell stage embryos and blastocysts with intact and/or artiﬁcial collapsed blas- tocoels as well as on gene expression of two growth related imprinted genes. Methods: Mouse two cell stage pre-implantation embryos were collected from naturally ovulating 6-8 week old CD1 females mated with NMRI males and allocated to 4 groups: I) control; two cell embryos were cultured to blastocyst stage followed by RNA extraction, II) two cell embryos were vitriﬁed and sub- sequently thawed before extraction of RNA, III) two cell embryos were cultured to the blastocyst stage and the blastocoel was mechanically collapsed using an ICSI injection needle before vitriﬁcation, thawing and RNA extraction, IV) same as III) but blastocoel cavities were left intact before vitriﬁcation. All em- bryo culture was done in micro-drops under oil and vitriﬁcation and thawing were performed using RapidVit™/RapidWarm™ Cleave/ Blast kit together with the cryoloop carrier system (All media from Vitrolife AB, Kungsbacka, Sweden). Post thawing, blastocysts were graded for re-expansion and two cell embryos were assessed for survival. Total RNA was extracted and reverse tran- scription was performed. Gene expression of imprinted genes H19 and IGF2 was analyzed by Q-PCR using TaqMan ® system (Applied Biosystems). Results: Embryos vitriﬁed at two cell stage showed reduced survival rate com- pared to embryos vitriﬁed at blastocyst stage with their blastocoel cavity collapsed (78% vs 80% P < 0.05). Blastocysts that vitriﬁed with their blastocoel cavity intact showed the lowest survival rate (52% P < 0.05) compared to the rest groups. H19 expression was reduced in two cell vitriﬁed embryos, increased in collapsed blas- tocysts and non-effected in non-collapsed blastocysts relative to the control. IGF2 expression was reduced at all groups compared to the control group. Conclusion: This data show that vitrifying embryos at the two cell or blastocyst stage with the blastocoel cavity mechanically collapsed, ensures high survival rates upon thaw. On the other hand, when considering long-term outcomes, blastocyst vitriﬁcation with intact blastocoels appear to be the optimum pro- cedure as it does not alter the expression of the H19 gene as compared to the control. IGF2 still manifests aberrant expression when embryos are vitriﬁed at both extremes of pre-implantation development. O-090 Paternity after gonadotoxic therapy as a result of cryopreserved or fresh semen J.M. Janssen 1 , J.C.M. Dumoulin 1 , G.A.J. Dunselman 1 , J.G. Derhaag 1 1 GROW – School for Oncology and Developmental Biology Maastricht Uni- versity Medical Centre, Dept. of Obstetrics & Gynaecology, Maastricht, The Netherlands Introduction: A considerable number of male cancer patients are diagnosed at an age at which they are starting a family. The ability to father children in the future is an important issue for approximately 60% of newly diagnosed patients. Subfertility or sterility can be associated with the disease itself or with its treatment. Radiation therapy and chemotherapy impair spermatogenisis at least temporarily and sometimes permanent. Pre-treatment cryopreservation of semen whether or not combined with ART in a later stage can decrease the rate of involuntary childlessness. The aim of our study was to evaluate to what ex- tent cryopreserved semen was used in trying to achieve a pregnancy. In addition we evaluated how many men fathered a child spontaneously. Materials and Methods: Patient characteristics, indication for cryopreserva- tion, paternity and use of cryopreserved semen of all male patients who banked semen for oncological reasons between 1985 and 2009 at the Maastricht Uni- versity Medical Center, Maastricht, the Netherlands were evaluated retrospec- tively. Patients were contacted by mail using a questionnaire. Results: Between 1985 and 2009 312 patients were referred to our department for sperm banking prior to gonadotoxic therapy. A total of 878 semen sam- ples from 296 men were preserved. From 94% of the approached patients data were collected. Fifty (16.9%) of these patients died without using their banked semen, 85 patients (28.7%) requested disposal of their cryopreserved semen. Twenty-nine men (9.8%) had used their cryopreserved semen to try to achieve a pregnancy. Eventually 10 of these men fathered one or more children (total number 16). Besides that 32 men fathered 40 children after natural conception. Conclusions: Although a small percentage of men used their banked semen in order to achieve a pregnancy, cryopreservation of semen is strongly rec- ommended in men undergoing potentially gonadotoxic treatment. It has to be noted that a remarkably high number of children are born after natural concep- tion. Both ﬁgures should be taken into account in counselling patients request- ing cryopreservation of semen for this indication. Material and Methods: A total of sixteen patients with normal semen had their fresh spermatozoa sample prepared by the layering method and concentration adjusted to 3x10 6 . Therefore, 1μl of the prepared sperm was deposited into a 10μl of PVP microdrop cover by oil in a Corning dish (Ref: 430166), at room temperature, for further sperm SHBF selection. SHBF selection was performed by using an inverted microscope (TE 300, Nikon, Japan) equipped with a cam- era, a C-mount and a 21-inch monitor at 2500x magniﬁcation. Two groups of sperm SHBF selection were evaluated: Group 1: spermatozoa with the presence of SHBF in their whole heads; i.e total SHBF (SHBF-T); Group 2: spermatozoa with the presence of SHBF in 50% of their heads (birefringence was localized in the post-acrosomal region); i.e parcial SHBF (SHBF-P). Experiment 1: A total of 921 spermatozoa with SHBF-T (n = 566) and SH- BF-P (n = 355) were deposited on slides in two different places for further DNA fragmentation assay. DNA fragmentation was determined by TUNEL; slides with the selected SHBF-T/P were air-dried and then ﬁxed at 4ºC in Carnoy’s solution and permeabilised with 0.1% Triton X-100. The smears were then pro- cessed for terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling. The number of DAPI (blue)-stained cells per ﬁeld was ﬁrst counted. Then, in the same ﬁeld, the numbers of red ﬂuorescent cells (TUNEL positive) were calculated. The same technician, blinded to subject identity, performed the whole experiment. Experiment 2: A total of 1476 spermatozoa with SHBF-T (n = 871) and SHBF-P (n = 605) were deposited on slides in two different places for further denatured DNA assay. Denatured DNA strand determination was performed by acridine orange ﬂuorescence. The slides with spermatozoa selected by SHBF- T/P were air-dried, ﬁxed overnight at 4ºC in Carnoy’s solution and then stained by acridine orange solution. Spermatozoa with denatured stranded DNA were determined under a ﬂuorescence microscope with 1000x magniﬁcation and 450–490nm excitation. Spermatozoa with double stranded DNA (normal) were ﬂuorescent green, and those with denatured DNA strands were ﬂuorescent red or yellow. The same technician, blinded to subject identity, performed the whole experiment. Results: Tables 1 and 2 show the results. The percentage of positive DNA frag- mentation in spermatozoa with SHBF-T (120/566; 21.2%) was signiﬁcantly higher (x 2 test; P < 0.0001) than in that in spermatozoa with SHBF-P (37/355; 10.4%). However, the percentage of denatured DNA (521/871; 59.8%) in sper- matozoa with SHBF-T was not signiﬁcantly different (x 2 test; P > 0.05) from that in spermatozoa with SHBF-P (380/605; 62.8%). Table 1. DNA fragmentation values in spermatozoa with total (SHBF-T) and partial (SHBF-P) head birefringence. DNA fragmentation (SHBF-T) (SHBF-P) Positive 120 37 Negative 446 318 P > 0.0001 Table 2. Denatured and double-stranded DNA evaluated by acridine orange ﬂuorescence in spermatozoa with total (SHBF-T) and partial (SHBF-P) head birefringence. DNA (SHBF-T) (SHBF-P) Denatured 521 380 Double-stranded 350 225 P > 0.05 Conclusion: In the present study, the data support a positive relationship be- tween spermatozoa with total SHBF in their heads and increase in DNA frag- mentation. O-089 The effect of vitriﬁcation on imprinted genes H19 and IGF2 in pre-implantation mouse embryos G. Koustas 1 , L. Shaw 1 , C. Sjoblom 1 1 Westmead Fertility Centre, Obstetrics and Gynaecology University of Sydney, Sydney, Australia Introduction: Cryopreservation of supernumerary embryos is an integral feature within an ART clinic, promoting single embryo transfer (SET), reducing multi- ple pregnancies and their associated risks. The use of a panel of different cryo- protectants, together with cooling either through slow-rate freezing or vitriﬁcation, are methods currently applied. Whilst slow-rate freezing is the protocol that has been principally employed to date, vitriﬁcation of embryos is becoming increas- ingly popular, as it has demonstrated a far superior outcome. We have evaluated the Downloaded from https://academic.oup.com/humrep/article-abstract/25/suppl_1/i35/590625 by guest on 06 June 2020

Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010 a given period of treatment, we and our patients are becoming liberated from the tyranny of current treatment paradigms. We are at last acknowledging the harm ovarian stimulation does to the embryo, the endometrium in which it must implant, and the woman undergoing treatment who must endure this assault on her physical, psychological and, too often, financial health. In this debate we will address these disadvantages, and explore the potential benefits of ending our love affair with ovarian stimulation for IVF, and embarking on a new relationship with nature. O-085 Con H. Fatemi1 1 UZ Brussel, Centre for Reproductive Medicine, Brussels, Belgium INVITED SESSION SESSION 22: PREVENTION OF MATERNAL DEATH IN EARLY PREGNANCY Monday 28 June 2010 O-086 17:00 - 18:00 Pregnancy and thromboembolism M. Greaves1 1 University of Aberdeen, Head of School of Medicine & Dentistry, Aberdeen, United Kingdom Maternal death from ectopic pregnancy and miscarriage J. Neilson1 1 University of Liverpool, School of Reproductive & Developmental Medicine, Liverpool, United Kingdom One of the three health-related UN Millennium Development Goals (MDGs) seeks to see a 75% reduction in maternal mortality worldwide by the year 2015. This is the MDG that is most off-target, and it is unlikely to be achieved. Progress in reducing maternal mortality can only be demonstrated by accurate population measurements – which can be extremely challenging in low-income countries – which is where the large majority of maternal deaths occur. The UK has had a continuous audit of maternal deaths since 1952. Identification of deaths has become very accurate by linking death certification with birth certification, nationally. The audit is ‘confidential’ in that assessment is made without knowledge of the names of the patients or clinicians or of hospitals. A similar model has been adopted elsewhere e.g. South Africa. Reports are published at 3 yearly intervals and generic messages in recent reports have included the importance of social exclusion and some ethnicities, the growing problem of obesity and its significance to maternal mortality, and the significance of suicide. This presentation will review trends in early pregnancy maternal deaths since the early 1950s, and focus on recent important findings. Deaths from ectopic pregnancy are more common than from miscarriage or from termination of pregnancy. A major concern has been the failure to suspect ectopic pregnancy in women with atypical clinical presentations, notably diarrhoea and vomiting. Deaths from sepsis may be an increasing problem, including deaths after miscarriage. The symptoms and signs of serious, pregnancy-related sepsis may be non-specific and deterioration can be extremely rapid. There is a need to reinforce the importance of basic clinical observations and encourage the use of monitoring tools such as the Modified Early Obstetric Warning Score (MEOWS). SELECTED ORAL COMMUNICATION SESSION SESSION 23: PARAMEDICAL LABORATORY Monday 28 June 2010 17:00 - 18:00 O-088 Relationship between DNA damage and sperm head birefringence C.G. Petersen1, L.D. Vagnini2, C.M. Junta2, A.L. Mauri3, F.C. Massaro3, L.F.I. Silva4, J. Ricci3, M. Cavagna5, A. Pontes6, R.L.R. Baruffi3, J.B.A. Oliveira1, J.G. Franco Jr.1 1 Centre for Human Reproduction Prof. Franco Jr /Paulista Centre for Diagnosis Research and Training / Department of Gynecology and Obstetrics Botucatu Medical School São Paulo State University - UNESP, Research Unit, Ribeirao Preto, Brazil 2 Paulista Centre for Diagnosis Research and Training, Research Unit, Ribeirao Preto, Brazil 3 Centre for Human Reproduction Prof. Franco Jr / Paulista Centre for Diagnosis Research and Training, Research Unit, Ribeirao Preto, Brazil 4 Centre for Human Reproduction Prof. Franco Jr / Paulista Centre for Diagnosis Research and Training / Postgraduate Fellow Department of Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University – UNESP, Research Unit, Ribeirao Preto 5 Centre for Human Reproduction Prof. Franco Jr, Research Unit, Ribeirao Preto, Brazil 6 Department of Gynecology and Obstetrics Botucatu Medical School São Paulo State University - UNESP, Research Unit, Botucatu, Brazil Introduction: Sperm head birefringence (SHBF) selection has been proposed to improve ICSI outcomes. Published studies have demonstrated that injection of spermatozoa with partial SHBF improves the development of viable embryos and rates of implantation and pregnancy in ICSI cycles. On the other hand, successful assisted reproduction techniques partly depend on the inherent integrity of sperm DNA, since high DNA fragmentation has been associated with increased rates of spontaneous abortion. The objective of this study was to evaluate the relationship between pattern human SHBF and DNA damage. i35 Downloaded from https://academic.oup.com/humrep/article-abstract/25/suppl_1/i35/590625 by guest on 06 June 2020 The two major complications observed in modern IVF are the occurrence of ovarian hyperstimulation syndrome (OHSS) and multiple pregnancies. In the United States of America and some European countries the multiple pregnancy rates after IVF have been reported to be more than 36%. Multiple pregnancies are the cause of preterm deliveries with low birthweight causing admission to neonatal unit care in more than 63% of those patients. Moreover, Low birth weight is associated with increased adult incidence of cardiovascular disease and associated disorders such as hypertension and diabetes mellitus. To avoid the mentioned complications, two approaches have been proposed: IVF in natural cycle or single stimulated GnRH antagonist cycle triggered by an agonist followed by single embryo transfer (SET) of cryo preserved embryos in natural cycle (FET). By performing IVF in natural cycle, hCG has to be administered for final oocyte maturation and oocyte retrieval. Otherwise, the oocyte retrieval can not be planned on an adequate way. However, by the administration of hCG, one can no longer speak about a natural cycle, but an “unstimulated” cycle. Moreover, hCG administrated in a natural cycle, seems to have a negative impact on the endometrium and the outcome. About a decade ago, GnRH antagonist protocols for the prevention of a premature LH surge were introduced, allowing final oocyte maturation to be triggered with a single bolus of a GnRH agonist (GnRHa). GnRHa is as effective as hCG for the induction of ovulation and apart from the LH surge a FSH surge is also induced. GnRHa triggering of final oocyte maturation does posses advantages over hCG triggering in terms of an almost complete elimination of OHSS and the retrieval of more mature oocytes. However, a poor clinical outcome is present, when GnRHa is used to trigger final oocyte maturation in IVF/ICSI antagonist protocols, due to an uncorrectable luteal phase deficiency. Since the impact of agonist trigger seems to be on the endometrium and not the oocyte/embryo quality, it has been suggested to cryopreserve the embryos and transfer in consecutive natural cycles. Vitrification is a cryopreservation technique that leads to a glass-like solidification, with rapid cooling of cells or tissues. vitrification of human zygotes at the pronuclear stage is a successful and reliable method with favorable outcomes and has been recommended as a routine technique for cryopreservation of human embryos. Moreover, Limited evidence is available regarding the optimal preparation of the endometrium for implantation in FET cycles. Recently, it was demonstrated that for patients with a regular cycle, FET in a spontaneous natural cycle has high implantation and ongoing pregnancy rates. In conclusion, by performing a single stimulation in a GnRH antagonist cycle triggered by a GnRH agonist followed by Cryo-thawed SET in consecutive natural cycles, the two major complications of COH for IVF could be eliminated almost completely without jeopardizing the outcome. O-087 Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010 Table 1. DNA fragmentation values in spermatozoa with total (SHBF-T) and partial (SHBF-P) head birefringence. DNA fragmentation (SHBF-T) (SHBF-P) Positive Negative 120 446 37 318 P > 0.0001 Table 2. Denatured and double-stranded DNA evaluated by acridine orange fluorescence in spermatozoa with total (SHBF-T) and partial (SHBF-P) head birefringence. DNA (SHBF-T) (SHBF-P) Denatured Double-stranded 521 350 380 225 P > 0.05 Conclusion: In the present study, the data support a positive relationship between spermatozoa with total SHBF in their heads and increase in DNA fragmentation. O-089 The effect of vitrification on imprinted genes H19 and IGF2 in pre-implantation mouse embryos G. Koustas1, L. Shaw1, C. Sjoblom1 1 Westmead Fertility Centre, Obstetrics and Gynaecology University of Sydney, Sydney, Australia Introduction: Cryopreservation of supernumerary embryos is an integral feature within an ART clinic, promoting single embryo transfer (SET), reducing multiple pregnancies and their associated risks. The use of a panel of different cryoprotectants, together with cooling either through slow-rate freezing or vitrification, are methods currently applied. Whilst slow-rate freezing is the protocol that has been principally employed to date, vitrification of embryos is becoming increasingly popular, as it has demonstrated a far superior outcome. We have evaluated the i36 effects of vitrification at two extremes of pre-implantation embryo development; two-cell stage embryos and blastocysts with intact and/or artificial collapsed blastocoels as well as on gene expression of two growth related imprinted genes. Methods: Mouse two cell stage pre-implantation embryos were collected from naturally ovulating 6-8 week old CD1 females mated with NMRI males and allocated to 4 groups: I) control; two cell embryos were cultured to blastocyst stage followed by RNA extraction, II) two cell embryos were vitrified and subsequently thawed before extraction of RNA, III) two cell embryos were cultured to the blastocyst stage and the blastocoel was mechanically collapsed using an ICSI injection needle before vitrification, thawing and RNA extraction, IV) same as III) but blastocoel cavities were left intact before vitrification. All embryo culture was done in micro-drops under oil and vitrification and thawing were performed using RapidVit™/RapidWarm™ Cleave/ Blast kit together with the cryoloop carrier system (All media from Vitrolife AB, Kungsbacka, Sweden). Post thawing, blastocysts were graded for re-expansion and two cell embryos were assessed for survival. Total RNA was extracted and reverse transcription was performed. Gene expression of imprinted genes H19 and IGF2 was analyzed by Q-PCR using TaqMan® system (Applied Biosystems). Results: Embryos vitrified at two cell stage showed reduced survival rate compared to embryos vitrified at blastocyst stage with their blastocoel cavity collapsed (78% vs 80% P < 0.05). Blastocysts that vitrified with their blastocoel cavity intact showed the lowest survival rate (52% P < 0.05) compared to the rest groups. H19 expression was reduced in two cell vitrified embryos, increased in collapsed blastocysts and non-effected in non-collapsed blastocysts relative to the control. IGF2 expression was reduced at all groups compared to the control group. Conclusion: This data show that vitrifying embryos at the two cell or blastocyst stage with the blastocoel cavity mechanically collapsed, ensures high survival rates upon thaw. On the other hand, when considering long-term outcomes, blastocyst vitrification with intact blastocoels appear to be the optimum procedure as it does not alter the expression of the H19 gene as compared to the control. IGF2 still manifests aberrant expression when embryos are vitrified at both extremes of pre-implantation development. O-090 Paternity after gonadotoxic therapy as a result of cryopreserved or fresh semen J.M. Janssen1, J.C.M. Dumoulin1, G.A.J. Dunselman1, J.G. Derhaag1 GROW – School for Oncology and Developmental Biology Maastricht University Medical Centre, Dept. of Obstetrics & Gynaecology, Maastricht, The Netherlands 1 Introduction: A considerable number of male cancer patients are diagnosed at an age at which they are starting a family. The ability to father children in the future is an important issue for approximately 60% of newly diagnosed patients. Subfertility or sterility can be associated with the disease itself or with its treatment. Radiation therapy and chemotherapy impair spermatogenisis at least temporarily and sometimes permanent. Pre-treatment cryopreservation of semen whether or not combined with ART in a later stage can decrease the rate of involuntary childlessness. The aim of our study was to evaluate to what extent cryopreserved semen was used in trying to achieve a pregnancy. In addition we evaluated how many men fathered a child spontaneously. Materials and Methods: Patient characteristics, indication for cryopreservation, paternity and use of cryopreserved semen of all male patients who banked semen for oncological reasons between 1985 and 2009 at the Maastricht University Medical Center, Maastricht, the Netherlands were evaluated retrospectively. Patients were contacted by mail using a questionnaire. Results: Between 1985 and 2009 312 patients were referred to our department for sperm banking prior to gonadotoxic therapy. A total of 878 semen samples from 296 men were preserved. From 94% of the approached patients data were collected. Fifty (16.9%) of these patients died without using their banked semen, 85 patients (28.7%) requested disposal of their cryopreserved semen. Twenty-nine men (9.8%) had used their cryopreserved semen to try to achieve a pregnancy. Eventually 10 of these men fathered one or more children (total number 16). Besides that 32 men fathered 40 children after natural conception. Conclusions: Although a small percentage of men used their banked semen in order to achieve a pregnancy, cryopreservation of semen is strongly recommended in men undergoing potentially gonadotoxic treatment. It has to be noted that a remarkably high number of children are born after natural conception. Both figures should be taken into account in counselling patients requesting cryopreservation of semen for this indication. Downloaded from https://academic.oup.com/humrep/article-abstract/25/suppl_1/i35/590625 by guest on 06 June 2020 Material and Methods: A total of sixteen patients with normal semen had their fresh spermatozoa sample prepared by the layering method and concentration adjusted to 3x106. Therefore, 1µl of the prepared sperm was deposited into a 10µl of PVP microdrop cover by oil in a Corning dish (Ref: 430166), at room temperature, for further sperm SHBF selection. SHBF selection was performed by using an inverted microscope (TE 300, Nikon, Japan) equipped with a camera, a C-mount and a 21-inch monitor at 2500x magnification. Two groups of sperm SHBF selection were evaluated: Group 1: spermatozoa with the presence of SHBF in their whole heads; i.e total SHBF (SHBF-T); Group 2: spermatozoa with the presence of SHBF in 50% of their heads (birefringence was localized in the post-acrosomal region); i.e parcial SHBF (SHBF-P). Experiment 1: A total of 921 spermatozoa with SHBF-T (n = 566) and SHBF-P (n = 355) were deposited on slides in two different places for further DNA fragmentation assay. DNA fragmentation was determined by TUNEL; slides with the selected SHBF-T/P were air-dried and then fixed at 4ºC in Carnoy’s solution and permeabilised with 0.1% Triton X-100. The smears were then processed for terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling. The number of DAPI (blue)-stained cells per field was first counted. Then, in the same field, the numbers of red fluorescent cells (TUNEL positive) were calculated. The same technician, blinded to subject identity, performed the whole experiment. Experiment 2: A total of 1476 spermatozoa with SHBF-T (n = 871) and SHBF-P (n = 605) were deposited on slides in two different places for further denatured DNA assay. Denatured DNA strand determination was performed by acridine orange fluorescence. The slides with spermatozoa selected by SHBFT/P were air-dried, fixed overnight at 4ºC in Carnoy’s solution and then stained by acridine orange solution. Spermatozoa with denatured stranded DNA were determined under a fluorescence microscope with 1000x magnification and 450–490nm excitation. Spermatozoa with double stranded DNA (normal) were fluorescent green, and those with denatured DNA strands were fluorescent red or yellow. The same technician, blinded to subject identity, performed the whole experiment. Results: Tables 1 and 2 show the results. The percentage of positive DNA fragmentation in spermatozoa with SHBF-T (120/566; 21.2%) was significantly higher (x2 test; P < 0.0001) than in that in spermatozoa with SHBF-P (37/355; 10.4%). However, the percentage of denatured DNA (521/871; 59.8%) in spermatozoa with SHBF-T was not significantly different (x2 test; P > 0.05) from that in spermatozoa with SHBF-P (380/605; 62.8%). Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010 O-091 The effect of culture under low oxygen tension on developmental competence of post-thawed human embryos S. Curnelle1, D. Nogueira1, V. Guitard1, F. Bonald1, N. Guillen1, J. Montagut1 1 IFREARES, Laboratoire de Biologie de la Reproduction, Toulouse, France INVITED SESSION SESSION 24: OVULATION AND FECUNDITY Tuesday 29 June 2010 08:30 - 09:30 O-092 Fecundity in couples trying to conceive naturally - the standard of reference for fertility treatments L. Schmidt1 1 University of Copenhagen, Department of Public Health Section of Social Medicine, Copenhagen K, Denmark Introduction: Fecundability is the probability of obtaining a clinically recognized pregnancy in a menstrual cycle among couples not pregnant in the previous cycle. Among young couples the fecundability is around 20%, and around 45%, 65% and 85% will conceive after 3, 6 and 12 cycles, respectively. Measures of fecundablity, time to pregnancy (TTP) or time to conceive (TTC) are studied either by collecting data retrospectively or prospectively among couples trying to achieve a pregnancy. O-093 Fecundity after ART and non-ART treatments - how does it compare to the reference of natural fecundity? A.N. Andersen1 1 Rigshospitalet, Rigshospitalet, Copenhagen, Denmark Introduction: Defining natural fecundity may be “retrospective” through analysis of time to pregnancy among fertile women who gave birth, or “prospective” studying the monthly probability of pregnancy in cohorts of couples planning pregnancy. Natural fecundity in young couples is around 20%, and 45%, 65% and 85% will conceive after 3, 6 and 12 cycles, respectively. Some treatments may be “curative”: ovulation induction in anovulatory infertility, intrauterine insemination with donor sperm or ICSI in couples with severe male infertility, IVF in tubal infertility or egg donation in WHO type III amenorrhea. Pregnancy rates in such cycles are high, but how do they compare with the efficiency of “natural conception”, based on relevant comparisons: Results: The probability for live birth per treatment cycle versus the natural fecundity (monthly chance of natural conception). Example. The live birth rates using modern ART with elective single embryo transfer in good prognosis cases and multiple embryo transfer to others, are equal to or exceeds those of natural conception, with delivery rates per initiated cycle in the range of 25 - 30% in females below 35 years. With multiple embryo transfer ART delivery rates are clearly higher (30 - 40%). Adding FER cycles further increase delivery rates per single stimulated cycle by 3 - 10%. The observed cumulative probability for live birth over a number of treatment cycles versus a comparable number of months attempting natural conception. Cohort studies shows that following 3 - 6 stimulated cycles ART may give delivery rates around 60% The estimated (Life table statistics) cumulative probability of live birth over a number of treatment cycles (or months) versus a number of months attempt of natural conception. Such statistics is based on the optimistic assumption that pregnancy rates are similar in those patients that continue treatment vs. those that drop out. If applied however, ART is projected to provide delivery rates around 80 - 90% after 6 cycles, indeed higher that natural conception rates (65%) after 6 months/cycles. In ovulation induction the cumulated live birth rates may be 50% over 12 month. Another possibility is to make a “censored or realistic estimate”, where the estimated prognosis of those that drop out are considered. Such delivery rates will be lower, but still higher that the observed. i37 Downloaded from https://academic.oup.com/humrep/article-abstract/25/suppl_1/i35/590625 by guest on 06 June 2020 Introduction: One of the effects that cryopreservation procedure may exert on embryos is the increase in oxidative stress. Oxidative stress is a potentially damaging process for proper cellular function. Culture of thawed embryos under low oxygen tension might alleviate cellular oxidative stress. The aim of this study was to evaluate the effect of culture of human post-thawed embryos in two different O2 concentrations on pre- and post-implantation development. Materials and Methods: This prospective study was performed between 2007 and 2009 and included consecutive patients aged ≤ 38 years old at cryopreservation date and having one or two embryos that survived post-thaw ( ≥ 50% of total number of cells intacted) with < 25% fragmentation. These embryos were frozen by slow method (Embryo Freezing Pack, Medicult) after culture for 2 or 3 days under 6% CO2 and air. Post-thawing (Embryo Thawing Pack, Medicult), embryos from 150 patients were singly placed in a 4-well Nunc dish containing 750 µl G-series Plus media (Vitrolife) and were prospectively randomized per patient in 6% CO2 incubators of two different conditions: ∼20% oxygen (20%-O2) or 5% oxygen (5%-O2). The rate of development was assessed before and after 20-22 hours. One to two embryos were transferred per patient. Only embryos containing at least ≥ 50% intact cells of the total number of cells at freezing were included in the analysis and were transferred. Embryo developmental rate, pregnancy and implantation rates were statistically analyzed. Results: The two groups were comparable regarding female age (5%-O2: 32.4 ± 3.4 versus 20%-O2: 31.3 ± 3.7) and mean number of embryos transferred (n = 113 in 5%-O2, mean: 1.4 ± 0.5; versus n = 107 in 20%-O2, mean: 1.5 ± 0.5). The mean number of cells at start of culture was 5.0 ± 2.0 in 5%-O2 and 4.7 ± 2.0 in 20%-O2 (P = 0.2). At the end of culture, the mean number of cells had significantly increased in both groups (P < 0.01). However, embryos cultured in 5%-O2 had higher number of cells than embryos cultured in 20%-O2 (6.4 ± 2.4 versus 5.7 ± 2.1; P < 0.03). The difference in the mean number of cells gained between the start and the end of culture did not reach statistical significance between 5%-O2 and 20%-O2 groups (1.4 ± 1.8 versus 1.0 ± 1.8 cells). Clinical pregnancy per ET were 25.6% versus 23.9% and implantation rates per gestational sac were 19.5% versus 16.8% for 5%-O2 and 20%-O2, respectively (P > 0.05). There was no difference in miscarriage rates between 5%-O2 and 20%-O2 (20.0 % versus 23.5 %) and delivery/ongoing pregnancy rates were 20.5% and 18.3%, respectively (P > 0.05). Conclusions: This preliminary result suggests a positive effect of culture under low oxygen tension on embryonic cleavage of thawed embryos. Further studies might clarify the role of low oxygen environment on pre-implantation embryonic survival rate. Substantiating, the current results could help in developing strategies for maintaining optimal microenvironment for the pre-implantation frozen-thawed embryo. Results: Retrospective studies are the most frequent and are often based on pregnancy-based study population or population-based studies. The advantages of retrospective TTP-studies are that it is possible to achieve a representative study population, information on TTP is easy to obtain, and the validity of the TTP data even for long-term recall has shown to be high. The disadvantage of using the pregnancy-based study populations is that sterile couples are excluded and infertile couples are systematically under-represented. Hence the TTPvalues are overestimated. TTP refers strictly only to periods of unprotected intercourse that end with conception, and hence do not take into account attempts that does not result in a pregnancy or to pregnancies achieved un-intentionally, e.g. from failure of a contraceptive method. Prospective studies include couples when they initiate their attempts of achieving pregnancy. The advantages is that it is possible to obtain detailed information of data having a possible influence on the fecundability, e.g. frequency and timing of sexual intercourses, ovulation, semen quality, and lifestyle and work exposures. The disadvantages of prospective studies is the need of highly motivated participants causing possible response bias. Which studies to compare with? A couples fecundability is among other things associated with the age of both partners, the frequency and timing of sexual intercourses, sexually transmitted diseases, body mass index, smoking, co-morbidity, and exposures at work. When comparing fecundity after medically assisted reproduction treatment with fecundity after non-treatment related conceptions it is of importance to compare TTP among study populations at least within identical age groups. Conclusion: As fecundability is age-dependent for both women and men it is of importance to compare fecundability of natural conceptions with fecundability after medically assisted reproduction in study populations based on similar age-groups. Furthermore, it is of importance to keep in mind that fecundability studies based on samples of pregnant/delivering women over-estimates TTP as couples not having achieved a pregnancy are excluded and infertile couples are systematically under-represented.

Memories of Civil War: Is a Multi-perspectival historical approach to Memory possible and helpful in understanding the History of the 20th century? Opposing and irreconcilable differences in the understanding of the history of the 20th century, as well as of the conflicting parties in the ideologically motivated war, which raged in the period between 1917 and 1945 (also called »European Civil War« by Ernst Nolte), are not typical only for Croatia. Contemplating the Spanish example can be useful if we want to understand better how Croatian society deals with this historical period. Starting from the question of how the Civil War in Spain (1936-1939) and its participants entered the collective memory of today’s Spain, where »memory wars« are still being fought, the text puts forth the idea that we can learn some lessons from the Spanish example. And not just learn, but use the knowledge to further understand similar examples from Croatian history, a country which sometimes seems to be suffocating from all its historical burdens. One area where a similar treatment can be useful regards the politics of memory in public spaces during the reigns of Franco and Tito. Both glorified the winning side, while the losing side was not even allowed to mark the graves of the victims, let alone build memorials and monuments. Both societies, Spanish as well as Croatian, are burdened by the existence of conflicting cultures of memory. The results of research into the process of »remembering and forgetting« can provide a necessary counterweight to help overcome these resulting mental patterns as currently exist. The paper advocates a comparative approach that would systematically investigate the way memory politics has been shaped in both Spain and Croatia. The comparison of the two could be useful because a traumatic historical legacy, as well as exclusive and mutually opposing collective memories, characterize both Spanish and Croatian society. Considerable periods of the 20th century can also be seen in Croatia as times of »forced utopia politics and mega-death« (Z. Brzezinski). The culmination of the conflict between formations that were in the service of competing totalitarian ideas took place in the territory of Croatia during the Second World War. This text expresses a belief that by sound understanding of the past, we can better understand our own present, especially through dealing with topics from the – for the most part – bloody Croatian 20th century. However, this is only genuinely possible if the sources one is using are relevant and credible, while at the same time being careful to contextualize the facts one comes across in these sources. Therefore, full availability of archival material is necessary; as one of the main requirements for scientific research. Furthermore, it is also necessary to distinguish images and ideas about the past from history arrived at as a result of methodical research. The author advocates for a scientifically based, and historically responsible way of researching so-called »controversial historical topics«. Only when historians speak about these issues of Croatian history by using the methods of critical historical science and »the language of facts and sources«, will it be possible to reach a clear-sighted and rational judgment of events that led to large-scale harassments in the name of totalitarian ideologies. The acceptance of a »multidirectional« collective memory (Michael Rothberg), based on the principles and values of a democratic constitutional order, seems to be a requirement for mutual understanding and reconciliation in society. This implies the acceptance of multiperspectivity and the obvious fact that disparate memories exist in Croatian society. Writing history according to the model of »winners and losers« does not allow the societies in question to get out of this vicious circle. What seems paramount in the research of Croatian history is to undergo a critical reconsideration of all politicized approaches in determining the number of victims of non-democratic regimes in Croatia. Also, it is a mere civilizational necessity to find and mark all the places of the mass graves of victims of all political regimes in the 20th century. In particular, an interdisciplinary approach seems the most suitable if we want to shed light on the complexity of different memories of the war and its impact on contemporary Croatian society. At the same time, multi-perspective historical memory represents an approach to history that promotes diverse perspectives and interpretations of past events and phenomena. Multiperspectivity as a methodological approach is a guard against the creation of one-sided and/or biased narratives about history, especially when it comes to so-called »controversial« or »sensitive« issues. It can contribute to the promotion of communication and reconciliation within society because it enables a conversation between different groups and individuals, which, in the best scenario, can lead to an understanding of the other’s perspective. A multi-perspective and combined approach to the most controversial issues of the Croatian past seems like a good way to develop a higher level of tolerance and a democratic culture of memory within Croatian society, which seems necessary for Croatian society to overcome inherited historical trauma. Analizirajući kako se Španjolski građanski rat (1936. – 1939.) i njegovi akteri poimaju u kolektivnome sjećanju današnje Španjolske u kojoj se još uvijek »ratuje sjećanjima«, u tekstu se problematizira stanje u Hrvatskoj koju također guše »povijesni balasti«, odnosno nastoje se izvući pouke vezane za povijesno nasljeđe i odnos prema nedemokratskim režimima. Kulminacija sukoba formacija koje su služile totalitarnim idejama dogodila se u Hrvatskoj u Drugome svjetskom ratu. Autor se zalaže za znanstveno, argumentirano i povijesno odgovorno istraživanje tzv. kontroverznih povijesnih tema. Prihvaćanje »višesmjernoga« (multidirectional) kolektivnog pamćenja (Michael Rothberg), utemeljenoga na načelima i vrednotama demokratskoga ustavnog poretka, čini se kao preduvjet za međusobno razumijevanja i pomirenje. To implicira prihvaćanje multiperspektivnosti i bjelodane činjenice da u hrvatskom društvu postoje pluralna sjećanja. Multiperspektivno povijesno pamćenje jest pristup povijesti koji promiče raznolike perspektive i interpretacije prošlih događanja i fenomena. Multiperspektivnost kao metodološki pristup brana je stvaranju jednostranih i/ili pristranih povijesnih narativa, posebice o tzv. »kontroverznim« ili »osjetljivim« pitanjima, i može doprinijeti dijalogu i pomirenju u društvu jer omogućuje dijalog različitih zajednica, a što u najboljemu slučaju rezultira razumijevanjem perspektive drugoga. Multiperspektivni i pluralni pristup i prema najkontroverznijim pitanjima hrvatske prošlosti čini se kao dobar put kojim bi se u hrvatskome društvu razvili viši stupanj tolerancije i demokratske kulture sjećanja, što se čini nužnim da društvo prevlada naslijeđene povijesne traume.

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Session 23: Paramedical Laboratory